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.

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.

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)

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.

In vitro maturation and in vitro fertilization of bovine oocytes cultured in a chemically defined, protein-free medium: effects of carbohydrates and amino acids

1999 ◽  
Vol 11 (2) ◽  
pp. 127 ◽  
Author(s):  
J. M. Lim ◽  
B. C. Lee ◽  
E. S. Lee ◽  
H. M. Chung ◽  
J. J. Ko ◽  
...  
Keyword(s):  
Amino Acids ◽  
In Vitro Fertilization ◽  
In Vitro Maturation ◽  
Essential Amino Acids ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Vitro Fertilization ◽  
Pronuclear Formation ◽  
Bovine Oocytes

This study was conducted to examine the effects of carbohydrates and amino acids on the maturation and fertilization of bovine oocytes. To evaluate the effect of each treatment without any unpredictable interference, oocytes were cultured in a simply defined medium (modified Tyrode’s medium; mT) without the addition of hormones and proteins. In Experiment 1, oocyte maturation to the metaphase-II stage was significantly (P<0.0001) enhanced after the addition of glucose (5.6 mМ), lactate (10 mМ) and/or pyruvate (0.5 mМ) to mT (37–74%) than after no addition (0%). In mT supplemented with glucose, the addition of 19 essential and non-essential amino acids (aa; 0, 0.01, 0.1, 1, 5 or 10%) did not further improve in vitro maturation (Experiment 2) or in vitro fertilization (Experiment 3) of oocytes. However, more (P<0.05) pronuclear formation after in vitro-insemination was found in oocytes matured in mT with 1% aa and glucose than in oocytes matured in mT with glucose alone (56% vs. 35%). Penetration of spermatozoa into the ooplasm was initiated at 3 h after insemination and pronuclear formation from 8 h (Experiment 4). When cultured inseminated oocytes were examined up to 192 h post insemination, a significant (P<0.05) increase in the number of 2-cell (18 v. 38%) and 8-cell embryos, (7 v. 20%) and morulae (0 v. 8%) was found after the addition of 1% aa to mT with glucose than after no addition (Experiment 5). A limited number of oocytes matured in mT with aa and glucose developed to the blastocyst stage (6%). These results indicate that exogenous carbohydrates and amino acids are prerequisites for the maturation and fertilization of bovine oocytes in vitro. Glucose alone promotes the nuclear maturation of oocytes, whereas amino acids aid the pronuclear formation of fertilized oocytes.

scholarly journals A defined medium for Leishmania culture allows definition of essential amino acids

2018 ◽  
Vol 185 ◽  
pp. 39-52 ◽  
Author(s):  
Archana Nayak ◽  
Snezhana Akpunarlieva ◽  
Michael Barrett ◽  
Richard Burchmore
Keyword(s):  
Amino Acids ◽  
Essential Amino Acids ◽  
Defined Medium ◽  
Definition Of

THE AMINO ACID REQUIREMENTS OF A PERMANENT STRAIN OF ALTERED UTERINE FIBROBLASTS (U12-705)

1958 ◽  
Vol 36 (1) ◽  
pp. 861-868
Author(s):  
H. E. Swim ◽  
R. F. Parker
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Horse Serum ◽  
Human Fibroblasts ◽  
Essential Amino Acids ◽  
Defined Medium ◽  
Isoleucine Leucine ◽  
Permanent Strain ◽  
Inhibition Of Growth ◽  
Amino Acid Requirements

A permanent line of altered human fibroblasts, strain U12-705, was found to require arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, and valine for growth in a defined medium supplemented with 2.5% (v/v) dialyzed chick embryo extract and 5% dialyzed horse serum. In the absence of any of the essential amino acids the cells not only fail to proliferate but undergo degenerative changes which increased with time. The omission of alanine, aspartic acid, glutamic acid, glycine, hydroxyproline, and proline either separately or collectively does not alter the rate of growth or result in changes in the appearance of the cells. Cysteine and glutathione are equally as effective as cystine in promoting the growth of U12-705. None of the D-enantiomorphs of the essential amino acids will effectively replace the corresponding L-isomer. Single D-amino acids are not inhibitory when added to the medium in 5 times the concentration of the L-amino acid. The minimum concentrations of essential amino acids which permit optimal proliferation under the conditions employed range from 0.005 to 0.5 mM. Essential amino acids with the exception of glutamine, isoleucine, leucine, threonine, and valine are toxic for U12-705 when employed at a concentration of 5 mM. Toxic manifestations vary with the amino acid and range from cytologic changes in the cells without a significant decrease in the growth rate to complete inhibition of growth and extensive cellular degeneration.

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