Synthetic Hydrogels for Stem Cell Propagation and Organogenesis

Technology

This invention is a synthetic hydrogel consisting of branched biodegradable polymers and one or more binders. The polymers are preferably multi-arm polyethylene glycol (PEG) macromers of various molecular weights while the binders include adhesion ligands, for example, integrin binding peptides derived from collagen, fibronectin, and/or laminin or extracellular matrix components (ECM) binders which are synthetic peptides with affinity for ECM components. The biodegradable polymers are crosslinked via synthetic peptides containing protease-, proteinase-, or transpeptidase- cleavable motifs so they can be dissolved on demand to yield intact organoids substantially free of hydrogel polymers. The method of forming the synthetic hydrogel involves a first step in which the PEG macromers and various peptide binders are functionalized. For example, PEG macromers may receive vinyl sulfone groups; the peptide binders or crosslinking agents may be synthesized with a free thiol group at the N-terminus. The hydrogels can also include cells, tissues, organs or combinations of these. Described in this invention is adjustment of the hydrogel’s biomechanical properties for support of organoids derived from human epithelial, endometrial or gastrointestinal cells.

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 and 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.