Leonard Hayflick

The serial cultivation of human diploid cell strains.

Abstract The isolation and characterization of 25 strains of human diploid fibroblasts derived from fetuses are described. Routine tissue culture techniques were employed. Other than maintenance of the diploid karyotype, ten other criteria serve to distinguish these strains from heteroploid cell lines. These include retention of sex chromatin, histotypical differentiation, inadaptability to suspended culture, non-malignant characteristics in vivo , finite limit of cultivation, similar virus spectrum to primary tissue, similar cell morphology to primary tissue, increased acid production compared to cell lines, retention of Coxsackie A9 receptor substance, and ease with which strains can be developed. Survival of cell strains at − 70 °C with retention of all characteristics insures an almost unlimited supply of any strain regardless of the fact that they degenerate after about 50 subcultivations and one year in culture. A consideration of the cause of the eventual degeneration of these strains leads to the hypothesis that non-cumulative external factors are excluded and that the phenomenon is attributable to intrinsic factors which are expressed as senescence at the cellular level. With these characteristics and their extremely broad virus spectrum, the use of diploid human cell strains for human virus vaccine production is suggested. In view of these observations a number of terms used by cell culturists are redefined.

THE LIMITED IN VITRO LIFETIME OF HUMAN DIPLOID CELL STRAINS

Abstract The time at which human diploid cell strains can be expected to cease dividing in vitro (Phase III) is not a function of the number of subcultivations but rather of the number of potential cell doublings. Each clonable cell within the population is endowed with the same doubling potential (50 ±10). Cells of the same strain, but with different “doubling potentials”, were mixed. Phase III in such mixed populations occurs at that time when the “youngest” cell component is expected to reach Phase III. The “older” component has no effect on the time at which Phase III is expected to take place in the “younger” component. An ancillary conclusion that Phase III cannot be explained by the presence of a latent virus, mycoplasma or media composition is confirmed. Human diploid cell strains derived from adult lung have a significantly lower doubling potential in vitro than do fetal strains. The Phase III phenomenon may be related to senescence in vivo. The cellular theory of aging must be related to normal cells in vitro and not to heteroploid cell lines. The former have a finite period of multiplication; the latter are indefinitely cultivable. In vivo experiments also indicate that transplanted normal tissue has a finite lifetime. Chromosome anomalies occurring in Phase III may be related to such anomalies occurring in the cells of older animals, including man. The survival curves obtained with human diploid cell strains are comparable to “multiple-hit” or “multiple-target” curves obtained with other biological systems where an initial threshold dose is required before an exponential form of the curve is established. Whatever cell component(s) may be involved in the finite lifetime of human diploid cell strains, the ultimate accumulation of nondividing cells could be the result of accumulated damage to a single cellular target or to inactivation of many targets.

The future of ageing

Advances in our knowledge of age-associated diseases have far outpaced advances in our understanding of the fundamental ageing processes that underlie the vulnerability to these pathologies. If we are to increase human life expectancy beyond the fifteen-year limit that would result if todays leading causes of death were resolved, more attention must be paid to basic research on ageing. Determination of longevity must be distinguished from ageing to take us from the common question of why we age to a more revealing question that is rarely posed: why do we live as long as we do? But if the ability to intervene in ageing ever becomes a reality, it will be rife with unintended and undesirable consequences.

Biological Aging Is No Longer an Unsolved Problem

Abstract:  The belief that aging is still an unsolved problem in biology is no longer true. Of the two major classes of theories, the one class that is tenable is derivative of a single common denominator that results in only one fundamental theory of aging. In order to address this complex subject, it is necessary to first define the four phenomena that characterize the finitude of life. These phenomena are aging, the determinants of longevity, age‐associated diseases, and death. There are only two fundamental ways in which age changes can occur. Aging occurs either as the result of a purposeful program driven by genes or by events that are not guided by a program but are stochastic or random, accidental events. The weight of evidence indicates that genes do not drive the aging process but the general loss of molecular fidelity does. Potential longevity is determined by the energetics of all molecules present at and after the time of reproductive maturation. Thus, every molecule, including those that compose the machinery involved in turnover, replacement, and repair, becomes the substrate that experiences the thermodynamic instability characteristic of the aging process. However, the determinants of the fidelity of all molecules produced before and after reproductive maturity are the determinants of longevity. This process is governed by the genome. Aging does not happen in a vacuum. Aging must be the result of changes that occur in molecules that have existed at one time with no age changes. It is the state of these pre‐existing molecules that governs longevity determination. The distinction between the aging process and age‐associated disease is not only based on the molecular definition of aging described above but it is also rooted in several practical observations. Unlike any disease, age changes (a) occur in every multicellular animal that reaches a fixed size at reproductive maturity, (b) cross virtually all species barriers, (c) occur in all members of a species only after the age of reproductive maturation, (d) occur in all animals removed from the wild and protected by humans even when that species probably has not experienced aging for thousands or even millions of years, (e) occur in virtually all animate and inanimate matter, and (f) have the same universal molecular etiology, that is, thermodynamic instability. Unlike aging, there is no disease or pathology that shares these six qualities. Because this critical distinction is poorly understood, there is a continuing belief that the resolution of age‐associated diseases will advance our understanding of the fundamental aging process. It will not. The distinction between disease and aging is also critical for establishing science policy because although policy makers understand that the funding of research on age‐associated diseases is an unquestioned good, they also must understand that the resolution of age‐associated diseases will not provide insights into understanding the fundamental biology of age changes. They often believe that it will and base decisions on that misunderstanding. The impact has been to fund research on age‐associated diseases at several orders of magnitude greater than what is available for research on the biology of aging. There is an almost universal belief by geriatricians and others that the greatest risk factor for all of the leading causes of death is old age. Why then are we not devoting significantly greater resources to understanding more about the greatest risk factor for every age‐associated pathology by attempting to answer this fundamental question—“What changes occur in biomolecules that lead to the manifestations of aging at higher orders of complexity and then increase vulnerability to all age‐associated pathology?”

The establishment of a line (WISH) of human amnion cells in continuous cultivation

Abstract 1. 1. A description is given of the events occurring in the alteration of a culture of primary human amnion cells to an established cell line (WISH). Photographic documentation of the event is presented. 2. 2. Characteristics of the cell line are given with respect to virus spectrum, preservation at −70 °C, and growth in suspended culture. 3. 3. Evidence is presented which suggests that the initial alteration occurred in vitro and not from a pre-existing altered cell in the amnion. 4. 4. A comparison is made between the events observed in the establishment of this cell line (WISH) and those described by other workers who have observed this phenomenon.

Motion of fatty acid spin labels in the plasma membrane of mycoplasma

Abstract The electron paramagnetic resonance (EPR) spectra of spin labeled fatty acid derivatives (1 (m,n)) in Mycoplasma laidlawii membranes showed a steep temperature dependence. The hyperfine splitting ( 2T m ) of these spectra decreased as the nitroxide radical was moved away from the polar head group of the fatty acid derivatives, demonstrating an increase in molecular motion of the nitroxide radical. The freedom of motion of the spin label I (12.3) in M. laidlawii membranes was higher in membranes containing cis-Δ 9 - octadecenoic acid than in membranes containing the corresponding trans isomer. A high freedom of motion was also observed in Mycoplasma membrane reggregates having a high lipid to protein ratio. Upon increasing the relative amount of protein in the reaggregates a decrease in the freedom of motion of the nitroxide radical was found. The freedom of motion of the spin label I (12,3) in reaggregates formed at a high Mg2+ concentration ( >20 mM ) was similar to that of the native membranes ( 2T m = 62.0 gauss ). Throughout the growth cycle of M. laidlawii a high freedom of motion of spin label 1 (12,3) was found in cell membranes from the early growth phase ( 2T m = 58.0 gauss ). These membranes had a low density ( d = 1.165 ) due a high amount of oleic acid in membrane polar lipids. Decreasing the growth temperature of the cells resulted in an increase in the amount of [14C]oleic acid and a decrease in the amount of [3H]cholesterol incorporated into the cell membrane. The freedom of motion of spin label 1 (12,3) in membranes from M. laidlawii cells grown at 15° was much higher ( 2T m − 58.0 gauss ) than that in membranes from cells growtn at 37° ( 2T m = 62.5 gauss ) when compared at the same temperature.

The Longevity of Cultured Human Cells

ABSTRACT: Research in aging is now becoming firmly rooted in the scientific method. If it receives the support it deserves, it may produce the next major advance in biology. This article summarizes some of the biological aspects of the field including hypotheses, the aging of cultured human cells, the inverse relationship between donor age and cell longevity, the senescence of cultured normal cells derived from different animal species, the latent period of explanted cells versus donor age, progeria and Werners syndrome, and the future of gerontological research. Life expectancy at birth has increased in recent years, but at age 65–70 it has remained virtually fixed. The human lifespan will not be changed significantly until the underlying biological causes of senescence are slowed or stopped. Since support for research in this field is almost non‐existent, it is more important to concentrate our efforts on increasing research support for gerontology than on increasing the mean lifespan of man. Without the former, the latter will never be improved.

The illusion of cell immortality.

Normal cultured cell populations are mortal but cells that are immortal are abnormal and most have properties of cancer cells. Nevertheless, this distinction becomes blurred because the terms ‘mortality’ and ‘immortality’ are subject to enormous variations in understanding. Forty years ago we showed that cell mortality and immortality are inextricably linked to longevity determination, ageing and cancer. We suggested that a counting mechanism existed in normal cells and that has now been identified as telomere attrition. This replicometer, in combination with the discovery of the enzyme telomerase, has gone very far in explaining why most normal somatic cells have a finite capacity to replicate both in vivo and in vitro and how immortal cancer cells circumvent this inevitability. It is suggested that telomere attrition may be better understood as a direct measure of longevity determination and to only have an indirect association with age changes.

Recent advances in the cell biology of aging

Cultured normal human and animal cells are predestined to undergo irreversible functional decrements that mimic age changes in the whole organism. When normal human embryonic fibroblasts are cultured in vitro, 50 +/- 10 population doublings occur. This maximum potential is diminished in cells derived from older donors and appears to be inversely proportional to their age. The 50 population doubling limit can account for all cells produced during a lifetime. The limitation on doubling potential of cultured normal cells is also expressed in vivo when serial transplants are made. There may be a direct correlation between the mean maximum life spans of several species and the population doubling potential of their cultured cells. A plethora of functional decrements occurs in cultured normal cells as they approach their maximum division capability. Many of these decrements are similar to those occurring in intact animals as they age. We have concluded that these functional decrements expressed in vitro, rather than cessation of cell division, are the essential contributors to age changes in intact animals. Thus, the study of events leading to functional losses in cultured normal cells may provide useful insights into the biology of aging.

Aging under glass

Abstract Like the virologists of the early 1950s and the cytogeneticists of the early 1960s, gerontologists of the early 1970s appear to be standing at the threshold of important new observations by exploring the behaviour of in vitro cultured cells. Several age-related properties of cultured normal cells have been described in the last decade and include (1) the finite lifetime (Phase III phenomenon) of cultured normal human diploid fibroblasts, (2) the inverse relationship to donor age of population doublings possible by such fibroblasts, (3) the direct correlation between donor age and the time necessary for the first cells to emigrate from tissue explants grown in vitro , and (4) characteristic age related changes in cell metabolism and population dynamics as normal cells approach Phase III. The predilection toward loss of cell division capacity or diminution of cell function both in vitro and in vivo may be a manifestation of deterioration in accurate message transcription or translation.