R. Shihman Chang

Continuous subcultivation of epithelial-like cells from normal human tissues.

Summary Epithelial-like cells from normal human conjunctiva, liver, kidney and appendix have been serially cultivated for 30, 26, 22, and 16 passages, respectively.SummaryEpithelial-like cells from normal human conjunctiva, liver, kidney and appendix have been serially cultivated for 30, 26, 22, and 16 passages, respectively.

Bicarbonate as an essential for human cells in vitro

Abstract Human conjunctival and Hela cells were grown in vitro in the presence or absence of HCO3−. In the presence of HCO3−, multiplication of both cell strains was rapid; whereas, in its absence, a decrease in the number of cells was found. Such a decrease was small if the medium was changed on alternate days, presumably because of transient exposure of the cells to CO2. At any time studied up to 14 days, cell multiplication was restored by the addition of HCO3− to the medium.

Carbon dioxide requirement and nucleic acid metabolism of HeLa and conjunctival cells.

Summary Over 90% of C14 fixed by HeLa or conjunctival cells growing in a trisbuffered nutrient medium containing 5 micro-curies of NaHC14O3 per 10 ml was recovered in the acid soluble and nucleic acid fractions. Most of this activity was found associated with the purines and the pyrimidines of such cells. The ribonucleosides were partially effective as a substitute for CO2 in supporting cell multiplication. A combination of ribosides and oxaloacetate provided conditions as good as or better than CO2 for multiplication of CO2-depleted conjunctival cells. It may be concluded that, under the described experimental conditions, the chief functions of CO2 in human cells are to provide specific precursors for syntheses of the purines, the pyrimidines and oxaloacetate.

Properties of a Transmissible Agent Capable of Inducing Marked DNA Degradation and Thymine Catabolism in a Human Cell.

SummaryThe appearance of a transmissible cytopathic agent during the first blind passage of pooled human “liver” cultures previously infected with 4 acute infectious hepatitis bloods is described. The infectivity of this agent in the “liver” culture was cell-associated. The infective degenerated “liver” cells retained their infectivity for at least 16 weeks at 4°C and 8 weeks at 36°C. Heating for 1 hour at 56°C failed to destroy completely the infectivity of infected mouse fibroblasts. One cycle of freeze-thawing or homogenizing, however, abolished the infectivity with occasional exceptions. Characteristic cytopathology consisted of cytoplasmic granularity, cell rounding followed by diminution of cell volume leading to morphologic picture of shrunken cells. During an intermediate stage of degeneration, characteristic intranuclear basophilic globules were seen regularly; the globules were Feulgen positive. The established cell line was more susceptible to the destructive action of this agent than primary c...Summary The appearance of a transmissible cytopathic agent during the first blind passage of pooled human “liver” cultures previously infected with 4 acute infectious hepatitis bloods is described. The infectivity of this agent in the “liver” culture was cell-associated. The infective degenerated “liver” cells retained their infectivity for at least 16 weeks at 4°C and 8 weeks at 36°C. Heating for 1 hour at 56°C failed to destroy completely the infectivity of infected mouse fibroblasts. One cycle of freeze-thawing or homogenizing, however, abolished the infectivity with occasional exceptions. Characteristic cytopathology consisted of cytoplasmic granularity, cell rounding followed by diminution of cell volume leading to morphologic picture of shrunken cells. During an intermediate stage of degeneration, characteristic intranuclear basophilic globules were seen regularly; the globules were Feulgen positive. The established cell line was more susceptible to the destructive action of this agent than primary cells. Preliminary immunological study failed to establish the identity of this agent.

Isolation of Nutritional Variants from Conjunctival and HeLa Cells.

SummaryA method used successfully in isolation of nutritional variants from the human conjunctival and HeLa cells has been described. Variants capable of continuous propagation, under prescribed experimental conditions, with d (+) xylose, d-ribose or sodium lactate as the sole source of added carbohydrate or intermediate have been isolated.Summary A method used successfully in isolation of nutritional variants from the human conjunctival and HeLa cells has been described. Variants capable of continuous propagation, under prescribed experimental conditions, with d (+) xylose, d-ribose or sodium lactate as the sole source of added carbohydrate or intermediate have been isolated.

Propagation of Conjunctival and HeLa Cells in Various Carbohydrate Media.

Summary 1) Under the described experimental conditions, both conjunctival and HeLa cells propagated satisfactorily in the following individual carbohydrates: starch, glycogen, maltose, d-glucose, d-fructose, d-mannose and d(+)galactose. Cell propagation in sucrose depended largely on source of dialyzed serum. Sodium pyruvate, sodium lactate, d-ribose and d(+)xylose were unable to support cell multiplication, though cell degeneration in the presence of these carbohydrates and intermediates was definitely retarded. L(+)rhamnose, l(-)fucose and l-sorbose appeared to delay the degeneration of the HeLa cells more effectively than that of the conjunctival cells. Lactose, l(-)xylose, d(-)arabinose, l(+)arabinose, d-2-deoxyribose, i-erythritol and dihydroxyacetone neither supported growth nor retarded degeneration. 2) Glucose at low concentration of 0.1 mM is capable of supporting cell growth for at least several months. Net increase in number of cells at a concentration of 0.1 mM is significantly lower than that in 10 mM glucose. When pyruvate and alanine were added to the medium, the concentration of glucose could be further reduced to 0.01 mM without resulting in cell degeneration. D-mannose and d(+) galactose both at 0.01 mM concentrations, and d-fructose at somewhat higher concentration, are effective substitutes for glucose.

Differences in Inositol Requirements of Several Strains of HeLa, Conjunctival and Amnion Cells.

Summary Under the described experimental conditions, qualitative differences in the inositol requirement of the following human cells have been found: 1) between several wild strains of HeLa cell obtained from several laboratories; 2) between a wild strain of conjunctival cell and a subculture propagating in a low glucose medium, and 3) between a stable laboratory strain and fresh explants of human amnion cells. The significance of these findings was discussed.SummaryUnder the described experimental conditions, qualitative differences in the inositol requirement of the following human cells have been found: 1) between several wild strains of HeLa cell obtained from several laboratories; 2) between a wild strain of conjunctival cell and a subculture propagating in a low glucose medium, and 3) between a stable laboratory strain and fresh explants of human amnion cells. The significance of these findings was discussed.

Macromolecular Growth Requirements of Human Cells in Continuous Culture

Summary The essential growth factors associated with serum proteins are present in cold ethanol fraction IV-4 for most human serum pools and in fraction II + III for 2 individual equine serums. Cytotoxic activity has been found in fraction II + III from most human serum pools. Further subfractionation of IV-4 and II + III by the established cold ethanol methods fails to yield active subfractions. The significance of this finding is discussed.

TEMPERATURE INDUCED CELLULAR RESISTANCE TO THE "LIPOVIRUS".

Summary The optimal temperature for development of specific degeneration in “lipo-virus”-infected cultures was 32-34°C. Partial and complete suppression was noted at 37.5°C and 38.5°C respectively. The “lipo-virus” was lethal to chick embryos if the virus was inoculated into the amniotic or allantoic sacs or onto the chorioallantoic membrane and the embryos then incubated at 33°C. Inoculation into the yolk sac or elevation of incubation temperature to 38°C nullified the lethal action of the “lipovirus” on chick embryos.

Meso-Inositol Requirement of HeLa and Human Conjunctival Cells.

Summary A concentration of meso-inositol of at least 4 μM was necessary for maximum multiplication of human conjunctival cells grown in vitro. Inositol was found to be essential for multiplication of HeLa cells.