Fine Structure of Healthy and TMV-Infected Tobacco Cells Grown in Tissue Culture

Three types of tobacco (Nicotiana tabacum cv. Havana 38) callus: 1) healthy stem callus, 2) TMV-infected stem callus, 3) TMV-infected leaf callus; and leaves differentiated from healthy stem callus, and from TMV-infected leaf callus were compared for fine structure. In addition, the fine structure was observed of plastids in cells of leaves differentiated from callus isolated from stem sections of TMV-infected hybrid tobacco plants (N. tabacum cv. Havana 38 ×N. glutinosa) grown under high temperature. The cytoplasmic organelles in tissue cultured cells were similar to those in cells of greenhouse-grown tobacco plants. Except for plastids, TMV infection did not noticeably affect morphologically other cellular organelles in tissue culture cells. In TMV-infected leaf callus, numerous small bodies were seen in plastid-like bodies, while vesicle-like structures were observed in the stroma of plastids in leaves differentiated from callus of hybrid tobacco inoculated with TMV. Morphological variations of mitochondria, such as swelling and vacuolization of the inner matrix, occurred frequently in TMV-infected leaf callus. Needle-like crystalline inclusions or looped inclusions composed of many fine, long filaments were considered TMV particles orientated parallel to each other. The TMV particles were detected in the cytoplasm of tissue culture cells.     

Rapid Staining and Preservation of Cells Grown in Plastic Tissue Culture Flasks

Cells grown in plastic tissue culture flasks may easily be fixed and stained in large batches by first binding the flasks in groups of 6-8 and then applying all fluids with an automatic syringe, followed by decanting as required After staining, the cells are mounted in a mixture of USP mineral oil in isoamyl alcohol (2:1). In this way the cells are impregnated with a clearing and mounting agent which does not cause discoloration of the stains, does not dissolve the plastic, and is nondrying.     

Trypsin Action on the Growth of Sendai Virus in Tissue Culture Cells III. Structural Difference of Sendai Viruses Grown in Eggs and Tissue Culture Cells

Polypeptides of egg-borne Sendai virus (egg Sendai), which is biologically active on the basis of criteria of the infectivity for L cells and of hemolytic and cell fusion activities, were compared by polyacrylamide gel electrophoresis with those of L cell-borne (L Sendai) and HeLa cell-borne Sendai (HeLa Sendai) viruses, which are judged biologically inactive by the above criteria. Densitometer profiles on the stained gels of egg Sendai resolved six polypeptides (virion protein [VP] 1 to VP6), in which VP2 and VP4 were identified as glycoproteins by PAS stain. Comparative electropherograms of both L Sendai and HeLa Sendai revealed that there were significantly larger amounts in the VP2 region of these viruses but VP4 was present only in greatly reduced amounts as compared to egg Sendai. It was also found that VP2 of L Sendai and HeLa Sendai consisted of two components, VP2a and VP2b, but the one of egg Sendai consisted of only VP2a. A mild trypsin treatment which converts both L Sendai and HeLa Sendai to a biologically active form selectively removed VP2b from these viruses and increased concomitantly the amounts of materials in the VP4 region. The same treatment of egg Sendai affected neither its biological activities nor its electropherogram. Consequently, gross polypeptide profiles on the stained gels of L Sendai and HeLa Sendai after trypsin treatment became favorably comparable to that of egg Sendai. Electrophoresis of labeled L Sendai and HeLa Sendai with a (3)H-amino acids mixture and (14)C-glucosamine resolved at least three glycoproteins, GP1, GP2, and GP3, each corresponding to VP2a, VP2b, and VP4, respectively. The trypsin treatment of these viruses removed almost all the radioactivity of GP2 and simultaneously increased the radioactive counts of GP3 and raised small amounts of rapidly moving heterogeneous glycoprotein, GP4. A possible relationship between the biological modification and the above characteristic polypeptide patterns of Sendai virus was discussed.     

Purification of Rabies Virus Grown in Tissue Culture

Extracellular rabies virus, grown in monolayer cultures of BHK21 cells in the presence of medium supplemented with bovine serum albumin, was purified by the following procedure. Virus was precipitated from infectious tissue culture fluid by zinc acetate and was resuspended in a solution of ethylenediaminetetraacetate. The suspension was filtered through a Sephadex column and was treated with ribonuclease and deoxyribonuclease. The virions were then pelleted by centrifugation at high speed and were resuspended in buffer solution. Banding of the virus by centrifugation in a sucrose density gradient was the final step in the purification procedure. Purified preparations contained bullet-shaped virus particles of variable length and little (up to 5%) contaminating host-cell material. Most of the virions were "complete", i.e., 180 nm long, but some virus particles were shorter. The length distribution of the virions was nonrandom. Shorter virions seemed to be noninfectious and showed markedly decreased hemagglutinating activity. The complement-fixing activity and the ribonucleic acid to protein ratio of the virions were not related to the length of the virus particles. Although the properties of extracellular and intracellular viruses were similar, the procedure was not suitable for purification of intracellular rabies virus.     

Freeze-Preservation of Rice Cells Grown in Suspension Culture

A simple procedure has been worked out for the freeze-preservation of rice (Oryza sativa L.) cells grown in suspension culture. The protocol differs in some interesting aspects from those established for other organisms. A peculiar feature of this procedure is that growth of freeze-recovered rice cells resumes after an extremely short lag period of 2–8 days and proceeds with a growth rate identical to that of untreated cells. This, together with data obtained with viability tests, rules out the possibility that selection of freeze-resistant mutant cells may occur, as postulated with other plant cells where growth resumption was considerably delayed in time. The viability of freeze-recovered rice cells, when assessed at time zero after thawing by measuring the mitochondrial respiratory efficiency, was 60–65% of that of untreated cells. However, the limits of this and other viability tests in determining the efficiency of the freeze preservation methods and the percentage of surviving cells were shown by experiments in which cell viability and cell growth were followed in cultures initiated with freeze-recovered rice cells.     

Effects of Malignant Effusions on the Mitotic Index of L Strain Mouse Cells Grown in Tissue Culture

By employing a clone of L strain mouse fibroblasts (L_E) which does not exhibit cell clumping and lysis (cytolytic antibody reaction), it was possible to screen for the presence of growth-regulating factors in human sera and effusions, exclusive of an antigen-antibody reaction. Under conditions of the test a mitotic index greater than 20% indicated the presence of a growth-promoting factor.A total of 11 pleural effusions was tested. Four of the eight malignant effusions possessed a growth-promoting factor, while none of the three non-malignant effusions or the one sample of human umbilical cord serum possessed such a factor. Overnight storage of the unfiltered effusions at 5° C. resulted in complete loss of the growthpromoting activity.     

Analysis of Methylglyoxal Metabolism in CHO Cells Grown in Culture

Recent evidence suggests that several unknown or ill-characterized factors strongly influence cell growth and function in culture. Isolating these factors is necessary in order to maximize culture productivities. Methylglyoxal (MG), a potent protein and nucleic acid modifying agent, has been identified as a player in the signaling pathways associated with cell death and is known to be detrimental to cultured cells. This compound is produced in all mammalian systems by spontaneous phosphate elimination from glycolytic pathway intermediates. A kinetic model that qualitatively describes the cellular distribution of protein-associated MG in the absence of enzymatic adduct formation predicted far lower levels of reversibly bound MG than measured in cultured CHO cells. This suggests that the targeted modification of proteins through enzymatically mediated mechanisms is a significant sink for cellular methylglyoxal. The model was validated with measurements of carbon flux through the glyoxalase pathway to d-lactic acid, a unique end product of MG metabolism in mammalian systems. Fluxes to d-lactic acid of up to 16.8 mmol ml-packed cells⁻¹ day⁻¹ were measured with CHO cells grown in batch culture or 100-fold more than found in normal tissues.     

The Effect of Carbohydrate and Nitrogen Concentration on Phenol Synthesis in Paul's Scarlet Rose Cells Grown in Tissue Culture

The effects of exogenous concentrations of glucose and nitrate on total ethanol extractable phenols and leucoanthocyanins were studied in Paul's Scarlet Rose cells grown in either liquid suspension or solid culture. Aliquots of liquid suspension cultured cells were harvested during logarithmic, early stationary, and late stationary periods of growth for determination of fresh weight, dry weight, total ethanol extractable phenols and leucoanthocyanins. Cells produced phenols during all phases of growth, but at stationary phase, the production was greatest. Increasing concentrations of exogenous glucose in the culture medium resulted in an increased synthesis of total phenols in logarithmic cells, and an increased synthesis of total phenols and leucoanthocyanin in stationary cells. Addition of increased concentrations of exogenous nitrate to the stationary cells grown in suspension culture markedly reduced synthesis of leucoanthocyanins although total phenol synthesis was not significantly affected. Similar observations were made in cells cultured on solid medium in respect to exogenous glucose concentration, however these cells differed from the suspension cultured cells by having increased amounts of total phenol synthesis and decreased synthesis of leucoanthocyanins in the presence of increasing concentrations of exogenous nitrate in the culture medium.     

Extraction of Enzymes from Cell Walls of Sugar Beet Cells Grown in Suspension Culture

Various glycosidases were extracted from cell walls of sugarbeet cells grown in suspension culture using three successiveprocedures employing saline, EDTA, and commercial cellulase.Saline was effective for extracting acid invertase, ß-galactosidase,and ß-glucosidase. EDTA extracted most of the -galactosidaseand some of the ß-glucosidase. It was most effectivebetween 27?C and 40?C. Commercial cellulase could extract mostof the -mannosidase in the cell wall when used at 27?C for 96h. These three procedures could not extract some of the acidinvertase and ß-glucosidase. The results suggest thatthe cell wall glycosidases are associated with different polysaccharides. These extraction procedures were also applied to the cell wallsof intact tissues, such as cotyledons, hypocotyls plus roots,and mature roots of sugar beets. EDTA as well as saline wasquite effective for extracting bound enzymes from the cell wallof intact tissue, which indicated that extraction with EDTAis useful for liberating bound enzymes from plant cell walls. (Received October 19, 1987; Accepted March 16, 1988)     

Blue Light Inhibits Mitosis in Tissue Culture Cells

Irradiation of the mitotic (prophase and prometaphase) tissue culture PK (pig kidney embryo) cells using mercury arc lamp and band-pass filters postponed or inhibited anaphase onset. The biological responses observed after irradiation were: (i) normal cell division, (ii) delay in metaphase and then normal anaphase and incomplete cytokinesis, (iii) exit into interphase without separation of chromosomes, (iv) complete mitotic blockage. Cell sensitivity to the light at wavelengths from 423 and 488 nm was nearly the same; to the near UV light (wavelength 360 nm) it was 5–10 times more; to the green light (wavelength >500 nm) it was at least 10 times less. To elucidate the possible mechanism of the action of blue light we measured cell adsorption and examined cell autofluorescence. Autofluorescence of cytoplasmic granules was exited at wavelengths of 450–490 nm, but not at >500 nm. In mitotic cells fluorescent granules accumulated around the spindle. We suppose blue light irradiation induces formation of the free radicals and/or peroxide, and thus perturb the checkpoint system responsible for anaphase onset.     

Life Cycle Analysis of Mammalian Cells: II. Cells from the Chinese Hamster Ovary Grown in Suspension Culture

A method for life cycle analysis in mammalian cells which utilizes the collection function has been applied to the Chinese hamster ovary grown in suspension. The following durations were found for the various parts of the life cycle: S, 4.13 hours; G1, 4.71 hours; G2, 2.81 hours; mitosis, 0.81 hours. The cell has a total generation time of 12.4 hours as opposed to 20.1 hours for the S3 HeLa cell. However, the relative lengths of each phase of the life cycle are identical within experimental uncertainty in the two cells.     

Cation Metabolism in Relation to Cell Size in Synchronously Grown Tissue Culture Cell

In randomly grown tissue culture cells (mouse leukemic lymphoblast, L5178Y) the number, volume, and Na⁺ and K⁺ content increase as an exponential function with a doubling time of 11.3 hr. In synchronously grown cells the volume increase of the population and of single cells follows the same exponential function as in randomly grown cells. In contrast, the cation content fluctuates during a single cell cycle. About 1½ hr after the cell division burst (at the beginning of the S period), a net loss of K⁺ occurs for a period of about 1 hr amounting to about 20% of the total K. Over the next 5 to 6 hr, the deficit in K⁺ is eliminated. The Na⁺ content shows a double fluctuation. It falls during the cell division burst, rises when the K⁺ content decreases, falls again when K⁺ content rises, and then increases again before the next cell division burst. The net fluxes of both Na⁺ and K⁺ are very small compared to the unidirectional fluxes (less than 5%), thus small changes in the balance of influx and efflux account for the changes in cation content during the growth cycle. Both unidirectional fluxes increase dramatically (by a factor of two) about 2 hr after the cell division burst, and then remain constant until after the next cell division. The pattern of electrolyte regulation during cell division does not follow a simple function such as cell number, cell surface, or cell volume, but must be related to specific internal events in the cell.     

A Model of Blebbing in Mitotic Tissue Culture Cells

A preliminary study of blebbing in tissue cultures has been made. The tubal epithelium of fetal mouse oviduct was cultured at 37°C in Rose chambers. A cinematographic record was obtained of phase microscope observations of mitotic cells. Measurements of the size of both cells and blebs were made on the film using a “traveling” microscope. The duration and the rise and decay times of blebs were determined simply by counting frames on the film. Detailed observations are reported on blebbing in four cells undergoing mitosis. The results indicate that the frequency of blebbing as well as the duration of individual blebs exhibits a maximum during telophase. A model is proposed to account for blebbing in mitotic cells. The model attributes to local regions of the cell membrane the property of constant tension independent of stretch over some restricted range of stretch. This property implies that the cell membrane is locally unstable. Further assumptions stated explicitly in the model are that (i) cell division occurs at constant volume, (ii) the cell membrane stretches during cleavage, (iii) there is a positive pressure drop across the cell membrane. Evidence is cited in support of these assumptions as well as independent evidence that the cell membrane may be locally unstable. A physical model is described which would be expected to exhibit blebbing given the above assumptions.     

Differentiation of Rat Lens Epithelial Cells in Tissue Culture

The growth and differentiation of rat lens epithelial cells in tissue culture were studied. Cells could be maintained for a number of generations in an undifferentiated state in suspension culture. When cultured as monolayers, they grew and differentiated in a series of six defined stages described here. These stages include morphological changes (elongation, followed by cell "spreading" or formation of cell aggregates), and biochemical changes (appearance of y-crystallin protein as detected by immunofluorescence). The process of differentiation appeared to be accelerated in the vicinity of elongated cells, occurred more rapidly at high cell density, and required frequent changes of medium. This suggests that cell-cell communication, and not medium factors, may be essential for promoting differentiation. The final morphology of the differentiated cells differed, depending on the embryonic age of the rats used as a source of lens epithelial cells. This implies that the programme for differentiation changes as a function of the embryonic age of the lens.     

Morphological Variants of Sindbis Virus Obtained from Infected Mosquito Tissue Culture Cells

Tissue-cultured Aedes albopictus cells infected with morphologically homogeneous Sindbis virus were found to produce progeny virions which could be divided into three classes based on size. The thickness of the envelope was constant on all three sizes of progeny virions suggesting that the variability in size rested with the viral nucleocapsid. It is suggested that the three classes of virions have icosahedral nucleocapsids composed of common subunits organized in decreasing triangulation numbers.     

Ultrafiltration as a Method for Concentrating Rift Valley Fever Virus Grown in Tissue Culture

Filtration by means of a Diaflo ultrafilter was used to concentrate three 1,000-ml lots and one 3,000-ml lot of tissue culture-grown Rift Valley fever virus. Quantitation of both infectivity and total protein was achieved. Water treatment with continued ultrafiltration of the virus concentrate provided a final virus product approximately 99.25% free of low-molecular-weight materials originally present in the growth medium.     

Validation of the Cardiosphere Method to Culture Cardiac Progenitor Cells from Myocardial Tissue

Background

At least four laboratories have shown that endogenous cardiac progenitor cells (CPCs) can be grown directly from adult heart tissue in primary culture, as cardiospheres or their progeny (cardiosphere-derived cells, CDCs). Indeed, CDCs are already being tested in a clinical trial for cardiac regeneration. Nevertheless, the validity of the cardiosphere strategy to generate CPCs has been called into question by reports based on variant methods. In those reports, cardiospheres are argued to be cardiomyogenic only because of retained cardiomyocytes, and stem cell activity has been proposed to reflect hematological contamination. We use a variety of approaches (including genetic lineage tracing) to show that neither artifact is applicable to cardiospheres and CDCs grown using established methods, and we further document the stem cell characteristics (namely, clonogenicity and multilineage potential) of CDCs.

Methodology/Principal Findings

CPCs were expanded from human endomyocardial biopsies (n = 160), adult bi-transgenic MerCreMer-Z/EG mice (n = 6), adult C57BL/6 mice (n = 18), adult GFP⁺ C57BL/6 transgenic mice (n = 3), Yucatan mini pigs (n = 67), adult SCID beige mice (n = 8), and adult Wistar-Kyoto rats (n = 80). Cellular yield was enhanced by collagenase digestion and process standardization; yield was reduced in altered media and in specific animal strains. Heparinization/retrograde organ perfusion did not alter the ability to generate outgrowth from myocardial sample. The initial outgrowth from myocardial samples was enriched for sub-populations of CPCs (c-Kit⁺), endothelial cells (CD31⁺, CD34⁺), and mesenchymal cells (CD90⁺). Lineage tracing using MerCreMer-Z/EG transgenic mice revealed that the presence of cardiomyocytes in the cellular outgrowth is not required for the generation of CPCs. Rat CDCs are shown to be clonogenic, and cloned CDCs exhibit spontaneous multineage potential.

Conclusions/Significance

This study demonstrates that direct culture and expansion of CPCs from myocardial tissue is simple, straightforward, and reproducible when appropriate techniques are used.