scholarly journals VARIATION IN THE LIFE-SPAN OF CLONES DERIVED FROM HUMAN DIPLOID CELL STRAINS

1974 ◽  
Vol 62 (1) ◽  
pp. 48-53 ◽  
Author(s):  
J. R. Smith ◽  
L. Hayflick
Keyword(s):  
Life Span ◽  
Mass Culture ◽  
Single Cells ◽  
Diploid Cell ◽  
Population Doubling ◽  
Mass Cultures ◽  
Single Mass ◽  
Cell Strains ◽  
Population Doublings

The doubling potential of several hundred clones derived from WI-38 and WI-26 cell cultures has been determined. Clones were isolated at various population doubling levels (PDLs) during the finite in vitro life-span of the mass (uncloned) cultures. In all cases, there was a large variation in population doubling potential (or life-span) among the clones isolated from a single mass culture. When clones were isolated from mass cultures which had undergone eight or nine population doublings, only about 50% of the clones were capable of more than eight population doublings. This percentage was further reduced when clones were isolated from mass cultures at higher PDLs. Mass cultures appear to be composed of two subpopulation classes: one with a low population doubling potential, and the other with a higher population doubling potential. Nevertheless, the highest doubling potential observed in clones isolated from any single culture was about the same as the doubling potential of the mass culture from which single cells were taken.

Absence of P21 Gene Augments Proliferation of Mesenchymal Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3039-3039
Author(s):  
Anthony Y. Tsai ◽  
Stephanie C. Filice ◽  
Elizabeth H. Javazon ◽  
Andrea T. Badillo ◽  
Alan W. Flake
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Kinase Inhibitor ◽  
Adipogenic Differentiation ◽  
Population Doubling ◽  
Facs Analysis ◽  
Competition Assay ◽  
Cell Counts ◽  
Population Doublings

Abstract Bone marrow derived mesenchymal stem cells (MSCs) have the capacity to differentiate into cells of the various mesodermal lineages. Efficiency of engraftment, however, remains a significant obstacle in MSC transplantation. P21 is a G1 checkpoint regulator and cyclin-dependent kinase inhibitor. In its absence, hematopoieitc stem cell (HSC) proliferation increases under normal homeostatic conditions. The aim of the study is to evaluate the effects of p21 deficiency on the proliferative capacity of MSCs. Therefore, MSCs from p21 KO and wildtype mice were maintained in culture in hypoxic and normoxic conditions. Cell counts and respective days in cultures were recorded from which population doubling (PD) times were calculated and compared. 1:1 Competition assays were performed between p21 KO MSCs transduced with DsRed and eGFP transduced WT MSCs as well as between DsRed transduced p21 KO MSCs and non-transduced WT MSCs. Osteogenic, chondrogenic, and adipogenic differentiation assays were performed on both cell populations using standard protocols. Sca-1, CD34, CD45, CD90, MHC Class I and II and other surface antigens were assessed using FACS analysis. In normoxic conditions, the p21 KO MSCs went through 25.68 population doublings in 60 days versus 25.43 population doublings in 103 days for the p21 WT MSCs. Under hypoxic conditions, the KO MSC population doubled 23.3 times in 58 days versus 14.78 times in 60 days for the WT MSCs. All population doubling times were taken from passage 10 cells. When the DsRed transduced KO MSCs (96.6-98.5% DsRed+) was placed in a 1:1 competition assay with eGFP transduced WT MSCs (99.3-99.9% eGFP+) or non-transduced WT MSCs (99.9–100% eGFP−), the normoxic results are as shown in Table 1. Similarly, the competition assay result between DsRed transduced KO MSCs (86.9–94.1% DsRed+) and eGFP transduced WT MSCs (99.7–100% eGFP+) or non-transduced WT MSCs (100% eGFP−) for the hypoxic condition are shown in Table 2. Both the p21 KO and wildtype MSCs populations were able to differentiate into ostenogenic, adipogenic, and chondrogenic lineages. No significant surface marker differences were observed between the 2 populations on FACS analysis. Our results clearly showed that p21 deficient MSCs have increased proliferative ability in vitro compared to normal MSCs. These findings have implications for expansion of MSC populations in vitro , and for the enhancement of competitive capacity of MSCs following in vivo administration. Table 1 - Normoxic Competition Assay KO MSC (DsRed) WT MSC (eGFP) KO MSC (DsRed) WT MSC (none) results analyzed by FACS Day 6 64.2±3.7% 34.5±4.3% 70.3±0.4% 19.7±0.6% Day 11 84.0±4.5% 13.6±4.6% 92.0±0.8% 6.4±0.7% Table 2 - Hypoxic Competition Assay KO MSC (DsRed) WT MSC (eGFP) KO MSC (DsRed) WT MSC (none) results analyzed by FACS Day 4 70.3±1.7% 30.0±1.7% 83.0±0.8% 18.6±0.9% Day 8 75.7±0.7% 25.8±0.7% 81.3±1.2% 20.6±1.2%

Establishment of a New Cell Line from Maxillary Sinus Carcinoma

1980 ◽  
Vol 89 (1) ◽  
pp. 24-28 ◽  
Author(s):  
Tadashi Nakashima ◽  
Kazumi Makishima ◽  
Ikuichiro Hiroto
Keyword(s):  
Maxillary Sinus ◽  
Cell Line ◽  
Single Cells ◽  
Latency Period ◽  
Soft Agar ◽  
Culture Cell ◽  
Population Doubling ◽  
Tissue Culture Cell ◽  
Population Doubling Time

We succeeded in deriving a long-term tissue culture cell line from human maxillary sinus carcinoma. This cell line, designated as MC, was passaged 100 times in vitro over a period of 18 months. The cells are globular in shape, grow as single cells in the culture medium, and the mean population doubling time is about 12 hours. The plating efficiency rate in soft agar is 78% and chromosomal analysis revealed the modal chromosome number to be between 47 and 51. These MC cells were transplanted into five nude mice, all of which developed a tumor after a latency period of 5 to 8 days and died within 39 days. Complete autopsy of all mice revealed no metastasis. Histopathological findings of the original and the transplanted tumor tissues showed a remarkable similarity.

scholarly journals IN VITRO STUDIES OF MAMMALIAN SOMATIC CELL VARIATION

1963 ◽  
Vol 117 (2) ◽  
pp. 267-284 ◽  
Author(s):  
Howard M. Cann ◽  
Leonard A. Herzenberg
Keyword(s):  
Mass Culture ◽  
Single Cells ◽  
Cytotoxic Action ◽  
Parent Cell ◽  
Multiple Exposures ◽  
Mouse Lymphoma Cells ◽  
Cell Variation ◽  
Mouse Lymphoma

When long term cultures of mouse lymphoma cells, known to possess the isoantigenic phenotype determined by the H-2d allele, are incubated with anti H-2d isoantibody and guinea pig complement, slightly more than 99 per cent of cells are killed under optimal conditions. Growth in mass culture and colony formation by single cells after incubation with isoantibody and complement are employed to assess the cytotoxic effect. The cytotoxic action of isoantibody is complement-dependent, for viability of cells exposed to antibody alone is unaltered. When excess isoantibody and optimum concentrations of complement are used, killing begins as soon as these reagents are mixed with the cells, and no further killing occurs after 5 to 15 minutes at 37°C. About 80 per cent of cells are killed with an isoantiserum containing antibody to two isoantigenic components of the H-2d complex. That the cytotoxic action is mediated through the H-2 isoantigen is shown by (a) isoantiserum containing only anti H-2d antibody produces maximal cell killing, and (b) isoantiserum from which anti H-2d antibody has been removed by absorption loses all cytotoxic activity. Variant cells resistant to the cytotoxic action of anti H-2d isoantibody were isolated from lymphoma cell populations surviving multiple exposures to isoantibody and complement. These variants can be distinguished morphologically from the isoantibody-sensitive parent cell line. Although variants are resistant to anti H-2d isoantibody, these cells possess H-2d isoantigen but in a lower concentration than found in cells of the parent line. The basis for resistance to cytotoxic isoantibody is discussed.

scholarly journals Effect of Fibroblast Growth Factor 2 on Equine Synovial Fluid Chondroprogenitor Expansion and Chondrogenesis

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Marta Bianchessi ◽  
Yuwen Chen ◽  
Sushmitha Durgam ◽  
Holly Pondenis ◽  
Matthew Stewart
Keyword(s):  
Growth Factor ◽  
Synovial Fluid ◽  
Fibroblast Growth Factor ◽  
Primary Cultures ◽  
Fibroblast Growth Factor 2 ◽  
Population Doubling ◽  
Fibroblast Growth ◽  
Monolayer Expansion ◽  
Population Doublings

Mesenchymal stem cells have been identified in the synovial fluid of several species. This study was conducted to characterize chondroprogenitor (CP) cells in equine synovial fluid (SF) and to determine the effect of fibroblast growth factor 2 (FGF-2) on SF-CP monolayer proliferation and subsequent chondrogenesis. We hypothesized that FGF-2 would stimulate SF-CP proliferation and postexpansion chondrogenesis. SF aspirates were collected from adult equine joints. Colony-forming unit (CFU) assays were performed during primary cultures. At first passage, SF-cells were seeded at low density, with or without FGF-2. Following monolayer expansion and serial immunophenotyping, cells were transferred to chondrogenic pellet cultures. Pellets were analyzed for chondrogenic mRNA expression and cartilage matrix secretion. There was a mean of 59.2 CFU/mL of SF. FGF-2 increased the number of population doublings during two monolayer passages and halved the population doubling times. FGF-2 did not alter the immunophenotype of SF-CPs during monolayer expansion, nor did FGF-2 compromise chondrogenesis. Hypertrophic phenotypic markers were not expressed in control or FGF-2 groups. FGF-2 did prevent the development of a “fibroblastic” cell layer around pellet periphery. FGF-2 significantly accelerates in vitro SF-CP expansion, the major hurdle to clinical application of this cell population, without detrimentally affecting subsequent chondrogenic capacity.

The Optimal Processing Time of Adipose-Derived Mesenchymal Stem Cell after Lipoaspiration.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4107-4107
Author(s):  
Jun Ho Jang ◽  
Kihyun Kim ◽  
Chul Won Jung ◽  
Keon Woo Park
Keyword(s):  
Processing Time ◽  
Population Doubling ◽  
Cell Surface Antigens ◽  
Optimal Processing ◽  
Cell Counts ◽  
Collagenase Treatment ◽  
Alternate Source ◽  
Initial Processing ◽  
Population Doublings

Abstract Background: Our aims were to characterize the phenotype of these cells, to investigate their immunoregulatory properties in vitro, and to determine the optimal processing time of AD-MSC after lipoaspiration. Methods: ASCs were isolated from lipoaspirated adipose tissues by treatment of collagenase A and cultured in a Dulbecco’s Modified Eagle’s Medium (DMEM). To evaluate the optimal processing time of ASCs, the nucleated cells were processed by collagenase treatment at 3, 6, 9, 12, 15, 24, 30 and 36 hours after lipoaspiration respectively. To know the characteristics of ASCs, the expression of cell surface antigens was analyzed by flow cytometry, and the proliferation potentials were estimated by colony forming abilities or capacities of population doubling. The differentiation potentials into adipocytes or osteoblasts were confirmed by accumulation of neutral lipid vacuoles stained with Oil-red O and expression of alkaline phosphatases. Results: When the nucleated cells were isolated by collagenase treatment after lipoaspiration, the mean cell yield was about 3.1 × 106 or 1.2 × 106cells per gram of lipoaspirate (n=8) processed. Nucleated cell counts were increased with time but plateau between 15h and 24h after initial processing and decreased after 24h. Flowcytometric analysis showed that Adipose tissue-derived stem cells (ASCs) have a marker expression that is similar to that of bone marrow stromal cells (BMSCs). ASCs expressed CD44, CD73, CD90, and CD105 and were absent for CD14, CD31 and CD45 expression. When primary cells were plated at 50 or 1000 cells/cm2 on 6-well plates, cumulative population doublings were about 50 times until passage 7 or 13 (approximately 130 days), respectively, and ASCs expanded to 1018 cells. ASCs were multipotent, differentiating along the adipocyte and osteoblast lineages. ASCs did not provoke in vitro alloreactivity of incompatable lymphocytes and, moreover, suppressed mixed lymphocyte reaction (MLR) and lymphocyte proliferative response to mitogen. Conclusion: The optimal processing time for AD-MSCs from lipoaspirates should before 24h after removal RBC and AD-MSCs can be a alternate source of multilineage MSCs for clinical use such as tissue repair and transplantation.

Spontaneous variability of single isolates of Phytophthora infestans. I. Cultural variation

1968 ◽  
Vol 46 (4) ◽  
pp. 329-348 ◽  
Author(s):  
C. E. Caten ◽  
J. L. Jinks
Keyword(s):  
Growth Rate ◽  
Phytophthora Infestans ◽  
Mass Culture ◽  
Cultural Variation ◽  
Phenotypic Characters ◽  
Original Mass ◽  
Mass Cultures ◽  
Single Mass ◽  
Selection For ◽  
Hyphal Tip

The variability in culture of mycelial isolates of Phytophthora infestans was studied by examining the variation among single zoospore, single sporangium, and single hyphal tip subcultures. Extensive variation in rate of growth and sporangium production on artificial medium was detected among the single zoospore progenies of three mass cultures. Differences in colony morphology and viability of zoospores were also apparent but were not studied in detail. Subcultures established by single sporangia or single hyphal tips were much more uniform than zoospore cultures, although significant differences in growth rate could still be detected. While, with continued culture, some of the single zoospore variants tended to revert to their parental type, others showed a remarkable degree of stability.Isolates established from single zoospores gave rise to as much variation in their asexual progenies as the original mass cultures. This persistence of variation was also observed with isolates whose derivation included two successive single zoospore propagations. Selection for high and low growth rate among the zoospore progeny of a single mass culture rapidly led to the production of populations of zoospore cultures with different mean growth rates. Such response to selection implies the existence of a genetic mechanism which allows the transmission of phenotypic characters from one asexual generation to the next.The origin of variation among single zoospore cultures is discussed with reference to five different asexual mechanisms of variation. It is suggested that variation is the result of cytoplasmic differences present in the original mass isolates, although the possibility of other mechanisms can not be completely dismissed. A review of the literature suggests that asexual variation associated with zoospore propagation is widespread in the genus Phytophthora.

scholarly journals Cultural Properties of Cryopreserved Thymic Multipotent Stromal Cells and Fetal Skin and Muscle-Derived Cells

2021 ◽  
Vol 31 (3) ◽  
pp. 249-257
Author(s):  
Valentina Nikolska ◽  
◽  
Yanina-Maria Semenova ◽  
Lyuba Taranukha ◽  
Ihor Nikolsky ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Culture Media ◽  
Lymphoid Cells ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Multipotent Stromal Cells ◽  
Average Population ◽  
Population Doublings ◽  
Adult Thymus

The paper provides a comparison of properties of cryopreserved fetal murine multipotent stromal cells (MSCs) of skin-muscular origin and those derived from adult thymus in culture in vitro. Fetal MSCs showed a 30% higher number of average population doublings within 24 hrs, and 41% lower average population doubling time. It was found that the fetal MSCs of the 4th passage had a 39% higher clonogenic activity than the adult thymus-derived ones. Fetal MSCs and those derived from adult thymus differentiated in osteogenic and adipogenic lineages with equal efficiency in special culture media. Fetal and thymus-derived MSCs were characterized by almost the same high ability of contact interaction with thymocytes, and the fibroblast-lymphocyte rosette (FLR) formation. They were far less active in FLR formation with lymph node cells. This indicated the presence of membrane affinity for immature lymphoid cells in both MSC subpopulations. The results showed the fetal MSCs to be significantly different from the adult thymus-derived MSCs by more active kinetics of growth and clonogenic potential. However, both cell subpopulations had virtually the same ability for linear differentiation and showed high activity during contact with immature lymphoid cells. Linear differentiation and the ability to interact with lymphocytes were found to be quite stable properties of MSCs, but a proliferative activity and in vitro colony formation distinguished significantly in different types of MSCs. This can be taken into account when choosing the cells for therapy, research and results assessment.

Nuclear transfer using clonal lines of porcine fetal fibroblasts with different sizes and population doubling rates

2008 ◽  
Vol 20 (8) ◽  
pp. 871 ◽  
Author(s):  
H. T. Cheong
Keyword(s):  
Cell Size ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Population Doubling ◽  
Developmental Potential ◽  
Fetal Fibroblasts ◽  
Clonal Lines ◽  
Population Doublings ◽  
Electrical Stimuli

The aim of the present study was to examine the development of pig embryos produced by somatic cell nuclear transfer (NT) using the clonal lines of fetal fibroblasts with different population doublings (PD) per day and sizes. Clonal lines were established by plating fetal fibroblasts from a Day 35 pig fetus into 96-well clusters, one cell to each well. Four clonal lines (L1–L4) were selected for NT according to their PD per day (1.1 ± 0.2 to 0.8 ± 0.2) and mean cell size (15.1 ± 2.0 to 20.1 ± 2.9). Donor cells were transferred into enucleated oocytes, fused and activated simultaneously with electrical stimuli (two pulses of 125 V mm–1 for 30 μs) and cultured for 6 days. The proportion of embryos that developed to the blastocyst stage in the L3 (19.6%) and L4 (25.3%) lines, which had a lower PD per day and larger cell size, were significantly higher (P < 0.05) than that of the L2 line (10.6%), which had a higher PD per day and the smallest cell size. The proportion of embryos developing to the blastocyst stage in the L1 line (17.3%), which had the highest PD per day and smaller cell size, was significantly lower (P < 0.05) than that of the L4 line. These results suggest that clonal lines with larger sized cell populations in mean and lower PD per day have a greater in vitro developmental potential following NT.

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