Mechanisms of Ageing and De velopment, 4 (1975) 159-166

159

Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

C H I C K E M B R Y O FIBROBLASTS S E N E S C E N C E I N V I T R O : P A T T E R N OF C E L L D I V I S I O N A N D L I F E SPAN AS A F U N C T I O N OF C E L L D E N S I T Y

PN1NA WEISSMAN-SHOMER and MICHAEL FRY

Department of Clinical Biochemistry, Technion, The Aba Khoushy School of Medieine in Hai]a, Haifa (Israel)

(Received August 6, 1974; in revised form January 27, 1975)

SUMMARY The pattern of cell division, ageing and death of cultured chick embryo fibroblasts inoculated at a wide range of cell densities is described. Cell populations seeded at densities of 2.1 10z to 3.1 104 cells per cm 2 double between 39 and 17 times respectively while their life span at all densities remains 57 :~ 3 days. Thus, under the experimental conditions employed, the life span of cultured embryonic chick cells is a function of calendar rather than mitotic time. We also find that the duration of phase III and the generation time of the cells during this period are proportional to the density of the culture. Moreover, the division rate of cells inoculated at low densities (4.2-31.0 10 z cells per cm 2) remains constant throughout their entire lifetime. It is suggested that the decline in the proliferative capacity of chick cells ageing in vitro is a function of both their density and their age.

INTRODUCTION Chick embryo fibroblasts l-a, as well as human diploid fibroblasts 4-8, embryonic heart cells of the marsupial Potorous tridactylis 9 and cells from Galapagos tortoise 10 have a limited life span in vitro. The pattern of cellular growth and senescence of normal cultured cells consists of an initiation period ("phase I") followed by a period of rapid multiplication of the cells ("phase II)" which evolves into a period of gradual decline in the proliferative capacity of the cells ("phase I l l ) " ultimately leading to death of the culture. Although the mechanism of this phenomenon has not yet been elucidated it may be regarded as an in vitro model system for cellular ageing2, 3, 5,1t and several theories have been offered in the recent years to explain the limited survival of cultured cell strains2,12,13A4. The pattern of growth of cultured fibroblasts poses several unresolved problems. One question is the interpretation of phase III as related to cell division. Cessation of cellular proliferation during phase III could result from a uniform increase in the generation time of all cells, alternatively it could be due to the appearance of an increasing population of nondividing cells, or could be due to both. The