In vitro and in vivo characterization of genetically engineered cells microencapsulated in a HEMA-MMA copolymer .

摘要

The encapsulation of genetically engineered cells in small-diameter ( ∼ 450 m m) poly(hydroxyethyl methacrylate-co-methyl methacrylate) (HEMA-MMA, 75% HEMA) microcapsules as a platform for the use of encapsulated cells in an alternate approach to autologous somatic gene therapy was explored. The effect of inclusion of a matrix in the core of the capsules on the behaviour of three different cell lines, namely human embryonal kidney (HEK) "293" cells transfected to secrete human hepatic lipase, mouse C2C12 myoblasts transfected to secrete human growth hormone, and mouse L929 fibroblasts transfected to secrete human placental alkaline phosphatase (SEAP) was investigated in vitro. Presence of ultralow gelling temperature agarose in the core of the capsule promoted the proliferation of HEK cells. The viability and transgene expression of transfected C2C12 cells was improved in microcapsules that contained MatrigelRTM, a reconstituted basement membrane extract from a mouse sarcoma. Co-encapsulation with a bovine dermal type I collagen successfully maintained the viability of L929 cells. In addition, reduction of the concentration of poly(HEMA-MMA) in the solution used to fabricate the capsules did not affect the rate or extent of proliferation of encapsulated HEK cells. The in vivo performance of poly(HEMA-MMA) encapsulated cells was investigated with transfected L929 cells that were implanted in the peritoneal cavity of C3H mice. The implantation of microcapsules that were suspended in a phosphate-buffered saline solution or an ultralow gelling agarose gel (SeaPrepRTM, which physically disintegrated) resulted in deformation, aggregation, and poor retrievability of the microcapsules. As a result, limited viability of the encapsulated cells was observed 3 weeks post-implantation. However, immobilization of the microcapsules in a low gelling temperature agarose gel (SeaPlaqueRTM) resulted in maintenance of viability of ∼ 50% of the encapsulated cells. Once the viable cells were released from retrieved microcapsules and regrown as monolayers, they expressed SEAP at a similar level to cells released from non-implanted microcapsules. Thus, the potential for the delivery of recombinant proteins with poly(HEMA-MMA) encapsulated genetically engineered cells was established in this study.