GROWTH OF RECOMBINANT FIBROBLASTS IN ALGINATE MICROCAPSULES

To develop a novel strategy of nonautologous somatic gene therapy, we now demonstrate the feasibility of culturing genetically modified fibr oblasts within an immunoprotective environment and the optimal conditi ons required for their continued survival in vitro. When mouse Ltk(-) fibroblasts transfected with the human growth hormone gene were enclos ed within permselective microcapsules fabricated from alginate-polylys ine-alginate, they continued to secrete human growth hormone at the sa me rates as the nonencapsulated cells. They also continued to prolifer ate in vitro for at least 1 month even though their viability graduall y declined to about 50%. The viability can be improved by controlling for (a) temperature during encapsulation, (b) duration of treatment wi th polylysine, (c) duration of liquefying the core alginate with sodiu m citrate, and (d) cell density at the time of encapsulation. The best conditions leading to improved survival and maximum proliferation of cells within the microcapsules were obtained by encapsulating the cell s at 4 to 10 degrees C instead of room temperature, coating the micros pheres with polylysine for 6 to 10 min instead of 20 min, liquefying t he core alginate by treating with citrate for 20 min instead of 6 to 1 0 min, and using a concentration of 2 x 10(6) cells/mL of alginate for encapsulation. Under such conditions, normally adherent and genetical ly engineered mouse fibroblasts survived and proliferated optimally wi thin the microcapsule environment. The encapsulated fibroblasts mainta ined their level of transgene expression while recombinant gene produc ts such as human growth hormone could diffuse through the microcapsule membrane without impediment. The demonstration that genetically modif ied fibroblasts can survive and continue to deliver recombinant gene p roducts from within these microcapsules and the optimization for their maximal viability and growth within microcapsules should increase the potential for success in using such microencapsulated recombinant cel ls for somatic gene therapy. (C) 1994 John Wiley & Sons, Inc. sothe fe a-