Convenient, reliable, Bias-Free dynamic patterning of multiple types of cells into precisely defined micropatterns for Co-Culture study

We present a simple and flexible mechanical approach for 2D micropatterning of multiple cell lines without surface modification. The principle is that during initial attachment cells only stick to unoccupied areas; we create such areas to selectively seed a subsequent cell line by using a stamp to erase previously attached cells at designed areas. By repeating this process, multiple types of cells were patterned on a single substrate. This strategy minimizes the disturbance to cell health, which might be a concern in printing-based patterning. Micropatterns of cells with resolution of.50 mm, and a co-culture of three cell types were easily achieved and studied. This method was found universal to at least MSC, HeLa, HepG2 and NIH 3T3. We also measured the invasion and cell growth under the co-culture of HeLa, MSC and HepG2. This method is useful for studying cell-cell interaction in high-throughput, free of the potential bias caused by selective surface modification in some previous methods.