Cell cultures derived from embryos and melanoma of poeciliid fish

Summary In poeciliid fish, melanoma of different degrees of malignancy can be produced by crossing specific genotypes. For a detailed investigation of the processes leading to proliferation or differentiation of the melanoma cells, it is necessary to establish cell cultures. The aim of the present study was to find out the optimal conditions for initiating and culturing poeciliid fish cells for the purpose of establishing cell cultures of melanoma. The optimal method was developed by using small pieces of late embryos as starting material and includes: (a) dispersion of tissue by mild stepwise treatment with a trypsin-EDTA mixture at low temperature; (b) culture of cells in the complex medium 199; (c) supplementation of medium with high percentage (20%) of fetal bovine serum; and (d) stabilization of pH by buffering the medium with HEPES. Under these conditions, primary and secondary cultures of embryonic cells have been initiated. An epithelial-like cell line has been subcultured for more than 80 passages. The method developed for embryonic tissues was used to start cell cultures from melanoma of platyfish-swordtail hybrids. Until now, only cells of rapidly growing malignant albino melanoma could be maintained in primary cultures. Secondary cultures could not be initiated since the melanoma cells tended to differentiate and stopped growing before a confluent monolayer was formed. Dedicated to Professor Dr. C. Kosswig on the occasion of his 75th birthday. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 103, Marburg, Zellenergetik und Zelldifferenzierung), and is part of the thesis of C. K.     

Control of lipoprotein lipase secretion in mouse macrophages

The regulation of secretion of lipoprotein lipase (LPL) was studied in in vitro-derived mouse bone marrow macrophages (BMM), peritoneal exudate and resident macrophages and in the macrophage-like tumor cell line J774.1. BMM in cultures initiated with low concentrations of bone marrow cells (LC-BMC cultures) secrete more LPL per cell than BMM in cultures initiated with high concentrations of bone marrow cells (HC-BMC cultures). The suppressed state of LPL secretion in HC-BMC cultures could be alleviated by the addition of a colony-stimulating factor source (L-cell-conditioned medium; L-CM) onto the culture medium or exchanging the medium of HC-BMC cultures with medium from LC-BMC cultures for short periods (4 h). Addition of L-CM increased LPL secretion also in LC-BMC cultures. Addition of L-CM to fresh culture medium had little or no effect, suggesting that, in addition to requirement for L-CM, optimal expression depended also on factors released by the growing cells, probably providing optimal growth conditions. L-CM enhanced LPL secretion by thioglycollate-elicited peritoneal macrophages and had no effect on LPL secretion by resident peritoneal macrophages. Secretion of LPL from adherent J774.1 cells showed a biphasic effect. Secretion increased with cell density up to the point when growth inhibition was observed. In dense cultures in which cell proliferation was almost arrested, LPL secretion was remarkably suppressed (80-90%). Change of medium of dense cultures to fresh medium or medium conditioned by sparse cultures (for the last 4 h of culture) led to enhancement of LPL secretion to levels similar to those optimally expressed by sparse cultures. L-CM did not enhance LPL secretion from J774.1 cells. Dense cultures of both BMM and J774.1 cells did not contain a stable inhibitor of LPL secretion and medium from sparse cultures did not contain an inducer of LPL secretion. The data suggest that proliferating macrophages secrete large amounts of LPL, whereas in nonproliferating, quiescent cells, this activity is much reduced. L-CM enhances LPL secretion in quiescent BMM and peritoneal exudate cells to levels expressed by proliferating cells. Since this effect is already expressed after a 4 h incubation period, it is not dependent on cell cycling but could be one of the early responses to this macrophage mitogen. In J774.1 cells, a change of medium is a sufficient signal for enhancement of LPL secretion in quiescent cells.     

Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor

It is shown that neokyotorphin (the -globin fragment 137–141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h. The results of flow cytometry analysis suggest that the effect of neokyotorphin on survival of L929 cells in serum-free culture medium is due to maintenance of cell proliferation in the absence of growth factors. Along with cell cycle progression the peptide induces reversible reduction of L929 cell size.     

Delayed reproductive death and ROS levels in the progeny of irradiated melanoma cells

The cell death and survival of proliferating (clonogenic) cells were investigated in two human melanoma cell lines to assess the optimal conditions for preparation of apoptotic bodies from melanoma cells. After 50 J/m2 UVB+UVC the maximal levels of apoptotic cells assayed by Trypan blue staining, nucleosomal DNA fragmentation, MTT, and TUNEL tests were observed within 2-3 d of radiation. In 100 Gy gamma-irradiated cultures these apoptosis indicators were delayed for up to 3 weeks. In addition, clonogenic cells were observed only in exponentially growing cultures irradiated with UV at high cell density but not in gamma-irradiated cultures. The response of melanoma cultures after high UV radiation doses contrasted to the response in lethally gamma-irradiated cultures. UV-irradiated melanoma cultures were recovered within two weeks. Most of the clonogenic cells in the recovered colonies contained micronuclei. ROS levels determined by DCF fluorescence and a modified MTT test were also normalized obviously due to the extensive antioxidant defense system of melanoma cells. UV radiation of tumor cells might be the preferential method for preparation of apoptotic bodies. The presence of clonogenic cells in the suspension of apoptotic bodies from melanoma cells used for pulsing of dendritic cells with tumor antigens might compromise this protocol for preparation of cell vaccines.     

In vitro culture of embryonic cells from the shrimp, Penaeus chinensis

Studies were carried out in our laboratory to develop cell cultures from embryonic tissue of Penaeus chinensis. Good yields of dissociated, uncontaminated, viable cell suspensions were obtained by physical disruption of harvested embryonic tissues. Primary cultures in the form of proliferating foci of cells were readily initiated using MPS medium (osmolality: 2.4%) with 20% heat-inactivated fetal bovine serum. Insulin-like growth factor-II (IGF-II) and basic fibroblast growth factor (bFGF) at different concentrations were also added into the culture medium. Passage of primary cultures resulted in rapid proliferation and good adherence of lymphocytes in the presence of IGF-II at 80 ng/ml and bFGF at 20 ng/ml. Cells maintained in subculture for up to 10 passages still had good cellular morphology and division rates.     

A Comparative Study of Protocols for Mouse Embryonic Stem Cell Culturing

Most stem cell laboratories still rely on old culture methods to support the expansion and maintenance of mouse embryonic stem (ES) cells. These involve growing cells on mouse embryonic fibroblast feeder cells or on gelatin in media supplemented with fetal bovine serum and leukemia inhibitory factor (LIF). However, these techniques have several drawbacks including the need for feeder-cells and/or use of undefined media containing animal derived components. Culture of stem cells under undefined conditions can induce spontaneous differentiation and reduce reproducibility of experiments. In recent years several new ES cell culture protocols, using more well-defined conditions, have been published and we have compared the standard culture protocols with two of the newly described ones: 1) growing cells in semi-adherence in a medium containing two small molecule inhibitors (CHIR99021, PD0325901) and; 2) growing cells in a spheroid suspension culture in a defined medium containing LIF and bFGF. Two feeder-dependent mouse ES (mES) cell lines and two cell lines adapted to feeder-independent growth were used in the study. The overall aim has not only been to compare self-renewal and differentiation capacity, but also ease-of-use and cost efficiency. We show that mES cells when grown adherently proliferate much faster than when grown in suspension as free-floating spheres, independent of media used. Although all the tested culture protocols could maintain sustained pluripotency after prolonged culturing, our data confirm previous reports showing that the media containing two chemical inhibitors generate more pure stem cell cultures with negligible signs of spontaneous differentiation as compared to standard mES media. Furthermore, we show that this medium effectively rescues and cleans up cultures that have started to deteriorate, as well as allow for effective adaption of feeder-dependent mES cell lines to be maintained in feeder-free conditions.     

Regulation of beta-adrenergic receptor expression in rat liver

To begin defining the factors regulating neurotransmitter receptor expression, we examined beta-adrenergic receptors in rat liver in vivo and in primary liver cultures under defined hormonal conditions. beta-receptors described a remarkable developmental profile in vivo, increasing fivefold between embryonic days 16 and 20, and decreasing tenfold by early adulthood. The developmental decrease reflected reduced receptor number without a change in receptor properties. The ontogenetic decrease appeared to be specific for beta-receptors; alpha-receptors developed in a hyperbolic fashion, reaching high plateau values by the third postnatal week. Adult rat liver cells plated into culture re-expressed high beta-receptor levels, exhibiting a 4-8-fold increase. A similar pattern of expression of the beta-receptors, having similar pharmacological properties, was observed in primary liver cultures maintained in serum-free medium, in a serum-supplemented medium or in several variations of a serum-free, hormonally defined medium designed for primary liver cultures. Thus, the degree of expression of the beta-receptors was not found affected by various hormones, by serum, or by any medium condition. By contrast, the degree of expression of the beta-receptors was markedly sensitive to cell density. High expression of the beta-receptors was observed at low cell densities (1-3 x 10(6) cells/150 mm dish), and low expression or no expression was observed in confluent cultures (10-20 x 10(6) cells/150 mm dish). Our experiments suggest that beta-receptor expression does not follow an immutable program, but may be regulated by density-dependent cell-cell interactions.     

Trypanosoma brucei brucei: In Vitro Production of Metacyclic Forms

ABSTRACT An in vitro method has been established to obtain metacyclic form populations of Trypanosoma brucei brucei . Trypanosome populations containing more than 98% of metacyclic forms were obtained from cultures which were: 1) initiated with bloodstream forms in primary cultures in the presence of Microtus montanus embryonic fibroblast-like cells (feeder cell layers); 2) maintained in glucose-free Eagle's minimum essential medium supplemented with 10 mM L-proline, 2 mM L-glutamine and 20% (v/v) fetal bovine serum at 27° C without medium change for five days; 3) subcultured in the absence of the feeder cell layers but in the presence of Cytodex 3 beads; 4) maintained for an additional nine days with medium changes on days 5, 8 and 11; and 5) harvested on day 14 by means of diethylaminoethyl cellulose column chromatography prior to the appearance of other infective forms. Most of the trypanosomes obtained under these conditions were morphologically similar to metacyclic forms derived from tsetse fly vectors, coated with variable surface glycoprotein and were infective for mice. In the primary cultures procyclic forms, epimastigotes and metacyclic forms appeared by day 8. When the duration of the subculture was prolonged to 17 days or more at 27° C, the metacyclic forms decreased in number while short trypomastigotes, long slender epimastigotes, and long slender trypomastigotes increased in number. These forms in such long-term cultures also appeared in diethylaminoethyl cellulose-isolated populations along with metacyclic forms.     

Trypanosoma brucei brucei: in vitro production of metacyclic forms.

An in vitro method has been established to obtain metacyclic form populations of Trypanosoma brucei brucei. Trypanosome populations containing more than 98% of metacyclic forms were obtained from cultures which were: 1) initiated with bloodstream forms in primary cultures in the presence of Microtus montanus embryonic fibroblast-like cells (feeder cell layers); 2) maintained in glucose-free Eagle's minimum essential medium supplemented with 10 mM L-proline, 2 mM L-glutamine and 20% (v/v) fetal bovine serum at 27 degrees C without medium change for five days; 3) subcultured in the absence of the feeder cell layers but in the presence of Cytodex 3 beads; 4) maintained for an additional nine days with medium changes on days 5, 8 and 11; and 5) harvested on day 14 by means of diethylaminoethyl cellulose column chromatography prior to the appearance of other infective forms. Most of the trypanosomes obtained under these conditions were morphologically similar to metacyclic forms derived from tsetse fly vectors, coated with variable surface glycoprotein and were infective for mice. In the primary cultures procyclic forms, epimastigotes and metacyclic forms appeared by day 8. When the duration of the subculture was prolonged to 17 days or more at 27 degrees C, the metacyclic forms decreased in number while short trypomastigotes, long slender epimastigotes, and long slender trypomastigotes increased in number. These forms in such long-term cultures also appeared in diethylaminoethyl cellulose-isolated populations along with metacyclic forms.     

Medium composition for effective slow freezing of embryonic cell lines derived from marine medaka (Oryzias dancena)

This study was conducted to identify optimal medium composition for freezing Oryzias dancena embryonic cell lines. Different freezing media consisting of various concentration of dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), and trehalose were prepared and long-term cultured embryonic cell line was frozen in each freezing medium by conventional slow freezing program for 7 days. Through measurement of viability and growth of post-thaw cells frozen in each freezing medium, it was determined that optimal composition of three components was 10 % DMSO, 20 % FBS, and 0.1 M trehalose. The post-thaw cells frozen in optimal freezing medium showed similar morphology and growth rate with non-frozen cells. Next, this condition was applied to two different sets of experiment; (1) freezing of the same cells during expanded period (57 days) and (2) freezing of short-term cultured cells from other batches for 7 days. The viability of post-thaw cells was significantly low and comparable in set 1 and 2, respectively, when compared with the result of long term-cultured cells frozen in optimal freezing medium for 7 days and similar morphology and growth rate with non-frozen counterparts were detected in the post-thaw cells from both sets. In conclusion, this study first reports the optimal medium composition for freezing O. dancena embryonic cells, which can contribute to fish species preservation as well as improvement of cell-based biotechnology by providing stable cell storage.     

Development of highly nutritive culture media

Summary A highly nutritive culture medium (MGM-464) was developed for insect cell primary culture. The new medium consists of 6 inorganic salts, 4 organic acids, 21 amino acids, 3 sugars, 10 vitamins, and 8 other chemicals, including natural substances. The complete medium was generated by adding 20 ml fetal bovine serum to 100 ml MGM-464. The detail of the composition of the medium is given in a table, and the protocol to prepare the medium is described in the text. Among the 15 kinds of cultures made with MGM-464, embryonic cells from a walking stick and ovarian cells from the common white were subcultured more than 70 times, and embryonic cells of a chrysomelid beetle were subcultured more than 15 times. Other cultures could not be subcultured. However, embryonic cells from the commercial silkworm and a cockroach, ovarial cells from the commercial silkworm and a sphingid moth, nervous cells from the commercial silkworm and two sphingid moths, and cells from the dorsal vessel plus surrounding tissue of the commercial silkworm survived for several mo. The cells from the honeybee embryos, aphid embryos, and planthopper embryos were rather short-lived, and deteriorated after about 1 mo.     

Conditions for initiating Lake Victoria haplochromine (Oreochromis esculentus) primary cell cultures from caudal fin biopsies

The global decline of freshwater fishes has created a need to cryopreserve biological materials from endangered species in an effort to conserve the biodiversity within this taxon. Since maternal gametes and embryos from fish are difficult to cryopreserve, somatic cells obtained from caudal fins have become an increasingly popular resource as they contain both maternal and paternal DNA ensuring valuable traits are not lost from the population. Somatic cells stored in cryobanks can be used to supplement endangered populations with genetically valuable offspring with the use of assisted reproductive technologies. However, initiating primary cell cultures from caudal fin biopsies of endangered species can be challenging as standardized protocols have not yet been developed. The objective of this study was to identify culture conditions, including antibiotic supplementation, biopsy size, and culture temperature, suitable for establishing primary cell cultures of ngege (Oreochromis esculentus), a critically endangered African cichlid. Six-millimeter caudal fin biopsies provided sufficient material to develop a primary cell culture when incubated at 25°C using standard fish cell culture medium containing 1× Primocin. Further investigation and application of these culture conditions for other endangered freshwater fishes is necessary.     

Derivation of a pluripotent embryonic cell line from red sea bream blastulas

A pluripotent cell line, sea bream embryonic stem‐like cells (SBES1), was developed from blastula‐stage embryos of the cultured red sea bream, Chrysophrys major . The SBES1 cells were cultivated in Dulbecco's modified eagles medium (DMEM) medium supplemented with foetal bovine serum, marine fish serum, fish embryo extract, selenium, basic fibroblast growth factor and leukemia inhibitory factor. They were small and round or polygonal, and grew actively and stabely in culture. The cells exhibited a positive alkaline phosphatase activity upon histochemical staining. When the cells were treated with all‐ trans retinoic acid, they differentiated into various types including neuron‐like, neuroglia‐like and muscle‐like cells, suggesting that the SBES1 cells remained pluripotent in culture. Chromosome analysis revealed that SBES1 cells had a normal diploid karyotype with 2 n  = 2 st  + 46 t . At present, SBES1 cells have been cultured for >180 days with more than 60 passages. High survival rate has been obtained after cryopreservation of cell cultures. This embryonic cell line may potentially be used for the production of transgenic red sea bream.     

Explanted Fish Neural Tubes Give Rise to Differentiating Neural Crest Cells

Neural tubes were explanted from the trunk of various embryonic stages of three teleost fish, Xiphophorus maculatus (platyfish), X. helleri (swordtail), and Oryzias latipes (Japanese medaka) with the aim to obtain in vitro differentiating neural crest cells. Outgrowth of cells was observed immediately after attachment of the explants on dishes coated with fibronectin. The outgrowing cells stained with the HNK-1 monoclonal antibody indicating that they were neural crest cells. Maximum cell outgrowth was obtained from explants of stage 9 of Xiphophorus and 19 of medaka, i.e., from stages characteristic of maximal neural crest cell segregation, and by the use of Leibovitz's (L-15) medium supplemented with 20% FBS. In this medium cells survived for more than two weeks; M199 also gave satisfactory results but DMEM allowed only poor cell growth and survival. Neuronal cells could be observed in all cultures after 48 hr, in some medaka cultures these cells were mixed with pigment cells but homogeneous pigment cell cultures were also observed. This in vitro system will be invaluable for the study of the developmental potential of fish neural crest cells and the contributions of extrinsic factors in neural crest cell fate.     

Differences in nutrient uptake between the fat body and embryonic primary cultures of silkworm （Bombyx mori）

Nutrition utilization and by-product formation in cultured insect cells has been investigated in several insect cells and has been of great interest to cell culturists and physiologists. In this research the biochemical changes in embryonic and fat body primary cultures of silkworm, Bombyx mori, have been compared. TC-100 medium supplemented with 10% and 20% FBS was used in embryonic and fat body primary cultures, respectively. Medium was renewed every week and the amount of glucose, uric acid, urea, total protein and alkaline phosphatase were measured in the samples from medium of primary cultures using spectrophotometeric methods. All biochemical macromolecules except uric acid showed significant changes. Glucose decreased in embryonic tissues, while in fat body culture its amount increased. Urea accumulation in embryonic culture was higher than in the fat body cultures. Since urea is a by-product, this accumulation could be due to higher utilization of amino acids. Total protein showed considerable changes and was consumed by embryonic culture more than the fat body＇ s. Alkaline phosphatase showed stronger activity in embryonic cells.     

Optimized mouse ES cell culture system by suspension growth in a fully defined medium

Mouse and human embryonic stem (mES and hES) cells have become one of the most intensively studied primary cell types in biomedical research. However, culturing ES cells is notoriously labor intensive. We have optimized current ES cell culture methods by growing mES cells in suspension in a defined medium. This protocol is unsurpassed in time efficiency and typically requires only 20 min of effective hands-on time per week. This protocol maintains a very high degree of pluripotent cells partly by mechanical separation of spontaneously differentiating cells. mES cells can be cultured for extended periods (>6 months) without the loss of pluripotency markers. High passage (>20) adherent mES cultures containing contaminating differentiated cells can be rescued and enriched in undifferentiated ES cells.     

Viability of dopaminergic neurones is influenced by serum and astroglial cells in vitro

Transplantation of embryonic nigral grafts into the striatum of Parkinson's disease patients is not optimal, mainly due to low survival of grafted neurones. Current strategies focus on enhancing neuronal survival by transplanting enriched neuronal cell populations. There is growing evidence for the importance of astroglia in neuronal survival.To characterise the effects of glial cells on dopaminergic neurones, 5-fluoro-2′-deoxyuridine was added to embryonic rat ventral mesencephalic cultures in the presence or absence of serum. The survival and morphology of glial fibrillary acidic protein immunopositive astroglia and tyrosine hydroxylase immunopositive dopaminergic neurones was examined. In serum-containing medium, astroglial cells predominated and 5-fluoro-2′-deoxyuridine had no significant effect on either astroglia or dopaminergic neurone survival. In serum-free medium, astroglial growth was attenuated and numbers were significantly lower in 5-fluoro-2′-deoxyuridine treated compared with untreated cultures. There was no significant difference in the numbers of dopaminergic neurones between 5-fluoro-2′-deoxyuridine treated and untreated cultures. However, by the eighth day in vitro, there were differences in the morphology of these neurones between treated and untreated cultures. This study shows that the use of 5-fluoro-2′-deoxyuridine and serum-free medium can produce a neurone-enriched culture. However, the dopaminergic neurone population present in these cultures appeared to be morphologically dissimilar to those found in control cultures as neurites were retracted and the cell somas of these cells appeared enlarged. These results provide information on the effects of astrocytes on dopaminergic neurones in ventral mesencephalic cultures and thus have implications for transplantation in Parkinson's disease.     

Viral transgenesis of embryonic cell cultures from the freshwater microcrustacean Daphnia

The aim of this study was to determine whether a recombinant vesicular stomatitis virus (VSV) vector encoding a transgene could be used to infect and express a foreign gene in embryonic primary cell cultures derived from the freshwater microcrustacean Daphnia, the most widely used ecotoxicological model organism. To facilitate the evaluation of gene transfer, a reproducible method for establishing primary cultures from Daphnia embryonic tissues was developed. Within 24 hr after infection, transgene expression could be detected in cell culture. VSV was found to replicate in the cells with no apparent cytopathic effect. Here we report the first evidence of gene transfer and foreign gene expression in cultures of Daphnia embryonic cells using a recombinant viral vector.     

A Cell Line (HEW) from Embryos of Haddock (Melanogrammus aeglefinius) and Its Capacity to Tolerate Environmental Extremes

Cell lines can be useful experimental tools for studying marine fish, which are often difficult to routinely obtain and maintain in the laboratory. As few cell lines are available from coldwater marine fish, cultures were initiated from late gastrula embryos of haddock (Melanogrammus aeglefinus) in Leibovitz's L-15 with fetal bovine serum (FBS). From one culture, a cell line (HEW) emerged that has been grown for close to 100 population doublings, was heteroploid, and expressed telomerase activity, all of which suggest HEW is immortal. Growth occurred only if FBS was present and was optimal at 12 to 18°C. Usually most cells had an epithelial-like morphology, but under some conditions, cells drew up into round central bodies from which radiated cytoplasmic extensions with multiple branches. These neural-like cells appeared within a few hours of cultures being placed at 28°C or being switch to a simple salt solution (SSS). At 28°C, cells died within 24 h. In SSS, HEW cells survived as a monolayer for at least 7 days. The sensitivity of HEW cells to morphological change and their capacity to withstand starvation should make them useful for investigating cellular responses to environmental stresses.