Synthetic Hydrogels for Stem Cell Propagation and Organogenesis

Technology

This invention uses multi-arm polyethylene glycol (PEG) macromers of various molecular weights; integrin binding peptide sequences derived from collagen type I and fibronectin; matrix-binding peptides that have an affinity for secreted collagen, laminin, and fibronectin; and peptides that are sensitive to matrix-metalloproteinases (MMPs). First, the PEG macromers and various peptides are functionalized (PEG macromers receive vinyl sulfone groups; the peptides are synthesized with a free thiol group at the N-terminus; the MMP sequence contains a thiol group at each terminus). Next, the PEG macromers, the matrix-binding peptides, and the cells are mixed together. Then, the dithiol-MMP peptides are added to begin crosslinking the hydrogel at 37°C and the Michael-type addition reactions finish in ~20 minutes at physiological pH (7.4), which is comparable to Matrigel™. The reagents used can be purchased off the shelf or made-to-order. Described in this invention is adjustment of the hydrogel’s biomechanical properties for support of intestinal stem cell proliferation from various regions of the gastrointestinal track for mouse and human origin.

Problem Addressed

Organoids are 3D cellular structures with organ-specific cell types and microarchitecture similar to the tissue of origin. They are grown typically by embedding stem cells in Matrigel™, then waiting for the stem cells to self-organize and undergo morphogenesis. However, Matrigel™ lot-to-lot variability and its complex composition prevents researchers from fully investigating the role of the matrix in stem cell organogenesis. The lot variability also contributes to unpredictability in drug discovery experiments. Lastly, extraction of the organoids for downstream analysis (e.g. transcriptomics) is difficult. This novel hydrogel enables control of the mechanical and biological properties allowing researchers to investigate the role of cell-matrix interactions in organogenesis, as well as provide greater consistency between batches for drug discovery. The inert polymer backbone of the synthetic matrix allows proteomic and transcriptomic analysis without removal of the organoid from the hydrogel.

Advantages

• Inexpensive and broadly accessible methods
• The hydrogel integrates easily into commercially available tools and methods
• High cell viability comparable to other gel types
• Broad support of intestinal cells from various regions of the intestinal track with the possibility of incorporating other cell types (intestinal myofibroblast, immune cells)
• Supports human and mouse stem cells
• Mechanically robust and easy to tailor

Publications

Oefner, et al. "Organoid Co-Culture Model of the Cycling Human Endometrium in a Fully-Defined Synthetic Extracellular Matrix Reveals Epithelial-Stromal Interactions." bioRxiv, September 30, 2021. https://doi.org/10.1101/2021.09.30.462577.

Chen, et al. "A Microenvironment-Inspired Synthetic Three-Dimensional Model for Pancreatic Ductal Adenocarcinoma Organoids." Nature Materials 21 (2021): 110-119. https://doi.org/10.1038/s41563-021-01085-1.

Engler, et al. "Engineering PEG-Based Hydrogels to Foster Efficient Endothelial Network Formation in Free-Swelling and Confined Microenvironment." Biomaterials 241 (2020): 119921. https://doi.org/10.1016/j.biomaterials.2020.119921.

Zhang, et al. "Engineering Helical Modular Polypeptide-Based Hydrogels as Synthetic Extracellular Matrices for Cell Culture." Biomacromolecules 21 (2020): 566-580. https://doi.org/10.1021/acs.biomac.9b01297.

Griffith, et al. "On-Demand Dissolution of Modular, Synthetic Extracellular Matrix Reveals Local Epithelial-Stromal Communication Networks." Biomaterials 130 (2017): 90-103. https://doi.org/10.1016/j.biomaterials.2017.03.030.