Showing papers in "In Vitro Cellular & Developmental Biology – Plant in 1972"

Species identity of insect cell lines.

Abstract: Three mosquito cell cultures designated as Suitor's clone ofAedes aegypti, Culiseta inornata andAedes vexans were shown to be moth by immunological, karyological, and isozyme analyses. The cells reacted with rabbit antimoth serum but not rabbit antimosquito serum. Chromosome analyses indicated Lepidopteran rather than Dipteran morphology, and three isozyme systems were confirmative. Any one of these assays would be sufficient to indicate that contamination had occurred and could be used as a periodic check for identity of cell cultures. Morphology and growth characteristics are also valid criteria to distinguish between these particular orders of insect cells.

Rat myocardial cells in vitro: mitosis and differentiated properties.

Abstract: Ventricular heart cultures were prepared from newborn rats and examined in Rose chambers by high resolution, phase-contrast optics. Details of the procedure are given for obtaining a high yield of myocardial or endothelial cells. Photographic records were obtained of living cells by using photomicrography, cinematography, and high speed filming to document the cytological differences between endothelial and myocardial cells, details of mitosis in the two cell types, and patterns of contractility. Myocardial cells can be distinguished from endothelial cells in the living state by the following criteria; dense cytoplasm; giant and pleomorphic mitochondria or sarcosomes: presence of small, round nuclei; binucleation; one or two round, dense nucleoli; a specialized Golgi apparatus around the nucleus; myofobrils; intercalated discs; myopodia; small, cell size; failure of cell migration; spontaneous contractions; formation of synchronized networks; and flattened appearance with adhesion to other cells during mitosis. Contracting myocardial cells are shown to undergo mitosis. Contractions become weaker at metaphase and then cease at anaphase. Daughter cells may resume contractions. Organized myofibrils are generally not detected during the mitotic periods when contractions are minimal or absent. It is proposed that myofibrils undergo a transient disorganization during mid-mitosis and become reorganized in the daughter cell(s). It is suggested that there is a competition for energy at the time of spindle changes and chromosome movements, so that priority is given to the mitotic process rather than to myofibrillar contractions. Myofibrillogenesis is considered to be a relatively rapid process. The average mitotic time is 2.5 hr fore endothelial cells and 6.1 hr for myocardial cells. Failure of cytokinesis frequently occurs in dividing myocardial cells and results in the formation of binucleated cells. A sequence, is presented of a binucleated myocardial cell which undergoes an abormal multipolar mitosis, leading to polynucleation. Myocardial cells incorporate tritiated thymidine into nuclear DNA. The question of mitosis and differentiation of myocardial cells is reviewed. It is concluded that myocardial cells can no longer be cited in support of the dogma, that differentiated cells do no divide. However, these is a temporary competition between the two phenomena at the metaphase-anaphase stages.

Totipotency and embryogenesis in plant cell and tissue cultures.

Abstract: An important development in the field of plant cell and tissue culture has been the demonstration in the past decade of the totipotency of higher plant cells. Isolated single cells were first successfully grown on a nurse tissue separated by a filter paper and gave rise to a callus tissue. Later, completely isolated single cells of tobacco were grown in microchambers to form small clumps of cells which then could be differentiated to form adult tobacco plants. Indirect evidence of the totipotency of higher plant cells has also been provided in a number of other plants. Embryo-like structures (or embryoids) or whole plants, or both, have been obtained from such highly differentiated cells as the pollen grains (gametic and haploid), photosynthetic palisade cells in leaves, epidermal cells from the hypocytyl, and the triploid endosperm cells; all of these cell types perform very highly specialized functions in the plant. Plant protoplasts (cell wall is digested with enzymes) have also been cultured to give rise to normal adult plants. In many instances embryoids have been produced in vitro from several species of flowering plants which do not show such asexual activity in nature. These embryoids are normally indistinguishable morphologically from embryos produced by gametic fusion, often follow the same pattern of cell divisions and differentiation as the developing zygote, and are economically important as they provide clonal populations. Early work in this area emphasized the necessity of dissociating tissues into single cells and providing a nutritional environment identical to that of the zygote in the embryo sac (usually by supplementing the medium with liquid endosperm from coconuts), before the cells could be released morphogenetically to express their totipotency by forming embryoids. Much of the recent work, however, has shown that perfect development of embryoids can be obtained in completely synthetic media in callus tissues as well as in suspension cultures.

Isolation of bacteriophages from commercial sera.

Abstract: Commercially available sera contain bacterial viruses. The possible origin of serum bacteriophages and the implication of this observation for tissue culture studies are discussed.

Amino acid utilization by L-M strain mouse cells in a chemically defined medium.

Abstract: The amino acid requirements of strain L-M mouse cells grown in a chemically defined medium (2×Eagle) containing only the 13 essential amino acids (EAA) were investigated. Medium and acid hydrolysate samples were analyzed for amino acid content by the method of ion exchange chromatography. The extent of utilization of the EAA differed;e.g. after 120 hr of cell growth without medium change, glutamine was exhausted from the medium; methionine, leucine, isoleucine, cystine, arginine, and valine were depleted 60 to 80%; other EAA were used to lesser extents. Although the EAA were used in excess of their requirements for protein synthesis, a correlation could generally be made between utilization and protein amino acid composition. Glutamine appeared to be, a growth-limiting factor. Use of U-14C-labeled glutamine indicated that over one-half of the metabolized glutamine was converted to carbon dioxide, 17% to cell material, and 15% was extracted from the amino acid pools. Nonessential amino acids (NEAA), viz. alanine, aspartic acid, glutamic acid, glycine, proline, and serine, were released into the medium during growth, and some were reutilized. Exogenous provision of these did not improve cell growth. In contrast to the other NEAA, only serine showed net utilization when provided exogenously. When glutamic acid largely replaced the glutamine in the medium, it exerted a sparing effect on the glutamine requirement for protein synthesis. Suggestions are given for the improvement of Eagle medium for cell growth.

The dye-exclusion test for cell viability: Persistence of differential staining following fixation

Abstract: A method is described whereby the differential staining of viable and nonviable unfixed cells, as observed by the dye-exclusion method, can be reproduced in glutaraldehyde-fixed preparations by staining with alcian blue. The results suggest that the differential staining is due, at least in part, to structural differences that are retained following aldehyde fixation.

Histochemical studies of rat ovarian follicular cells in vitro.

Abstract: Membrana granulosa cells were aspirated from large follicles of proestrous rat ovaries and were cultivated as monolayers. For histochemical identification of dehydrogenases, the monolayers were incubated in various steroid substrates, nicotinamide adenine dinucleotide, and Nitro Blue Tetrazolium. The presence of Δp5-3β-, 3α-, 17β- and 20α-hydroxysteroid dehydrogenases was demonstrated by the 4th day in vitro and was evident for as long as 20 days. Since none of these hydroxysteroid dehydrogenases is demonstrable in the membrana granulosa of intact follicles, it is concluded that the steroidogenic capacity of the cells, repressed in the preovulatory follicle in vivo, can be expressed upon mechanical removal from the follicle just as steroid synthesis occurs in these cells after normal ovulation.

Pyruvate kinase, hexokinase, and aldolase isozymes in rat liver cells in culture

Abstract: Isozyme patterns of the enzymes of ATP-hexose phosphotransferase, aldolase, and pyruvate kinase have been examined in a culture of cells derived from an adult rat liver. Normal adult liver characteristically contains glucokinase, aldolase B, and pyruvate kinase I isozymes. In contrast to this, the isozymes present in the cell line were found to be hexokinase, aldolase A, and pyruvate kinase III. Thus cells from an adult liver when taken into culture resemble fetal liver rather than adult liver. Attempts to induce in these cells the isozymes of ATP-hexose phosphotransferase and pyruvate kinase characteristic of the adult liver have been unsuccessful. The culture conditions were found to affect the nature of the kinetic properties of the pyruvate kinase isozyme in these cells. Cells maintained in the absence of glucose gave a sigmoidal-shaped substrate velocity curve for phosphoenolpyruvate, with aK m of 0.33mm. However, when the enzyme was extracted from cells grown in the presence of glucose, a hyperbolic-shaped substrate velocity curve was found, with aK m of 0.05mm. The two forms of this isozyme have been designated A and B, respectively. Form A can be converted to form B by preincubation with fructose diphosphate, whereas the B to A transition is mediated by ethylenediaminetetraacetate, ATP, or citrate. These two forms of the enzyme have distinctive properties in relation to inhibition by ATP, alanine, and phenylalanine, and to activation by fructose diphosphate. Their properties appear to resemble those of adipose tissue pyruvate kinase. The physiological significance of these properties is discussed.

Replication and differentiation in vitro of epidermal cells from normal human skin and from benign (psoriasis) and malignant (basal cell cancer) hyperplasia

Abstract: The normal steady state in human epidermis reflects a balance between the rates of cell proliferation and of cell differentiation (keratinization). In certain diseases, such as psoriasis and basal cell cancer, increased proliferative activity is associated with abnormalities in keratinization. These events have been thought to be directly related. However, studies in which the epithelial cells from normal epidermis, psoriasis, and basal cell cancer were grown in vitro suggest that abnormalities of the keratinization process do not directluy result from hyperproliferation. Alterations in keratinization probably result from abnormal dermal-epidermal interactions, independent of any dermal effects on mitosis.

Organ culture of fetal rat pancreas: quantitative analysis by linear scanning of islet and other tissue components.

Abstract: SUMMARY Pancreatic explants from rat fetuses of 20 days were grown in organ culture at the air-liquid interface of a cock serum-chick embryo extract medium. At 48-hr intervals through 10 days of incubation, cultures were fixed and prepared for light microscopic study. The tissue components comprising the explants were quantitated by the linear scan method. Scans were made at regular intervals throughout the tissue block, and the mass and volume percentages of each tissue component were calculated. Prior to culture, acinar tissue comprised 64% of the pancreas while islet tissue contributed less than 2%. During culture, there was a progressive increase in the percentage volume of both islet and duct epithelial tissues. Islet tissue reached a maximum of 11% of explant volume by 6 days of incubation, and duct epithelium 40% by 10 days. In contrast, there was a progressive loss of acinar tissue throughout incubation. By 6 days of culture, explants were devoid of acinar cells. The observations support the thesis that our culture conditions (a) promote the proliferation of duct epithelial cells and (b) are conducive to a selective differentiation and growth of islet cells but (c) are unfavorable for the maintenance or further differentiation of pancreatic acinar cells.

The differentiation of glial cells and glia limitans in organ cultures of chick spinal cord.

Abstract: Differentiation of glial cells and the glia limitans in organ cultures of chick spinal cord explanted at early neural tube stages, alone or with adjacent tissues, was studied by electron microscopy. Oligodendrocytes and astrocytes comparable to those seen in the chicken in vivo were observed, mainly in areas of good neuronal differentiation. A glia limitans with basal lamina, comparable to that in vivo, was found when spinal cord was bordered by normally adjacent tissues. When it was surrounded by vitelline membrane only, a characteristic limiting layer of glial processes, but no basal lamina, was seen. Contact with a filter membrane (Millipore) elicited excessive differentiation of glial filaments and modified cell fine structure; no glia limitans was formed.

Deoxyribonucleic acid synthesis, mitosis, and skeletal muscle differentiation.

Abstract: In the past, it was generally thought that processes of differentiation and proliferation in eukaryotic cells were antagonistic, the implication being that somehow the two processes cannot go on simultaneously within the same cell. There are, however, many examples of synthesis of deoxyribonucleic acid and of specialized cell proteins occurring simultaneously in the same cell (1-5). It has become clear in recent years that it would be more accurate to say that there are several differentiating systems in which special relationships exist between the events leading to overt differentiation and cell proliferation. Some of the better studied examples are in erythroid differentiation, where a programmed sequence of cell division occurs (6-9); in formation of tubular gland cells, in which division is required for the formation and subsequent synthesis of ovalbumin in the chick oviduct (10, 11); in viral transformation of cells, where cell division is required to establish this state (12-15); in the differentiation of mammary gland epithelium, where cell division must occur in the presence of hydrocortisone in order for prolactin to stimulate milk protein synthesis (16, 17); and in the differentiation of skeletal muscle cells, where cells synthesizing contractile proteins do not divide (18, 19). For several years we have been exploring the relationship of cell proliferation and differentiation in skeletal muscle cells differentiating in vitro. We describe this relationship and relate some of our general conclusions about deoxyribonucleic acid synthesis in differentiating myogenic cells. DISCUSSION

Growth of Chlamydia psittaci strain meningopneumonitis in mouse L cells cultivated in a defined medium in spinner cultures.

Abstract: L cells were grown in spinner cultures in a defined medium consisting of Waymouth medium MB752/1 (19) supplemented with 2 mg of fatty acid-free bovine serum albumin (BSA) per ml and 5 μg of oleate per ml (WO5 medium). Growth in WO5 medium was comparable to spinner L cell growth in two serum-containing media. The optimal concentration of oleate in the WO medium was 5 to 10 μg per ml. The use of 20 to 80 μg of oleate per ml of medium resulted in lower peak populations and earlier declines in viable cell counts. Cell death occurred rapidly in WO160 medium. Cell growth in WO medium containing 5 to 80 μg of oleate per ml was well above the level of growth observed when no oleate was present in the medium. Since the total lipid and fatty acid compositions of the BSA used in this study have been characterized by the authors, the WO medium may be considered a defined medium. L cells have been continuously maintained in spinner cultures in WO5 medium for over 50 passages with no major variation in the growth pattern. A 1000-fold increase inChlamydia psittaci strain meningopneumonitis, with a peak titer of 9.3×107 plaque-forming units per ml, was observed when the chlamydial agents were grown in spinner L cells in WO5 medium.

A method for in situ embedding of cultured cells grown on plastic surfaces.

Abstract: A method is described for the electron microscopic visualization of contact relationships between monolayer cells and the supporting surface of the plastic culture vessel

Dissociation of single cells from lung or kidney tissue with elastase.

Abstract: Experiments were conducted to determine the capacity of various enzyme preparations to dissociate single cells from guinea pig lung tissue. The number of cells separated from tissue progressively increased as the concentration of crude trypsin was increased from 25 to 250 mg per 100 ml. This action could be inhibited by soy bean trypsin inhibitor. Elastase, but not ethylenediaminetetraacetate (disodium salt), crystalline trypsin, nor chymotrypsin, dissociated cells from lung tissues. Crude trypsin (Trypsin 1∶300) was found to contain 3.0 Sachar units of elastase per mg. Elastase was also inhibited by soy bean trypsin inhibitor. Only some collagenase preparations dissociated cells from lung tissue. Impure bacterial proteases dissociated lung cells. Our data suggest that the term “trypsinization” to denote dissociation of cells from tissue with crude preparations of trypsin is misleading and should be discontinued.

Lysolecithin-induced fusion of fibroblasts.

Abstract: Lysolecithin has been used to induce cell fusion between two metabolically deficient mouse fibroblast lines, A9 and B82. Attempted fusion in suspension led to excessive cell clumping and complete loss of viability. Addition of lysolecithin solutions to confluent monolyers caused extensive detachment of cells from glass or plastic surfaces. At higher levels of lysolecithin few cells survived. When the conditions were controlled (50 to 250 μg per ml for up to 20 min), extensive polykaryocyte formation was observed. In the presence of selective medium (HAT) colonies of hybrid cells grew and a series of cell strains were isolated. The presence of inosinic acid pyrophosphorylase (absent in A9 cells) was demonstrated in the hybrid cells which were shown to have almost double the cell volume of the parent A9 and B82 cells. Unlike the parent cell lines, the hybrid cells grew well in the presence of HAT both as monolayers and in suspension.

The effects of the buffer hepes on the division potential of WI-38 cells

Abstract: N-2-Hydroxyethylpiperazine-N′-2-ethane sulfonic acid (HEPES) and N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES) have been found to be nontoxic substitutes for bicarbonate buffer in cell culture media. WI-38 embryonic human lung cells have been carried beyond the 60th passage with both HEPES and TES buffers.

Chemically defined media for mammalian eggs and early embryos.

Abstract: The purpose of this review is to bring together in a single article the chemically defined media used for the maturation, fertilization, development and low temperature storage of mammalian eggs and early embryos. The media have hitherto been scattered in the literature and in some instances their preparation was inadequately described. Thus the researcher did not have available a ready reference for preparing the media, nor did the commerical supplier have a definitive formula for his product. It is my hope that this review will help to overcome these difficulties, thereby advancing research in reproductive biology and fertility control. I have aimed for completeness. However, early versions of a medium which underwent improvement are omitted, unless the early version found wide acceptance. Formulas for the media and notes on their preparation are prefaced by brief summaries of their development and application. The amount of detail given regarding preparation necessarily varies with the data supplied by the author, either in published form or in response to letters which were sent to the principal investigators. I express my gratitude to all those who responded and supplied me with information beyond the scope of their original publications.

Effect of the gene lethal(1) myospheroid on drosophila embryonic cells in vitro

Abstract: The mutationlethal (1) myospheroid causes death in affectedDrosophila melanogaster embryos. The action of this gene was investigated by culturing normal and mutant embryonic cells in vitro. Under these culture conditions, normal myoblasts and neuro-blasts differentiated to yield myocytes and neurons. The action of the mutant gene was manifested in an altered differentiation of at least two cell types, myocytes and neurons. It prevented differentiation of all myocytes and a fraction of the neurons in vitro. Failure of these cells to differentiate in vitro suggests that the mutation affects the integrity of myocytes and neurons in vivo and contributes to themyospheroid lethal effect.

Classification of cell types: agglutination and chromosomal properties

Abstract: The properties of agglutination by plant lectins, along with chromosome patterns, were examined in a variety of mammalian cell types Untransformed adult and embryonic cells in culture, direct mouse spleen cell preparations, SV40-transformed 3T3 cells, and trypsinized 3T3 cells were all highly agglutinable with concanavalin A and with wheat germ agglutinin In contrast, untreated cells of the contact-inhibited 3T3 line were alone among the cells tested in their low agglutinability Chromosome analysis of the cultured cells showed that karyotypic variation from the diploid to an aneuploid state in mouse and rat embryo cultures was not accompanied by a change in agglutinability Adult rat lung, adult monkey kidney, and embryonic human lung cells, which were all highly agglutinable, showed the normal diploid pattern Thus, agglutination of cells by plant lectins appears to be a cellular property often associated with non-neo-plastic cells

Submerged organ culture: An improved method

Abstract: Explants of whole ovaries and oviducts from postnatal rats were completely submerged during cultivation as organ cultures in chemically defined medium. The oxygen concentration in the culture chamber was raised to over 90%, and the stainless steel platform, used for cultures at the surface of the medium, was abandoned and excluded from further use. Thus, all of the periphery of an explant had equal access to nutrients and oxygen. Throughout the ovarian explants the tissues appeared uniformly viable, and mitotic figures were distributed evenly. These observations are in contrast to those on organs cultivated at the surface of the medium where a lack of structural uniformity had been detected. The method has general application to a variety of organs.

Utilization of hexoses and synthesis of glycogen in two strains of HeLa cells.

Abstract: SUMMARY At 5 mM concentration, D-mannose, D-fructose, and D-galactose were not equivalent to glucose in their capacity to support growth of two strains of HeLa S3 cells. In addition, the amount of accumulated cell glycogen was much higher in cells grown in the glucose-containing medium than in media supplemented with other hexoses. In one of the cell strains, the glycolytic breakdown of mannose was relatively inefficient, resulting in toxic morphological manifestations and in markedly reduced growth. Similar toxic effects were observed in both cell strains when D-glucosamine and 2-deoxyglucose were added to the culture media. In one of the cell strains only D-glucosamine, 2-deoxyglucose, and D-mannose competed with glucose for glucose transport, while in the other cell strain some competition was observed also with D-fructose, D-galactose, and 3-O-methylglucose.

Brain histogenesis in vitro: Reconstruction of brain tissue from dissociated cells

Abstract: Comparative studies of the aggregative behavior of cells dissociated from different areas of embryonic chick and mouse brains show that each of the regionally differentiated lobes (cerebrum, optic tectum, and cerebellum), and the stem areas (diencephalon and medulla), form characteristic aggregates distinctive in size and shape. Bispecific co-aggregates are produced by commingling dissociated mouse cerebrum cells with chick cells from various brain regions, or from non-nervous tissues; the size of these co-aggregates and the extent of internal sorting out of cell types is closely related to the degree of homology between the interacting cell populations, e.g. co-aggregates of the closely homologous mouse and chick cerebral cell types contain homogeneous tissue fabrics of intermingled mouse and chick cells.

Some aspects of mouse mammary gland development from maturity to early pregnancy.

Abstract: Mammary epithelial cells in the mature, nonpregnant mouse gland neither proliferate nor develop, presumably because they are insensitive to insulin and serum factor (s). They can become sensitive in vitro in the absence of added hormones, and they become sensitive during pregnancy consequent to an action of prolactin. The sensitized cells must undergo a critical mitosis, under the influence of insulin or serum factor (s), or both, before they can be stimulated to make casein and α-lactalbumin.

Organ culture of the terminal abdominal segment of an adult female lepidopteron.

Abstract: Organ cultures containing the sex pheromone gland ofDiatraea saccharalis (F.) (sugarcane borer) were maintained for over 2 months. Juvenile hormone but not ecdysterone appeared to be essential for the maintenance of integrity of the sex pheromone gland in the organ culture system. Muscles associated with these cultures pulsated during hte entire culture period with or without juvenile hormone, suggesting that the hormone requirement was specific for the sex pheromone gland and not a general requirement of the cells of the organ-cultured segment.

A comparison of methods for morphological studies of cultured cells

Abstract: A number of fixation methods for different types of cells in culture were compared, and the best preservation of nuclear and cytoplasmic details was obtained by fixation with Bouin's solution for 15 min, prior to staining with hematoxylin and eosin. All of the fixatives, including Bouin's solution, damaged various structures, notably the peripheral glas-attached cytoplasm and the intercellular connections. Micrographs obtained by bright field, phase contrast, and interference contrast (Nomarski) microscopy are presented. Much more realistic pictures, bringing out details not observed after fixation and staining, were obtained by Nomarski microscopy of living, unfixed cultures. Most conspicuous were numerous thin, cytoplasmic, cilia-like extensions, concentrated on the glass-attached peripheral margins, which were also visible on other cell surfaces and as intercellular connections. These structures were most characteristic of SV40-transformed human amnion cells. Although fixation and staining emphasize certain cell components (for example, inclusion bodies), many aspects of cellular morphology are better demonstrated by observing living cells by interference microscopy or by Nomarski interference contrast microscopy. Surface features of unfixed cells, seen by Nomarski interference contrast microscopy, were similar to the surface features of glutaraldehyde-osmium tetroxide-fixed cells studied as metallic replicas in the electron microscope.

In vitro culture of somite stage rat embryos: a new technique for maintaining growth and continuously monitoring heart rate. Applications in metabolic and teratologic studies.

Abstract: Rat embryos removed during somite stages were attached to small rafts by using the technique described by D. A. T. New (J. Embryol. Exp. Morphol. 17: 513–525, 1967). The embryos were maintained in a viable growing state in circulating human serum. Twenty-four-hour cultures of day 11 rat embryos resulted in nearly normal increases in somite count, but length and protein increments were about one-half of in vivo values. After 24 hr of culture, the embryos were generally translucent and showed no apparent areas of gross or microscopic tissue necrosis. The apparatus is extremely compact and requires only about 9 ml of medium, which is sufficient to support three embryos. The small volume allows measurement of metabolites consumed or liberated with reasonable accuracy. By use of a technique of transillumination, using a laser and photomultiplying tube, a detailed and continuous record of heart rate can be made. Addition of drugs to, and sampling of, the culture medium is done easily.