Wright Dylan, Rajalingam Bimalraj, Selvarasah Selvapraba, Dokmeci Mehmet R, Khademhosseini Ali
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Lab Chip. 2007 Oct;7(10):1272-9. doi: 10.1039/b706081e. Epub 2007 Jul 25.
Many biological processes, such as stem cell differentiation, wound healing and development, involve dynamic interactions between cells and their microenvironment. The ability to control these dynamic processes in vitro would be potentially useful to fabricate tissue engineering constructs, study biological processes, and direct stem cell differentiation. In this paper, we used a parylene-C microstencil to develop two methods of creating patterned co-cultures using either static or dynamic conditions. In the static case, embryonic stem (ES) cells were co-cultured with fibroblasts or hepatocytes by using the reversible sealing of the stencil on the substrate. In the dynamic case, ES cells were co-cultured with NIH-3T3 fibroblasts and AML12 hepatocytes sequentially by engineering the surface properties of the stencil. In this approach, the top surface of the parylene-C stencil was initially treated with hyaluronic acid (HA) to reduce non-specific cell adhesion. The stencil was then sealed on a substrate and seeded with ES cells which adhered to the underlying substrate through the holes in the membrane. To switch the surface properties of the parylene-C stencils to cell adhesive, collagen was deposited on the parylene-C surfaces. Subsequently, a second cell type was seeded on the parylene-C stencils to form a patterned co-culture. This group of cells was removed by peeling off the parylene-C stencils, which enabled the patterning of a third cell type. Although the static patterned co-culture approach has been demonstrated previously with a variety of methods, layer-by-layer modification of microfabricated parylene-C stencils enables dynamic patterning of multiple cell types in sequence. Thus, this method is a promising approach to engineering the complexity of cell-cell interactions in tissue culture in a spatially and temporally regulated manner.
许多生物过程，如干细胞分化、伤口愈合和发育，都涉及细胞与其微环境之间的动态相互作用。在体外控制这些动态过程的能力对于制造组织工程构建体、研究生物过程以及指导干细胞分化可能具有潜在的用途。在本文中，我们使用聚对二甲苯-C微模板开发了两种在静态或动态条件下创建图案化共培养物的方法。在静态情况下，通过在底物上可逆密封模板，将胚胎干细胞（ES细胞）与成纤维细胞或肝细胞共培养。在动态情况下，通过设计模板的表面特性，将ES细胞依次与NIH-3T3成纤维细胞和AML12肝细胞共培养。在这种方法中，聚对二甲苯-C模板的顶面首先用透明质酸（HA）处理以减少非特异性细胞粘附。然后将模板密封在底物上，并接种ES细胞，这些细胞通过膜上的孔粘附到底层底物上。为了将聚对二甲苯-C模板的表面特性转变为细胞粘附性，在聚对二甲苯-C表面沉积胶原蛋白。随后，将第二种细胞类型接种在聚对二甲苯-C模板上以形成图案化共培养物。通过剥离聚对二甲苯-C模板去除这组细胞，从而能够对第三种细胞类型进行图案化。尽管先前已经用多种方法证明了静态图案化共培养方法，但微加工聚对二甲苯-C模板的逐层修饰能够依次对多种细胞类型进行动态图案化。因此，这种方法是以空间和时间调节的方式构建组织培养中细胞-细胞相互作用复杂性的一种有前途的方法。
