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The serial cultivation of human diploid cell strains

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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.
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... Normal human fibroblasts have a limited in vitro lifespan when cells enter a nondividing, senescent state, known as the Hayflick limit, which is one of the markers of senescence [45,46]. Neonatal foreskin CCD-1064Sk fibroblasts have approximately 54 population doubling level (PDL) that usually corresponds to cultured passage 25-28 for this specific cell line, while the rate of population doubling is three days. ...
... At the same time, in CCD-1135Sk fibroblast, 25 μM H2O2 caused 2-fold decline in the viability, while 50 μM-approximately three-fold, as compared to untreated control. This interesting finding showed that adult CCD-1135Sk dermal fibroblasts at passage 12, which according to the manufacturer, have already completed around 30-34 population doublings (PD), are almost in a pre-senescent state as they show some signs of senescence [46,47]. ...
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... Senescence was first described by Hayflick in isolated fibroblasts in culture [1,2]. In response to repeated replication, DNA damage, metabolic alterations, reactive oxygen species or cytotoxic drugs, cells enter permanent cell cycle arrest, change their morphology to more flat and large cells, express and secrete cytokines, chemokines, growth factors, bioactive lipids, and pro-apoptotic factors-the so-called senescence-associated secretory phenotype (SASP) and become positive for senescence-associated beta-galactosidase (SAβG) [3][4][5][6][7][8][9][10][11]. ...
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Thesis
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