Primary Fibroblasts from Human Adults as Target Cells forEx VivoTransfection and Gene Therapy

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Abstract
Diploid fibroblast (dFb) cultures were established from a total of 106 skin and serosa biopsies of human adults. Using an optimized enzymatic dissociation procedure, 1011 dFb/cm2 skin were obtained from patients younger than 60 years after an average time of 89 ± 8 days, with a mean population doubling time of 3.87 ± 1.4 days. Enzymatic dissociation of skin biopsies yielded cultures of significantly higher growth capacity of dFb than those prepared by mechanical dissociation followed by spontaneous outgrowth of cells. The plating efficiency that may be crucial for clonal selection of transfected cells was negligible when dFb were plated without feeder cells at low density, while it was enhanced to 9–24% by the addition of a feeder layer of irradiated human embryonal fibroblasts. DFb secreted various cytokines with spontaneous release of interleukin-6 (IL-6) in high quantities of up to 20 ng/106 cells/24 hr. In addition, one-third of the culture secreted substantial amounts of granulocyte-macrophage colony-stimulating factor (GM-CSF), while low amounts of tumor necrosis factor-α (TNF-α) were detectable in some cases after irradiation of the cells. Comparison of various transfection methods by a transient luciferase expression assay demonstrated that receptor-mediated gene transfer was approximately 10-fold more efficient than cationic lipofection of dFb, while electroporation resulted in substantially less expression of the reporter gene. We conclude that primary dFb can be obtained reproducibly from human adults and represent a suitable target cell population for receptor-mediated gene transfer and cationic lipofection. However, these cells are not biologically inert and the constitutive secretion of immunomodulatory cytokines may well affect in vivo therapeutic responses. Based on a specific dissociation protocol of skin biopsies, diploid fibroblast cultures from human adults could reproducibly be initiated and exhibited excellent growth potential. Characterization of these fibroblasts with respect to somatic cell therapy revealed constitutive production of substantial amounts of IL-6 and GM-CSF. Among several transfection methods tested, receptor-mediated gene transfer and cationic lipofection resulted in efficient transient expression of foreign genes.