Development of a Juxtracrine-Permeable Membrane for Compartmentalized Co-Culture.

摘要

Cell-cell interactions dictate a variety of physiological processes through an intricate balance of both contact-mediated (juxtacrine) and soluble signaling. Using in vitro co-culture models is one method that can help gain a better understanding of these processes. Ideally, co-cultures should include both contact and soluble signaling, while allowing separable populations of cells to determine cell-specific signals. Unfortunately, traditional compartmentalized co-cultures (media transfer, commercial membrane inserts) prevent cell contact, whereas re-sorting mixed co-cultures back into pure populations can be challenging. While mechanically configurable substrates permit separation after contact co-culture, there is a limited interface between the two populations and cross-contamination due to migration is a significant concern.;In this work, we address these limitations by creating thin, juxtacrine-permeable membranes that segregate cell populations during co-culture, while minimizing cell crossover by limiting pore size. High resolution membranes were fabricated at 1 mum thickness using simple photolithography on two different polymer materials, Parylene HT and 1002F negative photoresist. We achieved highly uniform arrays with pore sizes of 0.8, 1.2, 2, and 4 mum and porosities ranging from 5% to 40%. Cell viability tests indicated no significant difference on fabricated membranes in comparison to commercial membrane inserts and standard tissue culture plastic. Additionally, our membranes exhibited improved cell imaging, both in bright field and fluorescence microscopy, over commercial membranes.;Cell cross-migration was tested using HT-1080 metastatic cancer cells. We found less than 5% migration through our smaller pore-sized membranes, outperforming commercial membranes of similar pore size. Finally, we examined the ability for cell-cell contact across the membrane using three methods. First, confocal microscopy was used to examine spatial proximity of cells on membranes. Next, we utilized a receptor-ligand reporter system, which showed GFP expression upon successful binding between the Ly49H receptor and m157 ligand, both of which are cell membrane-bound. Lastly, to examine a more physiologically relevant system, we co-cultured rat hepatocytes with fibroblasts, a known contact-dependent interaction, and measured albumin production. Our findings indicate improved juxtacrine signaling on our fabricated membranes over commercial alternatives and offer the potential to enable compartmentalized contact-dependent co-culture studies.