Quantitative Studies of the Growth of Mouse Embryo Cells in Culture and Their Development Into Established Lines

Abstract

Disaggrcgatcd mouse embryo cells, grown in monolaycrs, undcrwcnt a progrcssivc dcclinc in growth ratc upon succcssivc transfer, the rapidity of the decline dcpcnding, among othcr things, on the inoculation density. Ncvcrthclcss, ncarly all culturcs dcvclopcd into cstablishcd lincs within 3 months of culture. Thc first sign of thc emcrgcncc of an established line was the ability of thc cells to maintain a constant or rising potential growth ratc. This occurred while thc cultures wcrc morphologically unchangcd. Thc growth rate continued to incrcasc until it cqualcd or cxcccdcd that of the original culturc. The carly cstablishcd cclls showed an increasing mctabolic autonomy, as indicated by dccrcasing dependence on ccll-to-ccll fccding. It is suggcstcd that the process of cstablishmcnt involvcs an altcration in ccll pcrmcability propcrtics. Chromosome studics indicatcd that thc cells rcsponsiblc for thc upturn in growth rate wcrc diploid, but latcr the population shifted to the tctraploid range, often very rapidly. Still later, marker chromosomes appcarcd. Different lines acquired diffcrcnt propcrtics, dcpcnding on thc culture conditions cmploycd; one line dcvclopcd which is cxtrcmcly scnsitivc to contact inhibition.

Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts

“…1a). As expected 5 , the cells grew well for approximately 1 week (2-3 population doublings) before proliferation began to decline. After 4-5 weeks (8-10 population doublings), the cultures senesced (arrow), showing no change in cell number for more than 1 week and a senescent morphology.…”

“…MEFs spontaneously overcome replicative senescence (immortalization) 5,6 , whereas human fibroblasts rarely do so. Moreover, MEF senescence relies predominantly on the p19 ARF /p53 tumour suppressor pathway, whereas human fibroblasts require loss of both p53 and retinoblastoma (Rb) tumour suppressor functions for Correspondence should be addressed to: J.C. (jcampisi@lbl.gov).…”

“…After 4-5 weeks (8-10 population doublings), the cultures senesced (arrow), showing no change in cell number for more than 1 week and a senescent morphology. Proliferation eventually resumed, owing to outgrowth of immortal variants 5 .…”

Targeting an E2F site in the mouse genome prevents promoter silencing in quiescent and post-mitotic cells

“…MEFs were rendered senescent by serial cultivation with the 3T3 protocol (Todaro and Green, 1963). Briefly, passage 2 MEFs were plated in duplicate at 1.5 Â 10 6 /10 cm plate and incubated for 3 days.…”

Cytogerontology since 1881: A reappraisal of August Weismann and a review of modern progress

“…Hayflick 1977), and it is clearly true for the two most extensively studied mammals, mice and humans. Diploid embryonic mouse fibroblasts can undergo only 14-28 population doublings in vitro (Rothfels et al 1963;Todaro and Green 1963), which, even allowing for the disparity in body size, is substantially less than the 50 or so for human fibroblasts. The maximum longevity of mice is 3.5 years compared with 110 years for humans.…”