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

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, 10(11) 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/10(6) 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-alpha (TNF-alpha) 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitogen Stimulation Cooperates with Telomere Shortening To Activate DNA Damage Responses and Senescence Signaling

“…As described in previous studies, telomeres were significantly shorter in latepassage cells of all three cell lines than in early-passage cells, but telomere length was stabilized by the overexpression of TERT ( Fig. 1; data not shown for WI38 cells) (21,27,34,36,56).…”

“…To analyze whether the senescence phenotype induced by telomere shortening is constitutively operative at the same level or is amplified by mitogenic stimuli, we first explored this phenomenon in human fibroblast cultures with three different cell lines (IMR90, WI38, and HK1) (27,36,56). As described in previous studies, telomeres were significantly shorter in latepassage cells of all three cell lines than in early-passage cells, but telomere length was stabilized by the overexpression of TERT ( Fig.…”

Biological and morphological characterization of human neonatal fibroblast cell culture B-HNF-1

“…They reached the stationary phase after 96 hours and the population doubling time was about 24 hours. Results obtained by other authors were quite diverse and they were dependent on the species origin and age of the tissue donor (Li et al 2009;Wu et al 2008;Veelken et al 1994).…”

The ubiquitin ligase APCCdh1 is required to maintain genome integrity in primary human cells

“…Cell culture U-2-OS human osteosarcoma cell lines and primary human fibroblasts (Veelken et al, 1994) were cultured in 90% Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, CA, USA)/10% fetal bovine serum (FBS) supplemented with 1% penicillin/streptomycin (Invitrogen), 1 mM sodium pyruvate (Invitrogen) and 2 mM L-glutamate (Invitrogen). Transduced U-2-OS and primary human fibroblasts were grown in the presence of 2.5 mg/ml puromycin (Sigma, St Louis, MO, USA).…”