Clonal Growth in Vitro of Human Cells with Fibroblastic Morphology

A methodology has been described for reliable cultivation in vitro of dispersed fibroblastic cells obtained from normal human organs. The procedure has permitted establishment of stable cell lines from almost every sample taken, among which the following organs were represented: skin, spleen, amnion, lung, liver, bone marrow, brain, muscle, and heart. Equally good growth has been achieved with cells from embryonic or adult tissues. The methods previously developed whereby single cells plated in Petri dishes grow into isolated macroscopic colonies can successfully be applied to the plating of human fibroblastic stocks. Plating efficiencies in the neighborhood of 50 to 60 per cent are readily achieved with such strains. The resulting colonies can be picked and clonal stocks established. Fibroblastic morphology is maintained in the colonies arising from every single cell of such clonal stocks. All of the single cells from epithelioid clonal strains also maintain their integrity throughout repeated subculture. Since the difference between clonal stocks of these two types is always maintained whenever the respective single cells are plated in the same medium, regardless of the previous history of these stocks, it may be concluded that a true genetic difference exists in these cell lines. In addition to the morphological differences between epithelioid and fibroblastic cell strains, the latter have more demanding nutritional requirements for single cell growth. Thus, single cells of fibroblastic lines almost never produce colonies with high efficiency unless the growth medium which is sufficient for epithelioid cells is supplemented with embryo extract, or a cell feeder layer. Fibroblastic cells are also more resistant to tryptic digestion of the bond uniting the cells to glass surfaces. By use of differential media, growth of both fibroblastic and epithelioid cells, respectively, has been obtained, from dispersed single cells obtained by trypsinization of a specimen of human embryonic lung.