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 Choline and Serum Protein Requirements of Mouse Fibroblast Cells (Strain LS) in Culture

1969 ◽  
Vol 5 (1) ◽  
pp. 135-142
Author(s):  
J. R. BIRCH ◽  
S. J. PIRT
Keyword(s):  
Bovine Serum Albumin ◽  
Population Density ◽  
Serum Albumin ◽  
Serum Protein ◽  
Cell Population ◽  
Bovine Serum ◽  
Choline Chloride ◽  
Mouse Fibroblast ◽  
Population Doubling ◽  
Maximum Cell

The maximum cell population density of mouse fibroblast (strain LS) cells growing in static suspension culture was found to be directly proportional to the dialysed calf serum concentration. This was due to choline limitation and the fact that serum protein was a major source of choline. The growth yield (Y) was 3.2 x 105 cells/µg choline chloride. Studies on the role of serum in the presence of excess choline showed the following. When protein was omitted from the medium, cell death occurred. Whole serum protein could be replaced by either (1) bovine serum albumin fraction V, or (2) crystalline bovine serum albumin + sodium pyruvate and α-ketoglutarate, or (3) polyvinylpyrrolidone + methylcellulose + pyruvate and α-ketoglutarate. The population doubling time was 24 h in the presence of whole serum protein and increased considerably with the substitutes (1-3). The increase in maximum cell population density (without medium changes) exceeded 2.9 x 106 cells/ml with either whole serum protein or substitutes (1) and (2). With serum substitute (3) the maximum increase in population density was reduced to 1.6 x 106 cells/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.

A microcomputer program for calculating cell population doubling time in vitro and in vivo

1995 ◽  
Vol 37 (3) ◽  
pp. 203-210 ◽  
Author(s):  
Jeffrey I. Zwicker ◽  
Robert T. Proffitt ◽  
C. Patrick Reynolds
Keyword(s):  
Cell Population ◽  
Doubling Time ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Microcomputer Program

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.

scholarly journals Expansion and characterization of bone marrow derived human mesenchymal stromal cells in serum-free conditions

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Samatha Bhat ◽  
Pachaiyappan Viswanathan ◽  
Shashank Chandanala ◽  
S. Jyothi Prasanna ◽  
Raviraja N. Seetharam
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Population Doubling ◽  
Seeding Density ◽  
Population Doubling Time ◽  
Differentiation Potential ◽  
Serum Free ◽  
Safety Issues ◽  
Free Media

AbstractBone marrow-derived mesenchymal stromal cells (BM-MSCs) are gaining increasing importance in the field of regenerative medicine. Although therapeutic value of MSCs is now being established through many clinical trials, issues have been raised regarding their expansion as per regulatory guidelines. Fetal bovine serum usage in cell therapy poses difficulties due to its less-defined, highly variable composition and safety issues. Hence, there is a need for transition from serum-based to serum-free media (SFM). Since SFM are cell type-specific, a precise analysis of the properties of MSCs cultured in SFM is required to determine the most suitable one. Six different commercially available low serum/SFM with two different seeding densities were evaluated to explore their ability to support the growth and expansion of BM-MSCs and assess the characteristics of BM-MSCs cultured in these media. Except for one of the SFM, all other media tested supported the growth of BM-MSCs at a low seeding density. No significant differences were observed in the expression of MSC specific markers among the various media tested. In contrary, the population doubling time, cell yield, potency, colony-forming ability, differentiation potential, and immunosuppressive properties of MSCs varied with one another. We show that SFM tested supports the growth and expansion of BM-MSCs even at low seeding density and may serve as possible replacement for animal-derived serum.

scholarly journals Effect of Inflammation on Gingival Mesenchymal Stem/Progenitor Cells’ Proliferation and Migration through Microperforated Membranes: An In Vitro Study

2020 ◽  
Vol 2020 ◽  
pp. 1-10 ◽  
Author(s):  
M. Al Bahrawy ◽  
K. Ghaffar ◽  
A. Gamal ◽  
K. El-Sayed ◽  
V. Iacono
Keyword(s):  
Pore Size ◽  
Progenitor Cells ◽  
Cellular Migration ◽  
Gingival Tissue ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Electron Microscopic ◽  
Healthy Human ◽  
Membrane Pores ◽  
Scanning Electron

Background. In the field of periodontal guided tissue regeneration, microperforated membranes have recently proved to be very promising periodontal regenerative tissue engineering tools. Regenerative periodontal approaches, employing gingival mesenchymal stem/progenitor cells in combination with these novel membranes, would occur mostly in inflamed microenvironmental conditions intraorally. This in turn entails the investigation into how inflammation would affect the proliferation as well as the migration dynamics of gingival mesenchymal stem/progenitor cells. Materials and Methods. Clones of human gingival mesenchymal stem/progenitor cells (GMSCs) from inflamed gingival tissues were characterized for stem/progenitor cells’ characteristics and compared to clones of healthy human GMSCs (n=3), to be subsequently seeded on perforated collagen-coated poly-tetra-floro-ethylene (PTFE) membranes with a pore size 0.4 and 3 microns and polycarbonic acid membranes of 8 microns pore size in Transwell systems. The population doubling time and the MTT test of both populations were determined. Fetal bovine serum (FBS) was used as a chemoattractant in the culturing systems, and both groups were compared to their negative controls without FBS. Following 24 hours of incubation period, migrating cells were determined on the undersurface of microperforated membranes and the membrane-seeded cells were examined by scanning electron microscopy. Results. GMSCs demonstrated all predefined stem/progenitor cell characteristics. GMSCs from inflamed gingival tissues showed significantly shorter population doubling times. GMSCs of inflamed and healthy tissues did not show significant differences in their migration abilities towards the chemoattractant, with no cellular migration observed in the absence of FBS. GMSCs from healthy gingival tissue migrated significantly better through larger micropores (8 microns). Scanning electron microscopic images proved the migratory activity of the cells through the membrane pores. Conclusions. Inflammation appears to boost the proliferative abilities of GMSCs. In terms of migration through membrane pores, GMSCs from healthy as well as inflamed gingival tissues do not demonstrate a difference in their migration abilities through smaller pore sizes, whereas GMSCs from healthy gingival tissues appear to migrate significantly better through larger micropores.

Characterization of a newly established uterine carcinosarcoma cell line featuring the sarcomatous phenotype of the tumor in vitro

2008 ◽  
Vol 18 (2) ◽  
pp. 339-344 ◽  
Author(s):  
H.-J. Schulten ◽  
J. Wolf-Salgó ◽  
C. Gründker ◽  
B. Gunawan ◽  
L. FÜZESI
Keyword(s):  
Cell Line ◽  
Hormone Receptors ◽  
Tumor Biology ◽  
Growth Factor Receptor ◽  
Population Doubling ◽  
Established Cell Line ◽  
Population Doubling Time ◽  
Uterine Carcinosarcoma ◽  
Specific Staining

We describe the newly established cell line CS-99 derived from a uterine carcinosarcoma retaining features of the sarcomatous phenotype in vitro. CS-99 cells exhibit a mesenchymal morphology with predominantly spindle-shaped cells at nonconfluence turning to pleomorphic appearance at confluence. The mesenchymal phenotype was evidenced immunohistochemically by strong vimentin and moderate SM-actin, which was similar to the sarcomatous component of the primary tumor. P53 was overexpressed in a subset of CS-99 cells. Epithelial membrane antigen was moderately expressed whereas other markers including pan CK, CK 5/6, CK 34, epidermal growth factor receptor, desmin, carcinoembryonic antigen, S100, KIT, ERBB2, and the hormone receptors, estrogen receptor and progesterone receptor revealed either weak or no specific staining in CS-99 cells. High self-renewal capacity corresponded to the population doubling time of 23 h in high passage. CS-99 cells were able to develop three-dimensional tumor spheroids in vitro. Cytogenetic analysis and multicolor fluorescence in situ hybridization of CS-99 demonstrated an almost stable karyotype including numerical changes +8, +18, and +20 and translocations, amongst others der(1)t(1;2), der(1)t(1;7), der(2)t(2;19), der(5)t(5;8), and der(5)t(5;14). Taken together, the cell line CS-99 exhibits strong growths dynamics and a complex but stable karyotype in higher passages, and can be further a useful in vitro model system for studying tumor biology of carcinosarcomas.

scholarly journals Sulodexide reduces glucose induced senescence in human retinal endothelial cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
A. Gericke ◽  
K. Suminska-Jasińska ◽  
A. Bręborowicz
Keyword(s):  
Endothelial Cells ◽  
High Glucose ◽  
Glucose Concentration ◽  
Chronic Exposure ◽  
Replicative Senescence ◽  
Population Doubling ◽  
Population Doubling Time ◽  
High Glucose Concentration ◽  
Retinal Endothelial Cells

AbstractChronic exposure of retinal endothelium cells to hyperglycemia is the leading cause of diabetic retinopathy. We evaluated the effect of high glucose concentration on senescence in human retinal endothelial cells (HREC) and modulation of that effect by Sulodexide. Experiments were performed on HREC undergoing in vitro replicative senescence in standard medium or medium supplemented with glucose 20 mmol/L (GLU) or mannitol 20 mnol/L (MAN). Effect of Sulodexide 0.5 LRU/mL (SUL) on the process of HREC senescence was studied. Glucose 20 mmol/L accelerates senescence of HREC: population doubling time (+ 58%, p < 0.001) β-galactosidase activity (+ 60%, p < 0.002) intracellular oxidative stress (+ 65%, p < 0.01), expression of p53 gene (+ 118%, p < 0.001). Senescent HREC had also reduced transendothelial electrical resistance (TEER) (− 30%, p < 0.001). Mannitol 20 mmol/L used in the same scenario as glucose did not induce HREC senescence. In HREC exposed to GLU and SUL, the senescent changes were smaller. HREC, which became senescent in the presence of GLU, demonstrated higher expression of genes regulating the synthesis of Il6 and VEGF-A, which was reflected by increased secretion of these cytokines (IL6 + 125%, p < 0.001 vs control and VEGF-A + 124% p < 0.001 vs control). These effects were smaller in the presence of SUL, and additionally, an increase of TEER in the senescent HREC was observed. Chronic exposure of HREC to high glucose concentration in medium accelerates their senescence, and that process is reduced when the cells are simultaneously exposed to Sulodexide. Additionally, Sulodexide decreases the secretion of IL6 and VEGF-A from senescent HREC and increases their TEER.

Sign in / Sign up

Export Citation Format

Share Document