(50f) Cellular Encapsulation and Therapeutic Protein Secretion in Mtg-Gels

A novel hydrogel that utilizes microbial transglutaminase to enzymatically crosslink gelatin (mTG-Gels) was developed as a cellular scaffold and encapsulation material for biohybrid artificial organ designs. The biocompatibility of this hydrogel was first demonstrated by encapsulating HEK293 cells and quantifying cellular proliferation with Hoechst 33342 staining. The viability of cells was further confirmed by releasing the cells from the hydrogel and allowing them to recolonize normal tissue culture flasks after three weeks of encapsulation. The thermal and enzymatic stabilities of the hydrogels were challenged by incubation in 37^oC PBS, Trypsin+EDTA, and Proteinase K baths. Because the hydrogels were covalently crosslinked, they were completely stable under physiological temperatures and resistant to harsh enzymatic proteolysis. The rate of degradation was also found to be tunable based on the gelatin content. To characterize the molecular diffusivity of these hydrogels, HEK293 cells were genetically engineered to secrete a model anti-cancer therapeutic protein, human interleukin 2 (hIL2) and encapsulated. Cells were also designed to express a non-fusional intracellular fluorescent protein marker, Destabilized DsRed-Express. In situ analysis of DsRed fluorescence showed that cells overlayed with a minimal height of hydrogel were the most metabolically productive as the hydrogel closely simulated a native 3-D extracellular matrix. Further increases in mTG-Gel overlay caused a reduction in fluorescence, which potentially signified a deprivation of oxygen and nutrients. hIL2 diffusion through the hydrogel into a top layer of media was found to be strongly influenced by the thickness of the overlay. Cells that were not overlayed with mTG-Gel were thus able to transport the most amount of hIL2 into the media despite their comparatively lower level of protein production. Diffusion cells employing various thicknesses of 4% mTG-Gels were used to derive an effective diffusion coefficient for hIL2. Utilizing this coefficient and the previous empirical data, a mathematical model was also developed to describe the diffusion of hIL2 through 4% mTG-Gels.