Cultured Cell Populations Research Articles

Quantitation of DNA Repair in Brain Cell Cultures: Implications for Autoradiographic Analysis of Mixed Cell Populations

Quantitation of DNA repair in the mixed cell population of mouse embryo brain cultures has been assessed by autoradiographic analysis of unscheduled DNA synthesis following UV-irradiation. The proportion of labelled neurons and the grain density over neuronal nuclei are both less than the corresponding values for glial cells. The nuclear geometries of these two classes of cell are very different. Partial correction for the different geometries by relating grain density to nuclear area brings estimates of neuronal and glial DNA repair synthesis more closely in line. These findings have general implications for autoradiographic measurement of DNA repair in mixed cell populations and in differentiated versus dividing cells.

Some observations of the morphology of the glial and neuronal cell populations in monolayer culture

Attempts have been made in the past to separate the glial and neuronal populations of cells for various studies. The homogeneity of cell populations achieved using these methods remains to be improved. Using a modified separation method and combined with subculturing, very pure glialand neuronal populations have been obtained. This paper reports the surface features and some properties of the two cell populations using the modified culture method.Ten day old chick embryo cerebra were removed aseptically and the meninges carefully excised. The tissues were washed in calcium and magnesium free (CMF) tyrode solution before incubation for 35 min. at 37°C in 0. 2% of trypsin in CMF. The cells were dissociated in E-II medium by gentle flushing of the tissues with a fine bore pipette for about 20 times. To each of the 35 mm culture dishes containing 2 ml of E-II medium and a poly-L-lysine coated coverslip, one ml of cerebral cell suspension was added and the cells were allowed to plate on a coverslip for 7 minutes at room temperature.

Differentiation in Culture of Myogenic Cells from Adult Mouse Muscle

Abstract Satellite cells isolated from skeletal muscle of adult mice undergo a process of differentiation in culture. Treatment of dissociated muscle fragments with collagenase, followed by three sequential treatments with trypsin, results in a cell suspension highly enriched in myogenic cells. This suspension is plated in collagen coated dishes and cultured in MEM supplemented with 10% horse serum and 3% embryo extract. Cell population in culture is mainly composed of myogenic cells which duplicate actively during the first two days of culture, then withdraw from the cell cycle and fuse to form multinucleated myotubes, with a concomitant increase of creatine phosphokinase (CPK) activity.

Differentiation of isolated calvarial cells into a mature heterogeneous bone cell population in culture

When isolated fetal calvarial cells were cultured in petri dishes without bone, they maintained a fibroblastoid appearance throughout the culture period and did not differentiate morphologically. In contrast, when similar cells were cultured on endosteal surfaces of devitalized bone, they differentiated into mature cells which resembled the heterogeneous collection of cells found in normal bone. Cells with the same morphologic appearance as osteoclasts appeared between 8 to 12 days of culture. At the same time that these large multinucleated cells appeared, mineral and matrix were released from the killed bones which had been previously labeled with 45Ca and 3H-proline. The resorption of dead bone by the cells was inhibited by colchicine (10 −7M) and cortisol (10 −6M), drugs which inhibit lysosomal enzyme release, but was not enhanced by parathyroid hormone or osteoclast activating factor.

Chapter 27 The Use of Complement-Mediated Cytolysis to Analyze Specific Cell Populations of Eukaryote Cells in Cultures

The complement-mediated antibody cytotoxicity assay has been used in studies on the changes in cell surface antigens during neoplastic transformation, the differentiation of mammalian cells, the chromosomal localization of specific genes for cell surface antigens, the removal of specific types of cells from mixtures so that biochemical analysis can be performed, and the detection and isolation of specific populations of lymphocytes. The development of antibody cytolysis techniques allows preparing antibodies against cell surface antigens, the capability to sort cells from populations which are quantitatively or qualitatively heterogeneous for the antigen under study. This can be done without extremely complex and expensive equipment. The chapter reviews the mechanism by which activation of complement is thought to lyse cells, the techniques for the use of complement-mediated antibody cytotoxicity to analyze populations of neoplastically transformed and untransformed cells, and how other investigators have used these techniques.

The role of hyperlipidemic serum on the proliferation and necrosis of aortic medial cells in vitro

The effects of hyperlipidemic rabbit serum on the proliferation and death of subcultured rabbit aortic medial cells were evaluated. The cultured cells were grown from the aortic media of normal adult male rabbits, prelabeled with [ 3H]thymidine, and then trypsinized, pooled, and subcultured. Either normal or hyperlipidemic rabbit serum was added to the culture media. Proliferative activity was measured by cell count and by the isotopic dilution of the prelabeled DNA, with corrections made for cell loss. Cell loss, presumed to represent mainly cell death, was assessed from the total loss of [ 3H]thymidine from the cultured cell population. The cells grown in basal medium of Eagle containing 10% normal serum showed a 25% loss in the first 3 days after trypsinization. The loss rate stabilized at 1.5% per day 5 days after the subculture. When these cells were exposed to hyperlipidemic serum, the rate of cell loss showed a 5.5-fold increase in the initial 3 days, and thereafter returned to the levels seen in the presence of normal serum. Cells exposed to hyperlipidemic serum also showed a 25–50% increase in proliferative activity. These alterations have been demonstrated in both the early proliferative phase and later stationary phase. Cells grown in hyperlipidemic serum were larger on the average and showed a 50% increase in protein content per cell.

Interacting cell populations in cultures of leukocytes from normal or leukemic peripheral blood

Kinetic studies in cultures containing 2 X 10(5) peripheral leukocytes from patients with acute myelobastic leukemia revealed extensive, radiation-sensitive increases in thymidine incorporation without parallel increases in cell number. Modest and variable stimulation of 3HTdR incorporation was seen with the addition of either leukocyte-conditioned medium prepared with phytohemagglutin (PHA) or PHA alone. However, using the method of limiting dilution, stimulation was always observed and ranged from 3- to 20-fold in individual patients. By mixing small numbers of intact cells with larger numbers of irradiated autologous cells, quantitative evidence was obtained for a cellular interaction between irradiated, PHA-stimulated populations capable of 3HTdR incorporation. Similar evidence for cell-cell interaction was obtained for normal leukocytes.

Interacting Cell Populations in Cultures of Leukocytes From Normal or Leukemic Peripheral Blood

Abstract Kinetic studies in cultures containing 2 X 10(5) peripheral leukocytes from patients with acute myelobastic leukemia revealed extensive, radiation-sensitive increases in thymidine incorporation without parallel increases in cell number. Modest and variable stimulation of 3HTdR incorporation was seen with the addition of either leukocyte- conditioned medium prepared with phytohemagglutin (PHA) or PHA alone. However, using the method of limiting dilution, stimulation was always observed and ranged from 3- to 20-fold in individual patients. By mixing small numbers of intact cells with larger numbers of irradiated autologous cells, quantitative evidence was obtained for a cellular interaction between irradiated, PHA-stimulated populations capable of 3HTdR incorporation. Similar evidence for cell-cell interaction was obtained for normal leukocytes.

Specificity of junctional communication between animal cells.

MANY types of animal cells, in culture and in vivo, form intercellular junctions which are freely permeable to small cellular ions and molecules but not to macromolecules1–10. These junctions are probably the gap junctions seen by electron microscopy11. Cells coupled by such junctions share their metabolites and small control molecules and respond jointly to changing demands on metabolism. Intercellular control of enzymic activity12,13 and metabolic interdependence causing controlled cell proliferation7 have both been observed in mixed cell populations in tissue culture. These integrative effects of junctional communication make a tissue behave as a homogenous unit rather than a collection of different cells.

Neuron culture from adult goldfish.

Neurons and glia from the central nervous system of the adult teleost Carassius auratus have been grown as explant cultures of minced brain tissue and as trypsin dissociated cells. These cultures exhibit extensive neurite growth from two neuronal types, have organotypic ultrastructure, and contain electrically active cells. Autoradiographic data indicate that these neurons do not divide in culture, and histological evidence suggests that some mature neurons survive explantation and regenerate processes. However, explantation of brain fragments not containing undifferentiated cells, localized in the ventricular and subventricular zones in the brains of fish, resulted in mesenchymal and glial cell cultures only. Therefore, a contribution to the population of cells in culture by undifferentiated cells must be considered. The cultured neurons remained viable for at least 19 weeks and ultrastructural and electrophysiological data indicate synaptic interaction between cells in explant cultures.

The Culture of Skin a Review of Theories and Experimental Methods

Two main criticisms can be leveled against the standard methods of skin culture: they are poorly quantifiable and the cultured cell populations are heterogeneous. A new technique based mainly on enzymatic dissociation allows specific cell types to be extracted from the skin before cultivation. In this way, separate cultures of epidermal keratinocytes and dermal fibroblasts can be obtained from the same piece of skin. These purified systems have been used to study the kinetics of epidermal cell growth and to quantify the effect of various chemically defined substances on the growth and differentiation of keratinocytes. With further refinements in technique, purified populations of melanocytes can be extracted. The co-culture of pigmented melanocytes with albino keratinocytes has been proposed as a model to study pigment donation in vitro. The usual organ culture technique, including the use of large explants of skin immersed in the culture fluid, has been modified to show that adult human skin partially regenerates in vitro and that mitotic activity goes on for months in the regenerated epidermis. The use of nucleic acid hybridization techniques, combined with skin cell cultures from human tumors, opens new avenues of research on human cancer.

Transepithelial transport in cell culture.

In cell culture a kidney epithelial cell line MDCK, forms a continuous sheet of identically oriented asymmetrical cells joined by circumferential occluding junctions. The reconstructed epithelial membrane has transport and permeability qualities of in vivo transporting epithelia. The cell layer can be readily manipulated when cultured on a freely permeable membrane filter and, when placed in an Ussing chamber, electrophysiological measurements can be taken. In the absence of a chemical gradient, the cell layer generates an electrical potential of 1.42 mV, the apical surface negative. It is an effective permeability barrier and lacks significant shunting at the clamped edge, as indicated by a resistance of 84 ohms-cm2, which increased when bulk flow from basolateral to apical was induced by an osmotic gradient or electroosmosis. The MDCK cell layer is cation selective with a relative permeability ratio, PNa/PCl, of 1.7. Net water flux, apical to basolateral, was 7.3 mul cm-2 hr-1 in the absence of a chemical gradient. The morphological and functional qualities of a transporting epithelium are stable in cell culture, and the potential use of a homogeneous cell population in cell culture would enhance studies of epithelial transport at the cellular and subcellular levels.

Production of epithelial and mesenchymal tumours with rat liver cells transformed in vitro.

Epithelial-like cells originating from the livers of 10-day and 8-week-old BD rats were established in culture. The cells were treated in vitro for 1 or 4 weeks with dimethylnitrosamine (DMN) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Although some structural changes were observed in treated cells, it was not possible to score for morphological transformation in vitro. Newborn syngeneic rats were injected with 1.5-2 times 10(6) treated or 1.5-5 times 10(6) control cells at various times up to 38 weeks from the beginning of treatment with the carcinogen. Following the injection of DMN-treated cells, a total of 32 of the 42 injected rat developed tumours, of which 17 were epithelial, 10 carcinosarcomas and 5 fibrosarcomas. Following the injection of the MNNG-treated cells into 61 rats, a total of 30 tumours were observed, including 8 carcinomas, 9 carcinosarcomas and 13 fibroscarcomas. Tumours, mainly of the mesenchymal type, were also observed in rats inoculated with control cells but at a lower frequency. The observation observed in rats inoculated with control cells but at a lower frequency. The observation of mesenchymal tumours is attributed to the presence of a mixed population of epithelial and mesenchymal cells in the orginal culture.

Modulation of murine granulocyte proliferation in diffusion chamber cultures

Normal mouse marrow cells were cultured in Millipore diffusion chambers for long periods of time and at a variety of cell concentrations. All cultures showed a pattern of granulocyte proliferation characterized by logarithmic growth followed by prolonged stabilization of cell number starting a 7 days thereafter. The height of this “plateau” varied in relationship to the level of cell input and characteristically was far lower than the maximum cell density that can be maintained in this culture system. Additional studies showed that the plateau represented a steady state of granulocyte turnover and was not due to alterations in the diffusion chambers or the host mice. Regulatory mechanisms intrinsic to the cultured cell population appeared to play a primary role in maintaining this stable plateau. Modulation of granulocyte proliferation was partly due to increasing cell density; particularly with high input concentrations. In addition, differential cell counts suggested that critical changes in the relationship between immature and mature granulocytes partially accounted for this apparent autoregulation of cell growth. The plateau period in diffusion chamber cultures in many ways resembles granulocyte proliferation in normal mouse bone marrow and is a useful model for the study of regulatory functions in granulocytopoiesis.

Modulation of Murine Granulocyte Proliferation in Diffusion Chamber Cultures

Normal mouse marrow cells were cultured in Millipore diffusion chambers for long periods of time and at a variety of cell concentrations. All cultures showed a pattern of granulocyte proliferation characterized by logarithmic growth followed by prolonged stabilization of cell number starting a 7 days thereafter. The height of this "plateau" varied in relationship to the level of cell input and characteristically was far lower than the maximum cell density that can be maintained in this culture system. Additional studies showed that the plateau represented a steady state of granulocyte turnover and was not due to alterations in the diffusion chambers or the host mice. Regulatory mechanisms intrinsic to the cultured cell population appeared to play a primary role in maintaining this stable plateau. Modulation of granulocyte proliferation was partly due to increasing cell density; particularly with high input concentrations. In addition, differential cell counts suggested that critical changes in the relationship between immature and mature granulocytes partially accounted for this apparent autoregulation of cell growth. The plateau period in diffusion chamber cultures in many ways resembles granulocyte proliferation in normal mouse bone marrow and is a useful model for the study of regulatory functions in granulocytopoiesis.

Nih gerontology research support: Policy evaluation of in vitro cell lineage studies

Populations of cultured human diploid cells, used extensively in research on aging, are not well understood in terms of kinetics and mitotic activity of constituent cells. NICHD has supported, by contract, cell lineage studies to elucidate such cell characteristics contributing to the cell population degeneration and death. The policy of NICHD (NIA) encouragement of such studies has been evaluated. A need for cell lineage and related data from culture-cell populations is recognized, and research necessary to achieve these data will be encouraged. Grant applications to this end are sought.

Studies of Leukemic Cell Populations in Culture

Abstract A suspension culture technique for the study of peripheral blood cells from patients with leukemia is described. Cells from patients in relapse were preserved by freezing with dimethylsulfoxide at -70°C to ensure the availability of the same population of cells for repeated experiments of varying designs. Cells from one AML patient proliferated only in the presence of phytohemagglutinin (PHA) or leukocyte-conditioned medium (LCM). Cells taken at a later date, after commencement of chemotherapy, failed to proliferate in response to PHA, although the responsiveness to LCM was retained. Proliferation of the cells obtained after chemotherapy could also be stimulated by culture supernatants from the original PHA-responsive cells. Eight other patients studied showed varying responses to LCM and PHA. It is proposed that cells from leukemic patients are made up of two interacting populations, one which produces stimulatory factors and another which responds to these factors by proliferation. Delineation of these two populations and the factors produced may provide a better understanding of leukemic cell proliferation.

Analysis of Interacting Cell Populations in Cultures of Marrow From Patients With Neutropenia

Abstract Utilizing an adherence separation procedure, marrow cells from six neutropenic patients were studied in cell culture for colony-forming ability (CFU-C), and for colony-stimulating function of the adherent cell (A-C) population. Assays on two patients showed reduced CFU-C and A-C function, while assays on three patients yielded normal CFU-C and A-C function; studies on the remaining patient exhibited reduced CFU-C in the presence of normal A-C function. The demonstration of these various abnormalities in marrow cell culture from neutropenic patients suggests that the in vitro techniques have relevance to the study of granulopoiesis in vivo.

SELECTIVE TRIGGERING OF HUMAN T AND B LYMPHOCYTES IN VITRO BY POLYCLONAL MITOGENS

Human lymphocytes from spleen and tonsils have been cultured with a variety of polyclonal mitogens. Cultures consisted of either unseparated T and B cells or alternatively purified T or B lymphocytes. The purity of the starting cell populations and the origin of activated lymphoblasts was analyzed with a panel of seven markers which discriminate between T and B cells. The selectivity of the lymphocyte responses was influenced by cell populations in a given culture, the mitogen used, and to a limited extent on culture conditions. Purified T lymphocytes from tonsil and spleen responded to phytohemagglutinin (PHA), pokeweed mitogen (PWM), and staphylococcal enterotoxin B (SEB). Purified B cells from spleen responded well to PWM, weakly to SEB and lipopolysaccharide, but not at all to PHA. Tonsil B cells responded weakly to PWM and SEB but not to PHA. Some B lymphocytes do respond to PHA in the presence of activated T cells. These results are discussed in relation to previously reported selective responses of human cells and parallel studies in animal species.

Selective proliferation of T cells in the mixed lymphocyte interaction

The contribution of avian thymus-derived (T) and bursa-derived (B 2) cells to the proliferating cell population in mixed lymphocyte cultures (MLC) was evaluated. When spleen cells of chickens containing chromosomally marked T and B 2 cells were stimulated in a one-way MLC by mitomycin C blocked allogeneic spleen cells, only T cells proliferated during a 4–9 day culture period. No evidence for significant recruitment of B 2 cells, expressed as proliferation of B 2 cells, was found. The initial viability and proliferative potential of B 2 cells was shown by a substantial and selective B 2 cell response to anti-immunoglobulin serum.