A Simple Aspect Ratio Dependent Method of Patterning Microwells for Selective Cell Attachment

Abstract

3-D culture has been shown to provide cells with a more physiologically authentic environment than traditional 2-D (planar) culture [1, 2]. 3-D cues allow cells to exhibit more realistic functions and behaviors, e.g., adhesion, spreading, migration, metabolic activity, and differentiation. Knowledge of changes in cell morphology, mechanics, and mobility in response to geometrical cues and topological stimuli is important for understanding normal and pathological cell development [3]. Microfabrication provides unique in vitro approaches to recapitulating in vivo conditions due to the ability to precisely control the cellular microenvironment [4, 5]. Microwell arrays have emerged as robust alternatives to traditional 2D cell culture substrates as they are relatively simple and compatible with existing laboratory techniques and instrumentation [6, 7]. In particular, microwells have been adopted as a biomimetic approach to modeling the unique micro-architecture of the epithelial lining of the gastrointestinal (GI) tract [8–10]. The inner (lumen-facing) surface of the intestine has a convoluted topography consisting of finger-like projections (villi) with deep well-like invaginations (crypts) between them. The dimensions of villi and crypts are on the order of hundreds of microns (100–700 μm in height and 50–250 μm in diameter) [11]. While microwells have proven important in the development of physiologically realistic in vitro models of human intestine, existing methods of ensuring their surface is suitable for cell culture are lacking. Sometimes it is desirable to selectively seed cells within microwells and confine or restrict them to the microwells in which they are seeded. Existing methods of patterning microwells for cell attachment either lack selectivity, meaning cells can adhere and migrate anywhere on the microwell array, i.e., inside microwells or outside of them, or necessitate sophisticated techniques such as micro-contact printing, which requires precise alignment and control to selectively pattern the bottoms of microwells for cell attachment [12, 13].