Cultivating cells under flow perfusion has two main advantages: constant medium renewal and reproduction of mechanical strain in vitro.
Media renewal is critical in microfluidic chips as cells are confined in microchamber. As a result, the media can be locally depleted in nutrients and saturated in toxins depending on the concentration cells. Constant perfusion smoothens local heterogeneities and assures that every cell is exposed to similar media composition.
In vivo, most cells are constantly exposed, actively or passively, to mechanical forces. Reproducing these physiological constraints in vitro is essential to induce the right phenotype to cells, finalize their maturation an maintain homeostasis. As an example, endothelial cells require to be cultured under stable constant shear strain.
To grow cells under perfusion, we recommend using the BE-Flow chip.
Please note that channels can be perfused independently or inter-connected to perfuse different cell types in series.
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. Biochemical stimulation is also central in oncology as it drives the migration and expansion of malign tumors.
Reproducing stable gradient in vitro is fundamental to elucidate the cellular pathways at play. Combining biochemical gradient with 3D hydrogel further increases the relevance of the model as the chemical stimulation is implemented in a physiological matrix.
To maintain stable gradient over time flow must be tightly regulated with high performances perfusion instruments.
To study chemotaxis in 3D hydrogel we recommend using BE-GRADIENT
Co-culture under perfusion is essential for various applications:
- to fully differentiate given cell types, coculture with another cell type is required
- in cosmetics or drug testing to reproduce interfaces link skin, lung or gut and monitor the uptake of active molecules
- to reproduce liquid-liquid or air-liquid interfaces.
The BE-Doubleflow chip best fits these application as a porous membrane separates the central chamber into two channels. Each channel can be flown independently. Each cell type can be cultured on one side of the membrane with its specific medium perfused at a physiological shear stress.
Transwell are classic devices widely used in standard cell culture to co-culture cell types, reproduce 2D-2D interfaces, 3D-2D interfaces, study cell invasion, assess monolayer permeability or active molecule uptake.
Replicating this tool in a microfluidic format enables to drastically reduce the volume of the underlying medium and thereby concentrate the molecule secreted by cells. Flow can also be implemented in the underlying channel to grow endothelial cells or constantly renew medium to make sure that the cells are grown in the same conditions during the whole experiment.
Cell seeding in 3D hydrogel is a delicate manipulation. Hydrogels are fragile and soft. They can easily fracture or detach from the chip if the flow is instable. Manual cell seeding is not recommanded as the discontinous flow results in heterogeneous cell seeding and can crack the hydrogel. The manual mode of the Flow EZ is perfect to circumvent these limitations as it allows to implement small flow rate increments while keeping an eye on the microscope.
Fluigent’s product have unprecedent performances in terms of stability and responsiveness.
High stability is particularly usefull for applications with constant shear stress like vascular perfusion.
High responsiveness is essential to reproduce complex flow patterns like aortic pressure variations. For complex flow pattern the orders can be set in flow or pressure variations.
In vivo, most cells are constantly exposed, actively or passively, to mechanical forces. Reproducing these physiological constraints in vitro is essential to induce the right phenotype to cells, finalize their maturation and maintain homeostasis.
Pressure based system are the finest systems to reproduce mechanical stimulation due to their excellent responsiveness and acute precision. Complex dynamic mechanical stimulation like diaphragm contraction of the lung, peristaltic motion of the gut or compression in cartilage can be easily reproduced and automated.
Cartilage on chip. Courtesy of S. LeGac, AMBER lab, Twente university.
Having access and controlling independently flow rate and pressure is a valuable advantage to:
- Monitor in live the pressure experienced by the cells. The pressure applied to maintain a constant flow rate may vary during the course of the experiment as the channel gets obstructed. Such variations may affect the cells and can vary between experiments and chips. Pressure based system are the only instrument on the market to provide this information.
- Directly implement living values. Physiological values in the body are measured pressure (occular, intracranial, arterial…). Pressure based systems allows to directly apply the right pressure instead of translating a pressure into flow rate or shear stress.
- Monitor the cell growth in live. As cells grow they obstruct the channel and thereby increase the chip resistance. Given the linear relationship between the pressure flow rate and resistance, monitoring pressure variations give direct access to the number of cells in the chip. This simple method avoid discarding a sample for each time point compared to traditionnal methods like trypsination or imaging.
Once the parameters (pressure, volume, flow rate) are set and optimized, protocol automation is the key to save time, limit contamination and decrease variability.
For protocols with sequential perfusion, Aria is the only system on the market to automate the delivery of up to 10 solutions in a sample.
For any protocol, valves, pressure and flow controller from Fluigent can be assemble to automate the protocol using a user-friendly software (MAT)