Anchorage-independent growth of normal human fibroblasts.

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Abstract
Normal human fibroblasts, considered to be entirely anchorage dependent for proliferation, have been grown in methylcellulose medium. The most important factor required for growth in suspension appears to be the use of high levels of serum and hydrocortisone. Newborn foreskin [HF1031, HF0814, HF1010 and HF0813] or fetal lung fibroblasts (GM2291] form colonies as large as 0.5 mm in diameter after 3 wk, with a colony-forming efficiency as high as 70%. Mouse 3T3 cells that do not form colonies in standard assays for anchorage-independent growth also grow under these conditions. Colony formation results after inoculation of as few as 100 cells/60 mm dish, and metaphase cells have been visualized with a fluorescent DNA stain, showing that colony formation is due to division rather than aggregation. Fibroblasts recovered from suspension and grown as monolayers retain a diploid karyotype and normal shape, do not form tumors upon injection into nude mice, and become senescent. Thus, the trait of anchorage-independent growth in vitro is clearly possessed by normal human fibroblasts and can be expressed under the proper conditions. [Also used in this study were: human cervical carcinoma HeLa D98OR cells; nontumorisenic fibroblast HeLa D98OR hybrid HSH5 cell and the tumorogenic SH5 segregant SH5T cell.].