Plating Efficiency Of Cells Research Articles

Erythroid colony formation in cultures of human marrow: effect of leukocyte conditioned medium.

A method, based on the differing capacities of cells to adhere to a column of polyester fibres, has been described for separating human bone marrow cells into a nonadherent and an adherent fraction. The effect of this cell separation procedure on colony formation by erythroid progenitor cells was investigated. In contrast to the unseparated population, it was found that erythropoietin-dependent erythroid colony formation by nonadherent cells could be considerably enhanced by the addition of leukocyte conditioned medium to the cultures. Similar erythroid enhancing activity was also detected in a partially purified preparation of granulocytic colony stimulating activity obtained from human embryo kidney culture supernatants. Erythroid colony formation in the absence of added erythropoietin, by non-adherent bone marrow cells from patients with polycythemia rubra vera, were also enhanced by the addition of LCM to the cultures. This finding suggests that the enhancing factor in LCM may not be dependent on the presence of erythropoietin in the cultures for its activity. While the cellular mechanisms by which leukocyte conditioned medium enhances erythroid growth remain to be determined, the data presented provides strong evidence for the view that the plating efficiency of erythroid progenitor cells is determined not only be the concentration of erythropoietin, but also by the presence of leukocyte conditioned medium in the cultures.

Growth behavior of ascites tumor cells in three-dimensional agar culture.

Growth behavior in vitro of ascites tumor cells was examined for a model study on the invasion in cancer. For this purpose three-dimensional agar culture was used. Seventeen strains of tumor cells were used. These tumor cells grow floating in ascites as single isolated cells (single cell strain) or forming different per cent of cell aggregates (island type strain). Plating efficiencies of tumor cells were variable, depending on the kinds of tumor stain. It was noted that FM3A cells of the cultured cell line showed almost 100% plating efficiency irrespective of the number of cells inoculated. Tumor cells formed 3 types of colony; that is solid type, mushroom-shaped and disk-shaped colony. These three types of colony were observed in all the tumor strains tested, depending on the position in agar layer. The cells of single cell strains showed the tendency to form loose colonies in which cell contact was loose and cells were easy to liberate from the periphery of the colony. The cells of island type strains showed the tendency to form packed colonies in which cell contact was tight. Growth behavior of cells of ascites tumor in agar medium showed good correlation with that in susceptible animals.

The effect of dibromodulcitol on resting and dividing lymphoid cells

The effect of dibromodulcitol (DBD) on the incorporation of labelled precursors into DNA and RNA fractions of PHA-stimulated human lymphocytes and of P388F lymphoma cells at various stages of their growth was studied. Both cell systems showed sensitivity to the drug within the concentration range of 1–10 μg/ml. When DBD was added before phytohaemagglutinin (PHA), human lymphocytes showed a DNA labelling that was more affected than RNA. In contrast, by adding DBD after PHA, RNA labelling was much more inhibited than DNA. In the latter case, the decrease in DNA labelling occurred only 24 h after drug treatment whereas RNA labelling was decreased 1 h after treatment. Levels of DBD which normally produced 30% inhibition in plating efficiency of P388F lymphoma cells affected uridine-5-T incorporation to a different extent at different stages of growth of the culture. Enhanced RNA labelling occurred in early exponential stage while at later stages of growth, RNA synthesis was depressed.

Toxicity testing in vitro. I. The effects of delta 9-tetrahydrocannabinol and aflatoxin B1 on the growth of cultured human fibroblasts.

The acute toxicity of delta 9-tetrahydrocannabinol (delta 9-THC) and aflatoxin B1 to two strains of cultured human fibroblasts has been studied. delta 9-THC had no effect on cell plating efficiency or on the growth of mass cultures at doses of 1 mug/ml (3.18 muM) or less; at 10 mug/ml plating efficiency was reduced by approximately half and at 20 mug/ml colony formation was zero. Aflatoxin B1 reduced plating efficiency at dose levels of 0.1 mug/ml (0.32 muM) and above; in mass clultures it retarded growth at 1 mug/ml and produced complete inhibition at 5 mug/ml. The potential usefulness of cultured human fibroblasts in toxicity testing is discussed and the importance of using normal diploid cells rather than aneuploid permanent lines is emphasized. The limitations of cell cultures in assessing toxicity, and possible solutions to these are considered.

Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity.

Various types of cells from the testes of mice and hamsters were separated according to differences in sedimentation velocity by centrifugal elutriation, a counterflow centrifugation technique. Approximately 3 times 10(8) cells, prepared from six mouse testes or from one hanster testis, were separated into 11 fractions in less than two hours as compared to the 4--5 hours required for sedimentation at unit gravity ("Staput"). Fractions enriched in elongated spermatids and spermatozoa (100%), stages 1--8 spermatids (69%) and pachytene spermatocytes (58%) were obtained from mouse testis dispersions. Similarly enriched fractions were obtained from hamster cells. A single fraction enriched in stages 1--8 spermatids (mouse) was prepared in less than 30 minutes. As many as 2 times 10(9) cells were separated in a single procedure. Spermatogenic cells exhibited no evidence of structural damage with trypan blud and phase microscopy, and recovery was essentially 100%. Centrifugal elutriation had no effect on sperm motility or on the plating efficiency of CHO cells.

The Factors Controlling Stem Cell Recirculation. I. Migration of Hemopoietic Stem Cells in Adrenalectomized Mice

The influence of bilateral adrenalectomy on hemopoietic stem cell (CFU) migration in mice has been studied. Formation of endogenous spleen colonies in lethally irradiated, leg-shielded mice was sharply increased by prior adrenalectomy, and this increase was not dependent on the volume of shielded bone marrow. Adrenalectomy was shown to increase endogenous spleen colony formation in sublethally irradiated mice as well. However, it had no affect on formation of spleen colonies in lethally irradiated mice injected with syngeneic bone marrow. The CFU content of murine bone marrow decreased acutely after removal of the adrenals, and this decrease was accompanied by a concomitant increase in the peripheral blood and splenic CFU. Thus, adrenalectomy appeared to have no affect on the splenic plating efficiency or proliferative rate of hemopoietic stem cells, but it did result in increased migration of stem cells from the bone marrow to the blood, and thence to the spleen. It is concluded that the adrenal steroids may be of physiologic importance in the regulation of ehmopoietic stem cell migration.

The factors controlling stem cell recirculation. i. Migration of hemopoietic stem cells in adrenalectomized mice

The influence of bilateral adrenalectomy on hemopoietic stem cell (CFU) migration in mice has been studied. Formation of endogenous spleen colonies in lethally irradiated, leg-shielded mice was sharply increased by prior adrenalectomy, and this increase was not dependent on the volume of shielded bone marrow. Adrenalectomy was shown to increase endogenous spleen colony formation in sublethally irradiated mice as well. However, it had no affect on formation of spleen colonies in lethally irradiated mice injected with syngeneic bone marrow. The CFU content of murine bone marrow decreased acutely after removal of the adrenals, and this decrease was accompanied by a concomitant increase in the peripheral blood and splenic CFU. Thus, adrenalectomy appeared to have no affect on the splenic plating efficiency or proliferative rate of hemopoietic stem cells, but it did result in increased migration of stem cells from the bone marrow to the blood, and thence to the spleen. It is concluded that the adrenal steroids may be of physiologic importance in the regulation of ehmopoietic stem cell migration.

Long term cell culture of rat liver epithelial cells retaining some hepatic functions

Summary Extensive studies of parameters conditioning high plating efficiency of epithelial liver cells at primary seeding allowed us to set up a technique for the routine culture of liver cells from rats of various ages (18 day-old pc to 7 month-old) in Ham F10 medium supplemented with 10 p. cent fetal calf serum and 10 p. cent human serum. Cultures, after several passages, or sometimes at primary seeding were free of fibroblasts. Cell lines were kept for over one year with a subculture splitting ratio of 1 to 10 each week. The generation time of the cells was 16–18 hours. Caryotype analysis showed a high majority of normal diploid and tetraploid cells. Various enzymes and metabolic pathways have been studied in primary culture and in cell lines: enzymes of the anaerobic metabolism of hexoses and metabolism of steroid hormones. Activity glucose-6-phosphatase was nearly lost in all cultures. Aldolase showed a specific liver activity with a cleavage ratio of phosphofructoses (F-1,6-diP/-1-P) equal to 1 or about 1 in several primary cultures and cell lines. Many metabolites arising from incubation of cell lines with 14C-labelled corticosterone, corticosterone-21-sulfate, testosterone and progesterone have been isolated and quantitated by gas liquid chromatography (GC) and mass fragmentography coupled to GC, using 14C/12C isotope ratio measurements. These metabolites indicate the presence in cultured cells of 3α/β-steroid-reductases, 4-enesteroid-reductases, 17β-steroid-oxido-reductase, 11β-steroid-oxido-reductase and ring hydroxylases.

Influence of oxygen and pH on plating efficiency and colony development of WI-38 and Vero cells

Clonal growth of human (WI-38) and adult monkey (Vero) cells was enhanced by lowering the O 2 content of the gas phase from atmospheric (18–20%) to 1%. The O 2 sensitivity of the low-passage diploid cells was significantly greater than that of the long-term heteroploid monkey line. Several culture media and serum sources consistently promoted superior colony development at 1% O 2, whereas variable growth and reduced plating efficiencies were observed in replicate cultures gassed with 20% O 2.

An in Vitro Assay to Measure the Viability of KHT Tumor Cells Not Previously Exposed to Culture Conditions

An in vitro colony assay which measures the viability of KHT fibrosarcoma cells is described. Single cell suspensions are prepared from tumors growing in C3H mice with no previous adaptation for in vitro growth. The assay involves incubating cells at 37° for 2 wk in Petri dishes containing a semisolid matrix of α medium, fetal calf serum, and Bacto agar. Colonies containing 20 or more cells are then counted under a microscope. The plating efficiency of the assay ranges from 5-40% based on the number of viable cells plated as determined by dye exclusion. However, this dye-exclusion technique may overestimate the number of viable cells since the plating efficiency of cells separated by velocity sedimentation ranges from 50-90%. Survival curves have been determined for cell suspensions irradiated in vitro in air or nitrogen. ${\rm D}_{0}\text{'}{\rm s}$ of 167 and 478 rads and extrapolation numbers of 1.6 and 1.4 were obtained for aerobic and anoxic curves, respectively. These numbers are in reasonable agree...

Einflu� von Licht und Medium auf die ?Plating Efficiency? isolierter Zellen aus Calluskulturen von Nicotiana tabacum var. ?Samsun?

Light increases the plating efficiency of single cells isolated from green callus cultures of Nicotiana tabacum var. “Samsun” and plated in densities of less than 100 cells ml-1 by more than 100%. At optimal light intensities plating efficiences between 80 and 90% were achieved with green cells, whereas the growth of isolated cells from chlorophyll free callus cultures was inhibited by light.

Growth and characteristics of monolayer cell cultures derived from benign hyperplasia of the prostate and carcinoma of the prostate (author's transl)

Monolayer cell cultures from 15 patients with benign nodular hyperplasia of the prostate and 7 patients with adeno-carcinoma of the prostate were established. Tissue culture methods are described. Cells obtained from carcinoma of the prostate showed a higher plating efficiency than those obtained from hyperplasia of the prostate. Electron microscopic studies revealed in cultures derived from adenocarcinoma of the prostate predominantly one epithelial cell type, which was characterized by close cell growth with many desmosomal junctions and atypical mitochondria. Testosterone in high concentrations decreased the plating efficiency of cells from hyperplasia of the prostate but had no effect in these concentrations on cells from carcinoma of the prostate. In low concentrations testosterone stimulated the growth of cells obtained from carcinomas.

Dermal Factors Affecting Epidermal Cells In Vitro

Collagen, acid mucopolysaccharides, and water soluble small molecular weight constituents from connective tissue were examined for their ability to affect epithelial cell growth in vitro . Collagen increased the plating efficiency of epithelial cells. Neither hyaluronic acid nor dermatan sulfate stimulated epithelial cells. Polar, small molecular weight materials extracted from connective tissue both markedly increased the survival time and stimulated the incorporation of 32 P into epithelial cells.

Inhibitory effect of carcinogenic aromatic hydrocarbons on transformation of 3T3 cells by SV 40.

SummaryThe carcinogenic polycyclic aromatic hydrocarbons 7,12-dimethylbenz[α] anthracene (0.0005 μg/ml) and 3-methylcholanthrene (0.005 μg/ml) did not have a toxic effect on the plating efficiency of 3T3 cells. The number of SV 40-transformed clones of 3T3 cells was decreased when these carcinogens were added 24 hr after virus inoculation and were in contact with the cells for 7 days. However, there was no effect when the carcinogens were in contact with the cells for 48 hr before or for 3 hr immediately after virus inoculation. No direct effect of the carcinogens on the infectivity of SV 40 was observed when the virus was incubated with from 0.002 to 200 μg/ml of the carcinogens and subsequently titered in the BSC-1 cell line. These findings confirm in vitro for another oncogenic virus the previously described inhibitory effect of these carcinogens on Friend and Rauscher virus leukemias. The most likely explanation seems to be an effect of the carcinogens on the synthesis of cellular DNA during the perio...

The use of nurse culture in the development of haploid clones in tomato

A nurse culture procedure and an appropriate medium are described for obtaining vigorous haploid clones originating from single tomato pollen grains. The grains are plated on filter-paper disks mounted over intact anthers placed on a modified Murashige and Skoog medium. The plating efficiencies of such pollen cells ranges between 0 and 60%. The variability in the plating efficiencies and the potential use of this technique over that of anther culture are discussed.

Separation of two factors affecting the aggregation kinetics from the conditioned medium.

Two effective factors contained in medium “conditioned” with a large number of cultured cells were separated through Sephadex G-25 gel, by assaying the plating efficiency and growth rate of chicken embryonic cells in secondary culture. The first factor, which is contained in the heavy molecular weight fraction α, can promote plating efficiency of the inoculated cells at low density. The second is contained in the light molecular weight fraction β, and promotes the growth rate of sparsely cultured cells. The effects of these factors upon the aggregation kinetics of singly isolated cells were studied. The factor in the fraction α promotes aggregation to result in fast flocculation. The factor in the fraction β, while not effective by itself, affects the aggregation of the cells treated with the factor in α. In the co-presence of both α and β, a “lag” was found after the initial fast flocculation. The possible mechanisms of the interaction of these two factors on the cell surface are discussed.

Studies with the schistosomacide hycanthone: Inhibition of macromolecular synthesis and its reversal

The new schistosomacide, hycanthone, was studied with regard to its effects upon macromolecular synthesis in mammalian (HeLa) cells. At concentrations as low as 5 × 10 −8 M, hycanthone reduces plating efficiency of HeLa cells. At 10 −6 M, effects on population growth are evident by the second generation. At 5 × 10 −6 M, RNA synthesis is markedly depressed while DNA and protein synthesis are relatively unaffected. New ribosomal RNA synthesis is rapidly inhibited by hycanthone at 5 × 10 −6 M, and its processing is markedly delayed. During hycanthone inhibition 28S RNA does not appear in the cytoplasm. Hycanthone inhibition was shown to be readily reversible by washing the cells in fresh medium. The case with which inhibition can be reversed in vitro suggests that it may be necessary to maintain blood levels of this compound if permanent eradication of schistosomal infection is to be attained. Further work must be done to establish this point.

In vitro studies of immune reactions against autochthonous and syngeneic mouse tumors induced by methylcholanthrene and plastic discs.

AbstractA colony inhibition (CI) technique was used to demonstrate lymph node cell (LNC) and serum‐mediated immune reactions against specific transplantation antigens of mouse sarcomas which were induced by methylcholanthrene (MCA) or by plastic disc implantation. Lymph node cells from mice whose primary MCA sarcomas had been removed 3–8 days before the test strongly reduced the plating efficiency of autochthonous target tumor cells. An approximately equal reduction was also obtained with LNC from mice with progressively growing MCA sarcomas or from those which had been inoculated once with untreated or heavily X‐irradiated (15,000 R) sarcoma cells. Cytotoxic antibodies were infrequently detected in serum from these mice.Repeated immunization with heavily irradiated tumor cells appeared to increase the level of cytotoxic humoral antibodies but did not augment the LNC‐mediated immune reactions.

Nature of the carrier state of bacteriophage SP-10 in Bacillus subtilis.

Although the association of phage SP-10 with Bacillus subtilis W-23-S(r) persists in heat- and antiserum-resistant form through the spore stage, it is unstable in vegetative cells and frequently terminates in loss of the carried phage or in lysis. On low-tonicity media, the plating efficiency of carrier cells is low. However, high concentrations of succinate or sucrose or a slowed growth rate preserve viability: on 0.48 m succinate-agar, the viable count per optical density unit is the same as that of a noncarrier control culture. Carrier clones retain phage on 0.48 m succinate-agar. At higher succinate levels, many colonies emerge free of phage; at 1 m succinate, all are cured, probably because high succinate inhibits reinfection. Growth of carrier cells in liquid medium with antiphage serum results in rapid curing; events in such cultures with and without succinate were studied quantitatively by tracing the emergence of sensitive cells, the multiplication and induction of carrier cells, and the sensitivity of carrier cells to superinfection with virulent phage. During log phase, 40 to 70% of the carrier cells became sensitive to virulent phage, although the same cells were insensitive during lag and stationary phase. Apparently, fluctuations in repressor levels are responsible. Spontaneous induction of carrier cells followed a qualitatively similar pattern, perhaps in response to changes in level of the same repressor. Production of sensitive segregants by carrier followed a different course, presumably because the repressor does not affect segregation. Many sensitive cells were found two to three divisions after inoculation in antiserum medium. This suggests that each inoculum cell contained one or only a few phage replicons. The data are compatible with the idea that the carrier state in media without antisera is maintained entirely by reinfection and without replication of phage in the latent state. Alternative models which involve replication of latent phage are not ruled out, however.

The incorporation of 3H-thymidine and 3H-uridine into chick and mouse embryo cells cultured on stainless steel

Accurate determination of the radioactivity of tritium-labelled materials plated on non-conducting surfaces such as glass is not possible using conventional gas-flow counters. During counting a positive electrostatic charge accumulates on the surface of the glass which results in a rapid and progressive decrease in the efficiency at which radiation from tritium-labelled materials is detected. The errors so caused preclude accurate measurement of the rates of synthesis of DNA and RNA in cells by determining the amounts of 3H-thymidine and 3H-uridine incorporated into monolayer cultures growing on glass coverslips. Squares of highly polished, high-grade stainless steel, of uniform area, have been shown to be acceptable substitutes for glass coverslips for these measurements. The growth and plating efficiency of chick and mouse embryo cells are not inhibited by stainless steel. 3H-Thymidine and 3H-uridine are rapidly incorporated into the DNA and RNA, respectively, of monolayer cultures of cells growing on stainless steel. The incorporation of 3H-thymidine and 3H-uridine is linear with time for 3 hr and the rate of incorporation is directly proportional to the number of cells in the monolayer culture within the range 1 to 4 × 10 5 cells per steel square (3.6 cm 2). The incorporation of either 3H-thymidine or 3H-uridine into replicate cultures is very reproducible, agreement within 10 per cent of the mean of each set of replicate cultures being readily attained. The design of the stainless steel squares is such that large numbers of replicate cultures can be manipulated rapidly without fear of damaging the monolayer cultures of cells.