(74d) Stable Microwell Arrays As Platforms for Long-Term Tissue Culture

Microtechnology tools have been widely used to fabricate microenvironments for high throughput analyses of a large variety of cell types. Our group has been using microtechnology tools to create microwell arrays to assay the prevalence of rare cells in complex cell mixtures and subsequently study their proliferation and migration behavior under specific biological conditions. The microwell arrays are formed via soft lithography, using polydimethylsiloxane (PDMS) molds. Poly(ethylene)glycol (PEG) hydrogel composes the walls of square microwell arrays which are bonded to a silane-functionalized glass substrate. PEG hydrogel was chosen due to its resistance to cell and protein adhesion, which prevents cell migration outside of its original microenvironment.  Our preliminary studies have shown that primary human endometrial stromal cells (ESCs) can be successfully isolated inside these microenvironments with relatively high platting efficiency. The dimensions of the microwells used in this study were 50x50um or 100x100um wells, 20um wall thickness and 40um height. We show that ESCs can proliferate in 100x100um microwells when cultured for up to 7 days. However, for long-term cell culture (> 3 days), optimization of the silanization protocol was necessary to ensure stable adhesion of the wells to the surface.  We tested 3 types of silane coupling agents and several solvents’ systems in order to achieve maximum durability of microwells under tissue culture conditions while maintaining high plating efficiency of endometrial cells. We also investigated the potential of varying mesh size of the PEG hydrogel, by varying synthetic parameters of the gel, for controlled molecule diffusion between wells.

  Atomic Force microscopy (AFM), contact angle measurements, optical microscopy, and cell adhesion assays were performed to characterize the substrates. Average mesh size of PEG hydrogels with various molecular weights was measured via a correlation with the equilibrium degree of swelling of the polymeric networks. Stability of the microwell arrays were verified every 2 days by imaging analysis, and the average of the delaminated area was calculated.  We demonstrate how we can use these arrays to perform long-term cell culture while isolating individual cells or a group of cells in order to study cell-cell interactions, cell migration and proliferation and several other facets of cell biology.