In situ micropatterning technique by cell crushing for co-cultures inside microfluidic biochips.

To perform dynamic cell co-culture on micropatterned areas, we have developed a new type of "on chip and in situ" micropatterning technique. The microchip is composed of a 200 microm thick PDMS (polydimethylsiloxane) chamber at the top of which are located 100 mum thick microstamps. The PDMS chamber is bonded to a glass slide. After sterilization and cell adhesion processes, a controlled force is applied on the top of the PDMS chamber. Mechanically, the microstamps come into contact of the cells. Due to the applied force, the cells located under the microstamps are crushed. Then, a microfluidic perfusion is applied to rinse the microchip and remove the detached cells. To demonstrate the potential of this technique, it was applied successfully to mouse fibroblasts (Swiss 3T3) and liver hepatocarcinoma (HepG2/C3a) cell lines. Micropatterned areas were arrays of octagons of 150, 300 and 500 microm mean diameter. The force was applied during 30 to 60s depending on the cell types. After cell crushing, when perfusion was applied, the cells could successfully grow over the patterned areas. Cultures were successfully performed during 72 h of perfusion. In addition, monolayers of HepG2/C3a were micropatterned and then co cultured with mouse fibroblasts. Numerical simulations have demonstrated that the presence of the microstamps at the top of the PDMS chamber create non uniform flow and shear stress applied on the cells. Once fabricated, the main advantage of this technique is the possibility to use the same microchip several times for cell micropatterning and microfluidic co-cultures. This protocol avoids complex and numerous microfabrication steps that are usually required for micropatterning and microfluidic cell culture in the same time.