Biomimetic Nanofibrous Mesh for Long-Term Preservation of Intestinal Epithelial Cells

Gastrointestinal GVHD is a major complication of allogeneic blood and marrow transplantation.  In vitro models for screening potential donor T cell reactivity to host intestinal epithelial cells (IEC) as a portent of GVHD have been hampered by difficulty in maintaining primary IEC cultures.  To approach this problem, conditional reprogramming (CR) technology was combined with tissue-engineering scaffolds composed of biocompatible polycaprolactone (PCL)/collagen nanofibers to enable a long-term preservation of primary murine IECs. Conditioned medium (CM) used for CR contains the Rho-associated kinase inhibitor Y-27632, an anti-apoptotic agent that can render CRIEC unsuitable for cell damage assays indicative of GVHD. Thus, we sought to create an enabling biomimetic extracellular matrix (ECM) platform that could support viable and functional CR murine-IEC (m-CRIEC) in the absence of Y-27632.

Methods: Small intestine-derived IEC were plated on fibronectin-coated coverslips with CM+Y-27632.   The m-CRIEC were removed from this media and seeded onto nanofibrous matrices in the presence of RPMI-1640 media. IEC normally reside on the thin fibrous basement membrane (BaMe) consisting of intermingled networks of laminins and type IV collagen, which provides cell anchoring and barrier functions. The BaMe interacts with cells through integrin receptors and other plasma membrane molecules, influencing cell phenotype and survival. Using electrospinning, BaMe-like fibrous meshes were prepared.  Slow degradable PCL was used as the fiber matrix phase in which Type I collagen (representing ECM molecules) was dispersed.

Results: Flow cytometric analysis and microscopic inspection of m-CRIEC growing ≤ 7 days under CM+Y-27632-depleted conditions presented with comparable viability and phenotypic display to that of m-CRIEC growing in CM+Y-27632, however only cells placed on nanofibers maintained cobblestone morphology and integrity (Figure 1).

Conclusions: We have demonstrated that nanofibrous meshes can provide a physiologically relevant ECM-like microenvironment for the ex vivo maintenance of IEC. This biomimetic approach should prove particularly beneficial for the assessment of intestinal GVHD potential elicited by donor T cells.