Microfluidic Drug Discovery
Microfluidic automated fluidic workflows as valuable tools for drug discovery
- Increased throughput and reproducibility
- Minimal reagent consumption
- Fast reaction times
- Automated fluidic workflow
Main applications in microfluidic drug discovery
Target selection and validation (drug synthesis)
When developing a drug target, protein structural and affinity studies are needed for target selection and validation, and protein interactions within cells must be studied. Single-cell protein quantitation, protein analysis in nanoliter droplets, and high-sensitivity ligand-binding interactions can be performed using microfluidic devices.
Hit identification and optimization (drug screening)
In microfluidics for drug development, the potential pool size of drug candidates is estimated to be of the order of 1063. Microfluidic drug screening devices, including high-throughput microfluidic multiplexed systems that contain thousands of micrometer chambers, microwell arrays, or microvalves perform high-throughput screening studies with higher sensitivity and shorter reaction times while decreasing reagents volumes and costs.
Preclinical studies (drug evaluation)
In recent years, alternatives to animal experimentation have become a research hotspot. In microfluidic drug discovery, organs-on- chips and organoids are widely used as alternatives as they emulate the architectural and functional complexity of native organs. These systems also enable the exploration of facets of human disease and development that are not accurately recapitulated by animal models.
Resources
Flow Control Performance and Enhanced Automation for Microfluidic Drug Discovery Applications
- Drug synthesis and droplet microfluidics: When it comes to droplet or particle generation, having control of the fluid delivery system is important. During droplet or particle production, the flow rate of each phase must remain constant and stable to allow the production of monodisperse droplets.
- As experts in fluid control for droplet microfluidics, Fluigent provides droplet generation packages and platforms for researchers. These seamlessly generate micrometer droplets for a wide range of applications. Custom systems are also available to integrate technological and industrial devices.
- Multiplexing for drug screening: More than twenty reagents can be tested simultaneously when performing multiplexing applications on microfluidic devices. In microfluidic drug discovery, automated fluidic workflows are a prerequisite for drug screening. Fluigent developed the Aria to automate multiple fluid deliveries. Reproducibility is improved through complete automation of the fluidic protocol. Fluigent is also able to develop a custom system to fit user specifications and requirements.
- Optimized shear stress and recirculation for organ on a chip studies and cell culture: When performing drug evaluation on 2D/3D cell culture and organs-on-chips, cell culture should be optimized through constant perfusion to enable nutrients/oxygen renewal and by inducing adequate passive stimulation through shear flow as the effect is substantial on cell properties. Sterility on the fluidic path is also a prerequisite. Fluigent develops fluid recirculating systems that perform unidirectional fluid recirculation, ensuring improved and more stable flow rates compared to peristaltic pumps. The system can run for 10 days. Custom systems are also available for microfluidic drug discovery technology integration into industrial devices.
Fluigent Solutions as an Alternative to Syringe Pumps for Better Performance and Automation
Flow rate stability and responsiveness is critical for the microfluidics for drug development applications. Syringe pumps are commonly used during this process. Depending on the model in use, syringe pumps show limited flow control, and the actual flow rate cannot be monitored. In addition, injection volumes are limited by the syringe, and automation can thus be difficult. An alternative to syringe pumps is pressure-based flow controllers. These show high-precision flow control, fast reaction time, and flow monitoring: parameters which are paramount for microfluidic drug discovery applications.
The benefits of choosing Fluigent for your flow control system:
- Best in class stability: < 0.5% thanks to our field-proven, patented FASTAB™ technology allowing optimal flow control with the robustness required in demanding industrial environments
- Workflow automation becomes straightforward with the Fluigent SDK and software included in the system
- Our engineering team members are experts in microfluidic design, mechanical and software integration, and biology applications
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A University College London (UCL) Paper
Paper: Awwad, S.; Ibeanu, N.; Liu, T.; Velentza-Almpani, A.; Chouhan, N.; Vlatakis, S.; Khaw, P. T.; Brocchini, S.; Bouremel, Y. Real-Time Monitoring Platform for Ocular Drug Delivery. Pharmaceutics 2023, 15 (5), 1444. https://doi.org/10.3390/pharmaceutics15051444
Part of the Faculty of Brain Sciences, the UCL Institute of Ophthalmology holds the top global ranking for ophthalmology studies (per CWUR Rankings by Subject 2017). Collaborating with Moorfields Eye Hospital, they form the largest co-located hub for eye research, education, and care worldwide. Through groundbreaking research in ophthalmology and eye health, their interdisciplinary approach unites scientists, clinicians, and patients, leading to tangible improvements in people’s lives.
In collaboration with UCL, Optceutics Ltd. manufactures advanced in vitro models of the human eye (PK-Eye™) that accelerate the development of longer-acting intraocular medicines. Optceutics Ltd. offers fee-for-service research and strategic collaborations, specializing in preclinical ophthalmic formulation optimization.
Challenges in Treating Posterior Eye Diseases
The posterior segment of the eye, where diseases like age-related macular degeneration, glaucoma, and diabetic retinopathy occur, poses significant challenges for treatment due to its complexity. These ocular diseases, which can lead to blindness, affect millions worldwide and are becoming more prevailing with the increase in the aging population.[1] To treat them, therapeutic antibodies and proteins have transformed the management of such conditions, often requiring direct injections into the eye for effective ocular drug delivery (figure 1). However, frequent intravitreal injections are burdensome for patients and healthcare systems, prompting the need for improved drug formulations to prolong efficacy and reduce risks. [2,3] Therefore, formulation strategies are essential to address these challenges, necessitating either progress preclinical evaluation in animal models or in vitro models.
Figure 1: Ocular routes for drug delivery.[4]
How Can in Vitro Models Overcome Limitations in Ocular Drug Delivery Compared to Animal Models?
Classical in-animal models present several limitations in research and drug discovery, including anatomical differences between animal and human eyes. In addition, the formation of anti-drug antibodies complicates the testing of biologics. Ocular tolerability and high costs associated with animal models further highlight the need for alternative testing techniques.[5]
In vitro models offer a promising solution, serving as alternatives or supplements to animal testing during preclinical development. These models not only reduce reliance on animal studies but also help validate dissolution specifications and demonstrate the bioequivalence of active pharmaceutical ingredients. They have been designed for various aims such as assessing protein stability in simulated vitreous fluids, understanding the impact of eye movements, and evaluating drug release/clearance times (figure 2). [6]
Despite significant progress in in vitro ocular formulation testing for posterior eye diseases, there is currently no approved model specifically designed to assess intraocular pharmacokinetics and still lack adequate representation of eye dynamics, such as flow rate and compartmentalization, crucial for understanding drug kinetics and stability within the eye.
Figure 2: Ultimate goals for In-vitro eye models to evaluate ocular drug delivery.[7]
PK-Eye™ Model: Example of Real-Time Monitoring Platform for Ocular Drug Development.
The PK-Eye™ model, designed and manufactured by Optceutics Ltd., emerged as a robust and user-friendly tool, addressing the limitations of in vivo models for developing long-acting intraocular medicines. Combining this in vitro model with real-time monitoring systems will facilitate more efficient preclinical testing by increasing data collection while minimizing manual intervention.[8]
In a paper published in Pharmaceutics (2023), a newly designed real-time monitoring platform was scaled and automated for ocular drug delivery. This platform aims to provide proof-of-concept for a fully automated and optimized PK-Eye™ model testing setup, to accelerate intraocular drug development by streamlining sample analysis and improving accuracy (figure 3).
Key features of the platform included monitoring flow, temperature, eye movements, and concentration evaluation of labeled protein molecules (figure 4).
Figure 3: Schematic of the PK-Eye™ model.[8]
Figure 4: Schematic of the PK-Eye™ platform including Fluigent’s FlowEZ.[7]
How to Scale up Real-Time Monitoring
Each PK-Eye™ model was connected to a FlowEZ microfluidic controller, with buffer (PBS, pH 7.4). These controllers were linked to an FLPG plus 2-bar pressure source. The pressure and flow were managed via the Fluigent A-i-O program (previous version of Oxygen). Scalability was tested with configurations running 1× LineUp Flow EZ controlling 1–6 models (figure 5). Experiments ran at a fixed flow rate of 2 μL/min at 37°C, with a 48-hour equilibration period before data recording. The flow rates were recorded with flow units S.
Figure 5: Experimental setup of flow rate control with 1:6 ratios using PK Eye models in PBS at RT with a pressure control of for 24h. [7]
Figure 6: Experimental setup used to select different flow lines to be analyzed by the concentration detection unit to monitor multiple PK-Eye™ models.[7]
To enable real-time monitoring of drug release profiles across multiple models or fluid inlets, microfluidic valves from Fluigent were integrated into the system. It included three 3-port/2-way microfluidic valves (2-SWITCH), an 11-port/10-position rotary valve (M-SWITCH), and a SWITCHBOARD (Previous version of SWITCH EZ) (figure 6). This platform facilitated the selective collection of outflows from different models or inlets into the concentration detector, allowing for varied release profile readouts.
Next, the integration of this platform with a concentration probe was demonstrated. A PK-Eye™ model with a membrane was connected to the 2-SWITCH, allowing switching between fluid outlets. PBS buffer was pumped through the model while the outflow was alternated between the model and a PBS reservoir. Alexa albumin was injected into the model, and the system toggled between the model and reservoir every 2 hours for real-time detection by the concentration probe.
Proof-of-Concept: Unlock Stability and Scalability Using Flow Controllers vs Peristaltic Pumps
Traditional syringe or peristaltic pumps are commonly used in pharmaceutical labs to impose a constant flow rate, but they often result in cyclical flow rate variations. Fluigent’s pressure-driven controller (FlowEZ) was introduced in this work to allow large-scale experiments, offering smooth flow rates with minimal variation and rapid response to pressure or flow rate changes. Troubleshooting was also easier with this system compared to peristaltic pumps (table 1).
Table 1: Comparison between the PK-Eye platform based on a peristaltic pump system or a flow controller system.
Model-Platform | Flow based on peristaltic pump (1st Generation PK-Eye™) | Flow based on pressure-driven controllers (Latest PK-Eye™ model) |
---|---|---|
Pressure-flow Monitoring | No | Yes (with graph readout) |
Simultaneous models testing (n) | 8 | 48 |
Circadian rhythm | No | Yes (with graph readout) |
Simulated vitreous fluids leakage monitoring | No | Yes (with graph readout) |
Eye movement monitoring | No | Yes, eye movements shown (with graph readout) |
Temperature monitoring | Thermometer | Temperature sensors |
Concentration readout | Manual sampling and HPLC analysis | Manual sampling and HPLC analysis, and use of concentration probe setup |
“A pressure-controlled flow system, as opposed to syringe pumps or peristaltic pumps, was introduced to allow large scale experiments by allowing a single pressure source to control several models simultaneously. The flow rate controlled by compressed air results in an extremely smooth flow rate with quasi-null flow rate variation”
S.Awwad et al., Pharmaceutics 2023,15, 1444.
In parallel systems, pressure remained constant, allowing multiple models to be controlled simultaneously from a single pressure source with the ability to run up to six models concurrently. Ratio experiments demonstrated this, with each model flowing within a natural aqueous humor flow rate range (between 1.5 µL/min and 2.7 µL/min), highlighting the uniformity of the setup (figure 7).
Figure 7: Flow rate and pressure drops within the six PK-Eye™ models in PBS at RT. [ 7]
Figure 8: Graph readout from the microfluidic system showing the flow and pressure from the models with SVF at 37°C. [7]
The microfluidic setup with the PK-Eye™ model ensured consistency and detects simulated vitreous fluids (SVF) leakage during testing, critical for maintaining quality control. Stability tests with the model showed consistent pressure and flow rates over 5 days, indicating successful SVF containment without leakage (figure 8). This highlights the robustness of both the microfluidic system and model setup throughout the experiment.
The integration of the 2-SWITCH/M-SWITCH setup facilitated continuous drug quantification across multiple model post-clearance from the PK-Eye™ model, utilizing a connected concentration probe through the M-SWITCH. The platform effectively selected different fluid outlets automatically from this combination of M-SWITCH and 2-SWITCH, enabling real-time monitoring of drug release between multiple flow outlets (figure 9).
Figure 9: Graph of the different flow rates in time (1.5,2.0 and 2.5 µL/min), and the M-Switch unit flow rate demonstrating the selection of the flow rate through the M-Switch.[7]
Figure 10: Continuous monitoring of Alexa albumin from the concentration probe connected to 2-SWITCH/M-SWITCH platform.[7]
As a proof-of-concept, the concentration-time profile of Alexa albumin injected into the PK-Eye™ was determined using a concentration probe setup. The platform successfully reproduced the drug release curve, reaching a maximum concentration of approximately 89 μg/mL at 20 hours. This demonstrates the automatic selection of channels flowing through the concentration probe (figure 10). Thus, it highlights the potential for automated redirection of different flows to record the concentration of cleared drugs, showcasing the real-time monitoring capabilities of the platform for ocular drug delivery.
Conclusion
The PK-Eye™ model, developed by Optceutics Ltd., fast-tracks intraocular drug development through real-time monitoring on an automated platform. This platform records pressure, flow, temperature, eye movement, and concentration in connected models, helping in ocular drug delivery studies. Computer-controlled microfluidics mimic ocular flow dynamics with high stability and responsiveness, while an eye movement platform studies their impact on drug clearance. The 2-SWITCH/M-SWITCH platform expands monitoring capacity for multiple models simultaneously.
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References:
(1) Ranta, V.; Urtti, A. Transscleral drug delivery to the posterior eye: Prospects of pharmacokinetic modeling. Adv. Drug Deliv. Rev. 2006, 58, 1164–1181.
(2) Jager, R.D.; Aiello, L.P.; Patel, S.C.; Cunningham, E.T. Risks of intravitreous injection: A comprehensive review. Retina 2004, 24, 676–698.
(3) Thrimawithana, T.; Young, S.; Bunt, C. Drug delivery to the posterior segment of the eye. Drug Discov. Today 2011, 16, 270–277.
(4) Souto, E. B.; Dias-Ferreira, J.; López-Machado, A.; Ettcheto, M.; Cano, A.; Camins Espuny, A.; Espina, M.; Garcia, M. L.; Sánchez-López, E. Advanced Formulation Approaches for Ocular Drug Delivery: State-Of-The-Art and Recent Patents. Pharmaceutics 2019, 11 (9), 460.
(5) Laude, A.; Tan, L.E.;Wilson, C.G.; Lascaratos, G.; Elashry, M.; Aslam, T.; Patton, N.; Dhillon, B. Intravitreal therapy for neovascular age-related macular degeneration and inter-individual variations in vitreous pharmacokinetics. Prog. Retin. Eye Res. 2010, 29, 466–475.
(6) Awwad, S.; Henein, C.; Ibeanu, N.; Khaw, P.T.; Brocchini, S. Preclinical challenges for developing long acting intravitreal medicines. Eur. J. Pharm. Biopharm. 2020, 153, 130–149.
(7) Awwad, S.; Ibeanu, N.; Liu, T.; Velentza-Almpani, A.; Chouhan, N.; Vlatakis, S.; Khaw, P. T.; Brocchini, S.; Bouremel, Y. Real-Time Monitoring Platform for Ocular Drug Delivery. Pharmaceutics 2023, 15 (5), 1444.
(8) Liu, T.; Ibeanu, N.; Brocchini, S.; Khaw, P. T.; Bouremel, Y.; Awwad, S. Development of an in Vitro Model to Estimate Mass Transfer from the Anterior Cavity. Front. Drug. Deliv. 2022, 2, 1025029.
A Queen’s University Belfast Paper
Paper: Weaver, E.; Sommonte, F.; Hooker, A.; Denora, N.; Uddin, S.; Lamprou, D. A. Microfluidic Encapsulation of Enzymes and Steroids within Solid Lipid Nanoparticles. Drug Deliv. and Transl. Res. 2023. https://doi.org/10.1007/s13346-023-01398-5.
The Lamprou lab, affiliated with Queen’s University Belfast, specializes in three main areas: nanoparticles for imaging and therapy, lab-on-a-chip technology, and therapeutic implants. Their interdisciplinary approach has driven innovation in healthcare since 2012, developing emerging technologies and novel drug delivery devices. The lab is led by Professor Dimitrios Lamprou, a leading expert in pharmaceutical technologies known for his significant contributions to 3D printing, microfluidics, and nanofibers, with over 150 peer-reviewed publications.The Lamprou lab, in collaboration with the Department of Pharmacy‑Pharmaceutical Sciences (University of Bari Aldo Moro) and Immunocore Ltd., used microfluidics to create eco-friendly lipid-based nanocarriers for encapsulating challenging active pharmaceutical ingredients (APIs).
What are the challenges for biologics and drug delivery?
Biologics are complex molecules derived from living sources, including mRNA vaccines like those used against COVID-19.1,2 Protecting biological drugs from the challenges posed by various inhospitable internal conditions in the body, such as proteases and pH variations, remains a difficult challenge. Since biologics are expensive to produce, optimizing the formulation process is crucial for mass production. There is therefore a growing interest in oral delivery to promote better patient compliance. One promising method is nanoformulation, specifically using solid lipid nanoparticles as a barrier for the active pharmaceutical ingredient post-administration. 3
What makes Solid Lipid Nanoparticles effective for various drug types?
Solid Lipid Nanoparticles have proven their effectiveness in delivering various types of drugs, including chemotherapy drugs, genetic material, and anti-inflammatory medications, through their stability, targeting capabilities, and size.4
SLNs present a special structure with a hydrophobic core and a hydrophilic external layer, enabling them to encapsulate both hydrophilic and hydrophobic substances. The solid core, consisting of solid lipids like waxes and glycerides, is responsible for containing the drugs, particularly hydrophobic ones. On the other hand, the surfactant layer on the outside of an SLN enhances stability and targeted drug delivery and can also contain hydrophilic drugs. The choice of drug significantly influences the components used in lipid-based nanocarrier formulation, with cationic lipid cores and surfactants being important for mRNA and DNA delivery.5
Lipid-based nanoparticles are not limited to SLNs alone, but also include liposomes, niosomes, and exosomes, each with their own distinct properties, as summarized in Table 1.
Figure 1: Structure of an SLN capable of encapsulating an API
Nanoformulation type | Formulation materials | Capable of encapsulating | Advantages | Disadvantages |
Solid Lipid Nanoparticles | • Waxes • Sterols • Surfactants | • Hydrophobic and hydrophilic | • Highly modifiable • High biocompatibility, including non-toxic degradation | • Require a cooling process for solidification • API leakage during storage • Complications caused by crystallisation |
Liposomes | • Phospholipids • Cholesterol | • Hydrophobic and hydrophilic | • Highly modifiable • Used for theranostic purposes • Simple synthesis | • Low skin permeability • Infrequently possess low mechanical strength |
Niosomes | • Non-ionic surfactants • Cholesterol | • Hydrophobic and hydrophilic | • Biocompatible and nonimmunogenic • Improve drug permeation through the skin • Less stringent storage requirements compared to liposomes | • Time-consuming to create • API leakage |
Exosomes | • Lipids • Proteins • Glycoconjugates | • Hydrophobic and hydrophilic • Genetic material | • Used for theranostic purposes | • Complex and expensive to artificially manufacture |
Table 1: Common lipid-based nanoformulations
How does microfluidics enhance SLN production for drug formulation?
Traditional bulk production methods for solid lipid nanoparticles, such as homogenization and microemulsification, have multiple drawbacks including unpredictable particle characteristics, low reproducibility and repeatability, and the environmentally harmful use of solvents.
To address these issues, microfluidics has emerged as a promising approach for lipid-based nanocarrier formulation and encapsulation of drugs.
Microfluidics has gained popularity in drug delivery due to its precise control of flow rates, device design, and mixing angles within submicron channels. These characteristics enable control over particle size, morphology, and encapsulation efficiency, as flow rates have a significant impact on these parameters. The microfluidic approach also improves time efficiency, allowing for continuous production within minutes, unlike traditional batch processes. As a result, microfluidics is particularly well-suited to formulations relying on self-assembly, like liposomes and SLNs, and has been employed for a wide range of APIs, including those used for gene therapy and addressing the COVID-19 pandemic.6,7,8
Figure 3 : Microfluidics, a promising approach for SLN nanoformulation.[9]
Aim of the study
To further explore this area of drug encapsulation, Edward Weaver et al. from the Lamprou Lab aimed to demonstrate microfluidics’ position at the forefront of solid lipid nanoparticle production in general, and for biologic SLNs specifically.
This paper examines the compatibility of trypsin (TRP) and testosterone (TES) for encapsulation in solid lipid nanoparticles using a microfluidic process integrating Fluigent’s Flow EZ. Testosterone was chosen as a positive non-biologic lipophilic API for comparison with previous bulk encapsulation research, while trypsin was evaluated for encapsulating a hydrophilic API in lipid-based nanocarriers. A combination of SLN materials, including tripalmitin (Tri-P), soybean lecithin (LEC), Tween 80 (T80), and cetyl palmitate (CP) in conjunction with Pluronic F68 (P68), was used for nanoformulation using the same experimental setup.
Solid Lipid Nanoparticle Production Method
Fluigent’s Flow EZ was used to synthesize various nanoformulations of solid lipid nanoparticles. The microfluidic device used was a Y-shaped inlet with etched herringbone channels. The overall system was maintained at a temperature of 60°C to ensure complete dissolution of the materials.
Eight different SLN formulations (F1-F8) were created, with materials dissolved at 60°C to match their final concentration (Table 2). For SLNs encapsulating APIs, TRP and TES were used at varying concentrations, with TRP in the aqueous phase and TES in the organic phase. The optimal flow rate ratio was determined to be 5:1 (aqueous to organic), and samples were collected. The ethanol excess was evaporated through vortex stirring, and SLN formulations were then refrigerated at 5°C for 24 hours.
The API-loaded SLNs were characterized by dynamic light scattering (DLS), zeta potential, atomic force microscopy (AFM), differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR).
Partial results: Stable and monodisperse SLNs for high encapsulation efficiency.
Solid lipid nanoparticles produced from various combinations and concentrations (F1-F8) were first evaluated by DLS. When comparing non-loaded SLNs to the ones with API (trypsin or testosterone), a slight increase in particle size upon encapsulation was observed. Formulations F2 (CP, P68) and F8 (Tri-P, LEC, Tween 80) presented the most favorable particle sizes (150 -180 nm) and were thus selected as model formulations for the study. The choice of API has a lesser impact on particle size compared to the choice of materials. In fact, the main factor affecting particle size appeared to be the interaction between the waxy core material and the surfactant layer.
Figure 6 : Particle size measurements for formulations F1–F8.
“However, it was also found in the current study that when using a commercially available chip and the Fluigent system, halving the required concentrations provided more opportune particle diameter. This factor indicates the importance of considering both the system and the microfluidic environment that is being used for formulation. ”
AFM results aligned with DLS analysis, confirming the size range, although slightly enlarged due to drying. It also verified the uniform dispersion of SLNs that was achieved, suggesting great potential for future development.
Figure 7: AFM images obtained for F8 SLN encapsulation: (a) TRP and (b) TES
Regarding stability studies, all of the formulations (F1-F8) appeared to function effectively. In particular, F2 and F8 demonstrated favorable stability characteristics, ensuring consistent and prolonged release.
FTIR spectroscopy confirmed the presence of TRP and TES in SLN formulations. Specific peaks in the spectra indicated the presence of these compounds in the formulations, thus demonstrating their encapsulation within the solid lipid nanoparticles.
Figure 8: Stability for TRP and TES-loaded F2 and F8 formulations, stored at a) 5 °C and b) 37 °C
Figure 9: FTIR spectra for all components of F2 TRP showing (a) F2 TRP-encapsulated SLNs, (b) TRP, (c) CP, and (d) P68
Figure 10: FTIR spectra for all components of F8 TRP showing (a) F8 TRP-encapsulated SLNs, (b) TRP, (c) LEC, (d) T80, and (e) Tri-P
Finally, the encapsulation efficiency (EE) and drug release with F2 and F8 formulation were evaluated. For testosterone, the encapsulation efficiency was high for both nanoformulations, showing a slight improvement over traditionally used encapsulation methods such as emulsification and homogenisation.10,11 Moreover, the release of TES from SLNs was around 65% for F2 and 45% for F8 within 72 hours, in agreement with the therapeutic range.
Regarding the encapsulation of TRP within solid lipid nanoparticles, the EE with the F2 formulation was 47% and showed controlled trypsin release, making it a suitable lead formulation compared to F8 (EE: 7%). This result is promising for future enhancement, taking into consideration that this encapsulation had not been attempted before with microfluidics.
Figure 11: Encapsulation efficiency for F2 and F8 formulations encapsulating both TRP and TES
Figure 12: Drug release displayed as % total release from encapsulated active pharmaceutical ingredient for (a) F2 and (b) F8
Conclusion
In this paper highlight, E. Weaver et al. from the Lamprou lab developed a microfluidic approach for solid lipid nanoparticle production and API encapsulation. Fluigent’s Flow EZ was used to provide controlled and constant flow rates, contributing to the creation of small homogeneous SLNs. Selecting appropriate SLN materials was essential to matching specific APIs due to their varying encapsulation capabilities. The nanoformulation which included low-concentration CP and P68 was shown to be the most promising.12 This microfluidic method was both reproducible and eco-friendly, making it possible to encapsulate previously challenging molecules.
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References
- (1) Anselmo AC, Gokarn Y, Mitragotri S. Non-invasive delivery strategies for biologics. Nat Rev Drug Discovery. 2019;18(1):19–40.
- (2) Schoenmaker L, et al. mRNA-lipid nanoparticle COVID-19 vaccines: structure and stability. Int Pharm. 2021;601: 120586 .
- (3) Mishra V, et al. Solid lipid nanoparticles: emerging colloidal nano drug delivery systems. Pharmaceutics. 2018;10(4):191.
- (4) Mura P, et al. Evaluation and comparison of solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) as vectors to develop hydrochlorothiazide effective and safe pediatric oral liquid formulations. Pharmaceutics. 2021;13(4):437.
- (5) Sommonte F, et al. The complexity of the blood-brain barrier and the concept of age-related brain targeting: challenges and potential of novel solid lipid-based formulations. J Pharm Sci. 2022;111(3):577–92.
- (6) Jaradat E, et al. Microfluidic paclitaxel-loaded lipid nanoparticle formulations for chemotherapy. Int J Pharm. 2022;628:122320.
- (7) Jain V, et al. Microfluidic device based molecular self-assembly structures. J Mol Liq. 2022: 119760.
- (8) Roces CB, et al. Manufacturing considerations for the development of lipid nanoparticles using microfluidics. Pharmaceutics. 2020;12(11):1095
- (9) Arduino I, et al. Preparation of cetyl palmitate-based PEGylated solid lipid nanoparticles by microfluidic technique. Acta Biomater. 2021;121:566–78.
- (10) Tajbakhsh M, et al. An investigation on parameters affecting the optimization of testosterone enanthate loaded solid nanoparticles for enhanced transdermal delivery. Colloids Surf A. 2020;589:124437.
- (11) Doktorovova S, Souto EB, Silva AM. Hansen solubility parameters (HSP) for prescreening formulation of solid lipid nanoparticles (SLN): in vitro testing of curcumin-loaded SLN in MCF-7 and BT-474 cell lines. Pharm Dev Technol. 2018;23(1):96–105.
- (12) Weaver, E.; Sommonte, F.; Hooker, A.; Denora, N.; Uddin, S.; Lamprou, D. A. Microfluidic Encapsulation of Enzymes and Steroids within Solid Lipid Nanoparticles. Drug Deliv. and Transl. Res. 2023.
The Lamprou Lab (Queen’s University Belfast)
The Lamprou lab, affiliated with Queen’s University Belfast, specializes in three main areas: nanoparticles for imaging and therapy, lab-on-a-chip technology, and therapeutic implants. Their interdisciplinary approach has driven innovation in healthcare since 2012, developing emerging technologies and novel drug delivery devices. The lab is led by Professor Dimitrios Lamprou, a leading expert in pharmaceutical technologies known for his significant contributions to 3D printing, microfluidics, and nanofibers, with over 150 peer-reviewed publications.
What are engineered nanoparticles?
Nanotechnology gained prominence after Richard P. Feynman’s 1959 famous lecture, “There’s plenty of room at the bottom”.1 However, the use of nanoparticles can be traced back to ancient times, as seen in the Romans’ incorporation of nanoparticles into glass manufacturing in the fourth century AD. The Lycurgus cup, an artifact from this period, notably displayed distinctive color changes under various lighting conditions due to the integration of nano-glass particles.2
In modern times, nanotechnology has developed into a comprehensive scientific discipline with diverse applications spanning multiple industries. From water purification and information technologies to drug development, environmental solutions, and the creation of robust yet lightweight materials, nanotechnology has emerged as a pivotal player.3,4
The fundamental units of nanotechnology are nanoparticles, defined as small particles ranging from 10 nm to 1000 nm in size. Engineered nanoparticles, specifically designed with dimensions under 100 nm, play a crucial role in manipulating materials at the molecular and atomic levels, demonstrating significant chemical, structural, electrical, biological and mechanical characteristics. They are classified into categories including ceramic, carbon-based, semiconductor, metal, lipid-based and polymeric nanoparticles.
What makes engineered nanoparticles promising in nanomedicine?
Engineered nanoparticles hold promise for various nanotechnology applications in medicine, including in-vivo and in-vitro diagnosis, drug delivery, and production of biocompatible materials. They are characterized by their high mass-to-surface area ratio, ability to adsorb and carry compounds, and quantum properties.5,6
These characteristics can benefit the drug delivery field, as its primary objective is to enhance the specificity of drug targeting, improve safety and biocompatibility, and reduce toxicity while maintaining therapeutic effects. Nanoparticles with dimensions less than 100 nm are considered excellent drug carriers due to their unique biological and physiological properties, allowing them to cross tissue and cell barriers effectively.7
Engineered nanoparticles also exhibit a higher likelihood of cellular uptake due to their larger surface area, enabling increased protein loading. The interaction of NPs with biological substances results in the formation of a “protein corona,” enhancing nanocarriers’ uptake by the reticuloendothelial system. This protein corona can serve as a functional carrier for targeted drug delivery, improving poorly soluble drug uptake, directing drugs to specific locations, and enhancing drug bioavailability.8
Overall, engineered nanoparticle applications in nanomedicine have been progressing recently, particularly in controlled drug delivery, nucleic acid-based treatment, cancer cell targeting, angiogenesis inhibition, and inflammation control.
How are engineered nanoparticles traditionally produced
The success of nanoparticles as drug carriers and in nanomedicine applications depends on a number of crucial factors, including NP fabrication strategies, physical properties, drug loading efficiencies, drug release potential, and especially the carrier’s toxicity.
Lipid-based nanoparticles exhibit low toxicity in in-vivo experiments. They can carry both hydrophilic and hydrophobic molecules, leading to prolonged half-life and controlled drug release. Here, we focus on two main classes of lipid-based nanoparticles: liposomes and solid lipid nanoparticles.
Liposomes
A liposome is a microsphere lipid constructed from one or multiple phospholipid bilayers, closely mirroring the structure of cell membranes. The liposome preparation process consists of three main stages: preparing aqueous and lipid phases, primary processing with lipids, and optional secondary processing steps. Most methods involve dissolving phospholipids in an organic solvent, with subsequent removal of the solvent through evaporation—a critical step in liposome formation. Two straightforward methods of liposome synthesis are film hydration and solvent injection.
Liposomes generated through film hydration tend to be polydisperse. Parameters like the duration of rotary evaporation, mixing speed, and temperature after hydration affect liposome quality, emphasizing the need for careful monitoring.10,11
The major factors to consider for use of the solvent injection method are the temperature during injection and the injection rate. These factors will affect the size, shape, and polydispersity of the liposomes produced. The solvent injection method involves certain challenges, with continuous exposure of therapeutic substances to high temperatures and organic solvents affecting liposomal product stability and safety.
This results in high polydispersity and a non-homogeneous formulation.12
Solid Lipid Nanoparticles
Solid lipid nanoparticles (SLN) have emerged as a promising alternative to other lipid formulations like liposomes or polymeric nanoparticles. These spherical colloidal particles, ranging from 10 to 1000 nm in size, offer controlled drug release due to limited drug mobility in the solid lipid. SLNs provide advantages such as targeted drug delivery, controlled release, increased stability, scalability in production, and avoidance of organic solvents. Comprising a solid lipid core in an aqueous medium with a surfactant, SLNs use different lipids (steroids, fatty acids, triglycerides, etc.) and require stabilizing agents like surfactants or emulsifiers. The drug insertion process depends on drug hydrophobicity, solid lipid category, and polymeric alterations in the lipid. Production techniques include high-pressure homogenization, solvent evaporation, ultrasonication, hot homogenization, microemulsion, and others.13,14
Table: comparison of the different manufacturing methods used for polymeric NPs and lipid-based NPs
Nanoparticles Type | Manufacturing Method | Advantages | Disadvantages |
---|---|---|---|
Lipid formulation | Film hydration | – Established method – Understood method | – High consuming of the organic solvents – High PDI – Lack of reproducibility – Need for additional downsizing step – Difficulties in scaling-up |
Lipid formulation | Solvent injection | – Simple and fast – Scaling-up possibility | – Exposing to organic solvent – High PDI – Stability problems |
Lipid formulation | Extrusion | – Uniform and homogenous formulation | – Possible clogging of the membrane pores. – Difficulties in scaling-up |
Lipid formulation | High pressure homogenization | – Scaling-up possibility – Uniform formulation | – High energy consumption – Multiple steps – Bulky system |
Lipid formulation | Microemulsion | – Small particle size – Homogenous formulation | – Difficulty in removing the excess water – Use high concentration of surfactants |
Nanoparticles Type | Manufacturing Method | Advantages | Disadvantages |
---|---|---|---|
Polymeric | Emulsification-salting out | – Avoids surfactants and chlorinated solvents | – Need for purification steps – Encapsulate lipophilic drugs only |
Polymeric | Emulsification solvent diffusion | – Scaling-up possibility – Batch-to-batch reproducibility | – The possible diffusion of the hydrophilic drug into the aqueous phase – The need to eliminate high volume of aqueous phase from the colloidal dispersion |
Polymeric | Emulsification- evaporation | – Simple and versatile | – Risk of nanodroplets coalescence during the evaporation process – Time consuming |
Polymeric | Dialysis | – Effective and simple method – Produce polymeric nanoparticles with narrow distribution | – Time consuming – Use of high amount of dialyzing medium, which stimulate the premature release of NPs content |
Polymeric | Nonparticipation | – Simple and established method – Use low concentrations of surfactant | – Restricted for lipophilic drugs – Low polymer concentration obtained |
How does microfluidics enhance the properties of engineered nanoparticles
The principal innovation of microfluidics is the ability to transfer the traditional bulk technique to microscale fluidic chips. Solvents can be mixed within microchannels by a pumping system with continuous laminar flow. This type of flow offers high mixing quality and enhances the performance of microscale devices. The ability to adjust the flow rate ratio (FRR) and total flow rate (TFR) allows for continuous production of monodisperse and homogenous engineered nanoparticles.
Fluigent’s pressure-driven controllers and flow sensors enable this high stability and fast response in fluid flow, which is highly challenging to achieve with traditional pumping methods like peristaltic or syringe pumps. Overall, this method offers high reproducibility and low batch-to-batch variations. In addition, the method’s versatility makes the encapsulation process faster while keeping encapsulation efficiency high.
Microfluidics offers a fast, simple, single-step technique for liposome manufacturing, but challenges for large-scale production currently hinder its wide-scale implementation due to high costs.15,16
Most microfluidics-based solid lipid nanoparticle manufacturing follows a similar procedure involving dissolving lipids and drugs in an organic solvent, which is then introduced into the microfluidic device alongside an aqueous phase with a surfactant. Control over lipid-to-drug concentration, flow rate, and velocity influences the final nanocarrier characteristics.
Solid lipid nanoparticles produced by microfluidic processes have smaller particle sizes, better homogeneity, and higher encapsulation efficiency compared to bulk methods. In addition, cytotoxic studies demonstrate potent anti-proliferative effects of microfluidic SLNs in cancer cell lines.
However, research on microfluidic-produced SLNs is limited, and challenges exist, particularly with regard to the material used for microfluidic chips, with PDMS-based chips being sensitive to organic solutions.17
Conclusion
In this paper highlight, we presented the promise and challenges of microfluidics technology for the design and formulation of nanomedicines. While traditional methods face limitations in producing small engineered nanoparticles with desirable characteristics, microfluidic systems offer a one-step, controllable process with improved outcomes.
Comparative studies have shown that nanocarriers produced by microfluidics exhibit superior properties. Future advancements in microfluidic systems, combined with complementary tools like process analytical technology and molecular imaging technologies, are expected to optimize NP production and expand their medical applications.
Microfluidics holds promise for shaping the future of engineered nanoparticle research and development.
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Microfluidics Article Reviews Solid lipid nanoparticles for biologics and drug encapsulation Read more
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Microfluidics Article Reviews A mRNA encapsulation platform integrating Fluigent’s FlowEZ Read more
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Expert Reviews: Basics of Microfluidics Microfluidics for vaccine development Read more
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Microfluidic Application Notes Liposome Nanoparticle Synthesis Read more
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Expert Reviews: Basics of Microfluidics Flow control for droplet generation using syringe pumps and pressure-based flow controllers Read more
References
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(2) Bayda, S.; Adeel, M.; Tuccinardi, T.; Cordani, M.; Rizzolio, F. The History of Nanoscience and Nanotechnology: From Chemical-Physical Applications to Nanomedicine. Molecules 2019, 25, 112. Schoenmaker L, et al. mRNA-lipid nanoparticle COVID-19 vaccines: structure and stability. Int Pharm. 2021;601: 120586 .
(3) Grobert, N.; Hutton, D. Nanoscience and nanotechnologies: Opportunities and uncertainties. Lond. R. Soc. R. Acad. Eng. Rep.2004, 46, 618.
(4) Thiruvengadam, M.; Rajakumar, G.; Chung, I.M. Nanotechnology: Current uses and future applications in the food industry.3 Biotech 2018, 8, 74.
(5) Duncan, R. The dawning era of polymer therapeutics. Nat. Rev. Drug Discov. 2003, 2, 347–360.
(6) De Jong, W.H.; Borm, P.J. Drug delivery and nanoparticles:applications and hazards. Int. J. Nanomed. 2008, 3, 133–149.
(7) LaVan, D.A.; McGuire, T.; Langer, R. Small-scale systems for in vivo drug delivery. Nat. Biotechnol. 2003, 21, 1184–1191.
(8) Nel, A.E.; Mädler, L.; Velegol, D.; Xia, T.; Hoek, E.M.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Understanding biophysicochemical interactions at the nano-bio interface. Nat. Mater. 2009, 8, 543–557.
(9) Mitchell, M.J., Billingsley, M.M., Haley, R.M. et al. Engineering precision nanoparticles for drug delivery. Nat Rev Drug Discov 20, 101–124 (2021).
(10) Jaradat, E.; Weaver, E.; Meziane, A.; Lamprou, D. A. Microfluidics Technology for the Design and Formulation of Nanomedicines. Nanomaterials 2021, 11 (12), 3440.
(11) Alam, S.; Mattern-Schain, S.; Best, M. Targeting and triggered release using lipid-based supramolecular assemblies as medicinal nanocarriers. In Comprehensive Supramolecular Chemistry II; Elsevier: Oxford, UK, 2017; pp. 329–364.
(12) Maherani, B.; Arab-Tehrany, E.; Mozafari, M.R.; Gaiani, C.; Linder, M. Liposomes: A review of manufacturing techniques and targeting strategies. Curr. Nanosci. 2011, 7, 436–452.
(13) Mehnert, W.; Mäder, K. Solid lipid nanoparticles: Production, characterization and applications. Adv. Drug Deliv. Rev. 2001, 47,165–196.
(14) Uner, M.; Yener, G. Importance of solid lipid nanoparticles (SLN) in various administration routes and future perspectives. Int. J.Nanomed. 2007, 2, 289–300.
(15) Weaver, E.; Uddin, S.; Cole, D.K.; Hooker, A.; Lamprou, D.A. The Present and Future Role of Microfluidics for Protein and Peptide-Based Therapeutics and Diagnostics. Appl. Sci. 2021, 11, 4109.
(16) Guimarães Sá Correia, M.; Briuglia, M.L.; Niosi, F.; Lamprou, D.A. Microfluidic manufacturing of phospholipid nanoparticles: Stability, encapsulation efficacy, and drug release. Int. J. Pharm. 2017, 516, 91–99.
(17) Arduino, I.; Liu, Z.; Rahikkala, A.; Figueiredo, P.; Correia, A.; Cutrignelli, A.; Denora, N.; Santos, H.A. Preparation of cetylpalmitate-based PEGylated solid lipid nanoparticles by microfluidic technique. Acta Biomater. 2021, 121, 566–578.
Table of contents
I. Droplet-based microfluidics, a microfluidic chip application
Microfluidic generation of droplets has attracted a lot of interest, enabling high throughput experiments by generating millions of micro-reactors and chambers in a few seconds. This microfluidic chip application method produces highly monodispersed droplets of very small volumes (μL to fL) of fluids with high frequency (up to hundreds of kHz), providing better control of processes like mixing, encapsulation, sorting, and sensing. Microfluidic-based droplets have many diverse and varied applications, such as particle synthesis3 and chemical analysis4. Highly controlled droplet production also enables single-cell analysis or drug testing 5,6.
Microfluidic-based droplet generation and control allow for:
- Highly monodispersed (<2% size variation) droplet production, with potentially high generation rate (up to hundreds of kHz)
- Highly reproducible complex structures (water-in-oil-in-water emulsions, multiple encapsulations…)
- Single droplet manipulation as an individual pL scale biochemical reactor
- Miniaturization of production and bioanalytical devices
With these characteristics, droplet microfluidics has a large value, including bio(chemical) analysis, and nano and microscale generation of materials7. Several microfluidic chip designs exist to generate droplets for a various field of applications. A common design is the T-junction, where the dispersed phase is injected from a channel that is perpendicular to the channel carrying the continuous phase (figure 1).
Application of microfluidic chip using droplet microfluidic concepts, components, and processes are now being adopted and leveraged by end-users to enable new science and innovation. Real-world success is now evidenced through a range of mainstream commercial products that are applied to key biological and healthcare-related problems (e.g., 10X Genomics, Drop-seq, and nucleic acid quantification via Droplet DigitalTM PCR systems)8.
Today, it is possible to produce multiple emulsions with complex droplet morphologies. Production of multi-cored droplets (droplets that contain a controlled number of inner droplets at one or more hierarchical levels), Janus droplets (i.e. biphasic or triphasic droplets with two or three physically and chemically distinct surface domains) is now possible using droplet microfluidics9 (figure 2).
Figure 2: Types of multiple emulsions from the simple encapsulation (a), double emulsion (b), and so on (c,d) to bi- and tri-phasic structures (e to h), multiple encapsulations (I to k), and hybrid microjet achievable using droplet-based microfluidics9
Microfluidic chip application for the production of highly reproducible PLGA microparticles
In recent years, biodegradable microspheres/microparticles have gained widespread importance in the delivery of bioactive agents10. The copolymers of Poly (D, L-lactic-co-glycolic acid) (called PLGA)/poly (lactic acid) PLA microparticles are one of the most successful new drug delivery systems (DDS) in labs and clinics. Because of their biocompatibility and biodegradability, they can be used in various areas, such as long-term release systems, vaccine adjuvant, and tissue engineering11.
In PLGA microparticle production for drug release and delivery, microparticle size is a key parameter as it is directly related to the microparticles degradation rate and the drug release rate12. Although PLGA microparticle synthesis appears to be a successful drug delivery system, the current processes and tools to produce PLGA microparticles have many limitations, such as wide microparticle size distribution, poor repeatability, and aggressive chemical preparation conditions11. To solve these problems, droplet-based microfluidics application chip offers an efficient method for improvement.
Fluigent provides the Raydrop: a new breakthrough technology leading to outstanding particle size monodispersity and production flexibility. The Raydrop works as a co-flow focusing principle (figure 3). The nozzle and outlet capillary are aligned in a continuous phase chamber, the dispersed phase comes through the nozzle to create the microparticles into the continuous phase and exits by the outlet insert. Using this application of microfluidic chip method, PLGA microparticles ranging from 15 to 50 µm diameters have been successfully generated.
The PLGA microparticle production station allows excellent reproducibility and significantly improved monodispersity (CV < 2%) as compared to other methods on the market. It allows one to continuously produce PLGA microparticles (up to 10 000 droplets/s) without unwanted interruption for long-term experiments.
Apllication notes:
A microfluidic chip application for single-cell mRNA-seq sequencing using droplets: Drop-seq technology
The production of highly monodispersed emulsions or more complex structures (water-in-oil-in-water emulsions, multiple encapsulations…) makes microfluidic chip applications an excellent approach for single-cell analysis or single-cell culture. The technique allows for droplet-based single-cell RNA-sequencing, such that one can characterize complex tissues with many cell types and states under diverse conditions. One of the pioneering microfluidic chip application methods is Drop-Seq technology, which entraps a single cell and a single primer-barcoded bead in each droplet (figure 4).
Cells are thus separated into nanoliter-sized aqueous droplets, with a different barcode associated with each cell’s mRNAs. The primers on beads contain a barcode consisting of three sequences. One sequence is for PCR amplification and is common to all the beads. The second sequence consists of hundreds of individual primers that also share the same ‘‘cell barcode’’.
Finally, the third part has different unique molecular identifiers (UMI), enabling mRNA transcripts to be digitally counted13 (figure 5). The droplets are sequenced altogether, allowing quick profiling of thousands of individual cells from a heterogeneous population.
The power of this microfluidic chip application technology resides in the fact that during sequencing, one can distinguish where the original information came on a cell to cell basis.
This allows one to make a gene expression map of the cell, or even to distinguish cell populations within a tissue.
Apllication notes:
II. Microfluidic cell culture for a better cell behavior understanding
Microfluidic cell culture is another application of microfluidic chip that has significant advantages over macroscopic culture in flasks, dishes, and well-plates14 (figure 6). The microfluidic chip fabrication process allows great flexibility in the design of microfluidic devices, permitting one to understand and control interactions between cells, substrates, and the surrounding medium, physically as well as biochemically15.
This microfluidic chip application technology offers new possibilities to accurately reproduce the cellular environment and enables the analysis of biological processes that were not accessible before. Morphology-wise, chips can be structured at the cell scale to reproduce the mechanical constraints experienced by cells. Biochemically, stable gradients can be implemented with a high spatial resolution (typically, micrometer resolution).
Finally, constant perfusion enables the continuous renewal of nutrients and oxygen to promote cell growth and maintain optimal activity during long-term cell culture. Cost reduction due to volume reduction is also a major benefit15.
Microfluidic chip application model of a tumor microenvironment
The physical microenvironment of tumors is characterized by heterotypic cell interactions and physiological gradients of nutrients, waste products, and oxygen. This tumor microenvironment has a major impact on the biology of cancer cells and their response to chemotherapeutic agents. Despite this, most in vitro cancer research still relies primarily on cells grown in 2D and in isolation in nutrient and oxygen-rich conditions.
Ayuso et al. presented an easy-to-use microfluidic chip application device that can mimic the three-dimensional architecture of multicellular spheroids, while at the same time generating a visible, live “tumor slice” that allows easy monitoring of cells in different regions of the microenvironment in real-time as well as their response to different drugs17 (figure 7).
In this application of microfluidic chip setup, tumor cell behavior in different regions of the microdevice was studied and analyzed in conjugation with measurements of hypoxia and glucose concentrations across the device. The differential cellular response to several well-known drugs in different parts of the microdevice emphasizes the potential of this technology for analyzing the impact of microenvironmental parameters on drug response.
The figure presents microdevices in a Petri dish containing a central culture chamber and 6 channels. To better understand how the chip works, picture B shows one microdevice filled with (yellowish) collagen hydrogel flowing to the microchamber from the right middle channel and blue-colored water perfused through the two lateral microchannels.
In experimental conditions, the culture medium perfused through the lateral microchannels provides nutrients and oxygen creating physiological gradients across the device. Cells near the ‘surrogate’ blood vessels are viable, whereas oxygen-poor cells in the center of device start to die creating a ‘necrotic core’ similar to the necrotic regions of tumors. It is possible to monitor cells with fluorescent dye in microdevice17 (picture D).
Application notes & expertises:
- Assess Cell Proliferation Using Pressure as a Tool
- Creating a Microfluidic Cancer-on-Chip Platform
- Cancer Cell Analysis Made Easy with Aria: cell Capture and Labeling
- Passive and active mechanical stimulation in microfluidic systems
- Mimicking in-vivo environments: biochemical and biomechanical stimulation
III. Organ on a chip, a cutting edge application of microfluidic chip
Many efforts are devoted to the development of cancer metastasis models that can help in understanding the disease and the development of innovative therapeutic strategies. Current in vitro and in vivo cancer models are incapable of satisfactorily predicting the outcome of various clinical treatments on patients18. Therefore, new application of microfluidic chip methods and approaches for drug discovery and health research are being developed. The concept of mimicking the organ-level function of human physiology or disease using cells inside a microfluidic chip application setup was first published in 2004. In 2010 that the term organ-on-a-chip (OOAC) was invented by Ingber, et. al., who developed a microfluidic chip model to capture organ-level functions of the human lung19.
Microfluidic chip applications enable one the unique ability to control the cellular microenvironment with high spatiotemporal precision and to present cells with mechanical and biochemical signals in a more physiologically relevant context19. The manipulation of the micro-liter volumes of liquids has made these models a platform where scaling, and dynamic crosstalk between cells can be achieved. Microfluidic chips can now use geometries and structures to permit the use of physiological length scales, concentration gradients, and the mechanical forces generated by fluid flow to mimic the in vivo microenvironment experienced by cells20.
These biomimetic applications platforms overcome many drawbacks encountered with conventional tissue culture models. OOAC engineering microfluidic chip application has attracted enormous interest and attention from the pharmaceutical industry, regulatory agencies, and even national defense agencies. This is demonstrated by the increase of OOAC research papers and by the emergence of at least 28 organ-on-a-chip companies in less than seven years21.
A microfluidic chip design to reconstitute organ-level lung functions
To demonstrate that it is possible to engineer a microsystem that replicates the complex physiological functionality of living organs, Huh et al. developed a multifunctional microdevice that reproduces key structural, functional, and mechanical properties of the human alveolar-capillary interface, which is the fundamental functional unit of the living lung19. The microfluidic chip application device consisted of compartmentalized PDMS microchannels to form an alveolar-capillary barrier on a thin, porous, flexible PDMS membrane coated with the extracellular matrix and human alveolar epithelial cells and human pulmonary microvascular endothelial cells is cultured on opposite sides of the membrane (figure 8).
In fact, the device recreates physiological breathing movements (shown in Figure 8.B) by applying vacuum to the side chambers and causing mechanical stretching of the PDMS membrane forming the alveolar-capillary barrier. The device is made of 3 PDMS layers that are bonded to form two sets of three parallel microchannels. (Figure 8.C) PDMS etchant is flowed through the side channels in order to form the two large side chambers. (Figure 8.D). Figure 8.E represents an image of an actual lung-on-a-chip microfluidic device19.
To put it in a nutshell, air is subsequently introduced into the compartment to create an air-liquid interface to mimic the lining of the alveolar air space19 .
Using this microfluidic chip application platform, the authors demonstrated that breathing motions, simulated by the organ-on-chip platform, might greatly accentuate the proinflammatory activities of silica nanoparticles and contribute substantially to the development of acute lung inflammation19. This behavior could not be determined using existing in vitro models.
Application notes & expertises:
- Peristaltic Pump vs Pressure-Based Microfluidic Flow Control for Organ on Chip applications
- Long-term fluid recirculation system for Organ-on-a-Chip applications
- CNRS/UTC: study of a liver-on-a-chip model
- Development of a human gut-on-chip to assess the effect of shear stress on intestinal functions
- Cartilage-on-a-chip, an example of complex mechanical stimulation using Fluigent’s technology
- Creating a Microfluidic Cancer-on-Chip Platform using Fluigent’s High Throughput Cell Perfusion Pack
IV. Particle and cell sorting applications using cell sorter chips
Efficiently isolating and organizing cells from complex mixtures is a crucial task in various fields such as biology, biotechnology, and medicine. Microfluidic chips play a pivotal role in this process, commonly employed to enrich or purify cell samples, thereby enhancing efficiency in research and development22.
Traditionally, optical methods like FACS (Fluorescent Activated Cell Sorting) are used for cell detection. FACS utilizes a laser beam, with the scattered light providing characteristic information about cells and their components. Automated and robust, FACS platform has been a gold standard for cell sorting. However, current commercial platforms face limitations in sample throughput and processing speeds, posing challenges for generating clinical-scale samples.
In contrast, platforms with microfluidic chip offer affordability, simplicity, and a smaller footprint. These chips employ various techniques for cell sorting, each with specific speeds and efficiencies. The chip’s dimensions accommodate diverse cell sorters, ranging from large-volume to precise single-cell sorters.
Additionally, microfluidic cell sorting can integrate various fluidic operations within a single chip, making it versatile for lab-on-a-chip applications, diagnostics, and therapeutics. This approach holds promise in both academic and industrial labs.
Cell sorter in microfluidic devices relies on determining specific cell parameters, such as size, shape, density, or surface markers. Heterogeneous cell solutions are injected into the microfluidic chip, where cells with different properties experience varying forces, leading to their separation into different streamlines and exits.
Microfluidic cell sorting can be categorized into three categories:
- Fluorescent label-based,
- Bead-based
- Label-free cell sorting22
Label-free sorting is perhaps the most studied and comprises both active systems (relying on external fields for sorting) and passive systems that don’t use fluorescent labels or beads. Instead, these methods leverage inherent differences in cellular morphology between cell groups 22.
Inertial microfluidics for continuous particle separation in spiral microchannels
Kuntaegowdanahalli et al. developed a simple inertial microfluidic device for continuous multi-particle separation, using the Dean-coupled inertial migration principle in spiral microchannels.
In this innovative design, dominant inertial forces, combined with the Dean rotational force arising from the microchannel’s curved geometry, cause particles to settle at a single equilibrium position near the inner microchannel wall. The specific position of particle equilibrium is determined by the ratio of inertial lift to Dean drag forces.
The researchers applied this principle to create a spiral lab-on-a-chip, showcasing size-dependent particle focusing at distinct equilibrium positions across the microchannel cross-section from a multi-particle mixture23. Randomly dispersed particles equilibrate at different positions along the inner wall of the spiral microchannel, influenced by lift and drag forces (see Figure 9 – insert 2). As the particles reach the end of the spiral, they align and separate into different channels (see Figure 9 – insert 3).
A notable advantage of this microfluidic chip application is its high throughput, reaching 1.5 mL/min, achieved without the need for sheath flow or sequential cell manipulation. This feature is particularly beneficial for processing native biological fluids and applications in flow cytometry.
Application notes & expertises:
V. Micromixers an application using microfluidic chips
Micromixers play a crucial role in lab-on-chip devices for various microfluidic chip applications, including drug delivery, sequencing, amplification, and biochemical reactions. They can be broadly classified into two categories based on the actuation method: passive and active.
Passive mixing relies on the microfluidic chip’s geometry and fluid properties without external sources. In laminar flow, typical in microfluidics, mixing primarily occurs through diffusion. This property allows for precise tuning of mixing by employing lamination, where two or more liquids flow in parallel, enabling diffusion to take place. For emproved and faster mixing, chaotic advection can be induced by modifying the microfluidic chip’s geometry, altering channel shapes for splitting, folding, stretching, or disrupting fluid flow.
On the other hand, active mixing involves external perturbation. Various methods are employed in the microfluidic chip application field for active mixing. Dielectrophoresis mixing uses an electric field to move particles toward or away from an electrode, creating chaotic advection. Acoustic wave energy can also mix fluids by generating strong acoustic waves that interfere with each other, creating advection 24. Additionally, adjusting the microfluidic chamber temperature can enhance mixing, as the diffusion coefficient of a liquid is temperature-dependent25.
Submillisecond organic synthesis using a serpentine-shaped microfluidic chip application
In chemical synthesis, it is important to explore the synthetic pathways of an intermediate. To fully observe these pathways, control over its lifetime and mixing time is required.
The reaction mixture had to be well-mixed within the lifetime of the reactive intermediate. An efficient means of prolonging the lifetime is to lower the reaction temperature (-78°C to -100 °C), above the melting point of many organic solvents. Using microfluidic devices, mixing can be extremely fast, with mixing times unattainable by batches26. However, mixing time is increased at low temperatures, as the solvent viscosity exponentially increases.
To circumvent this issue, the authors used a three-dimensional serpentine-shaped microfluidic chip, allowing improved mixing by chaotic advection. The conceptual scheme of the 3D serpentine microchannel fabricated by lamination is represented in figure 10.A.
The figure 10.B represents a detailed scheme for a nanoliter reaction space of rectangular 3D serpentine channels with three inlets and the optical image showing from the top the nanoliter reaction space schematized in B. The optical images of respectively the chip reactor module and assembly are illustrated in Figure 10.D and E.
The utilization of this platform for the application enabled submillisecond mixing.
VI. Microvalves and microfluidic chip applications with reduced internal volume
In the past 10 years, efforts have been devoted to the development of microfluidic platforms capable of performing several assays using programmable fluidic operations within an array of microvalves. Similar to programmable logic circuits where multiple electronic computing routines are executed on a single microdevice, programmable microfluidic platforms have been implemented27, allowing one to perform fluidic operations such as mixing, sampling, washing, and reacting automatically within a single microfluidic chip by modifying the sequence/order of fluidic operations using the software.
The primary advantage of microvalves over their macroscale counterparts is the significantly reduced dead volume, which is important in many microfluidic chip applications that require precise flow control at small flow rates28. They are useful in biological and chemical applications, such as quantitative metabolic biomarker and genetic analysis29,30, protein-based biomarker detection31, or small molecule chemical and environmental analysis32. These microfluidic chip and valve application platforms usually consist of a 2D array of microvalves that permit flow regulation, on/off switching and sealing of liquids, gases, or vacuums33.
Several microvalves have been developed using pneumatic, electrokinetic and electrochemical actuators. Among these mechanisms, pneumatic actuation is often recognized as the most reliable method due to the simplicity of fabrication, ease of use, scalability, reliability, and a high degree of accuracy. Pneumatically actuated microvalves utilize the deflection of an elastomer (typically PDMS) membrane to control fluid flow34.
A fully integrated multilayer microfluidic chemical analyze for automated sample processing, labelling, and analysis
Capillary zone electrophoresis (CZE), is a powerful tool for chemical analysis and is widely used for environmental monitoring, astrobiology, and biosensing32. CZE assays usually require complex and manual sample processing.
Commercial platforms for automated CZE have been implemented to address this concern, but are expensive, and large, thus hardly field deployable in challenging environments. Microfluidic application chips and devices allow miniaturization, automation, and reduction in sample volume requirements for chemical and biochemical sensing.
Using membrane microvalve technology, it is possible to automate metering, transporting, routing, and mixing operations. Kim et al. introduced a microfluidic platform consisting of pneumatically actuated “lifting gate” microvalves integrated with a glass CZE microchip, providing extremely low dead volumes between components.
All the procedures, including buffer filling, labeling, and dilution, can be automated. The microchip was used to analyze diverse compound classes, such as amino acids, and oxidized biomarker compounds, like aldehydes/ketones and carboxylic acids in less than 30 min.
VII. Wearable microfluidics: an emerging application
An emerging application of microfluidic chip, is the use of microfluidic concepts for wearable device applications36. Here, microfluidics presents several key value propositions. The microstructures store or handle fluids and are the core of the sensing device. Using microchannels of the microfluidic chip, precise liquid amounts can be manipulated, allowing for highly accurate and reliable devices and being useful for bodily fluids that are often secreted or extracted in limited quantities.
Also, wearable microfluidic devices could also stock a specific drug for precise dispensing at controlled intervals. Innovations in flexible microfluidics and electronics have led to numerous applications. Typically, a wearable microfluidic device will collect a fluid, transfer it to a localized site where detection or measurement is performed. Blood and sweat are common analytes as they provide insights into physiological states such as temperature, pH, and hydration36. Wearable microfluidics finds applications in the pharmaceutical, food, sportswear, and cosmetic fields.
A wearable microfluidic device for the capture, storage and colorimetric sensing of sweat
A wearable microfluidic device for the capture, storage and colorimetric sensing of sweat
As mentioned previously, sweat is an analyte of interest because of its rich content of important biomarkers. It is easy to collect compared to blood. In situ quantitative analysis of sweat is of great interest for monitoring physiologic health status (for example, hydration state) and for the diagnosis of disease37.
Existing systems for sweat collection and analysis are confined to laboratories, where standard analytical technologies can be performed. Though highly precise, the analysis is time-consuming and costly.
To address this issue, Kho et al. developed a soft wearable microfluidic system than can directly harvest sweat from pores on the surface of the skin37.
The device routes the sample to different channels and reservoirs for multiparametric sensing of markers of interest, with options for wireless interfaces to external devices for image capture and analysis.
The device can measure total sweat loss, pH, lactate, chloride, and glucose concentrations by colorimetric detection using wireless data transmission. As it is a simple, low-cost, and fast analysis point of care device, it could be used to accumulate data from individual users over time, and this could serve as an analytical approach for interpreting trends in marker concentrations, potentially providing warning signs when performing physical activity.
Conclusion
Since the introduction of microfluidics, the scope of microfluidic chip applications has kept extending over the years. The first applications were focused on analytical chemistry, but today the field of life science and specifically point of care is in the core of microfluidics. We have presented applications where microfluidic chips show great advantages compared to conventional systems. The importance of these applications was illustrated by showing research papers related to these applications. Some important applications of microfluidic chip were introduced here. Microfluidics covers a wide range of applications such as microreactors, bioprinting, fuel cells, and many more.
Expertises
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Microfluidics case studies Creating a Microfluidic Cancer-on-Chip Platform using Fluigent’s High Throughput Cell Perfusion Pack Read more
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Microfluidic Application Notes Long-term fluid recirculation system for Organ-on-a-Chip applications Read more
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Microfluidics case studies CNRS/UTC: study of a liver-on-a-chip model Read more
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Microfluidic Application Notes Alginate Microcapsule Synthesis Read more
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Microfluidic Application Notes Agarose Microcapsules Synthesis Read more
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Microfluidic Application Notes PLGA nanoparticle synthesis using 3D microfluidic hydrodynamic focusing Read more
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Microfluidic Application Notes Development of a human gut-on-chip to assess the effect of shear stress on intestinal functions Read more
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Microfluidic Application Notes PLGA microcapsules synthesis Read more
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Microfluidic Application Notes Peristaltic Pump vs Pressure-Based Microfluidic Flow Control for Organ on Chip applications Read more
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Microfluidic Application Notes Double Emulsion Generation Read more
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Microfluidic Application Notes Microfluidic Chitosan Microcapsules Production Read more
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Microfluidic Application Notes Alginate Microbeads Production Read more
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Microfluidic Application Notes Single cell sorter microfluidic platform Read more
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Microfluidic Application Notes Assess Cell Proliferation Using Pressure as a Tool Read more
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Microfluidic Application Notes Cartilage-on-a-chip, an example of complex mechanical stimulation using Fluigent’s technology Read more
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Expert Reviews: Basics of Microfluidics Mimicking in-vivo environments: biochemical and biomechanical stimulation Read more
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Organ-on-a-chip (OOAC) is the concept of mimicking the organ-level function of human physiology or disease using cells inside a microfluidic chip. Microfluidics provides the unique ability to control the cellular microenvironment with high spatiotemporal precision and to present cells with mechanical and biochemical signals in a more physiologically relevant context. The ability to manipulate micro-liter volumes of liquids has made these models a platform where scaling and dynamic crosstalk between cells can be achieved.
Microfluidic chips can now use geometries and structures to permit the use of physiological length scales, concentration gradients, and the mechanical forces generated by fluid flow to mimic the in vivo microenvironment experienced by cells. These biomimetic platforms overcome many of the drawbacks encountered with conventional tissue culture models.
Applications of OOAC cell culture
Therapy development
Organ-on-chip cell culture platforms have proven potential in providing tremendous flexibility and robustness in drug screening and development by employing engineering techniques and materials. More importantly, there is a clear upward trend in studies that utilize human-induced pluripotent stem cells (hiPSC) to develop personalized tissue or organ models. For this purpose, the use of cell culture chips allows users to study complex culture configurations by joining a culture well with a microfluidic channel via a porous membrane. This is the optimal device for Air Liquid Interface (ALI) culture, endothelium/epithelium barrier and crosstalk studies.
Drug discovery
The development of emerging in-vitro tissue culture platforms can be useful for predicting the human response to new compounds. Recently, several in-vitro tissue-like microsystems, also known as “organ-on-a-chip studies”, have emerged to provide new tools for better evaluating the effects of various chemicals on human tissue.
Organ-on-chip cell culture models can therefore be used for accurate prediction and mechanistic investigation of dose-limiting human toxicities of prospective drugs, as well as for the exploration of new therapeutic approaches to mitigate the observed toxic effects. In the drug discovery pipeline, predictions made by these models could inform and facilitate early efforts to identify, modify and optimize lead compounds, thereby developing safer drugs with an increased likelihood of success in clinical trials.
Regenerative medicine
The development of emerging in-vitro tissue culture platforms can be useful for predicting the human response to new compounds. Recently, several in-vitro tissue-like microsystems, also known as ‘organ-on-a-chip studies’, have emerged to provide new tools for better evaluating the effects of various chemicals on human tissue.
Organ-on-chip cell culture models can therefore be used for accurate prediction and mechanistic investigation of dose-limiting human toxicities of prospective drugs, as well as for the exploration of new therapeutic approaches to mitigate the observed toxic effects. In the drug discovery pipeline, predictions made by these models could inform and facilitate early efforts to identify, modify and optimize lead compounds, thereby developing safer drugs with an increased likelihood of success in clinical trials.
Omi, the new automated organ-on-chip platform
Discover Omi, the new automated platform for OOAC applications developed by Fluigent. Equipped with state-of-the-art technologies, this platform will enable you to carry out any perfusion protocol. It has the ability to customize and automate any protocol, including simple perfusion, recirculation, sampling and injection. It meets the needs of beginners in organ-on-chip cell culture research and advanced organ-on-chip researchers looking for automation and reproducibility.
This versatile, automated organ-on-a-chip platform can perform long-term OOAC cell cultures under flow to control shear stress conditions. Its two-hour battery life and WiFi connectivity ensure easy, intuitive control. It can be easily transported from incubator to microscope for live cell imaging while maintaining cell perfusion under battery power. You can also monitor your experiment from anywhere using the Omi app.
Towards the next generation of organ-on-a-chip cell culture platforms
Fluigent and Beonchip are partnering to offer a complete solution for organ-on-chip cell culture.
This webinar will first introduce Beonchip, their chips and the numerous applications that can be performed with them.
The second part will focus on flow control systems. Although often considered secondary, flow control is as important as chip design, as cells are highly sensitive to mechanical forces. Results demonstrate how cells are affected by peristaltic pumps compared to pressure control systems.
Organ-on-chip research is an emerging field that offers substantial benefits compared to conventional cell culture. In many labs, considerable effort is put into choosing the right chip design, but the impact of flow control is still undetermined.
It is our intent to create awareness of the importance of flow and its effects on studies.
Read our expertise page to learn more about the benefits of flow control in cell culture and about our products and those of our partners (Beonchip), and to reach out to our team of experts to find the solution that best meets your needs.
Resources
-
Microfluidic Application Notes Long-term fluid recirculation system for Organ-on-a-Chip applications Read more
-
Microfluidics case studies CNRS/UTC: study of a liver-on-a-chip model Read more
-
Expert Reviews: Basics of Microfluidics Why is a controlled shear-stress a key parameter of your microfluidic experiments? Read more
-
Microfluidic Application Notes Development of a human gut-on-chip to assess the effect of shear stress on intestinal functions Read more
-
Microfluidic Application Notes Peristaltic Pump vs Pressure-Based Microfluidic Flow Control for Organ on Chip applications Read more
-
Microfluidic Application Notes Assess Cell Proliferation Using Pressure as a Tool Read more
-
Microfluidic Application Notes Cartilage-on-a-chip, an example of complex mechanical stimulation using Fluigent’s technology Read more
-
Expert Reviews: Basics of Microfluidics Passive and active mechanical stimulation in microfluidic systems Read more
-
Expert Reviews: Basics of Microfluidics Prostate Organoid Culture in Microbeads Read more
-
Expert Reviews: Basics of Microfluidics Mimicking in-vivo environments: biochemical and biomechanical stimulation Read more
-
Expert Reviews: Basics of Microfluidics MYOCHIP | H2020 European project Read more
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Expert Reviews: Basics of Microfluidics Application of microfluidic chip technology Read more
Importance of fluid handling for organ-on-a-chip cell culture
In many labs, considerable effort is put into choosing the right chip design, but the impact of flow control is still undetermined. It is our intent to create awareness of the importance of flow and its effects on one’s studies.
Fluigent, in partnership with Beonchip, offers a wide range of cell culture chips according to the field of application: to study cell culture under flow, to explore the crosstalk between different 2D and 3D cultures in biomimetic environments, to apply electrochemical gradients to 3D cell cultures, and to study complex culture configurations by joining a culture within a microfluidic channel via a porous membrane.
Optimized cell culture activity: Constant perfusion enables the continuous renewal of nutrients and oxygen to promote cell growth and maintain optimal activity during long-term cell cultures.
Biomechanics: Organ on chip cell culture technology has paved the way for investigating the impact of mechanical strains in cell biology research by reproducing key aspects of an in-vivo cellular microenvironment. Combining microfluidics and microfabrication enables one to reproduce mechanical forces experienced by living tissues at the cell scale.
Passive stimulation induced by shear flow: Liquid flow usually induces shear stress on cells or tissues cultured on the device. This is called shear flow, and the effect is substantial on cell growth, phenotype, and genetic expression.
Active mechanical stimulations originate from the function of the organ. Organs like lungs, muscles and intestines are in active motion. Cells in those organs are mainly subjected to compression and stretching. Organ-on-a-chip cell culture can simulate these environments, allowing for more realistic and detailed results to be obtained.
Biochemical studies: Cells are constantly exposed to biochemical stimulation from the early embryonic stage to adult life. The spatiotemporal regulation of these signals is essential as it determines cell fate, phenotype, metabolic activity as well as pathological behaviors. The fast response and high stability of Fluigent instruments make them the best solution available on the market to reproduce these complex variations in-vitro.
Read our expertise page to know more about the benefits of flow control in cell culture, learn more about our products and those of our partners (Beonchip), and reach out to our team of experts to find a solution that best meets your needs.
Flow control systems and Fluigent’s added value
Implementing perfusion and automated fluid delivery in organ on chip cell culture protocols offers major advantages. Compared to manual pipetting, Automation increases reproducibility, saves time, and improves the level of control in the experiment as all the parameters are tightly regulated (time of delivery, volume, and speed of injection).
Multiple flow control technologies are available for sub-millimeter range fluid management. As demand for microfluidic pumps with higher flow stability, fast response time, versatility, and automation capabilities have increased, pressure controllers have become the device of choice.
Response time and stability
The working principle of such pumps is to pressurize the sample reservoirs to control the pressure drop between the inlet and the outlet of the microfluidic system. The responsiveness of the generated flow rate depends on the responsiveness of the pressure pump.
Microfluidics in Pharmaceutics
Drug delivery
Microparticles and emulsions are used for a wide variety of pharmaceutical products including intravenous, intramuscular, ocular, or orally delivered compounds. Emulsions are also used as templates for polymer microparticles, lipid nanoparticles, or microcapsules. These are later used for drug delivery, with the emulsion being the active pharmaceutical ingredient (API) itself, or as an adjuvant for co-administration.
Droplet microfluidics technology produces multiunit drug delivery systems with precise dosage control, targeted release, and homogeneous distribution. Conventional methods struggle to achieve monodispersity (<5%) required for efficient drug delivery. Our technology revolutionizes drug delivery, offering improved treatment options and personalized therapies and advancing pharmaceutical development and patient care.
To learn more about droplet microfluidics for pharmaceutical applications, read our white paper about droplet microfluidics.
Disease modeling and characterization
Animal studies are the current standard for evaluating potential treatments, but they often struggle to correlate with human outcomes due to physiological differences and genetic variations. These studies are costly, time-consuming, and raise ethical concerns. Human clinical studies face challenges due to individual diversity and research complexity. Microfluidic models in an organ-on-chip format offer a promising alternative, providing an efficient, cost-effective, and ethical approach for drug discovery and personalized medicine. This technology advances the field of pharmaceutics by enabling the development of predictive methods to evaluate new compounds and therapeutics. It then helps in disease modeling and characterization, enhancing the drug discovery and development process.
To delve deeper into the topic of microfluidics for pharmaceutical applications using organ-on-chip systems, read our OOC white paper.
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Looking for another market?
From the life sciences to the food industry, many applications require the use of fluids driven at flow rates from nanoliters to milliliters per minute. At such low flows, the success of these applications strongly depends on the level of control and automation of the fluidic operations.
These applications require flow control systems that are adapted for ensuring their success.
Introduction
Erasmus MC is a leading international academic hospital at the forefront of the medical field. Its staff, volunteers, and students collaborate to provide healthcare for patients with complex disorders, rare conditions, and urgent medical needs. Recognized as a world-class scientific research organization, Erasmus MC strives to improve their understanding of diseases and disorders, working towards prediction, treatment, and prevention. The institution’s guiding principle revolves around the integration of biomedical research, clinical research, and health sciences to form a comprehensive approach to the advancement of healthcare.
Bi/ond, an innovative and international biotechnology company based in the Netherlands, was created with the primary aim of harnessing the potential of microchips to drive biological innovation. Working in close partnership with biologists, the company focuses on the creation of reproducible and accurate biological models that help develop inclusive and precise cures for medical issues. By drawing on its expertise in microelectronics and its in-depth knowledge of biological solutions, Bi/ond succeeds in bridging the gap between the fields of biology and engineering.
These two entities have combined their knowledge and technologies to develop an innovative Cancer-on-Chip (CoC) platform for assessing response to treatments using Fluigent’s high throughput cell perfusion pack.
Learn more about the Erasmus MC Cancer Institute
Learn more about Bi/ond
Testimonial
“We started out as a novice to the field of organ-on-chip cultures. Fluigent was very helpful at this stage to get started and keep the system up and running. They were always there to answer questions. Therefore, the precision pumping system was a thing we did not have to worry about, and we could concentrate on our own expertise, the biological materials in the culture device.”
Dr. D.C. (Dik) van Gent PhD
Universitair Hoofd Docent – Molecular Genetics
Why develop a Cancer-on-Chip (CoC) device to predict drug response?
Overcome the challenges in cancer treatments
Searching for personalized therapy treatment for individual patients is a challenging process. The crux of the problem lies in precisely defining the optimal treatment regimen for each individual. Although many molecular biomarker-based treatment strategies have been employed in cancer therapy, their ability to reliably predict individual responses to chemotherapy remains limited in most cases. This means there is a pressing need for ex vivo bioassays capable of effectively predicting a patient’s response to specific treatments, thus facilitating the selection of the most appropriate and effective therapeutic approach to optimize life expectancy and quality of life.
Historically, cancer cell lines and animal models have played a key role in assessing the efficacy of chemotherapies. However, when it comes to predicting tumor sensitivity in individual patients, these preclinical models fall short. Their main utility lies in studying the general characteristics of specific tumor types or stages of disease, which fails to consider the heterogeneity found in cancer and undermines their predictive power for responses to individualized treatment. These models are also time-consuming to set up and use, which limits their usefulness.
One potential solution lies in the direct assessment of drug responses using patient tumor tissue slice cultures. These ex-vivo cultures maintain the entire tumor microenvironment, including immune cells, and preserve the original tissue’s architecture. However, the development of long-term ex vivo culture systems, particularly those lasting more than 7 days, remains a major hurdle. The difficulty lies in managing mechanical stress on tissue slices, which can lead to the disruption of tissue integrity and non-physiological behavior, as well as in ensuring optimal culture conditions.
Consequently, it is crucial to invest in the development of more physiologically relevant ex vivo tissue slice culture systems, such as a cancer-on-chip platform, which enables the prolonged culture of tumor slices under precisely controlled conditions. Such advances could revolutionize personalized medicine and dramatically improve cancer treatment by enabling more precise and effective therapies, tailored to each patient’s specific needs.
The development of the organ-on-chip platform, an innovative technology
Despite considerable progress in computational and in vitro biology and toxicology over the past two decades, the failure rate of experimental drugs in clinical trials remains high, with over 80% of drugs failing to reach the market. Of these failures, 60% are attributed to lack of efficacy and 30% to toxicity. This situation has given rise to growing concerns about rising costs, wasted time, and ethical problems associated with animal experimentation, which often proves inadequate for predicting human reactions in a clinical context.
In addition, traditional live-cell experiments using cells grown on 2D substrates coated with serum or extracellular matrix molecules present limitations. Although they promote cell proliferation, they often fail to reproduce tissue-specific functions. Human organs, with their diverse functions, rely heavily on complex interactions between specialized cell types at well-defined interfaces, arranged in complex geometries and responding to specific microenvironments. Due to these issues, there is an urgent need for new modeling and testing platforms capable of better predicting human responses.
Organ-on-a-chip (OoC) technology represents a significant advancement in drug discovery and development, offering new tools for disease modeling and characterization, as well as potentially more accurate methods for assessing the toxicity and efficacy of new compounds and therapies. The organ-on-a-chip concept involves reproducing the functions of human physiology or disease at organ level in microfluidic chips, using different cell types.
Microfluidics plays a crucial role in enabling precise control of the cellular microenvironment, presenting cells with mechanical and biochemical signals in a more physiologically relevant context. Working with liquid volumes in the microliter range, these models enable dynamic scaling and interaction between cells. In addition, microfluidic chips can use geometries and structures to mimic physiological length scales with concentration gradients and mechanical forces generated by fluid flow, thus recreating the in vivo microenvironment faced by cells. This biomimetic approach, involved in the cancer-on-chip platform used in this study, overcomes many of the limitations encountered with conventional tissue culture models. [1]
How to combine OOAC and therapy assessments
The CoC platform provides continuous media perfusion, nutrient supply, waste removal, and the ability to collect samples for analysis. The aim of this platform is to develop a reproducible culture system for assessing the sensitivity of (breast and prostate) tumors to chemotherapy, using living material that closely resembles the original tumor and allows long-term culture without significant changes in viability or tissue characteristics. In addition, the system must enable a direct assessment of response to treatment through microscopic imaging and analysis based on fluid sampling.
In this article, the Cancer-on-a-Chip (CoC) microfluidic platform described uses an 6-well plate with silicon-based microfluidic chips. These chips offer greater flexibility than glass-based culture systems, as they allow for the easy integration of sensors for pH detection, metabolite screening, and oxygen sensing. In addition, the silicon-based design enables parallelization, taking advantage of semiconductor technology to improve scalability, reproducibility, and cost-effective large-scale production.
One notable application of this new Cancer-on-a-chip platform is personalized medicine. It facilitates the in vitro culture of tumor tissue slices under precisely controlled conditions, enabling the prediction of in vivo tumor responses to therapy in individual patients. The platform has been successfully used to grow tumor slices, including patient-derived xenografts (PDX), and faithfully mimicked tumor cells.
Use of the high throughput cell perfusion pack in the Coc platform to predict drug responses
Mimicking the in vivo cancer tissue on a microfluidic chip
In this study, the microfluidic chips from Bi/ond are made of polydimethylsiloxane (PDMS) film with embedded microfluidic channels, supported by a silicon (Si) frame. The chips’ top plates serve as an interface connecting the inlet and outlet, enabling media diffusion through the tissue-supporting membrane. The chip features four microfluidic fittings for the external pumping systems. The bottom part of the chip is designed to ensure compatibility with microscopes and oxygenation, achieved through PDMS window openings under the chips.
The microfluidic chips containing tumor tissue slices are housed in a ComPLATETM. The ComPLATETM, designed by Bi/ond, is a smart, compact, and reusable well-plate specifically tailored for cultivating complex tissues. The plate is comprised of a black 6-wells bottom plate, a transparent top plate to cover the chip, and a white fixation ring. This design helps create independent cultivation and the analysis of individual tumor slices.
The top plate of the ComPLATETM provides the option for single or double flow of media. Each well has sufficient space to accommodate the tissue slice’s growth over time. The microchannel and top interface facilitate constant perfusion and nutrient replenishment, enabling maintenance of the tissue slices while removing waste products. Furthermore, oxygenation of the tissue slices is enhanced through a gas exchange via the PDMS layer of the optical window.To monitor fluid flow rates inside each well throughout the culture period, the entire Cancer-on-a-Chip platform is connected to a Fluigent Microfluidic Flow Control System.
A, Top view of the microfluidic chip illustrating its components: the PDMS film in which the microfluidics are embedded, and the silicon frame, which includes the inlet and outlet to the channels in the film. B, Vertical cross-section of the microfluidic chip. C, Representation of the CoC platform. D, ComPLATETM device. E, Cross-section of CoC illustrating the diffusion and perfusion toward the tissue slice.
Using Fluigent technology to ensure a high throughput cell perfusion
The Fluigent Cell Perfusion Pack is specially designed for high-throughput experiments. It has been carefully optimized for maximum efficiency in multiple-chip perfusions, enabling the simultaneous growth of multiple organ models in one incubator, which is ideal for the cancer-on-a-chip platform. The package includes a compact 8-channel pressure controller, a flow platform, and reservoir support that can be easily integrated into an incubator track. The user-friendly interface makes it easy to set up and operate the system, creating the proper physiological conditions for effortless long-term experiments. The system is highly reproducible and scalable.
To achieve continuous perfusion, an FLPG Plus pumping system was used as the pressure source. The flow rate was then maintained using the MFCSTM-EZ pressure-controlled microfluidic flow control system. Flow sensors (FLOW UNIT-S) were used to monitor flow throughout the culture using Fluigent software. A precise inlet flow rate of 5 µl/minute was used to perfuse PDX tissue slices through the chip’s upper and lower channels. CoC tissue was cultured under optimal conditions in a humidified atmosphere with 5% CO2 at 37°C, and the culture medium was renewed every 3 days for up to 2 weeks.
Comparison with an ex-vivo model
One of the aims of this study is to compare this innovative cancer-on-chip platform with a more traditional ex-vitro model. This traditional model consists of a cell culture in a 3mL customized culture with medium 6-well standard plates on an orbital shaker at 60 rpm. This will demonstrate the added value of using a platform based on Organ-On-a-Chip technology compared to more conventional methods. This will be made possible by carrying out various tests and comparing the data obtained by these two methods. These tests consist of studying the treatment response of tumors, long-term tumor tissue slice culture, and gene expression analysis.
Partial results
Treatment response of tumors
To assess the validity of the platform, a crucial question is whether in vivo treatment responses can be predicted by treatment responses. This validation was carried out using the cisplatin treatment on PDX breast cancer tumors (cisplatin-sensitive and cisplatin-resistant) with three biological replicates each.
To verify that the platform retained the essential features of tumor-associated cell morphology and proliferative capacity, untreated tumor slices at day 0 and day 7 were evaluated.
The effect of cisplatin treatment on cell proliferation and death was assessed in tissue slices grown under normal ex vivo conditions and in the CoC device. Cisplatin-sensitive tumor slices in the platform showed a significant increase in apoptotic cells and a notable decrease in replicating cells upon cisplatin treatment. In contrast, cisplatin-resistant PDX tissue slices showed no significant changes in either signal compared with untreated controls. [2]
The response to cisplatin treatment observed in the Cancer-on-a-Chip platform correlates with known tumor responses in in vivo and ex vivo cultures, suggesting its reliability for drug response analysis. Interestingly, breast PDXs cultured in the CoC platform showed a more robust response to cisplatin treatment compared with the ex vivo culture method, indicating better drug delivery in tumor slices with the platform.
To evaluate the performance of the Cancer-on-chip device in another tumor type, the PC82 androgen-dependent prostate tumor was used under the same conditions. The results led to the same conclusion as the breast tumor slices. [3]
In conclusion, CoC cultures accurately reproduced tumor responses to two different treatments (prostate and breast) in breast and prostate tumor models known to be sensitive in vivo.
Figure 3: Prediction of therapy response using cisplatin-sensitive and -resistant PDX in ex vivo and CoC platforms. A, Representative EdU (proliferation) and TUNEL (apoptose) staining of cisplatin-sensitive breast PDX. B, Quantification of the fraction of EdU-positive and TUNEL-positive cells showing breast PDXs were sensitive to cisplatin. C, Representative EdU and TUNEL staining of cisplatin resistant breast PDX. D, Quantification of the fraction of EdU-positive and TUNEL-positive cells showing breast PDXs were insensitive to cisplatin therapy, thereby validating the application of CoC for therapy response for patient tumors. E, Analysis of DNA damage response in cisplatin-sensitive and -resistant PDX treated with cisplatin. Cisplatin treatment induced more double-strand breaks in cisplatin-sensitive PDX than in cisplatin-resistant PDX. F, Scatter plot showing 53BP1 foci count per cell in cisplatin-sensitive and -resistant PDX.
Long-term tumor tissue slice culture
Longer culture times are essential for studying therapeutic responses that require longer incubation periods-more than one week-such as the development of therapeutic resistance or clonal outgrowth. The main limitation observed in ex vivo culture is the preservation of optimal proliferative capacity and tissue architecture.
To address this, a study was carried out using breast tumor slices from five independent patient-derived xenografts. These slices were cultured for 14 days in the Cancer-on-a-Chip platform and, in parallel, in the ex vivo 6-well plate. After 7 days of culture, similar rates of cell proliferation in the CoC device compared with day 0 were observed, but slightly slower proliferation in the ex vivo condition. TUNEL staining revealed a slight increase in cell death in the ex vivo system compared with the CoC platform at day 7.
Notably, these differences became more pronounced in extended 14-day cultures. The platform showed better preservation of tumor tissue architecture and cell proliferation than the ex vivo culture system. In contrast, the ex vivo condition showed a significant decrease in proliferation at day 7 compared with PDX tumors at day 0, whereas no significant difference was observed between day 7 and day 0 for the platform, indicating slightly slower cell proliferation in ex vivo culture compared with CoC.
These results underline the superiority of the Cancer-on-chip device for prolonged culture times (beyond 7 days) of tumor tissue slices compared to the ex vivo system (better preservation of cell integrity and cell proliferation).
Figure 4: Breast PDX tumor tissue slices cultured in ex vivo condition and in CoC device for up to 14 days. A, Quantification of the fraction of EdU-positive and TUNEL-positive cells for 5 breast PDX tissue slices cultured for up to day 7 (B) and day 14 (C). D, Representative image showing breast PDX tumors labeled with geminin (red nuclei) and DAPI (blue nuclei). E, QIBC analysis of three independent breast PDX tumors with more than 3,000 cells analyzed for each are shown in each condition. F, Quantification of geminin-positive cells showed CoC at day 7 had similar cell proliferation profile as in day 0 than ex vivo condition.
Gene expression analysis
To assess the impact of CoC culture on gene expression changes, analysis using RT-PCR and whole transcriptome sequencing was performed. Tumor-specific gene pathways in PDX breast tumors cultured ex vivo and in the Cancer-on-a-Chip platform were examined. Surprisingly, there were no statistically significant changes in these pathways, suggesting minimal alterations in tumor growth characteristics under both conditions. Next, whole transcriptome sequencing of PDX breast tumors was performed. Genes were identified as differentially expressed on day 7 ex vivo and on days 7 and 14 under CoC culture conditions. 150 human genes are differentially expressed in day 7 ex vivo tumor slices, far more than the 30 human genes differentially expressed in day 7 CoC and the 14 human genes in day 14 CoC.
To understand the reasons for the differences observed, various tests studying cell cycle progression and apoptosis were carried out. They led to the conclusion that ex vivo culture conditions induced greater immune activation and DNA damage after 7 days, making the CoC system a more accurate representation of the original tumor and the preferred choice for studying responses to therapies.
Conclusion
The researchers developed a microfluidic CoC platform capable of maintaining cell viability, proliferation and tissue structure in breast cancer PDX slices for at least 14 days. This platform successfully predicted responses to cisplatin therapy for breast cancer and antiandrogen therapy for PDX prostate cancer tumor slices. To fully establish its potential as an in vitro diagnostic test for therapy selection, it will require clinical validation using biopsies from patients receiving the same chemotherapy.Although the current study has focused on PDX models of breast and prostate cancer, this Cancer-on-a-Chip platform also holds promise for other solid tumors. Its ease of use and small footprint make it a versatile tool for ex vivo studies, including functional genomics, drug screening and personalized medicine research.
Expertises & Resources
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Expert Reviews: Basics of Microfluidics Microfluidic pressure control for organ-on-a-chip applications: A comprehensive guide Read more
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Microfluidic Application Notes Long-term fluid recirculation system for Organ-on-a-Chip applications Read more
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Microfluidics case studies CNRS/UTC: study of a liver-on-a-chip model Read more
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Interviews & Testimonials Panel Discussion & Interviews – Microfluidics & Organ-On-Chips Read more
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Microfluidics White Papers Microfluidic white paper – A review of Organ on Chip Technology Read more
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Microfluidic Application Notes Peristaltic Pump vs Pressure-Based Microfluidic Flow Control for Organ on Chip applications Read more
Related Webinars
Related products
References
[1] Microfluidic white paper – A guide to Organs-on-Chips technology, Fluigent
[2] Naipal KA, Verkaik NS, Sanchez H, van Deurzen CH, den Bakker MA, Hoeijmakers JH, et al. Tumor slice culture system to assess drug response of primary breast cancer. BMC Cancer 2016;16:78
[3] van Weerden WM, van Steenbrugge GJ, van Kreuningen A, Moerings EP, de Jong FH, Schr€oder FH. Assessment of the critical level of androgen for growth response of transplantable human prostatic carcinoma (PC-82) in nude mice. J Urol 1991;145:631–4
What can droplet microfluidics be used for?
Drug delivery
In recent years, biodegradable microcapsules/microparticles have gained widespread importance in the delivery of bioactive agents. Polymer-based microcapsules/microparticles are one of the most successful new drug delivery systems.
They can be used in various areas such as long-term release systems, vaccine adjuvant, and tissue engineering.
Droplet-based microfluidics produce highly monodispersed droplet and microcapsule/microparticle production opposed to batch emulsion methods and provide an “In-line” continuous droplet production process.
Formulation
The encapsulation of active ingredients to create flavors or fragrances for cosmetics and food products is a key part of their formulation. A challenge of droplet generation applied to encapsulation is to prevent the leakage of the encapsulated species.
The possibility to encapsulate these compounds allows users to control the release of the compound and improve pharmacokinetics. Modern drug encapsulation methods allow efficient loading of drug molecules inside nanoparticles, thereby reducing systemic toxicity associated with drugs.
Targeting nanoparticles can enhance the accumulation of nanoencapsulated drugs at the diseased site.
Next generation sequencing (NGS)
Encapsulation of a single cell inside a droplet increases NGS efficiency. The ability to study cells at single cell level using droplet based systems combined with NGS techniques allow for the sequencing of mRNA from a large number of cells.
The power of this technology, combined with droplet generation, resides in the fact that during sequencing, one can distinguish where the original information came on a cell-to-cell basis. This allows one to make a gene expression map of the cell, or even to distinguish cell populations within a tissue.
Drug discovery
In-vitro cell culture is a fundamental component of biological production systems and biotechnological research. The ability to grow cells outside of their natural environment offers many industry solutions from the high quantity production of enzymes to cell toxicity studies and drug discovery.
Droplet generationallows one to encapsulate single or multiple cells into tiny droplets of pL volume which are generated at a rate of approximately one thousand per second.
Diagnostics
A key measurement challenge in diagnostic research involves identifying small changes in nucleic acid sequences that are commonly associated with genetic diseases such as Down’s syndrome and many cancers.
Digital PCR (dPCR) carries out a single reaction within a sample as standard PCR, however the sample is separated into a large number of partitions where reactions take place in each partition individually. This is an excellent solution to partition a sample, and dPCR technology that makes use of droplet microfluidics is often called droplet digital PCR (ddPCR).
How to control droplet size and volume using fluid handling technology ?
When it comes to particle or droplet generation, having control of the fluid delivery system is important. During particle or droplet production, the flow rate of each phase must remain constant and stable to allow the production of monodisperse droplets.
The ability to control the flow rate of each phase allows for more control over the process, precisely and easily regulating the size of the droplet or particle generated.
Which flow control system to generate monodisperced droplet ?
Flow rate stability is critical for having repeatable reactor volumes and reproducible results. Syringe pumps are commonly used for generating droplets in microfluidic experiments. Depending on the model in use, syringe pumps show limited flow control.
As a consequence, the droplet size (proportional to the flow rate), is affected. The actual flow rate cannot be controlled with syringes or peristaltic devices. The flow rate value is displayed on the device, but no information on the time required for reaching a set flow rate is given.
The time for flow equilibrium may vary depending on the microfluidic setup, and flow rate can oscillate depending on the instrument. An alternative to syringe pumps for the generation of droplets are pressure-based flow controllers. These show high-precision flow control and fast response times. Read more on the expertise page.
How are liquid droplets created with microfluidics pressure controllers?
To underline the importance of pressure control, our application note compares the production of water-in-oil emulsions using microfluidic syringe pumps to pressure-based flow controllers. Droplet size, stability, and the time required to reach several droplet diameters are dependent on each instrument.
Resources
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Microfluidic Application Notes Encapsulation of Cells In Small Double Emulsions Read more
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Microfluidic Application Notes Encapsulation of multiple emulsions in a single droplet Read more
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Microfluidics case studies The Hebrew University: Encapsulation and culture in 3D hydrogels for Single cell sequencing Read more
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Microfluidics case studies University of Cambridge: Microfluidic GUV production and testing Read more
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Expert Reviews: Basics of Microfluidics The Raydrop | A new droplet generation device based on non-embedded co-flow-focusing Read more
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Microfluidic Application Notes Double Emulsion Generation Read more
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Microfluidic Application Notes Microfluidic Chitosan Microcapsules Production Read more
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Microfluidic Application Notes Alginate Microbeads Production Read more
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Microfluidic Application Notes PLGA Microparticles Synthesis Read more
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Microfluidic Application Notes UV-Crosslinking of Microcapsules Read more
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Microfluidic Application Notes UV-Crosslinking of Microparticles Read more
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Expert Reviews: Basics of Microfluidics Microfluidic Droplet Production Method Read more
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Expert Reviews: Basics of Microfluidics Flow control for droplet generation using syringe pumps and pressure-based flow controllers Read more
Founded in 1972, the Compiègne University of Technology (UTC) is a research and educational institute with the aim of positioning technology at the center of interdisciplinary research projects. Among the 3 main research axes, the “technologies for healthcare” field is of particular importance as bringing technologies to biomedical research allows for a better understanding of the ways to treat pathologies, improved diagnostic methods,developnew in-vitro models for drug testing, and more..
UTC, in partnership with various academic and industrial partners, relies on 8 research units. Among them is the Biomechanics and Bioengineering CNRS research center, where the interactions between fluids and biological structures are heavily studied. In this field of research, microfluidic systems are continuously developed, as they are perfectly suited to make miniaturized, robust, and controllable systems involving fluid handling and biological functions, such as human liver-on-a-chip models. Learn more about UTC.
Testimonial
“FLUIGENT pressure controllers were used before my arrival at BMBI laboratories to control the fluid circulation when studying fluid structure interactions. The goal of these experiments was to measure the deformation of soft flowing object in order to evaluate their mechanical properties. An accurate knowledge of the flow strength is key to the success of this approach.
I was the first in the team to use pressure control for cell culture experiments. I compared the behavior of cells in dynamic culture in biochips combined with pressure controllers and peristaltic pumps. Thanks to FLUIGENT MFCS systems, I could monitor the pressure applied at the inlet reservoir and demonstrate that it is correlated with the number of cells inside the biochip. This kind of information cannot be obtained when a peristaltic pump is used. Cell growth inside the chip may eventually lead to the clogging of the flow circulation.
This clogging can be anticipated when pressure-controlling the fluid circulation in the system, while when using a peristaltic pump, it might remain undetected until the detachment of the chip. In addition, the use of pressure-controlled flow is crucial when working with pressure sensitive cells such as endothelial cells which require a precise monitoring of the pressure variations. During my work at BMBI laboratory, I saw an expansion of the use of FLUIGENT pressure controllers. Thanks to their simplicity, user friendly interface and their accuracy, different applications are being explored.
New research projects are continuously being launched and the use of FLUIGENT pressure controllers to induce the flow is strongly proposed thank to the non-invasive feedback that such technology confer.”
Taha Messelmani, PHD student at Université de Technologie de Compiègne | UTC · BioMécanique et BioIngénierie (BMBI)
Why develop a microfluidic model for liver tissue culture?
Limitation of animal models for drug discovery
Drug development is a long process that can take up to 15 years [1] and include several requirements. Some drug development processes such as animal testing, are subject to strong debates like the ethical relevance of involving animals in the process. In addition, the final product can fail to receive authorization for commercialization due to toxicity or insufficient efficacy [2]. Animal models not only are being strongly discussed due to ethical questions, but they also show limitations for drug development as there are many differences between animal and human biology [3].
Advantages of microfluidic organ on–a-chip models
To overcome these limitations, in-vitro models are being developed to better mimic the human environment and hence accurately determine the human response to drugs. In particular, organ-on-a-chip models are of special interest, as they allow the culture tissue to be studied under dynamic and regulated conditions, reproducing physiological shear-stress with controlled molecule concentrations and3D structures like the in-vivo.
UTC developed an in-vitro liver-on-a-chip model, one of the most promising organ-on-a-chip technologies for drug screening and biological assays, in which they investigate the growth of hepatic cells under dynamic conditions in a microfluidic chip (the biochip) composed of a chamber filled with a 3D hydroscaffold structure. In these conditions, the cells grow and gather to form spheroids, which are not observed in conventional 2D static cultures. Figure 1 summarizes the experiment and illustrates its main results: the self-organization of cells into spheroids and the functionality of liver cells are assessed by their level of secretion of urea and albumin. The organ functions of this human liver-on-a-chip model were found to be improved as compared to traditional culture methods, indicating the viability of the in-vitro model.
Figure 1: Graphical abstract of the liver-on-a-chip model
Use of Fluigent products for characterizing the microfluidic culture of liver tissue
In this study, a 3D hydroscaffold composed of hyaluronic acid was built to embed the cells and provide them with a suitable growth environment. Then, the cells are grown in this hydroscaffold, and due to medium providing shear-stress conditions and a 3D structure, they tend to form spheroids that increase in size over time (fig 2).
Figure 2: Spheroids growth in the chip at several timepoints
For the cells to grow efficiently and form a viable liver-on-a-chip model, the chip is constantly perfused with a culture medium. However, the 3D hydroscaffold and cell proliferation can have an impact on the ability of liquid to flow through the system due to potential clogging.
To monitor this clogging effect, a microfluidic setup consisting of Fluigent pressure-based flow controllers, MCFS-Ex and Flow units M, was created to not only impose the desired flow rate but also measure the pressure level in the chip at several timepoints (fig 3). The flow controller was connected to both the inlet and the outlet of the microfluidic chip, allowing the measurement of the pressure at each side of the liver-on-a-chip model. The flow unit was placed on the fluidic path to ensure a constant flowrate of 10µL/min, and the pressure difference was determined. An increase of pressure difference would mean clogging appeared in the system.
Figure 3: Fluigent setup used for pressure measurement
Partial results
The first step was to investigate the clogging effect with hydroscaffold in the chip. To do this, the pressure level was measured on the chip with and without the integrated hydroscaffold and compared between the 2 conditions. In Figure 4, we see that the pressure values are similar between the 2 conditions for various tested flowrates, demonstrating no clogging due to the presence of the hydroscaffold in the human liver-on-a-chip model.
Figure 4: Pressure comparison between the empty biochip and the biochip filled with 3D hydroscaffold for various flow-rates
After cells were seeded in the biochip filled with the 3D hydroscaffold, the pressure difference was measured at 10µL/min at several timepoints (fig 5). During days 0 to 11, the pressure values were found stable, around 60mbar, with spheroid growing up to 450µm diameter. Despite the increase in cell numbers, the hydraulic resistance remains stable, and the medium can circulate between the spheroids. From day 14 on, a strong proliferation was observed (fig 2), and spheroids began to form large aggregates, occupying most of the biochip. Eventually, the aggregates caused clogging at day 21, demonstrated by the pressure rise seen in figure 5.C. The pressure then goes back to normal when the aggregate is flushed away. This pressure increase, up to 1.5 bar, caused chip leakage and experimental failure. The chip failure gave information on the time of viability of the liver-on-a-chip model.
Figure 5: Pressure evolution during the 14 first days of culture (a), and pressure measurement inside the chip at days 14 and 21 (b,c)
References
[1] Hughes, J.P.; Rees, S.; Kalindjian, S.B.; Philpott, K.L. Principles of early drug discovery. Br. J. Pharmacol. 2011, 162, 1239–1249. [Google Scholar] [CrossRef]
[2] Freyer, N.; Knöspel, F.; Strahl, N.; Amini, L.; Schrade, P.; Bachmann, S.; Damm, G.; Seehofer, D.; Jacobs, F.; Monshouwer, M.; et al. Hepatic differentiation of human induced pluripotent stem cells in a perfused three-dimensional multicompartment bioreactor. Biores. Open Access 2016, 5, 235–248. [Google Scholar] [CrossRef]
[3] Merlier, F.; Jellali, R.; Leclerc, E. Online hepatic rat metabolism by coupling liver biochip and mass spectrometry. Analyst 2017, 142, 3747–3757. [Google Scholar] [CrossRef]
Expertises & Resources
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Microfluidics case studies A microfluidic Artery-on-a-Chip using Fluigent’s Microfluidic Flow Control System, the MFCS Read more
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Microfluidics case studies Creating a Microfluidic Cancer-on-Chip Platform using Fluigent’s High Throughput Cell Perfusion Pack Read more
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Microfluidic Application Notes Automating Neuronal Cell Immunofluorescence in Microfluidic Chips Read more
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Expert Reviews: Basics of Microfluidics Microfluidic pressure control for organ-on-a-chip applications: A comprehensive guide Read more
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Microfluidics case studies University of Rochester: A tissue chip platform for real-time sensing of secreted inflammatory markers using ARIA Read more
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Microfluidics White Papers Microfluidic white paper – A review of Organ on Chip Technology Read more
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Expert Reviews: Basics of Microfluidics How to choose a microfluidic chip Read more
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Microfluidic Application Notes Peristaltic Pump vs Pressure-Based Microfluidic Flow Control for Organ on Chip applications Read more