A reproducible procedure for primary culture and subsequent maintenance of multiple lines of human skin fibroblasts

Abstract

Cultured fibroblasts are a valuable tool to study many cellular processes and their modification by aging. Fibroblasts are a useful cell type in which to study many diseases, including those of the nervous system, in which a strong genetic component is suspected. Fibroblasts permit the study of multiple, dynamic processes in living cells, while avoiding the effect of the dying process and post-mortem artifacts that limit other approaches. For results to be comparable across time in one laboratory or consistent between laboratories, the detailed culture techniques require meticulous care and replicability. Lack of attention to detail in initial stages can lead to selection of different cell populations. Small variations in othe variables such as batches of serum can significantly alter growth rates and comparisons of cells from controls and Alzheimer patients. The aim of this paper is to present a detailed protocol for comparison of multiple cell lines from many patients. An example of using this approach to study growth and phase out (i.e., senescence) of cells from Alzheimer patients is presented. This procedure represents a modification of an earlier protocol (Cristofalo and Charpentier, 1980).

Assessment of Membrane Potentials of Mitochondrial Populations in Living Cells

“…A fibroblast cell line GM8399 from human skin was purchased from the Coriell Cell Repository (Camden, NJ). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 1 g/L glucose supplemented with 10% fetal bovine serum in a humidified 5% CO 2 incubator at 37°C (17). Five to 7 days before experiments, the cells were subcultured onto DeltaT glass bottom dishes from Bioptechs (Butler, PA) at 500 cells/cm 2 .…”

Serial passage of MC3T3‐E1 cells down‐regulates proliferation during osteogenesis in vitro

“…Once cultures were initiated in T-25 flasks at a seeding density of 1.25 × 10 3 /cm 2 , the cells were subcultured every 4 days at the same seeding density. The population doubling (Gibson et al . 1998) at each passage was calculated using the total number of cells seeded and the total number of cells harvested according to the following equation:…”

Glyceraldehyde 3-Phosphate Dehydrogenase Abnormality in Metabolically Stressed Huntington Disease Fibroblasts

“…Fibroblasts were obtained from the NIGMS Human Genetic Mutant Cell Repository (Camden, N.J., USA) and meticulously cultured as described [Sheu et al, 1994;Gibson et al, 1998]. The familial HD group (accession No., sex of donor and age in years) included GM04281 (F, 20); GM03621 (F, 29); GM04737 (M, 20); GM04847 (M, 31); GM04719 (F, 39); GM04877 (F, 40); GM01187 (M, 43) and GM01169 (M, 50) (mean B SEM = 34 B 4 years).…”