ArticlePDF Available

The serial cultivation of human diploid cell strains

Authors:

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.
A preview of the PDF is not available
... Senescent cells, although metabolically active, are especially harmful as they disrupt tissue repair and regeneration by altering the wound microenvironment ( Figure 1) (10). Cellular senescence, first described by Hayflick in 1961, is a state of stable cell cycle arrest characterized by distinct secretory properties and altered metabolism, playing a key role in aging and chronic diseases (11). Senescent cells arise in response to various stresses, such as oxidative stress and mitochondrial dysfunction, leading to cell proliferation arrest, resistance to apoptosis, and altered gene and protein expression profiles (12,13). ...
... Senescent cells, although metabolically active, are especially harmful as they disrupt tissue repair and regeneration by altering the wound microenvironment ( Figure 1) (10). Cellular senescence, first described by Hayflick in 1961, is a state of stable cell cycle arrest characterized by distinct secretory properties and altered metabolism, playing a key role in aging and chronic diseases (11). Senescent cells arise in response to various stresses, such as oxidative stress and mitochondrial dysfunction, leading to cell proliferation arrest, resistance to apoptosis, and altered gene and protein expression profiles (12,13). ...
Article
Full-text available
Chronic or non-healing wounds, such as diabetic foot ulcers (DFUs), venous leg ulcers (VLUs), pressure ulcers (PUs) and wounds in the elderly etc., impose significant biological, social, and financial burdens on patients and their families. Despite ongoing efforts, effective treatments for these wounds remain elusive, costing the United States over US$25 billion annually. The wound healing process is notably slower in the elderly, partly due to cellular senescence, which plays a complex role in wound repair. High glucose levels, reactive oxygen species, and persistent inflammation are key factors that induce cellular senescence, contributing to chronic wound failure. This suggests that cellular senescence may not only drive age-related phenotypes and pathology but also be a key mediator of the decreased capacity for trauma repair. This review analyzes four aspects: characteristics of cellular senescence; cytotoxic stressors and related signaling pathways; the relationship between cellular senescence and typical chronic non-healing wounds; and current and future treatment strategies. In theory, anti-aging therapy may influence the process of chronic wound healing. However, the underlying molecular mechanism is not well understood. This review summarizes the relationship between cellular senescence and chronic wound healing to contribute to a better understanding of the mechanisms of chronic wound healing.
... Senescence, which results from permanent cell-cycle arrest and is a hallmark of aging, was first defined by Hayflick and Moorhead in the early 1960s (Hayflick & Moorhead, 1961). Cellular senescence is observed not only with aging, but also in organismal development, maintaining tissue homeostasis, tissue remodeling and repair, acute injury, and in wound healing (Demaria et al., 2014;Muñoz-Espín et al., 2013;Ramakrishna et al., 2012;Storer et al., 2013;Wiley & Campisi, 2021;Yun et al., 2015). ...
Article
The Midwest Aging Consortium (MAC) has emerged as a critical collaborative initiative aimed at advancing our understanding of aging and developing strategies to combat the rising prevalence of age-related diseases. Founded in 2019, MAC brings together researchers from various disciplines and institutions across the Midwestern United States to foster interdisciplinary geroscience research. This report summarizes the highlights of the Fourth Annual Symposium of MAC, which was held at Iowa State University in May 2023. The symposium featured presentations on a wide array of topics, including studies on slow-aging animals, cellular senescence and senotherapeutics, the role of the immune system in aging, metabolic changes in aging, neuronal health in aging, and biomarkers for measuring the aging process. Speakers shared findings from studies involving a variety of animals, ranging from commonly used species such as mice, rats, worms, yeast, and fruit flies, to less-common ones like naked mole-rats, painted turtles, and rotifers. MAC continues to emphasize the importance of supporting emerging researchers and fostering a collaborative environment, positioning itself as a leader in aging research. This symposium not only showcased the current state of aging biology research but also highlighted the consortium’s role in training the next generation of scientists dedicated to improving the healthspan and well-being of the aging population.
Preprint
Background: Hemolytic uremic syndrome (HUS) is a primary thrombotic microangiopathy that can cause acute renal failure, particularly in children, and is commonly triggered by Shiga toxin-producing Escherichia coli (STEC) infection. Although previous in vitro studies have shown that Shiga toxin (STX) can directly affect the endothelium, gram-negative bacteria such as E. coli also contain lipopolysaccharide (LPS) which is known to independently activate endothelial cells. Furthermore, studies in mice and baboons have shown a synergistic effect of LPS and STX in inducing HUS when administered simultaneously. Objective: Determine how LPS, STX, or combined LPS/STX treatment affect the gene expression program of endothelial cells. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with STX, LPS or a combination of STX and LPS for either 4 or 24 hours, after which unbiased RNA sequencing analysis was performed. Results: Four-hour treatment with LPS resulted in a greater number of differentially expressed genes as compared to treatment with STX. In contrast, 24-hour treatment with LPS or STX induced similar numbers of differentially expressed genes. Dual treatment with both toxins resulted in a synergistic effect, showing significant up- or downregulation of multiple transcripts that were not observed in either single treatment group, particularly at the 24-hour timepoint. Conclusion: Our data provide a resource for assessing the role of endothelial cell gene expression in the pathogenesis of STEC infection and suggest that the responses of these cells to LPS, STX, or combined LPS/STX treatment are unique and temporally regulated.
Article
Introduction Progressive aging, or senescence, of mesenchymal stem/stromal cells (MSCs) is a major obstacle faced when trying to culture potent stem cells for use in therapy. Senescent cells are irreversibly nondividing cells that cease performing critical functional effects. Elimination of senescent cells using biochemical means, such as the use of senolytic drugs like dasatinib, may be useful in retaining the viable and proliferating populations of the cells. Methods An in vitro approach was used to investigate the effect of dasatinib on phenotypic, genotypic, and immunomodulatory functionality of osteogenic and adipogenic differentiated MSCs. Replicative senescence was achieved through multiple sub-culturing in vitro, then senescent and non-senescent cultures were treated with a standard dosage of dasatinib. MSCs were then differentiated into osteogenic, adipogenic or chondrogenic cultures using conditioned media to be tested for the three criteria being investigated. Results Significant changes were observed in these criteria, indicated by evidence gathered from proliferation and indoleamine 2,3 dioxygenase activity assays. Phenotypic results of dasatinib were shown to reduce the population of senescent MSCs while allowing non-senescent MSCs to continue differentiating and proliferating without interference from senescent cells. Genotypic results showed no change to upregulation in markers associated with osteogenic and adipogenic cells when exposed to dasatinib. Indoleamine Dioxygenase activity showed insignificant differences in cells exposed to dasatinib versus control groups, providing evidence against compromised cellular immune function. Conclusion This investigation provides insight into how dasatinib effects MSCs functional ability and provides a better understanding of the function of senolytic agents.
Article
Full-text available
Cutaneous wound healing is a complex multi-step process that is highly controlled, ensuring efficient repair to damaged tissue and restoring tissue architecture. Multiple cell types play a critical role in wound healing, and perturbations in this can lead to non-healing wounds or scarring and fibrosis. Thus, the process is tightly regulated and controlled. Cellular senescence is defined as irreversible cell cycle arrest and is associated with various phenotypic changes and metabolic alterations and coupled to a secretory program. Its role in wound healing, at least in the acute setting, appears to help promote appropriate mechanisms leading to the complete restoration of tissue architecture. Opposing this is the role of senescence in chronic wounds where it can lead to either chronic non-healing wounds or fibrosis. Given the two opposing outcomes of wound healing in either acute or chronic settings, this has led to disparate views on the role of senescence in wound healing. This review aims to consolidate knowledge on the role of senescence and aging in wound healing, examining the nuances of the roles in the acute or chronic settings, and attempts to evaluate the modulation of this to promote efficient wound healing.
Article
Full-text available
The deoxyribonucleic acid (DNA) content of Feulgen-stained interphase nuclei from human amnion epithelium and liver parenchyma as well as the DNA content of their sex chromatin or heterochromatic bodies was determined histophotometrically. In female nuclei the ratio between the nuclear DNA content and that of their sex chromatin or heterochromatic bodies remains constant irrespective of the polyploid state of the nucleus. Thus, in polyploid nuclei the heterochromatic bodies double their DNA content with each duplication of the diploid nuclear DNA content. Therefore, it is assumed that the large or multiple heterochromatic bodies of female polyploid nuclei are conjugated or multiple sex chromatin bodies. Some male diploid nuclei show a distinct heterochromatic body whose DNA content is about one-half that of a sex chromatin. About one-half of the polyploid male nuclei have heterochromatic bodies whose DNA content is approximately one-half of those of female nuclei of the corresponding polyploid class. This would indicate that in male diploid nuclei the single X chromosome sometimes leaves a heterochromatic rest one-half the size of a sex chromatin and in polyploid male nuclei the two or more X's may leave larger heterochromatic bodies. However, many male nuclei, even when polyploid, do not have distinct heterochromatic bodies. Possibly the heterochromatic portions of the X's have failed to join and form a sex chromatin-like body; or the Y inhibits the X from leaving a heterochromatic rest. The proposal of other authors that the sex chromatin is derived from but one of the X chromosomes of the female nucleus, the X in the male leaving no heterochromatic rest, is also considered. The DNA content does not vary significantly between nuclei with and without sex chromatin or heterochromatic bodies. It remains clear that female nuclei with abnormally large or multiple sex chromatin bodies and male nuclei with large distinct heterochromatic bodies are always polyploid.
Article
In 1951 Gey (7) succeeded in developing for the first time & strain of human cells (HeLa) that could be cultivated indefinitely in vitro. This strain has since been used widely as a laboratory tool. In the few years following Gey's achieve ment, other investigators have applied themselves to establishing lines of human cells from normal and cancer tissue. Many of these efforts have been successful. In the fall of 1955, twenty different strains of human cells were reported in answer to a questionnaire circulated by the Tissue Culture Association. Established strains of human cells are being used in a variety of areas in experimental
Article
A strain of rabies fixed virus has been successfully cultivated in tissue cultures of hamster kidney cells. This confirms an earlier report by Kissling. In the experiments here recorded a special culture tube incorporating a dialyzing membrane made it possible to maintain the cells in continuous culture for many weeks. By using this technique it was possible to obtain culture fluids of high infectivity.
Article
Zusammenfassung Der Verfasser gibt eine kurze Übersicht über die neueren Methoden derin vitro-Kultur von Säugerzellen. Eingehend wird die Entwicklung von stabilen Kulturen diploider Zellen und Untersuchungen über die spezifischen Wachstumsfaktoren isolierter menschlicher Zellen besprochen. Auch die Wirkung der Ultraviolettstrahlung auf solche Zellen wird diskutiert.
Article
THE contamination of tissue culture cell lines by pleuropneumortia-like organisms has become an increasingly important problem1-4. The contamination itself is insidious and cannot be detected ordinarily by gross changes in the appearance of the media macroscopically or microscopically. The tissue culture cells themselves, when observed microscopically, may or may not exhibit slight cytopathology as evidenced by intracytoplasmic granularity. Properly stained preparations may reveal intracytoplasmic particles, presumably the contaminating pleuropneumonia-like organisms5-7.