(5ak) Tissue and Metabolic Engineering of Biohybrid Artificial Organs

In an effort to develop a biohybrid artificial organ, mammalian cells were engineered with several properties to make them adept in secreting therapeutic proteins and surviving low oxygen levels in an encapsulated environment. Three cell lines, C2C12, Jurkat, and HEK293, were investigated for their ability to secrete human interleukin 2 (hIL2), which can serve as an anti-cancer agent. An intracellular red fluorescent protein marker was independently expressed using an internal ribosome entry site sequence so that hIL2 remained active and free for secretion. DsRed fluorescent protein markers were used since red light is known to be transmissible through mammalian tissue. Transient transfection of all three cell lines proved that internal red fluorescence measurements were indeed linearly correlated with the concentration of hIL2 secretion. To increase the survivability of encapsulated cultures, cells were engineered with an anti-apoptotic gene, bcl-2Δ, placed under the control of a hypoxia sensitive promoter. This protective system was found to lessen both hypoxia induced necrosis and apoptosis. To complete the biohybrid system a novel hydrogel (mTG-Gel), utilizing microbial transglutaminase to enyzmatically crosslink gelatin, was developed as a biocompatible cellular scaffold for encapsulating the engineered cells. Specifically, HEK293 cells that were metabolically engineered with all the previous characteristics were encapsulated in mTG-Gels. In situ analysis of DsRed fluorescence showed that cells overlayed with a minimal height of gel were the most productive. Further increases in mTG-Gel overlay caused a reduction in fluorescence. Human IL2 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.