A 3D Protein-Engineered Matrix for Stem Cell Transplantation

Transplantation of stem cells is a promising therapeutic strategy for a variety of injuries and degenerative diseases. Poor and unpredictable cell transplantation to and retention at the injury site currently constitute major roadblocks in the development of these therapies. Using hydrogels as cell-carriers is a potential solution to this problem, by protecting and localizing cells such that they can aid in healing. Unfortunately, commercially available hydrogels often require non-physiological triggers for gelation, potentially damaging cells during encapsulation. In addition, few exhibit thixotropic properties, which are necessary for hand injection through a syringe needle and rapid gel recovery. To address this, we have designed a novel family of protein hydrogels: Mixture-Induced Two-Component Hydrogels (MITCH) that are recombinantly engineered to undergo gelation at constant physiological conditions, shear thinning, and rapid regelation at the injection site. Cells are encapsulated in MITCH by pre-mixing with component one followed by mixing with component two, causing network formation through hetero-assembly of the two polymers. This results in a gel with cells evenly distributed throughout. Encapsulated cells are cultured in growth or differentiation media. ATP and DNA quantification assays, immunocytochemistry, and confocal imaging are used to evaluate viability, distribution, and morphology. Rheological measurements describe the thixotropic mechanics of the gel. Cell-gel constructs are injected into the subcutaneous dorsa of athymic mice, and cell localization to the injection site is assessed through bioluminescence imaging (BLI) and histological analysis. MITCH are reproducible cell encapsulation and injection systems. They support the growth and uniform 3D distribution of relevant cell types, including human and mouse adipose-derived stem cells (hASCs, mASCs). Cultured in osteogenic differentiation medium, hASCs morphologically resemble osteoblasts.  Rheology confirms gelation upon mixing to produce a range of moduli, 10 to 50 Pa, dependent on the choice of molecular recognition binding partners. Rheology also shows that gels undergo thinning under shear force and re-form upon force removal. The injection of MITCH-encapsulated mASCs into mice results in significantly improved cell retention over two weeks when compared to collagen or saline. MITCH successfully demonstrate that a molecular recognition hetero-assembly strategy enables cell encapsulation at constant physiological conditions. In addition, shear-thinning and self-healing properties allow for effective hand injection of gel-encapsulated cells, resulting in improved retention at the injection site. These outcomes, coupled with the ability to further tune MITCH due to the protein-engineered synthesis strategy employed, make MITCH promising for a myriad of stem cell clinical therapies.