Growth of human diploid cells (strain MRC-5) in defined medium; replacement of serum by a fraction of serum ultrafiltrate

1979 ◽  
Vol 35 (1) ◽  
pp. 381-392
Author(s):  
K. Lambert ◽  
S.J. Pirt
Keyword(s):  
Amino Acids ◽  
Active Material ◽  
Maximum Yield ◽  
Calf Serum ◽  
Defined Medium ◽  
Cell Yield ◽  
Maximum Cell ◽  
Macromolecular Component ◽  
Human Diploid Cells ◽  
Diploid Cells

A calf serum ultrafiltrate fraction permitted growth for at least 3.5 generations, including one subculture, of MRC-5 cells in defined medium in the absence of whole serum. The active material has a molecular weight of 10 000 Daltons or less. This suggests that there may be no requirement for a large macromolecular component of serum. The ultrafiltrate was assayed by maximum cell yield from a serum-limited inoculum in a defined medium containing non-limiting amounts of vitamins, amino acids, glucose, a 68-component supplement, iron and methylcellulose. The levels of vitamins, amino acids and glucose were based on quantitative measurements of uptake and the levels of the other components by minimum amount required for maximum yield in defined medium without ultrafiltrate or serum. With excess ultrafiltrate maximum cell yield was limited by the defined part of the medium, probably the supplement. The cell doubling time in defined medium with ultrafiltrate fractions was 70 h compared with 27 h in the medium with serum. Excess ultrafiltrate did not inhibit growth. The lowered growth rate is attributed to a nutritional deficiency in the supplement.

The quantitative requirements of human diploid cells (strain MRC-5) for amino acids, vitamins and serum

1975 ◽  
Vol 17 (3) ◽  
pp. 397-411
Author(s):  
K. Lambert ◽  
S.J. Pirt
Keyword(s):  
Amino Acids ◽  
Growth Factor ◽  
Inorganic Compounds ◽  
Calf Serum ◽  
Essential Amino Acids ◽  
Defined Medium ◽  
Cell Populations ◽  
Maximum Cell ◽  
Human Diploid Cells ◽  
Diploid Cells

The uptakes of all essential amino acids, vitamins (except riboflavin), glucose and serum during growth of human diploid cells (MRC-5) were determined. The amino acid uptakes varied considerably with the conditions of culture. The glucose requirement is several times greater than that for mouse LS or human HeLa cells. These analytical results were used to modify the medium so as to ensure that an excess of all defined medium constituents was present and pH was not limiting during study of the serum requirements. It was then found that maximum cell populations were directly proportional to the serum concentration. Hence the growth was limited by the supply of an unknown growth factor in serum. The serum growth factor was not replaced by a mixture of over 60 vitamins, co-enzymes, hormones and other organic and inorganic compounds considered to be possible growth factors, although this mixture did not lower the growth rate and somewhat (22%) increased the yield from the serum growth factor. The unit of serum growth factor is precisely defined in terms of the amount in a standard batch of calf serum. This standard contains 10 units/ml whereas the other batch of serum used contained only 5 units/ml.

157 EFFECTS OF FETAL CALF SERUM OR PHENAZINE ETHOSULFATE AND FRUCTOSE OR GLUCOSE ON EMBRYONIC DEVELOPMENT AND LIPID ACCUMULATION OF BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 186
Author(s):  
M. Barcelo-Fimbres ◽  
G. Seidel Jr
Keyword(s):  
Amino Acids ◽  
Lipid Accumulation ◽  
Lipid Droplets ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Essential Amino Acids ◽  
Defined Medium ◽  
Bovine Embryos ◽  
Cell Embryo ◽  
Phenazine Ethosulfate

Slaughterhouse oocytes (n = 6222) were maturated in a chemically defined medium (CDM) similar to SOF plus 0.5% fatty acid-free BSA (FAF-BSA) and hormones (M-CDM) for 23 h at 38.5°C in 5% CO2 in air. Oocytes and frozen-thawed sperm, centrifuged through a Percoll gradient, were co-cultured for 18 h in F-CDM (CDM + heparin). Zygotes were cultured at 38.5°C in 5% CO2/5% O2/90% N2 in CDM-1 (CDM + nonessential amino acids, 10 μM EDTA, 0.5% FAF-BSA, and 0.5 mM fructose or glucose in Expt 1 and glucose in Expt 2). In both experiments, after 48 h, 8-cell embryos were cultured 135 h in CDM-1 (CDM-1 + essential amino acids, no EDTA, and 2 mM fructose or glucose). A factorial design with two hexoses and three additives in CDM-2 (control; 10% fetal calf serum (FCS); and 0.3 μM phenazine ethosulfate (PES), an electron acceptor that oxidizes NADPH) was used for both experiments, each replicated eight times. For Expt 1, Day 7.5 blastocysts were fixed and stained with Sudan Black B to quantify cytoplasmic lipid droplets. A digital photo at 600× of the equatorial part of the embryo was evaluated by classifying lipophilic droplet diameters as small (S, <2 μm), medium (M, 2 to 6 μm), or large (L, >6 μm), reported as number of lipid droplets (LD) per 1000 μm2. Data were analyzed by ANOVA. For Expt 1, 8-cell embryo production per oocyte matured was not affected by fructose or glucose (P > 0.1) (70 vs. 68%, respectively); however, blastocyst rates per oocyte matured (B/O) and per 8-cell embryo (B/E) were higher (P < 0.01) for fructose than glucose (Table 1). There were no differences between control, PES, and FCS (P > 0.1) for B/O, or B/E. For Expt 2, B/O and B/E were higher (P < 0.01) for fructose than for glucose. No differences were found for additives (P > 0.1) control, FCS, or PES for B/O or B/E. There was an interaction (P < 0.05) between additives and hexoses for blastocyst production, because the benefit of fructose compared to glucose was greater for controls than for FCS or PES (means not presented). Accumulations of each size of LD were less for PES (P < 0.05) than for control and FCS. Control and PES were lower than FCS (P < 0.05) for S, M, and L droplets. There was no effect of fructose or glucose (P > 0.1) on numbers of S, M, or L droplets (Table 1). In conclusion, fructose produced more blastocysts than glucose after 8-cell development, but there was no hexose effect either before this stage or in lipid accumulation. PES reduced and FCS increased lipid accumulation relative to controls. Table 1. Main effects of additives and hexoses on development of bovine embryos (±SE)

Improvements in a Chemically Defined Medium for the Growth of Mouse Cells (Strain LS) in Suspension

1970 ◽  
Vol 7 (3) ◽  
pp. 661-670
Author(s):  
J. R. BIRCH ◽  
S. J. PIRT
Keyword(s):  
Amino Acids ◽  
Population Density ◽  
Cell Population ◽  
Defined Medium ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Free Medium ◽  
Average Maximum ◽  
Maximum Cell ◽  
Medium Change

In the medium used in previous work, both growth rate and maximum cell population density were reduced in protein-free medium. This was due to iron limitation. In a medium supplemented with iron, the average minimum population doubling time was reduced from 78 to 37 h and the average maximum cell population density increased from 1.6x106 to 3.3x106/ml. In the improved medium described, sodium pyruvate, α-ketoglutaric acid, methylcellulose and polyvinylpyrrolidone are no longer required although methylcellulose prevents an initial fall in population which occurs in polymer-free medium. The present study indicates that amino acids now limit maximum cell population density. By increasing the concentration of the relevant amino acids we have obtained maximum cell populations in excess of 5.106 cells/ml without medium change. Finally we describe a method for measuring the growth of the LS cell in the defined medium by an opacity method.

The Effect of Medium Changes on the Growth and Metabolism of the Human Diploid Cell, W1-38

1971 ◽  
Vol 8 (1) ◽  
pp. 43-52
Author(s):  
J. B. GRIFFITHS
Keyword(s):  
Protein Synthesis ◽  
Contact Inhibition ◽  
Cell Yield ◽  
Unexpected Result ◽  
Inhibitory Interaction ◽  
Inhibition Of Growth ◽  
Rna And Protein Synthesis ◽  
Medium Change ◽  
Human Diploid Cells ◽  
Diploid Cells

It has been established that although an inhibitory interaction occurs when a culture of human diploid cells become crowded together (contact inhibition of growth), multiple-layered cell sheets are obtained by using a continuous medium perfusion culture. A similar effect is obtained when the culture medium is changed at frequent intervals, and this paper reports the effects of a medium change on cell growth and metabolism. A direct relationship was found between cell yield and the number of medium changes given to a culture. This was an unexpected result because normally when a culture is prolonged by additional feeding the cell yield shows a diminishing return. The amino acid and glucose uptakes and growth yields (the ratio of the amount of cell dry weight produced to substrate used) were determined and they also showed that a unit amount of growth occurred per medium change, and that cessation of growth was accompanied by cessation of nutrient uptake and metabolism. Medium changes had a profound affect on cellular metabolism, especially on DNA and protein synthesis. As a culture approached confluency, DNA, RNA and protein synthesis were sequentially inhibited. After a medium change there was a sequential stimulation of DNA, RNA and protein synthesis in the same order as they were inhibited. The inhibitory mechanism that is affected by cell crowding is obviously reversed by a medium change. The results presented in this paper suggest that contact inhibition of growth primarily affects DNA synthesis and that if the cell is able to take up a sufficient supply of nutrients in a crowded culture then this inhibition can be overcome.

scholarly journals Production of High-Titered Interferon in Cultures of Human Diploid Cells

1972 ◽  
Vol 2 (6) ◽  
pp. 476-484 ◽  
Author(s):  
E. A. Havell ◽  
J. Vilcek
Keyword(s):  
Human Diploid Cells ◽  
Diploid Cells

Results of chromosome analysis of rabies virus infected human diploid cells (WI-38)

1977 ◽  
Vol 54 (3) ◽  
pp. 255-257 ◽  
Author(s):  
M. Majer ◽  
Annelies Herrmann ◽  
R. Mauler
Keyword(s):  
Rabies Virus ◽  
Chromosome Analysis ◽  
Human Diploid Cells ◽  
Diploid Cells

The Glucose, Insulin and Glutamine Requirements of Suspension Cultures of HeLa Cells in a Difined Culture Medium

1971 ◽  
Vol 9 (2) ◽  
pp. 529-537
Author(s):  
G. J. BLAKER ◽  
J. R. BIRCH ◽  
S. J. PIRT
Keyword(s):  
Amino Acid ◽  
Hela Cells ◽  
Growth Yield ◽  
Dry Weight ◽  
Defined Medium ◽  
Cell Populations ◽  
Serum Supplement ◽  
Protamine Sulphate ◽  
Protamine Zinc Insulin ◽  
Maximum Cell

The serum supplement in a defined medium for the growth of HeLa cells could be replaced by protamine-zinc-insulin (0.2 u./ml). Insulin (0.4 u./ml) replaced the growth-stimulatory properties of protamine-zinc-insulin, whilst protamine sulphate (5 µg/ml) was found to be toxic to the cells. The addition of insulin to cultures depleted of insulin increased both cell growth rates and maximum cell populations. In the defined medium, HeLa cells could only utilize glutamate when a small amount of glutamine was included. Glucose, at a level of 2 mg/ml, was shown to limit maximum cell populations. The growth yield from glucose was 295 µg cell dry weight/mg glucose. When the medium glucose concentration was increased to 4 mg/ml, HeLa cell populations in excess of 16 x 105 cells (i.e. 640 µg dry weight)/ml were routinely achieved in the defined medium supplemented with insulin. Growth is then limited by the amino acid supply. Increasing the amino acid concentration of the medium by 50% raised the maximum cell population to 23.5x105 cells (i.e. 940 µg dry weight)/ml.

Efficient transfer of cloned DNA into human diploid cells: protoplast fusion in suspension

1984 ◽  
Vol 4 (11) ◽  
pp. 2549-2552
Author(s):  
P Litzkas ◽  
K K Jha ◽  
H L Ozer
Keyword(s):  
Simian Virus 40 ◽  
Cell Types ◽  
Simian Virus ◽  
Selective Medium ◽  
T Antigen ◽  
Human Diploid Fibroblasts ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Human Diploid Cells ◽  
Diploid Cells

A method for fusion of protoplasts bearing amplified plasmids and human diploid fibroblasts or other cell types in suspension is described. Transient expression of plasmid-encoded proteins occurs in up to 50% of the human cells, as demonstrated for simian virus 40 T antigen by immunofluorescence and the Escherichia coli xanthine-guanine phosphoribosyl transferase by autoradiography. In contrast, frequencies of stable transformants were similar to those obtained by the CaPO4 coprecipitation technique. However, experiments with both methods involving the recombinant pRSVneo (in which the Rous sarcoma virus long terminal repeat regulates expression of the antibiotic-inactivating aminoglycoside phosphotransferase) revealed a much higher frequency of colonies in G418 selective medium with constructions in which the early region of simian virus 40 DNA was present as well. We propose a role for the simian virus 40 T antigen in enhancing stable transformation in this system.

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