Development of a human gut-on-chip to assess the effect of shear stress on intestinal functions
Fluid flow and shear stress have previously been reported to promote proper intestinal cell differentiation, formation of villus-like 3D structures and enhanced intestinal barrier function.
Our gut-on-chip system made of BEOnChip microfluidic chips coupled to pressure controllers offers a way to control the microenvironment of in vitro cultured intestinal epithelium that is closer to the physiological state.
Introduction to gut-on-chip
The intestine is a primary and intricate organ responsible for the uptake of nutrients and water while performing a critical immunological function. It’s also the main route for drug absorption and houses a large number of symbiotic microorganisms (microbiota) that support digestion and absorption of nutrients. Intestine dysfunction may cause severe and chronic gut diseases including Inflammatory Bowel Disease (IBD) (e. g., ulcerative colitis and Crohn’s disease), celiac disease or Irritable Bowel Syndrome. Although the incidence of intestinal disorders, especially in the case of IBDs, is increasing globally, their etiology is not fully understood. A complex interaction between the host immune system, genetics, microbiota and environmental factors is the most accepted causative agent, in which the imbalance between pro-inflammatory and anti-inflammatory cytokines and alterations of the composition and function of the gut microbiota (dysbiosis) play a pivotal role. Nowadays, the gut-microbiome research community commonly uses laboratory mice to study these diseases. However, animal models often fail when extrapolated to humans due to the differences in microbiota composition and immune system.
In recent years, organ-on-chip (OOC) technology has emerged as a very promising tool to overcome animal model limitations by recapitulating tissue-and-organ level physiology and function into biologically inspired microfluidic in vitro devices. OOC devices emulate relevant conditions of the microenvironment found in vivo, which is otherwise not possible with conventional cell cultures typically based on 2D monoculture plates. Due to the complex gut dynamics, host-microbiome interactions, and differences between species, gut-on-a-chip (GOC) systems are especially necessary models to advance the knowledge in the intestinal physiology and diseases etiology.
Gut-on-chip was developed to simulate the structure, function, and microenvironment of the human gut and isbecoming a powerful tool to construct physiological models of the human gut, perform drug testing and development, and investigate host–microbiome interactions (1). Commensal microbes present in the digestive system are mostly anaerobic and require low levels of oxygen.
The BE-FLOW microfluidic chip is made of cyclic olefin polymer (COP), a material impermeable to gases that allows the control of gas concentration within the devices.
In addition, peristalsis of the intestinal wall is known to affect the differentiation of the cells that line the digestive tract. Therefore, devices that generate fluid flow and shear stress have been reported to promote accelerated differentiation of intestinal epithelial cells, formation of three-dimensional villus-like structures, and enhanced intestinal barrier function, mimicking the complex functions of the normal human intestine.
Compared with conventional model systems, gut-on-chip offers the capabilities of real-time observation, simulation of the intestinal microenvironment, adjustable fluid flow shear stress , and host–microbiome interface (1).We propose a complete system to set up a model of human gut-on-chip, combining BE-FLOW chips with Fluigent pressure-based flow controllers. This system provides a way of controlling the intestinal epithelium’s microenvironment in a manner that is more in line with the physiological state.
Development of a human gut-on-chip: Materials
BEOnChip device: BE-FLOW
The design of the BE-flow chip consists of two independent channels with a threaded inlet and outlet enabling the insertion of connectors and tubing coupled to fluidic controllers. It’s possible to apply an independent flow rate in both channels using any perfusion system. To avoid culture media evaporation during the onset of the experiment (before perfusion has started), water-filled reservoirs are positioned next to the medium reservoir (cf scheme).
Fluigent Equipment for gut-on-chip experiments
Fluigent positive pressure controllers use small air pressures (mbar) to force a solution to rise through an output tube, precisely controlling the flow rate of a microfluidic system.
The fluidic system configuration is represented in the illustration:
- Pressure compressor RX
- FlowEZ (1000 mbar)
- FlowUnit S
Development of a Human Gut-on-Chip: Method
BE-FLOW cell culture
Before seeding, pre-warm the Be-Flow standard device in the incubator overnight to minimize the formation of air bubbles.
- Fill the channel with 100μl of collagen at 0.1 mg/ml (in PBS 1X) and incubate at 37°C for 30min. Wash the channel by adding 100μl of PBS 1X into the inlet and by aspirating it at the outlet using P200 pipette. Repeat this step three times.
- Aspirate PBS 1X completely before seeding.
- Seed 1.106 Caco-2 cells resuspended in 50μl of culture media.
- Incubate at 37°C, 5% CO2 until the cells have properly spread (2-4h).
- Once cell spreading has occurred, add 300μl of culture medium into the medium reservoirs. Add H2O to the evaporation reservoirs and cover.
If connection to a perfusion system is not possible for more than 2 hours following cell seeding, keep the device in the incubator and renew the culture medium using a rocker as long as needed.
Microfluidic gut-on-chip Set Up
Before setting the flow up:
- Sterilize and pre-warm the tubes and the fluidic elements overnight at 37°C.
- Set the system in a laminar flow cabinet.
- The channels and inlet/outlet wells should never be depleted of culture medium.
- Both inlets and outlets are designed to be able to use connectors (1/4’’- 28).
To work with cells, the assembly of the circuit must be carried out under sterile conditions under a biosafety cabinet. Before use, autoclave the CAPs, pneumatic tubes, threaded connections and ferrules to be used.
- Connect all circuit components except the microfluidic device.
- Establish a flow of ethanol (70%) for at least 15 min to sterilize the sensor and then wash with abundant sterile H2O. Dry by passing air through at maximum pressure. The sensor CANNOT be autoclaved.
- Prime the tubes of the circuit with culture medium until there are no air bubbles
- Remove the culture medium from the chip reservoirs (not from inlet/outlet wells) and connect the tubes to the outlet/inlet using the threaded connections and ferrules.
- The ferrules should be manipulated with the help of sterile forceps.
- Ensure that the tube is perfectly fixed.
- Remove the displaced medium from the reservoirs.
5. Once the system is closed, switch the flow on at 5.8µl/min (shear stress 0.02 dyn/cm²) for 4 days to promote villi formation.o Observe the system under perfusion for a few minutes to check that there are no leaks.
For more information, download the application note.
Development of a Human Gut-on-Chip: Results
After 4 days of perfusion, flow is stopped and the gut-on-chip system is placed under an inverted microscope to monitor cell behaviour/differentiation. As observed in Figure 1, cell density is increased after 4 days of culture under flow conditions. Furthermore, Caco-2 started to form 3D structures-features not seen under static conditions.
Figure 1. Phase contrast images of Caco-2 monolayer in a BE-FLOW channel (A) 24h post cell seeding
before the addition of flow, and after 4 days under shear stress conditions (B), or static conditions
(C). Scale bar = 200 μm.
(1). Ashammakhi, N., Nasiri, R., Barros, N., Tebon, P., Thakor, J., & Goudie, M. et al. (2020). Gut-on-a-chip: Current progress and future opportunities. Biomaterials, 255, 120196. doi: 10.1016/j.biomaterials.2020.120196
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