Parameters of growth in primary cultures and cell lines established from Drosophila imaginal discs

We have further characterised our tissue culture system for the growth in vitro of Drosophila imaginal disc cells, including the culture medium requirements for optimum growth and we have adjusted the protocol recommended for the initiation of cultures. Many imaginal disc fragments become organised into vesicles, and some of these secrete extracellular material into the lumen. Sensory axons differentiate in primary disc cultures, in the absence of bristle formation. The early stages of cell division to form a cell line are recorded.     

Differential heat shock response of primary human cell cultures and established cell lines

The influence of hyperthermia on the cellular growth and protein synthesis pattern from primary human brain tumour cells and skin fibroblasts was compared with established and experimentally transformed tumour cell lines. Primary cell cultures did not show any visible morphological changes after 42 degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized a protein with an apparent molecular mass of 70 kDa and an isoelectric pH of 7.0 as early as 3 h after the initial hyperthermal treatment.     

Localization of eukaryotic initiation factor 2 in neuron primary cultures and established cell lines

Eukaryotic initiation factor 2 (eIF-2) is a heterotrimeric protein with subunits α, β and γ that forms a ternary complex with Met-tRNA and GTP. It promotes the binding of Met-tRNA to ribosomes and controls translational rates via phosphorylation/dephosphorylation mechanisms. By means of immunofluorescence and post-embedding immunocytochemistry of intact cells and quantitative immunoblotting of cell extracts, the cellular distribution of the initiation factor has been examined in primary neuronal cultures as well as in two established cell lines: PC12 phaeochromocytoma cells and rat pituitary GH4C1 cells. Our results indicated that the initiation factor is located not only in the cytoplasm but also in the nuclei of the cultured neurons and cell lines. In the cytoplasm, immunocytochemical studies reveal that the factor is present mainly in those areas that are rich in ribosomes. In the nucleus, the immunolabelling of eukaryotic initiation factor 2 verified the presence of gold particles in both nucleolar and extranucleolar areas. The specific distribution of this factor on both sides of the nuclear envelope suggests that it might have some nuclear-related function(s) besides its already known role in the control of translation     

Localization of eukaryotic initiation factor 2 in neuron primary cultures and established cell lines

Eukaryotic initiation factor 2 (eIF-2) is a heterotrimeric protein with subunits α, β and γ that forms a ternary complex with Met-tRNA and GTP. It promotes the binding of Met-tRNA to ribosomes and controls translational rates via phosphorylation/dephosphorylation mechanisms. By means of immunofluorescence and post-embedding immunocytochemistry of intact cells and quantitative immunoblotting of cell extracts, the cellular distribution of the initiation factor has been examined in primary neuronal cultures as well as in two established cell lines: PC12 phaeochromocytoma cells and rat pituitary GH4C1 cells. Our results indicated that the initiation factor is located not only in the cytoplasm but also in the nuclei of the cultured neurons and cell lines. In the cytoplasm, immunocytochemical studies reveal that the factor is present mainly in those areas that are rich in ribosomes. In the nucleus, the immunolabelling of eukaryotic initiation factor 2 verified the presence of gold particles in both nucleolar and extranucleolar areas. The specific distribution of this factor on both sides of the nuclear envelope suggests that it might have some nuclear-related function(s) besides its already known role in the control of translation     

Density dependent regulation of growth in suspension cultures of L-929 cells

Suspension cultures of L-929 fibroblasts grown to densities of 6 to 10 × 10⁶ cells/ml through daily centrifugation and resuspension in fresh media, have been maintained for periods up to five months without change in viability or cell size. DNA synthesis and mitosis in these cultures is limited to 5% of the cells per day, a fraction very nearly equal to the fraction of cells rendered nonviable, most likely during the manipulations associated with medium renewal. The kinetics of the flow of cells into the S and M periods following (a) renewal of the medium and (b) dilution of the high density cultures, suggest that the large majority of the cells are in a G₀ or early G₁ phase, resuming growth readily in response to decreased cell density. This is further indicated by the sequence of the marked shifts occurring in the cell volume distribution spectrum of the high density cultures after dilution. Long term, steady state regulation of growth with retention of intact viability was thus demonstrated in the case of a long established aneuploid cell line. The fact that this occurs in suspension but not in attached cultures, supports the concept that impairment of growth control in such cells affects predominantly regulatory mechanisms located at the cell surface rather than those concerned with intracellular synthesis and metabolism.     

Flow cytometric cell cycle analysis of somatic cells primary cultures established for bovine cloning

An important factor governing developmental rates of somatic cloned embryos is the phase of the cell cycle of donor nuclei. The aim of this experiment was to investigate the distribution of cell cycle phases in bovine cumulus and fibroblast cells cultured using routine treatment, and under cell cycle-arresting treatments. The highest percentages of cumulus cells in the G0 + G1 stage were observed in uncultured, frozen/thawed cells originating from immature oocytes (79.8 +/- 2.2%), fresh and frozen/thawed cells from in vitro matured oocytes (84.1 +/- 6.2 and 77.8 +/- 5.7%, respectively), and in cycling cells (72.7 +/- 16.3 and 78.4 +/- 11.2%, respectively for cumulus cells from immature and in vitro matured oocytes). Serum starvation of cumulus cultures markedly decreased percentages of cells in G0 + G1, and prolonged starvation significantly increased (P < 0.05) percentages of cells in G2 + M phase. Culture of cumulus cells to confluency did not increase percentages of cells in G0 + G1. Contrary to findings in cumulus cells, significantly higher percentages of cells in G0 + G1 were apparent when fibroblast cells were cultured to confluency or serum starved, and significantly increased (P < 0.01) as the starvation period was prolonged. It is concluded that for particular cell types specific strategies should be used to attain improvements in the efficiency of cloning procedures.     

The ultrastructure of imaginal disc cells in primary cultures and during cell aggregation in continuous cell lines

We have examined the ultrastructure of cellular vesicles in primary cultures of wing imaginal disc cells of Drosophila melanogaster. These cells maintain the apico-basal polarity characteristic of epithelial cells. The apical surfaces secrete extracellular material into the lumen of the vesicle from plasma membrane plaques at the tip of microvilli. During the course of one passage, cells from the established cell lines grow to confluence and then aggregate into discrete condensations joined by aligned bridges of cells. Cells in these aggregates are tightly packed, and there appears to be a loss of the epithelial polarity characteristic of the vesicle cells. Elongated cell extensions containing numerous microtubules are found in aggregates, and we suggest that these may be epithelial feet involved in the aggregation process. Virus particles are commonly found both within the nucleus and the cytoplasm of cells in the aggregates.     

Localization ofα 2-macroglobulin in human primary sarcomas and synthesis in established cell linesin human primary sarcomas and synthesis in established cell lines

Summary The presence ofα ₂-macroglobulin was detected with the avidin-biotin technique in more than 20-yr-old paraffin blocks from human sarcomas.α ₂M was found mainly in the cytoplasm of the tumor cells, and almost all tumor cells were positive. This serum glycoprotein, which is a major plasma proteinase inhibitor with a wide specificity, was also shown to be synthesized and secreted by all three cell lines derived from primary sarcomas but was not detected in cultures of the autologous skin fibroblasts. For the detection ofα ₂M in situ and in vitro an antiserum to tumor-associatedα ₂-macroglobulin was used. Our work was supported by grant no. 55-B86-21XB, from the Swedish Cancer Society.     

Voltage dependent calcium channels in cerebellar granule cell primary cultures

Voltage activated calcium channels were studied in rat cerebellar granule cells in primary culture. Macroscopic currents, carried by 20mM Ba2+, were measured in the whole-cell configuration. Slowly inactivating macroscopic currents, with a maximum value at a membrane potential around 5 mV, were recorded between the 1st and the 4th day in culture. These currents were completely blocked by 5mM Co2+, partially blocked by 10 microM nifedipine, and increased by 2 to 5 microM BAY K-8644. Two types of channels, in the presence of 80 mM Ba2+, were identified by single channel recording in cell-attached patches. The first type, which was dihydropyridine agonist sensitive, had a conductance of 18 pS, a half activation potential of more than 10 mV and did not inactivate. This type of channel was the only type found during the first four days in culture, although it was also present up to the 11th day. The second type of channel was dihydropyridine insensitive, had a conductance of 10 pS, a half activation potential less than -15 mV, and displayed voltage dependent inactivation. This second type of channel was found in cells for more than four days in culture.     

Density dependent growth of corneal endothelial cells cultured in vitro

Using the cornea of macaque monkey, we demonstrated the relationship between cell density and growth of endothelial cells in vitro. Corneal endothelial cells in a cell sheet grow most actively in regions with cell density of 1000 to 1800 cells/mm2, in explant cultures and cell sheets and in concentrated inocula dissociated cells. Cell morphology was well sustained in these cultures. Cells cultured at a higher cell density retained their potential to proliferate actively, showing clear contrast to cells cultured at a density lower than 200 cells/mm2. When dissociated cells were cultured at a low density and maintained for more than 4 weeks, they gradually lost their growth potential, altered into polymorphonuclear giant cells and eventually dedifferentiated. In addition, cells with no contact with each other did not express growth potential. Density dependent growth was confirmed by measuring the mitotic index against the cell density per square mm from the center to the peripheral regions in cultured explants. It is concluded that the growth pattern of corneal endothelial cells is closely related to cell density, and that growth of these cells might be regulated through intercellular communications.     

Density dependent regulation of growth in L cell suspension cultures. 3. Elevation of specific activity of acetylcholinesterase

High density L cell suspension cultures were previously shown to remain viable for indefinite periods of time and to exhibit marked inhibition of DNA synthesis and mitosis while the fraction of total protein synthesis represented by collagen is increased. The present study demonstrates that regulation in this system extends to the activity of acetylcholinesterase found to be approximately 100-fold greater in the high density populations than in low density exponentially growing cultures. Kinetic studies of the increase of the activity, its fluctuation over an extended period of time and its decrease upon resumption of exponential growth after dilution of the cultures were performed. The data obtained indicate that the enzyme does not accumulate in high density populations merely as a result of the absence of net protein synthesis and cell division but that changes of its rates of synthesis and possibly degradation are involved. The expression of regulated acetylcholinesterase activity in a cell line of connective tissue origin is considered in relation to phenotype reprogramming and to cell membrane associated growth control mechanisms.     

Generation of stable cell lines by spontaneous immortalization of primary cultures of porcine granulosa cells

We report the generation of stable cell lines obtained by spontaneous immortalization of primary cultures of porcine granulosa cells. Three hundred stable cell lines were obtained from three independent immortalization trials. Two of these cell lines retained the steroidogenic capabilities characteristic of granulosa cells, such as de novo synthesis of progesterone and conversion of androstenedione into estradiol-17beta. All the stable cell lines expressed the P450arom and 3betaHSD genes, confirming their granulosa origin. Moreover, the steroidogenic stable granulosa cells also expressed StAR and P450scc genes. Stable cells were developed in cultures using Medium 199 supplemented with 5% newborn calf serum (NBCS). The surviving cells overcame the senescent phase and entered a stage of continuous growth for over one hundred generations. No stable colonies were obtained from cultures grown in MEM or DMEM or media supplemented with 10% NBCS or 5 and 10% fetal calf serum (FCS). Medium 199 is a formulation richer in nutrients compared to MEM or DMEM and the cell growth capability of NBCS is lower than that of FCS, probably due to deficiency of growth factors. We speculate that spontaneous immortalization of granulosa cells may be facilitated by using a rich culture formulation supplemented with low concentrations of serum deficient in growth factors. We have validated the stable cell lines for studying the effect of hormonal steroids on granulosa cell steroidogenesis and the expression of the steroidogenic genes. Therefore, we believe that they are useful models to study the molecular mechanism involved in granulosa cell differentiation and steroidogenesis.     

Computer analysis of organelle translocation in primary neuronal cultures and continuous cell lines

Organelle translocation in a number of cell types in tissue culture as seen by high-resolution Zeiss-Nomarski differential interference contrast optics was filmed and analyzed by computer. Principal cell types studied included primary chick spinal cord, chick dorsal root ganglion, ratbrain, and various clones of continuous cell lines. Organelle translocations in all cell types studied exhibited frequent, large changes in velocity during any one translocation. The appearance of particles as seen with Nomarski optics was correlated with their fine structures in one dorsal root ganglion neurite by fixing the cell as it was being filmed and obtaining electron micrographs of the region filmed. This revealed the identity of several organelles as well as the presence of abundant neurotubules but no neurofilaments. Primary cell cultures exhibited more high-velocity organelle movements than continuous cell lines. The net progress of an organelle in a given direction was greater in primary neuronal cells than in fibroblasts or continuous cell lines. These findings are correlated with the literature on organelle translocation and axoplasmic transport.     

Reversible release of chick embryo fibroblast cultures from density dependent inhibition of growth

Initiation of proliferation in density-inhibited chick embryo fibroblast cultures induced by insulin or trypsin was partially reversed by replacing the medium with supernatants from parallel non-stimulated cultures. Growth stimulation by neuraminidase, pokeweed mitogen, bacterial lipo polysaccharide or purified tuberculin was less, or not at all, affected by this procedure. Medium change per se caused some proliferation in non-stimulated cultures. Increased rate of sugar uptake in insulin-stimulated cultures returned to the level of that in non-stimulated cultures within a few hours after medium change. This reversion took place apparently irrespective of the phase of the cell cycle. Replacing the medium with supernatants from non-stimulated cultures induced a rapid decline in subsequent thymidine incorporation during the first S-phase, and completely abolished the second peak of DNA synthesis. The fraction of cells irreversibly committed to mitosis increased when the time after stimulation increased. Less than three hours' incubation with insulin or trypsin was needed to initiate proliferation of a significant fraction of the cell population. It is concluded that reversion of the initiated cycle of a given cell is no more possible after the cell has entered the S-phase.     

Prostaglandin actions in established insect cell lines

Prostaglandins (PGs) are oxygenated metabolites of arachidonic acid (AA) and two other C20 polyunsaturated fatty acids that serve as biochemical signals mediating physiological functions. We reported that PGs influence protein expression in insect cell lines, which prompted the question: do PGs influence cell proliferation or viability in insect cell lines? Here, we report on the outcomes of experiments designed to address the question in cell lines from three insect orders: Hemiptera (squash bug, Anasa tristis, BCIRL-AtE-CLG15A), Coleoptera (red flour beetle, Tribolium castaneum, BCIRL-TcA-CLG1), and Lepidoptera (tobacco budworm, Heliothis virescens, BCIRL-HvAM1). Treating the insect cell lines with PGA₁, PGA₂, or PGD₂ led to dose-dependent reductions in cell numbers. All three cell lines were sensitive to PGA₁ and PGA₂ (IC₅₀s = 9.9 to 26.9 μM) and were less sensitive to PGD₂ (IC₅₀s = 31.6 to 104.7 μM). PG treatments also led to cell death at higher concentrations, as seen in mammalian cell lines. PGE₁, PGE₂, and PGF_2α treatments did not influence AtE-CLG15A or HvAM1 cell numbers at lower concentrations, but led to dose-related reductions in TcA-CLG1 cells at higher concentrations. Similar treatments with pharmaceutical inhibitors of PG biosynthesis also led to reduced cell numbers: MAFP (inhibits phospholipase A₂), indomethacin (inhibits PG biosynthesis), and esculetin (inhibits lipoxygenase). Because these pharmaceuticals are used to relieve inflammation and other medical issues in human medicine, they are not toxic to animal cells. We infer PGs are necessary in optimal quantities for ongoing homeostatic functions in established cell lines; in quantities outside the optimal concentrations, PGs are deleterious.     

Rat liver epithelial cell cultures in a serum-free medium: primary cultures and derived cell lines expressing differentiated functions

Rat liver epithelial cells explanted in a serum-free medium (SFM) composed of Ham's F10 basal medium plus free fatty acids adsorbed on bovine albumin gave successful rise to primary cultures and then to long-term cell lines that expressed liver functions; induction of L-tyrosine aminotransferase by glucocorticoids, hepatic pattern of progesterone metabolism, and biosynthesis of murine primary bile acids; chenodeoxycholic and cholic acid common to higher vertebrates and alpha-muricholic acid specific of the rat bile.