Necessary amino acids and vitamins for the growth of human diploid fibroblasts

1979 ◽  
Vol 40 (1) ◽  
pp. 281-291
Author(s):  
J. Litwin
Keyword(s):  
Amino Acids ◽  
Growth Rate ◽  
Folic Acid ◽  
Cell Growth ◽  
Calf Serum ◽  
Life Time ◽  
Lung Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Support Cell

Only 2 amino acids and one vitamin were found to be essential for the growth of human embryonic diploid lung fibroblasts when 10% undialysed calf serum was used as a medium supplement. These amino acids were either glutamine + cysteine or serine + homocysteine. Replacing cysteine or homocysteine with cystine or homocystine, respectively, reduced growth. The growth rate in the glutamine + cysteine medium was slightly less than that in Eagle's medium, but the in vitro life time was similar. Folic acid was the only vitamin needed to support cell growth in vitro. The addition of other vitamins had no stimulatory effect with the possible exception of nicotinamide. When other amino acids were added to glutamine + cysteine none showed stimulatory effects but tryptophan was either toxic or inhibitory for the 3 human diploid strains examined. Serine was inhibitory for WI-38 but not for MRC5 cells. Subtle nutritional differences appear to exist between fibroblasts of the same type obtained from different embryos.

scholarly journals Growth of Human Diploid Fibroblasts in Media with Different Amino Acid Composition

1974 ◽  
Vol 14 (3) ◽  
pp. 671-680
Author(s):  
J. LITWIN
Keyword(s):  
Growth Rate ◽  
Cell Attachment ◽  
Calf Serum ◽  
Lung Fibroblasts ◽  
Good Growth ◽  
Initial Exposure ◽  
Human Diploid Fibroblasts ◽  
Constant Ph ◽  
Almost All ◽  
Better Than

Human embryonic diploid lung fibroblasts were capable of growing in a medium consisting of Eagle's MEM vitamins plus 0.1 µg/ml biotin, Earle's salt solution, 4 mM glutamine, 0.4 mM cystine, 0.5 mM tyrosine, 10 % undialysed calf serum and 1 mM pyruvate. The growth rate and percent cell attachment in this medium was slightly less than in Eagle's MEM but the longevity was about the same. Addition of 0.5-1.0 mM histidine stimulated growth but addition of O.2 mM phenylalanine, methionine or valine decreased longevity markedly. Maintaining a constant pH of about 7.5-7.6 with 20 mM of the organic buffers HEPES, TES or TRICINE yielded a growth rate equal to or better than that of cells growing in unbuffered medium. Some buffers gave good growth initially but eventually the longevity decreased (20mM BIS-TRIS, 20 mM BICINE, 10 mM TRIS). In almost all cases the initial exposure to the buffers yielded the best growth results.

The uptake of amino acids by mouse cells (strain LS) during growth in batch culture and chemostat culture: the influence of cell growth rate

1967 ◽  
Vol 168 (1013) ◽  
pp. 421-438 ◽  
Keyword(s):  
Amino Acids ◽  
Growth Rate ◽  
Amino Acid ◽  
Cell Growth ◽  
Glutamic Acid ◽  
Batch Culture ◽  
Amino Acid Uptake ◽  
Essential Amino Acids ◽  
Growth Yields ◽  
Acid Uptake

The uptake of thirteen essential amino acids by mouse LS cells in suspension culture was determined by bacteriological assay methods. Chemostat continuous-flow cultures were used to determine the effect of different cell growth rates on the quantitative amino acid requirements for growth. The growth yields of the cells ( Y = g cell dry weight produced/g amino acid utilized) were calculated for each of the essential amino acids. A mixture of the non-essential amino acids, serine, alanine and glycine increased the cell yield from the essential amino acids. The growth yields from nearly all the essential amino acids in batch culture were increased when glutamic acid was substituted for the glutamine in the medium. The growth yields from the amino acids in batch culture were much less at the beginning than at the end of the culture. The highest efficiencies of conversion of amino acids to cell material were obtained by chemostat culture. When glutamic acid largely replaced the glutamine in the medium the conversion of amino acid nitrogen to cell nitrogen was 100 % efficient (that is, the theoretical yield was obtained) at the optimum growth rate (cell doubling time, 43 h). The maximum population density a given amino acid mixture will support can be calculated from the data. It is concluded that in several routinely used tissue culture media the cell growth is limited by the amino acid supply. In batch culture glutamine was wasted by (1) its spontaneous decomposition to pyrrolidone carboxylic acid and ammonia, and (2) its enzymic breakdown to glutamic acid and ammonia, but also glutamine was used less efficiently than glutamic acid. Study of the influence of cell growth rate on amino acid uptake rates per unit mass of cells indicated that a marked change in amino acid metabolism occurred at a specific growth rate of 0.4 day -1 (cell doubling time, 43 h). With decrease in specific growth rate below 0.4 day -1 there was a marked stimulation of amino acid uptake rate per cell and essential amino acids were consumed increasingly for functions other than synthesis of cell material.

Exogenous glycosaminoglycans (GAG) are able to modulate avian skin differentiation (epithelial keratinization and feather formation

Development ◽  
1984 ◽  
Vol 82 (1) ◽  
pp. 25-40
Author(s):  
E. Becchetti ◽  
G. Stabellini ◽  
A. Caruso ◽  
P. Carinci
Keyword(s):  
Hyaluronic Acid ◽  
Chick Embryo ◽  
Synthetic Medium ◽  
Calf Serum ◽  
Epithelial Differentiation ◽  
Dermal Fibroblasts ◽  
Conditioned Media ◽  
Lung Fibroblasts ◽  
Skin Explants

Several reports have suggested that mesenchymal glycosaminoglycans (GAG) may be involved in the regulatory role of epithelial differentiation. Some researchers have pointed out that exogenousGAG affects extracellular GAG accumulation. We have therefore examined the effect of added GAG on two typical processes of avian skin differentiation: keratinization and feather formation. Glycosaminoglycans, either obtained from fibroblasts cultures (conditioned media) or purified commercially available GAG were administered to 5/6-day chick embryo back skin explants. Control cultures were supported with 199 synthetic medium, chick embryo extract or calf serum. Explants have been examined by histological and histochemical procedures. Skin explants maintained in vitro for 7 days exhibited an epithelial differentiation and a dermal histochemical reactivity which were related to the composition of the culture medium. In conditioned media from dermal fibroblasts, but not from heart or lung fibroblasts, explants always exhibited keratinization. In purified-GAG-containing media, keratinization was observed with condroitinsulphates and not with hyaluronic acid. Keratinization was always related toprevalent accumulation of hyaluronic acid in the underlying mesenchyme whereas feather formation was in relation to deposits of condroitinsulphates in dermis pulp. The above findings demonstrate that exogenous GAG is able to modulate avian skin differentiation and that this regulation is linked to an influence on the mesenchymal GAG pattern.

The Contribution of Caseins to the Amino Acid Supply for Lactococcus lactis Depends on the Type of Cell Envelope Proteinase

1998 ◽  
Vol 64 (6) ◽  
pp. 1991-1996 ◽  
Author(s):  
Benedicte Flambard ◽  
Sandra Helinck ◽  
Jean Richard ◽  
Vincent Juillard
Keyword(s):  
Amino Acids ◽  
Growth Rate ◽  
Amino Acid ◽  
Lactococcus Lactis ◽  
Cell Envelope ◽  
Essential Amino Acids ◽  
Genetically Engineered ◽  
Amino Acid Requirements ◽  
Rate Limiting

ABSTRACT The ability of caseins to fulfill the amino acid requirements ofLactococcus lactis for growth was studied as a function of the type of cell envelope proteinase (PI versus PIII type). Two genetically engineered strains of L. lactis that differed only in the type of proteinase were grown in chemically defined media containing αs1-, β-, and κ-caseins (alone or in combination) as the sources of amino acids. Casein utilization resulted in limitation of the growth rate, and the extent of this limitation depended on the type of casein and proteinase. Adding different mixtures of essential amino acids to the growth medium made it possible to identify the nature of the limitation. This procedure also made it possible to identify the amino acid deficiency which was growth rate limiting for L. lactis in milk (S. Helinck, J. Richard, and V. Juillard, Appl. Environ. Microbiol. 63:2124–2130, 1997) as a function of the type of proteinase. Our results were compared with results from previous in vitro experiments in which casein degradation by purified proteinases was examined. The results were in agreement only in the case of the PI-type proteinase. Therefore, our results bring into question the validity of the in vitro approach to identification of casein-derived peptides released by a PIII-type proteinase.

137 EFFECT OF THE ADDITION OF FOLIC ACID TO MATURATION AND CULTURE MEDIA ON DEVELOPMENT OF THE BOVINE BLASTOCYST AND ITS SURVIVAL RATE AFTER FREEZE-THAWING

2017 ◽  
Vol 29 (1) ◽  
pp. 177
Author(s):  
S. Sato ◽  
O. Dochi ◽  
K. Imai
Keyword(s):  
Folic Acid ◽  
Survival Rate ◽  
Culture Media ◽  
Cell Damage ◽  
Calf Serum ◽  
Cleavage Rate ◽  
Chi Square ◽  
Exact Test ◽  
Chi Square Test

Reactive oxygen species (ROS) are the main causes of cell damage in bovine embryos in vitro. Folic acid (FA) is an antioxidant that protects cells from ROS. We studied the effect of the addition of FA to maturation and culture media on development of bovine blastocysts and their survival rate after freeze-thawing. Cell-oocyte complexes (COC) were allowed to mature in HEPES (25 mM)-buffered TCM199 (TCM199) supplemented with 5% calf serum (CS), 0.02 AU mL−1 of FSH, and FA (0, 2.5, 25, and 50 µM) for 20 hours (20–25 COC/100-µL droplet of the medium). After 6 hours of gamete co-culture (5 × 106 sperm/mL), presumptive zygotes were cultured in CR1aa medium supplemented with 5% CS and FA (0, 2.5, 25, and 50 μM) for 9 days (day of fertilization = Day 0). Expanded blastocysts that developed from Day 7 to 9 were frozen for further study. Each embryo was frozen in Dulbecco’s PBS (D-PBS) supplemented with 20% CS, 1.5 M ethylene glycol (EG), and 0.1 M sucrose (SUC). Embryos were equilibrated with their freezing medium for 15 min and loaded individually into a 0.25-mL straw. These straws were put into the cooling chamber of a programmable freezer precooled at −7°C. After 2 min, straws were seeded and held for 13 min at −7°C. Next, straws were cooled to −30°C at −0.3°C/min before being plunged into liquid nitrogen. Frozen embryos were thawed by allowing straws to stand in air for 7 s and warming them in a 30°C water bath for 20 s. Thawed embryos were washed twice with D-PBS supplemented with 20% fetal calf serum (FCS), which was warmed to 38°C. They were immersed into the same medium at 38°C for 10 min, and each embryo was cultured in a 20-μL droplet of TCM199 supplemented with 10% FCS and 0.1 mM β-mercaptoethanol (TCM-199-βME) for 72 h. Embryo cleavage rate was observed at 55 h post-insemination. Blastocyst rates were analysed at 9 days post-insemination. Rates of embryos developing into reexpanded, hatching, and hatched blastocyst stages were determined after 72 h of thawing. All data were analysed by the chi-square test and Fisher’s exact test. Cleavage and blastocyst rates after insemination at 55 hours and 9 days, respectively, were not significantly different among media containing 0 μM (n = 278; 74.1% and 39.9%), 2.5 μM (n = 260; 74.2% and 45.8%), 25 μM (n = 258; 75.6% and 45.7%), and 50 μM (n = 253; 76.3% and 42.7%) FA. Survival and hatching rates of frozen and thawed expanded blastocysts after 72 h in culture were 62.5% and 56.3%, respectively, in 0 μM FA (n = 16); 85.2% and 74.1% in 2.5 μM FA (n = 27); 66.7% and 62.5% in 25 μM FA (n = 24); and 68.0% and 64.0% in 50 μM FA (n = 25). Blastocysts cultured in media containing 2.5 μM FA tended to have a higher survival rate than those cultured in media containing 0 μM FA, although this difference was not significant (P = 0.09). Inclusion of FA did not appear to influence development or post-thaw survival of bovine blastocysts produced in vitro.

scholarly journals EFFECT OF THE AMINO ACIDS AND DIALYZABLE CONSTITUENTS OF EMBRYONIC TISSUE JUICE ON THE GROWTH OF FIBROBLASTS

1926 ◽  
Vol 44 (3) ◽  
pp. 397-407 ◽  
Author(s):  
Lillian E. Baker ◽  
Alexis Carrel
Keyword(s):  
Amino Acids ◽  
Cell Migration ◽  
Active Cell ◽  
Embryonic Tissue ◽  
Tissue Extract ◽  
Considerable Part ◽  
Support Cell ◽  
Cell Life ◽  
Growth Promoting

The ultrafilterable constituents of embryonic tissue extract are unable to support cell life in vitro. They stimulate cell migration and possibly multiplication, without increasing the mass of the tissue. Embryonic tissue extract freed from amino acids by dialysis still retains a considerable part of its growth-promoting properties. The area of growth of tissues in embryonic tissue extract free from amino acids is appreciably less than that with the whole extract, probably owing to the denaturation of part of the protein, or perhaps to the inactivation or loss of an enzyme. The addition of either the ultrafilterable components or an artificial mixture of amino acids to this dialyzed extract increases the area of cell migration but does not restore all the activity lost on dialysis. The observed differences in growth of tissue, due to the addition or removal of dialyzable and ultrafilterable constituents of the extract, prove that the amino acids produce a more active cell migration and possibly multiplication, but no building up of new protoplasm.

9-β-D-ARABINOFURANOSYLADENINE AS AN INHIBITOR OF METABOLISM IN NORMAL AND NEOPLASTIC CELLS

1965 ◽  
Vol 43 (1) ◽  
pp. 1-15 ◽  
Author(s):  
John J. Brink ◽  
G. A. LePage
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Growth ◽  
Survival Time ◽  
Liver Cells ◽  
Tumor Bearing ◽  
Ascites Tumors ◽  
Soluble Fractions

Treatment of L1210 ascites tumors in vivo with combinations of 9-β-D-arabinosyladenine and 6-methyladenine nucleosides repressed cell growth by approximately 30%. This repression was measured by the volume of accumulated cells after 6 days of treatment with these compounds. No significant increase of the survival time of L1210 tumor-bearing mice was obtained when the mice were treated under identical conditions. Incubation of TA3 or L1210 ascites cells in vitro with 6-methyIadenosine inhibited the deamination of added arabinosyladenine by 95% for TA3 and 50% for L1210. The cleavage of adenosine or deoxyadenosine in the presence of arabinosyladenine was unaffected. Incorporation of C14-adenine or C14-uracil into the DNA of TA3 cells was inhibited when arabinosyladenine and the four natural deoxynucleosides were incubated in vitro. This inhibition was relieved specifically by adenosine but not by deoxyadenosine. Arabinosyladenine affected the uptake of C14-amino acids into the proteins of TA3 cells and liver. Elevation or depression of incorporation varied with the amino acid used. Incorporation of arabinosyladenine-C14into the RNA of subcellular components of TA3 and liver cells was greatest in the nuclear and soluble fractions.

scholarly journals Importance of pfkA for Rapid Growth ofEnterobacter cloacae during Colonization of Crop Seeds

2000 ◽  
Vol 66 (1) ◽  
pp. 87-91 ◽  
Author(s):  
D. P. Roberts ◽  
P. D. Dery ◽  
I. Yucel ◽  
J. S. Buyer
Keyword(s):  
Amino Acids ◽  
Growth Rate ◽  
Growth Rates ◽  
Rapid Growth ◽  
Sweet Corn ◽  
Natural Soil ◽  
Transposon Insertion ◽  
Carbon Compounds ◽  
The Impact

ABSTRACT Enterobacter cloacae A-11 is a prototrophic, glycolytic mutant of strain 501R3 with a single transposon insertion inpfkA. The populations of strain A-11 on cucumber and radish seeds were smaller than the populations of strain 501R3 in natural soil, but the populations of these two strains on pea, soybean, sunflower, and sweet corn seeds were similar (D. P. Roberts, P. D. Dery, I. Yucel, J. Buyer, M. A. Holtman, and D. Y. Kobayashi, Appl. Environ. Microbiol. 65:2513–2519, 1999). The net effect of the mutation in pfkA in vitro was a shift from rapid growth on certain carbohydrates detected in seed exudates to much slower growth on other carbohydrates, amino acids, and organic acids. The impact of the mutation in pfkA was greatest on the growth rate of E. cloacae on the seeds that released the smallest quantities of fructose, other carbohydrates, and amino acids. Corn, pea, soybean, and sunflower seeds released total amounts of carbohydrates and amino acids at rates that were approximately 10- to 100-fold greater than the rates observed with cucumber and radish seeds for the first 24 h after inhibition began. The growth rate of strain A-11 was significantly less (50% less) than the growth rate of strain 501R3 on radish seeds, and the growth rate of strain A-11 was too low to estimate on cucumber seeds in sterile sand for the first 24 h after inhibition began. The growth rate of strain A-11 was also significantly lower on soybean seeds, but it was only 17% lower than the growth rate of strain 501R3. The growth rates of strains 501R3 and A-11 were similar on pea, sunflower, and corn seeds in sterile sand for the first 30 h after imbibition began. Large reductions in the growth rates of strain A-11 on seeds were correlated with subsequent decreased levels of colonization of seeds compared to the levels of colonization of strain 501R3. The strain A-11 populations were significantly smaller than the strain 501R3 populations only on radish and cucumber seeds. The mutation in pfkA appears to decrease the level of colonization by E. cloacae for seeds that release small quantities of reduced carbon compounds by decreasing the size of the pool of compounds that support rapid growth by this bacterium.

scholarly journals In Vitro Assessment of Antifungal Caspofungin on Leishmania donovani Culture Isolation

2017 ◽  
Vol 2017 ◽  
pp. 1-4
Author(s):  
Narayan Raj Bhattarai ◽  
Keshav Rai ◽  
Suman Rijal ◽  
Basudha Khanal
Keyword(s):  
Growth Rate ◽  
Leishmania Donovani ◽  
Inhibitory Concentration ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Fungal Growth ◽  
Antifungal Drug ◽  
Fungal Contamination ◽  
In Vitro Assessment

Leishmania parasite isolation from the human aspirates is always challenging due to most probability of the fungal contamination and the use of antifungal drug which could support the selective growth of the Leishmania parasite. In this study, we examine the effect of antifungal drug caspofungin on the promastigote stage of Leishmania donovani. Promastigote parasite was cultivated in M199 + 20% heat-inactivated fetal calf serum and plated in 96-well plates. Seven different concentrations of caspofungin (512 µg/ml to 8 µg/ml) were exposed to parasites, and 50% inhibitory concentration (IC50) was calculated. Candida spp. was used in the experiments to know the efficacy of caspofungin to inhibit fungal growth. The IC50 values of Leishmania strains ranged from 23.02 to 155.80 µg/ml (mean 90.25 ± 39.01 µg/ml), and it was significantly higher (P value = 0.02) than IC50 values of Candida spp. (ranged from 0.001 to 0.12 µg/ml, mean = 0.05 ± 0.05 µg/ml). The reduced growth rate of the parasite was found with exposure to 50 µg/ml of caspofungin. Growth inhibition of Leishmania donovani is significantly lower with caspofungin and could be used to protect the parasite cultivation from fungal contamination.

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