Engineering the intestinal basement membrane microenvironment using PEG-based hydrogels

Abstract

Event Abstract Back to Event Engineering the intestinal basement membrane microenvironment using PEG-based hydrogels Victor Hernandez-Gordillo1, Gi Hun Choi1, Mollie Smoak2, Rebecca Carrier3 and Linda Griffith1 1 Massachusetts Institute of Technology, Biological Engineering, United States 2 Louisiana State University and Agricultural Center, Department of Biological and Agricultural Engineering, United States 3 Northeastern University, Department of Chemical Engineering, United States Introduction: Intestinal organoids or “mini-guts” are multilobulated structures composed of budding crypts, epithelial cells and well-defined lumens. Mini-guts have gained interest as a tool to study intestinal biology and as a platform for drug screening because they resemble aspects of the intestinal complexity found in vivo[1]. Mini-guts are cultured embedded in ill-defined Matrigel-based hydrogels. Lot-to-lot variability and the presence of residual growth factors in Matrigel can negatively impact the biological outcomes. Thus, the development of a well-defined matrix that can recapitulate aspects of the basement membrane for intestinal organoid culture is of vital importance. Poly(ethylene glycol, PEG)-based hydrogels are good candidates as synthetic matrices. They can be tailored to different applications by varying the stiffness, degradability and cell adhesion properties. Here we report our efforts to develop PEG-based hydrogels functionalized with a fibronectin-derived peptide (SYNK-RGDS) or a collagen-derived peptide (GFOGER) for intestinal organoid culture. Materials and Methods: Hydrogels were prepared by first reacting a 40 KDa, 8-arm PEG-vinyl sulfone with cell adhesive peptides containing single free thiols groups. Then cross-linked using matrix metalloproteinase sensitive peptides harboring two free thiols groups. We incorporated two fibronectin-derived peptides (RGDS and SYNK-RGDS) and one derived from collagen (GFOGER). Single intestinal stem cells were embedded in these hydrogels either directly during hydrogel assembly or indirectly using a two-step encapsulation. The two-step encapsulation allowed us to modulate the stiffness and cell adhesion properties of the bottom and top hydrogel. Results and Discussion: Preliminary results suggest that direct encapsulation of single cells obtained from mouse organoids in these PEG-based hydrogels results in formation of organoids similar to those that form in Matrigel (Fig. 1b). The PEG-GFOGER hydrogel showed higher number of organoids when compared to PEG-Synk-RGDS hydrogel. A two steps encapsulation procedure with varying stiffness and cell adhesion properties resulted in a completely different morphology. Mouse cells developed multicellular growth structures that resemble the villi of the intestine (Fig. 1c). Further analysis will be needed to confirm the identity of these structures. Figure 1. a) Overall strategy to created PEG-Based hydrogels. b-c) Representative confocal image of mouse intestinal stem cells directly embedded b) or two-steps embedding strategy c) after 7 days of culture in PEG-GFOGER hydrogel systems. Actin is depicted in green and nuclei in blue. Conclusions: We were able to identify hydrogel culture conditions that allow single mouse cells to develop into three-dimensional structures that resemble mouse organoids in direct cell encapsulation or multicellular growth that resembles the villi when using two stem cell encapsulation. DARPA; NIH; Boston Children Hospital