IN VIVO AND IN VITRO OBSERVATIONS OF LEPTOSPIRA POMONA BY ELECTRON MICROSCOPY.

Miller, Norman G. (University of Nebraska College of Medicine, Omaha) and Richard B. Wilson. In vivo and in vitro observations of Leptospira pomona by electron microscopy. J. Bacteriol. 84:569-576. 1962.-Leptospira pomona 3341 was observed by electron microscopy, after the preparation of thin sections from culture material and from infected hamster tissue. The external membrane of low electron density envelops the entire leptospire and appears to be quite flexible, as suggested by its many folds. The spiral protoplasmic body is tubular in structure with a relatively dense wall and a central area of low electron density. Occasionally, very dark circumscribed bodies were seen imbedded in the protoplasmic wall. Detailed morphology is presented of a knoblike structure located at the end of the axial filament. Bifurcation of the axial filament could be demonstrated in leptospires from cultures. Leptospires were observed free or enclosed in vesicles within the cytoplasm of liver parenchymal and renal tubule cells. Erythrocytes located in kidney tissue also contained leptospires within the cytoplasm. The appearance of intracellular leptospires is much the same as those seen extracellularly or from culture.     

Variability of the effects of serum-free medium,dibutyryl-cyclic AMP or theophylline on the morphology of cultured new-born rat astroblasts

The effects of serum deprivation, of dibutyryl-cyclic AMP (dBcAMP) and of theophylline on the morphology of cultured new-born rat astroblasts have been studied using Eagle's basal medium (BME) or Eagle's minimum essential medium (MEM) as culture media. Serum deprivation had no effect on cells cultured in BME, while in MEM, deprivation induced a rapid morphological transformation involving the appearance of multiple processes. This phenomenon was rapidly reversible when serum was again added. In serum-supplemented BME, dB-cAMP (1 mM) and theophylline (1 mM) had no effect. In serum-supplement MEM, theophylline (1 mM) had no effect while dB-cAMP (1 mM) induced a slower and poorly reversible morphological alteration. On the other hand cells in serum-free BME showed multiple processes after addition of dB-cAMP (1 mM) or theophylline (1 mM). This rapid alteration was completely reversed either by removal of dB-cAMP and theophylline or by addition of serum.     

Growth factors and hyperthermia. II. Viability of Chinese hamster ovary HA-1 cells during serum starvation and hyperthermia

We tested the possibility that hyperthermia kills HA-1 cells in a manner analogous to growth factor deprivation. HA-1 cells were inactivated by serum starvation when incubated in Eagle's MEM at a density of 40 cells/cm2 or less. Cells became resistant to the absence of serum when the cell density was greater than 400 cells/cm2 or when lethally irradiated HA-1 feeder cells were present. The feeder cells exerted their effect through a diffusible factor. In addition, a 1:1 mixture of Eagle's MEM and Ham's F-12 enabled HA-1 cells to remain viable without serum. Ten days growth in Eagle's MEM + Ham's F-12 without serum resulted in the formation of microcolonies of cells. This indicated that growth factor deprivation was not lethal to HA-1 cells, and it suggested that they may have been partially transformed. The presence of the growth factors insulin, transferrin, and fibroblast growth factor (FGF) reduced cell killing by a small amount during conditions of serum starvation. After hyperthermia, the presence of growth factors again diminished cell killing by a modest amount (approximately twofold). Feeder cells also improved cell survival after hyperthermia. The effect of feeder cells was greatest when cells were trypsinized immediately after hyperthermia. When cells were not trypsinized after heating, feeder cells increased survival less than twofold. In summary, the absence of growth factors was not lethal to HA-1 cells, and therefore the cytotoxic effects of hyperthermia could not be explained fully by the failure to bind growth factors. HA-1 feeder cells secreted undefined, growth-promoting substances, but feeder cells exerted only a small positive effect on cell survival after hyperthermia when cells were not trypsinized after heating.     

Restricted growth of rat kidney proximal tubule cells cultured in serum-supplemented and defined media

Proximal tubules suitable for in vitro culture were prepared from rat kidney cortex by a Ficoll-gradient centrifugation technique which yielded greater than 94% purity. The tubules were seeded into culture dishes, and cell growth was monitored in both Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum and in a defined medium consisting of 50:50 Ham's F12 and Dulbecco's supplemented with insulin, transferrin, and hydrocortisone. Growth in serum-containing medium was continuous; however, the specific activity of the brush border enzyme alkaline phosphatase decreased rapidly with time, and the culture morphology became fibroblastic by 6 days. Neither collagen-coating of the dishes nor addition of the differentiation inducer hexamethylene-bisacetamide had any significant effect on growth or enzyme activity of the cultured cells. Theophylline, another inducer of differentiation, proved cytotoxic. Growth of proximal tubule cells in defined medium proceeded for 4 days before irreversible growth arrest occurred. Alkaline phosphatase activity and epithelial morphology remained relatively constant throughout the culture period. Additions of the growth factors triiodothyronine, prostaglandin E2, and epidermal growth factor were unable to unblock the growth arrest. If cells cultured in defined medium for 3 days were switched to serum-supplemented medium, continuous growth occurred, but both alkaline phosphatase activity and epithelial morphology were rapidly lost. As a test of the culture method, rabbit proximal tubule cells were cultured under similar conditions in defined medium. Growth was prolific and continuous for up to, but not exceeding, 30 days, and differentiated properties were retained. It was concluded that both rat and rabbit proximal tubule cells have a limited proliferative capacity in vitro but that the capacity of the rat cell to divide is much reduced relative to the rabbit cell.     

Effect of growth factors on the differentiation of chick precardiac mesoderm in vitro

We have found that precardiac mesoderm extirpated from chicken blastoderm at stage 5 fails to differentiate into beating tissue when cultured in Eagle's minimum essential medium (MEM), while it can pulsate provided either the endoderm is present or serum is added to the MEM. To identify the factor(s) which influence early myocardial differentiation, we examined the effect of insulin-like growth factor 1 (IGF-1), activin A and basic fibroblast growth factor (bFGF). All these growth factors showed a stimulating effect on myocardial differentiation and it is conceivable that these factors exhibit the same effect in vivo. Correspondence to: Y. Yamazaki     

Effect of insulin-like growth factor I (IGF-I) on Sertoli cell metabolism in the pubescent rat]

The influence of Insulin-like Growth Factor I (IGF-I) on some metabolic functions of Sertoli cells from peripubertal rats was investigated. Sertoli cells were isolated from the testes of 24-day-old animals and cultured at 32 degrees C in Eagle's MEM with or without 1 nM IGF-I. Sertoli cells cultured in the presence of IGF-I showed increased nuclear RNA polymerase activity (+80%) and augmented protein synthesis (+50%).     

The enumeration of Leptospira interrogans serovar pomona by a bioluminescence ATP assay

A rapid method for enumerating viable Leptospira interrogans serovar pomona cells was investigated using a bacterial adenosine triphosphate (ATP) assay. The ATP was assayed by the luciferin-luciferase bioluminescence reaction. Samples of serovar pomona grown in liquid polysorbate 80-bovine albumin (P80-BA) medium for 1-3 days were analysed for ATP content, culture density (nephelometry), direct cell count and most probable number of viable cells (MPNVC) as determined by the dilution tube technique. A linear relationship was found between ATP content and the number of viable cells over the range of 4 X 10(8) to 8 X 10(9) leptospires/ml. Over this range the correlation coefficient for ATP content versus viable cells (0.96) was similar to the coefficient for culture density versus the number of viable cells. The coefficient for direct counts versus the number of viable cells was smaller. The bioluminescence assay of bacterial ATP is a promising method for enumerating viable leptospires in pure culture.     

Cell cycle progression delay in conditioned medium does not play a role in the repair of X-ray damage in Chinese hamster V79 cells

We tested our hypothesis that the lower survival of X-irradiated cells in growth medium (GM) relative to that in conditioned medium (CM) is due to differences in nutrient concentration levels rather than to differential effects on cell progression and growth. Chinese hamster V79 cells in log and unfed plateau phase, grown in Eagle's minimal essential medium (MEM) with 15% serum (100% GM), were irradiated. Before plating, cells were incubated in situ in various concentrations of MEM with serum (GM, normal cell progression) or MEM without serum or in CM (no cell progression). Cell survival was the lowest in 100% MEM with or without serum and increased with the decrease in MEM and serum concentrations, reaching a plateau in 40% MEM or 40% growth medium (40% MEM with 6% serum), similar to that in conditioned medium. Growth kinetics was the same in 40 and 100% growth medium, but the D0 of cells in 40% growth medium was higher than that of cells in 100% GM. Similarly, the D0 of cells in 40% MEM was higher than that of cells in 100% MEM, although cell progression was absent in both media. The radiation sensitivity of cells was the same in 40% GM with progression and in 40% MEM and CM with no progression. Cells in low-nutrient media were flatter than those in 100% MEM or GM. There was a correlation between the nutrient concentration in the medium postirradiation and the D0. This correlation was independent of the presence or absence of serum and thus independent of cell cycle progression. The cell morphology which is dependent on the nutrient concentration appears to influence the ability of a fraction of cells to repair their radiation damage.     

Class Specificity of Avian and Mammalian Sera in Regards to Myogenic Cell Growth in vitro

Chick myogenic cells grew in the presence of a small amount of avian serum in a culture medium composed of Eagle's minimum essential medium (MEM) and horse serum. Mammalian sera, except for fetal bovine serum at high concentrations, could not substitute for the avian serum.
Rat myogenic cells grew in the presence of a small amount of mammalian serum in a culture medium composed of MEM and chick serum: avian sera, except for dove serum at high concentrations, could not substitute for the mammalian serum.
Serum from animals of the class from which the myoblasts were obtained was needed for cell growth. It is thus concluded that there is a class specificity among sera in regards to myogenic cell growth. The only exceptions to this hypothesis found so far were fetal bovine and dove sera.     

Transforming growth factor-beta and platelet-derived growth factor synergistically stimulate contraction by testicular peritubular cells in culture in serum-free medium

We report investigations on factors influencing contractility by testicular peritubular cells (PC) maintained in culture in a three-dimensional collagen gel system, and the behavior of PC in culture on a two-dimensional system. At low and moderate cell densities, PC embedded in collagen gels in serum-free Eagle's minimal essential medium (MEM) have a lesser degree of contractility than PC in culture in MEM containing calf serum. The contractility by PC, measured by determining changes in diameter of the collagen gel, was increased by addition of transforming growth factor-beta (TGF-beta) to serum-free MEM, and this was further enhanced by supplementing the medium with platelet-derived growth factor (PDGF). In the absence of TGF-beta, however, PDGF had no detectable effects on PC contractility. Other growth factors examined (epidermal growth factor, insulin, and fibroblast growth factor) did not influence the degree of contractility of PC in serum-free MEM in the presence or absence of TGF-beta. PC maintained in MEM supplemented with platelet-poor serum (PPS) have a lesser degree of contractility than their counterparts in MEM containing 2.5% calf serum. The addition of TGF-beta and PDGF to PPS-supplemented MEM restored contractility by PC to a level comparable to that observed by PC in MEM containing complete serum. The addition of nonpurified bovine serum albumin (BSA) to MEM greatly increased PC contractility. By contrast, highly purified BSA had no such effect, suggesting that one or more components adsorbed to the impure BSA was implicated. Polyclonal antibody against fibronectin did not influence the contractility of PC in collagen gels in the presence or absence of serum. Antiserum against TGF-beta partially blocked the enhancement of contractility of PC in MEM containing non-purified BSA. In PC plated on top of a collagen gel lattice, the attachment, spreading, and cell shape were greatly influenced by the presence of TGF-beta and PDGF, both singly and together. Data presented are interpreted to indicate that effects elicited by serum on the properties of PC in culture, and on the contractility of PC, can be attributed in part to the combined influences of TGF-beta and PDGF in serum.     

A simple method for cloning and replica plating of mammalian cells using multicellular spheroids.

V 79/4 Chinese hamster cells or HeLa cells grow in Eagle's MEM supplemented with 25 microgram/ml dextran sulphate to form clonal multicellular spheroids. These cell clones, consisting of 5-10(2) cells, are easy to separate, to transfer from one culture vessel into another and grow as normal monolayer colonies on Dederon cloth circles after subculture in Eagle's MEM without dextran sulphate. A simple replica technique is described by which 500 clones can be transfered onto at least 3 replica cloth circles, 10 cm in diameter, with a replica plating efficiency of approximately 100%.     

Growth stimulation of bovine endothelial cells by vitamin A

Retinol at concentrations of 10(-6) and 10(-5) M stimulated growth of bovine aortic endothelial cells maintained in Eagle's MEM supplemented with delipidized serum. In addition to retinol, retinal, retinoic acid, and retinyl acetate were also growth stimulatory. At very low inoculum densities (4-40 cells/cm2) the growth promoting effect could be demonstrated only in the presence of conditioned medium from macrophage-like culture P388D1. When added to media containing whole (nondelipidized) serum, retinol was growth inhibitory at 10(-6) and 10(-5) M concentrations.     

Evaluation of culture media for effects on cell cycle kinetics and incidence of chromosomal aberrations in human blood cells

Peripheral blood samples from 17 apparently healthy male volunteers were set up in duplicate cultures using three commercially available media: Eagle's MEM, RPMI 1640, and TC 199. BUdR (5-bromo,2-deoxyuridine) (10 micrograms/mL) was added to one of the cultures from each person in each medium after 24 h of culture initiation. All cultures were harvested at 72 h of incubation in the presence of colcemid. RPMI 1640 stimulated the highest mitotic activity in both BUdR-treated and untreated cultures. Higher numbers of first division metaphases corresponded with the higher frequency of chromosome-type aberrations in cultures with Eagle's MEM as compared with RPMI 1640 media. On the other hand, higher numbers of chromatid-type aberrations were present in cultures with TC 199 as compared with those with Eagle's MEM. When the chromosome- and chromatid-type aberration data were pooled to score total cytogenetic abnormalities, an influence of the medium was demonstrable. While cultures with Eagle's MEM and TC 199 had the greater number of first division cells, third of subsequent division cells were most prevalent in RPMI 1640 cultures. It is inferred that the length of the cell cycle, the mitotic index, and to some degree the incidence of spontaneous cytogenetic abnormalities are variable attributes of culture media.     

Chicken kidney cell culture in medium without serum

When chicken kidney cell (CKC) culture in a petri dish was prepared in medium with or without serum and incubated in a humidified incubator at 38 degreesC with no addition of CO2, monolayers of CKCs were formed completely on the 5th day of cultivation. Growth medium used for CKC culture was Eagle's minimum essential medium containing 0.3% of dehydrated tryptose phosphate broth. The number of cells in both cultures prepared in medium with or without serum was the same when measured on the 5th day of cultivation. Monolayers of CKC culture prepared in medium with or without serum were maintained up to 21 days of cultivation, while maintenance medium was changed every 4th day. The time of appearance and degree of cytopathic effect, plaque-forming ability, and propagation of some avian viruses were similar in both cultures prepared in medium with or without serum.     

Growth and morphogenesis of mouse prostate epithelial cells in collagen gel matrix culture

Epithelial cells were isolated from the ventral prostate gland of the mouse after prolonged incubation in a mixture of collagenase, Dispase and hyaluronidase followed by extensive pipetting. The isolated epithelial cells were then embedded in collagen gels. After cultivation in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, epidermal growth factor, 5 alpha-dihydrotestosterone and cortisol, stimulation of growth and branching morphogenesis of the epithelial cells were observed. Under these culture conditions, growth of contaminating fibroblastic cells was rarely seen. These observations suggest that hormones including androgen directly stimulate the growth and morphogenesis of mouse prostate epithelial cells in culture.     

Restricted amino acid nutrition induces attachment to glass and spreading of Ehrlich ascites tumour cells

Ehrlich ascites tumour (EAT) cells were cultured in vitro in Eagle's MEM and Medium 199 with a lowered amino-acid content. Under these conditions EAT cells lose their rounded shape typical of highly malignant cancer cells, and begin to spread on the substratum. The changes in EAT cell morphology are preceded by a decrease in the rate of protein synthesis. These changes were maintained for three days after returning the cells to Eagle's MEM with a normal amino-acid content, but the return to control media did not cause reasumption of growth in the once spread cells. The increase in glucose content (up to five-fold) or the presence of inhibitors of DNA synthesis did not prevent the attachment and flattening of EAT cells in media with a lowered amino acid content. Several possible mechanisms of the influence of restricted amino-acid availability on the changes in EAT cell surface properties are pointed out and the need for study of cancer cell responses to restricted nutrition is discussed.     

Requirement of serum components for the preservation of primordial germ cells in testis cords during early stages of testicular differentiation in vitro in the mouse

Mouse gonadal primordia were isolated from embryos on the 11th day of gestation and cultured in vitro. They developed into either testes or ovaries after 7 days of culture in Eagle's minimum essential medium (MEM) supplemented with horse serum, whereas they did not differentiate in MEM alone. We studied how serum components are required for testicular development in vitro. When gonadal primordia were cultured in MEM alone for the first 1-3 days and subsequently in MEM supplemented with serum, testis cords developed while germ cells disappeared or only a few remained in the testis cords. In contrast, when serum was present in the medium during the first day of culture and omitted thereafter, germ cells were retained within testis cords. These results suggested that some serum component(s) is specifically required by germ cells independent of testis cord organization. Of more than 10 serum components tested, low and very low density lipoprotein fractions increased the number of germ cells in testicular explants.     

EFFECTS OF CULTURE MEDIA ON THE "FOREIGN" DIFFERENTIATION OF LENS AND PIGMENT CELLS FROM NEURAL RETINA IN VITRO

Neural retinal cells of 3.5-day-old quail embryos were cultured as a monolayer to examine their potentials for differentiation in vitro. The "foreign" differentiation into lentoid and pigment cells was much affected by the choice of medium (Eagle's MEM and Ham's F–12); in Eagle's MEM, neural retinal cells differentiated extensively into lentoid bodies and pigment cells, as previously reported in cultures of chick neural retinal cells, while in Ham's F–12, though the cells proliferated as well as in Eagle's MEM, the "foreign" differentiation is inhibited. When primary cultures were transferred to secondary cultures, the occurrence of "foreign" differentiation did not depend on the medium used for the primary culturing, but wholly on the medium used for secondary cultures. This difference in differentiation in two different media was quantitatively substantiated by measuring the amounts of α-, δ-crystallins and melanins of cultured cells.     

Biological Effects of Leptospiral Lipids

Lipids were extracted from virulent Leptospira pomona and were purified. These lipids fixed complement in the presence of antiserum to L. pomona but did not stimulate the production of homologous agglutinins in rabbits, mice, or hamsters. When subsequently challenged, all of the mice and hamsters were fully susceptible to L. pomona. The lipid material was neither dermonecrotic nor lethal for mice or hamsters, but 382 mug of lipid from virulent or avirulent leptospires inhibited the growth of normal mice. Leptospiral lipids were toxic for peritoneal macrophages maintained in vitro, and when administered simultaneously with a million lethal doses of L. pomona, the lipids hastened death of the hamsters, presumably by inhibiting phagocytosis early in the course of the infection.