A method for growing stationary plant suspension cell cultures

Summary Stationary culture of plant cell suspensions has been achieved. Slurries, produced when small amounts of agar (0.1–0.4%) were added to culture media, were used to suspend plant cells. Growth proceeded more slowly than in standard shake culture, but cells remained viable for months of culture. This method of growing plant cells in stationary culture should be useful for general applications including long-term cell culture, shipment of cultures, and physiological, molecular biological, and pathological studies. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable. Editor’s Statement This procedure for growing stationary suspension cultures in an agar slurry should be useful for shipping suspensions and for long-term maintenance of little used or back-up cultures.     

Isolation, culture and immortalisation of hepatic oval cells from adult mice fed a choline-deficient, ethionine-supplemented diet

Oval cells have great potential for use in cell therapy to treat liver disease, however this cannot be achieved until the factors which govern their proliferation and differentiation are better understood. We describe a method to establish primary cultures of murine oval cells, and the derivation of two novel lines from these. Primary cultures from the livers of wildtype or TAT-GRE lacZ transgenic mice subjected to a choline-deficient, ethionine-supplemented diet comprised up to 80% oval cells at day 7 based on A6 or CK19 staining. Cell lines were clonally derived, which underwent spontaneous immortalisation following prolonged maintenance in culture. Immunostaining and RT-PCR demonstrated they express hepatocytic and biliary markers and they were therefore termed “bipotential murine oval liver” (BMOL) cells. Under proliferating culture conditions, BMOL or BMOL-TAT cells abundantly expressed oval cell and biliary markers, whereas mature hepatocytic markers were upregulated when the growth conditions were changed to facilitate differentiation. Hepatic differentiation of BMOL-TAT cells could be traced by measuring the expression of their lacZ transgene, which is driven by a promoter element from tyrosine aminotransferase (TAT), a marker of adult hepatocytes. Interestingly, haematopoietic markers were upregulated in superconfluent cultures, indicating a possible multipotentiality. None of the cell lines grew in semi-solid agar, nor did they form tumours in nude mice, suggesting they are non-tumourigenic.

These novel murine oval cell lines, together with a reliable method for isolation and culture of primary oval cells, will provide a useful tool for investigating the contribution of oval cells to liver regeneration.     

Comparative antibiotic eradication of mycoplasma infections from continuous cell lines

Accumulating data implicate mycoplasma contamination as the single biggest problem in the culture of continuous cell lines. Mycoplasma infection can affect virtually every parameter and functional activity of the eukaryotic cells. A successful alternative to discarding infected cultures is to attempt to eliminate the contaminants by treatment with specific and efficient antimycoplasma antibiotics. The addition of antibiotics to the culture medium during a limited period of time (1-3 wk) is a simple, inexpensive, and very practical approach for decontaminating continuous cell lines. Here, we examined the effectiveness of several antibiotic treatment protocols that we have employed routinely in our cell lines bank. On an aggregate, 673 cultures from 236 chronically mycoplasma-positive cell lines were exposed to one of the following five antibiotic regimens: mycoplasma removal agent (quinolone; a 1-wk treatment), enrofloxacin (quinolone; 1 wk), sparfloxacin (quinolone; 1 wk), ciprofloxacin (quinolone; 2 wk), and BM-Cyclin (alternating tiamulin and minocycline; 3 wk). The mycoplasma infection was permanently (as determined by three solid mycoplasma detection assays) eliminated by the various antibiotics in 66-85% of the cultures treated. Mycoplasma resistance was seen in 7-21%, and loss of the culture as a result of cytotoxically caused cell death occurred in 3-11% of the cultures treated. Overall, 223 of the 236 mycoplasma-positive cell lines could be cured in a first round of antibiotic treatment with at least one regimen. Taken together, 95% of the mycoplasma-infected cell lines were permanently cleansed of the contaminants by antibiotic treatment, which validates this approach as an efficient and technically simple mycoplasma eradication method.     

Tissue culture of endod (Phytolacca dodecandra L'Herit): growth and production of ribosome-inactivating proteins

Summary Leaves and stems from endod (Phytolacca dodecandra L'Herit), known to produce the 29 kDa ribosome-inactivating protein (RIP) dodecandrin, were initiated into tissue culture. Callus and suspension cultures were maintained on modified Murashige and Skoog medium plus 1.0 mg/l 2,4-dichlorophenoxyacetic acid. Six callus and two suspension cell lines were screened for dodecandrin production by western blots with affinitypurified antiserum. Antiribosomal activity of culture extracts was tested by in vitro translation assays. One suspension cell line was found to be free of immunoreactive proteins and a ribosome inhibitor. All other cell lines contain a ribosome inhibitor, although only two callus cell lines show detectable amounts of immunoreactive proteins at the same M_r as dodecandrin. Other immuno-reactive proteins were detected in callus (M_r 31000, 33000, 41000 and 43000) and in suspension cells (M_r 23000 and 43000), and may be ribosome inhibitors related to dodecandrin—either other RIPs or dodecandrin at various stages of processing.     

Elimination of mycoplasmas from cell cultures by a novel soft agar technique

Summary Mycoplasmal infection of cell cultures remains a significant threat to diagnostic and research procedures. In certain defined situations, curing of mycoplasmal infected cultures is a reasonable exercise. Four methods of curing were compared: treatment with BM-cycline, 5 bromouracil, use of specific antisera and treatment of infected cells suspended in soft agar with antibiotics. Antisera treatments were of low efficiency of curing: 50%. None of nine infected cell lines treated with 5-bromouracil were consistently cured of mycoplasmas. The use of BM-cycline was effective for some, but not all lines and required long periods of treatment, 12–21 days. 35 naturally or deliberately infected cultures were treated in soft agar a total of 119 times. This procedure which consisted of suspending infected cultures in soft agar containing appropriate antibiotics resulted in successful mycoplasmal elimination 118/119 times. This soft agar technique took 1–3 days. In separate studies, it was shown that certainMycoplasma fermentans strains were resisted to this and other curing methods. This may be due to their intracellular location. Such strains may be more amenable to antibiotics that penetrate mammalian cells. It is concluded that the soft agar technique is a rapid, efficient and reliable method to eliminate cell culture mycoplasmas. These studies were supported in part by grant 15748 from the National Institute of Allergy and Infectious Diseases and the W. W. Smith Charitable Trust.     

In vitro induction of pollen embryos and plantlets in Aesculus carnea Hayne through anther culture

Uninuclear microspores in red horse chestnut anther cultures formed pollen embryos and plantlents in MS agar medium supplemented with varying 2,4-D concentrations (1.0, 1.5 or 2.0 mg l^-1) and 1.0 mg l^-1 Kin. The highest number of embryogenic anthers (38%) was obtained in MS medium containing 1.0 mg l^-1 of each 2,4-D and Kin. The ability of pollen embryos to germinate was closely correlated with normal embryo morphology and was influenced by hormone content in the medium (MS+;1.0 mg l^-1 IAA+1.0 mg l^-1 GA₃+0.1 mg l^-1 Kin+400 mg l^-1 glutamine). Pollen embryos and plantlets had the haploid chromosome number (x=n=40). Cytological examinations demonstrated pollen dimorphism of this Aesculus species.Abbreviations AC activated charcoal - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin 6-furfurylaminopurine - GA₃ gibberellic acid - MS Murashige and Skoog     

Factors influencing efficiency of somatic embryogenesis of Gentiana kurroo (Royle) cell suspension

In this paper, we would like to show unexpected morphogenic potential of cell suspensions derived from seedling explants of Gentiana kurroo (Royle). Suspension cultures were established with the use of embryogenic callus derived from seedling explants (root, hypocotyl and cotyledons). Proembryogenic mass proliferated in liquid MS medium supplemented with 0.5 mg l⁻¹ 2,4-D and 1.0 mg l⁻¹ Kin. The highest growth coefficient was achieved for root derived cell suspensions. The microscopic analysis showed differences in aggregate structure depending on their size. To assess the embryogenic capability of the particular culture, 100 mg of cell aggregates was implanted on MS agar medium supplemented with Kin (0.0–2.0 mg l⁻¹), GA₃ (0.0–2.0 mg l⁻¹) and AS (80.0 mg l⁻¹). The highest number of somatic embryos was obtained for cotyledon-derived cell suspension on GA₃-free medium, but the best morphological quality of embryos was observed in the presence of 0.5–1.0 mg l⁻¹ Kin, 0.5 mg l⁻¹ GA₃ and 80.0 mg l⁻¹ AS. The morphogenic competence of cultures also depended on the size of the aggregate fraction and was lower when size of aggregates decreased. Flow cytometry analysis reveled luck of uniformity of regenerants derived from hypocotyl suspension and 100% of uniformity for cotyledon suspension.     

Two new C3H mouse ascites tumor cell lines capable of proliferation in vivo and in suspension culture: Morphological,karyological, kinetic,and immunological properties

Summary The establishment of mouse tumor cell lines capable of proliferating in vivo and in suspension culture was undertaken. The MM-46 tumor line, initiated from primary mammary carcinoma arising in a C3H/He mouse, was maintained for over 100 generations in the peritoneal cavities of syngenic mice. At the 50th generation of the tumor suspension, cultures were initiated. The established cell lines, designated TMT-1 and TMT-2, were characterized in vitro and in vivo. The morphological finding indicated that TMT-1 and TMT-2 cells from mice closely resembled the MM-46 tumor cells. The oncogenic potential of the cultured cells was comparable to that of the original ascites tumor. The population doubling time of TMT-1 and TMT-2 cell lines was about 12 hr in mice, whereas the population doubling time of both cell lines lengthened to 20 hr in suspension culture. The increase of doubling time in culture was due to the prolongation of the G₁ period. The cell lines, TMT-1 and TMT-2, whether from culture or mice, possessed colony forming ability in soft agar medium. The colony forming ability of the cells decreased gradually through in vivo passages but it recovered upon recultivation of the cells from mouse to culture. Chromosome analysis and cytotoxicity test by anti-MM antiserum indicated that TMT-1 and TMT-2 cell lines closely resembled and had been derived from MM-46 tumor line. Therefore, it is possible to assay cell survival in vitro after in vivo experiments on these cells.     

Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines

Mycoplasma contamination of cell lines is one of the major problems in cell culturing. About 15-35% of all cell lines are infected with a limited number of mycoplasma species of predominantly human, swine, or bovine origin. We examined the mycoplasma contamination status in 495 cell cultures by polymerase chain reaction (PCR) assay, microbiological culture method, and deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization, and in 103 cell cultures by PCR and DNA-RNA hybridization, in order to determine the sensitivity and specificity of the PCR assay in routine cell culture. For those two cohorts, results for the three or two assays were concordant in 92 and 91% of the cases, respectively. The sensitivity (detection of true positives) of this PCR detection assay was 86%, and the specificity (detection of true negatives) was 93%, with positive and negative predictive values (probability of correct results) of 73 and 97%, respectively. PCR defined the mycoplasma status with 92% accuracy (detection of true positives and true negatives). The mycoplasma contaminants were speciated by analyzing the PCR amplification fragment using several restriction enzymes. Most of the cultures (47%) were infected with Mycoplasma fermentans, followed by M. hyorhinis (19%), M. orale (10%), M. arginini (9%), Acholeplasma laidlawii (6%), and M. hominis (3%). To sum up, PCR represents a sensitive, specific, accurate, inexpensive, and quick mycoplasma detection assay that is suitable for the routine screening of cell cultures.     

Effects of some chemical factors on flagellation and swarming ofVibrio alginolyticus

Vibrio alginolyticus strains recently isolated from Dutch coastal seawater changed flagellar organization when cultivated in the presence of certain chemical agents. On agar media with more than 4.0% (w/v) NaCl the number of lateral flagella per cell decreased with increasing salt concentration. Both on agar media and in broth cultures with 6.0–9.0% (w/v) NaCl, cells with polar tufts of 2–4 sheathed or unsheathed flagella were frequently found. Cells grown on agar media with 7.3–9.8% (w/v) Na₂SO₄ had drastically reduced numbers of lateral flagella, but lacked polar tufts. EDTA suppressed growth, but did not affect flagellar arrangement. In the presence of 0.1–0.3% boric acid or 0.05–0.1% aluminium hydroxide, cells in liquid media tended to produce lateral, in addition to the polar flagella normally observed in broth cultures. Of a number of surface-active agents tested, Tween 80 and Na-taurocholate, even in high concentrations, did not affect flagellation. Bile salts (0.1%) and Na-deoxycholate (0.05%) strongly reduced the number of both polar and lateral flagella. In agar cultures, Na-lauryl sulphate (0.01–0.1%) inhibited the formation of lateral, but increased the incidence of polar flagella. Teepol (0.05–0.2%) had a similar effect and also it had a deteriorating effect on the sheaths of the polar flagella. Concomitant with the reduction in the number of lateral flagella, induced by these agents, swarming on agar media was inhibited.     

Production of molecular hydrogen with immobilized cells ofRhodospirillum rubrum

Summary Cells ofRhodospirillum rubrum have been immobilized in various gels and tested for photobiological hydrogen production. Agar proved to be the best immobilizing agent with respect to production rates as well as stability. Agar immobilized cells were also superior compared to liquid suspension cultures. Growth conditions of the cells prior to immobilization, e.g. cell age, light intensity or nutrient composition, were of primary importance for the activity in the later immobilized state. A reactor with agar immobilized cells has been operated successfully over 3000 h with a loss of the activity of about 60%. Mean rates for hydrogen production for immobilized cells in this work during the first 60 to 70 hours after immobilization were in the range of 18 to 34 μl H₂ mg⁻¹ d.w. h⁻¹ and thus by a factor of up to 2 higher than liquid cultures under the same conditions. Maximal rates of hydrogen production (57 μl H₂ ml⁻¹ immobilized cell suspension) were reached in agar gel beads with cells immobilized after 70 h growth in liquid culture in the light and a cell density of 1.0 mg ml⁻¹, 70 h after immobilization.     

Neoplastic potential of rat tracheal epithelial cell lines induced by 1-nitropyrene and dibenzo(a,i)pyrene.

Our previous study showed that both 1-nitropyrene (1-NP) and dibenzo(a,i)pyrene (DBP) induced enhanced growth variants (EGVs) in primary cultures of rat tracheal epithelial (RTE) cells exposed in vivo. Cell lines were established from some of the EGVs. Further studies, using anchorage-independent growth in soft agar and tumorigenicity in athymic nude mice, were performed to determine the neoplastic potential of EGVs induced by 1-NP and DBP. Results show that three of five from DBP- and five of five from 1-NP-induced cell lines displayed anchorage-independent growth. The colony forming efficiency (CFE) from DBP-induced cell lines was 0.067 per thousand and CFE from 1-NP-induced cell lines was 0.151 per thousand. There is a significant difference between the two CFEs (mu = 12.08, P<0. 01). Two of five DBP- and five of five 1-NP-induced cell lines produced squamous cell carcinomas (SCC) in nude mice. The rate of tumorigenicity counted by injected sites was 20% (6/30) for DBP-induced cell lines and 57% (17/30) for 1-NP-induced cell lines. There is a significant difference between the results of tumorigenicity from the cell lines induced by the two different compounds (chi(2)=8.53, P<0.01). Neither of the two cell lines from spontaneously developed foci grew in soft agar or produced SCC in nude mice. It seems that the neoplastic potential of transformed RTE cells induced by 1-NP was higher than that of DBP.     

Toxicity of parathion-methyl to cells, akinetes and heterocysts of the cyanobacterium Cylindrospermum, sp. and the probit analysis of toxicity

The toxicity of a commercial formulation of the insecticide parathion‐methyl to the N2‐fixing filamentous cyanobacterium (blue‐green alga) Cylindrospermum, sp. was studied. A concentration of parathion‐methyl of 0.5 ppm caused growth increase in liquid growth media. The minimum inhibitory concentration of parathion‐methyl for both types (N₂, fixing and nitrate supplemented) of liquid and solid media was 1.0 ppm. LC₅₀ values were: 4.4 ppm (liquid, N₂, fixing), 5.5 ppm (liquid, nitrate supplemented), 3.3 ppm (agar, N₂‐fixing) and 4.0 ppm (agar, nitrate supplemented). LC100 values for N₂‐fixing liquid and both types of agar media were 10.0 ppm, while for the liquid nitrate supplemented medium the LC₁₀₀ was 12.0 ppm. Both akinete (spore) formation and germination were inhibited below the highest permissive concentration of 8.0 ppm, with the insecticide incorporated in the agar media. In soil, the LC50 and LC100 values for parathion‐methyl were 13.6 and 30 ppm, respectively. Both the dehydrogenase activity of heterocysts (monitored by 2,3,5‐triphenyl tetrazolium chloride reduction) and the nitrogen concentration of cultures (estimated by the micro‐Kjeldahl method) were affected by the insecticide, but the latter (N₂‐fixation) was more sensitive. The Kruskal‐Wallis H test on the numbers of vegetative cells in the filaments revealed that the insecticide significantly affected the division of vegetative cells. The cyanobacterium could detoxify the growth medium containing high levels (30 and 40 ppm) of the insecticide in short‐term exposures at the expense of cell viability.     

Carbon Dioxide as an Essential Requirement for Cultured Sycamore Cells

Carbon dioxide (optimum concentration c. 1.0%) is essential to the initiation of the growth in suspension culture or on agar plates of cultured sycamore cells. By effective flushing of the cultures with CO₂-free air it is possible to demonstrate this requirement with initial cell densities up to 50 × 10³ cells ml^?1. This growth-promoting activity of carbon dioxide is not related to any effect it may have on the pH of the culture medium. The cells fix applied carbon dioxide into organic and amino acids but attempts to replace the carbon dioxide requirement by non-toxic levels of organic or amino acids have not been successful.     

Sodium Valproate Facilitates the Propagation of Granulocytic Ehrlichiae (Anaplasma phagocytophilum) in HL-60 Cells

As already shown, some inducers of the differentiation of promyelocytic cells along the granulocytic pathway, such as dimethylsulphoxide (DMSO) or all-trans retinoic acid, can enhance propagation of granulocytic ehrlichiae in HL-60 cell cultures. This study was conducted to prove whether sodium valproate, a salt of di-n-propylacetic acid (VPA) known to trigger cellular differentiation in several solid and hematopoietic malignancies is similarly efficient in ehrlichial cultures. Two cell lines derived from HL-60, that is, low-passage undifferentiated HL-60 (HL-60F) and high-passage HL-60 spontaneously differentiated towards monocytic phenotype (HL-60J) were grown in RPMI 1640 medium supplemented with 10% FBS. The respective HL-60F and HL-60J IC₅₀-values for NaVPA were estimated to be 0.8 and 2.2 mM under these culture conditions; to stimulate the differentiation, the respective doses of 0.3 and 1.2 mM were then applied. When the NaVPA-treated cells of both lines were challenged with an ehrlichial laboratory strain (HGE), maintained in splenectomized NMRI mice, the respective 1–2 and ≤0.1% primary infection rates in HL-60F and HL-60J cultures were observed 3 days post-inoculation. In comparison, only rare (≤0.1%) infected HL-60F and no infected HL-60J cells were recorded under the same experimental conditions in untreated control cultures. HGE continuously propagated in NaVPA-supplemented HL-60F cultures remained infectious to mice at least up to the 95th passage (12 months). NaVPA can thus facilitated continuous propagation of granulocytic ehrlichiae in cell cultures without a substantial loss of infectiveness. This revised version was published online in July 2006 with corrections to the Cover Date.     

Somatic embryogenesis in Vicia faba L.

The paper describes a method of somatic embryo induction in callus and suspension cultures of Vicia faba L. Callus was induced from immature cotyledons (green maturity stage) of white-flowering horse bean lines cultured on L2 medium (Phillips and Collins 1979) supplemented with 1% sucrose, 0.7% agar and different concentrations of 2,4-dichlorophenoxyacetic acid. The medium with 2.5 M 2,4-Dichlorophenoxyacetic acid was found optimum for embryogenic callus induction. Somatic embryos developed after transfer of the callus to media lower or zero 2,4-Dichlorophenoxyacetic acid and increased level of sucrose (2.5%). The release of somatic embryos from the callus was more apparent after transfer to liquid medium. There were various stages of somatic embryo development, i.e. globular, heart-shaped and torpedo ones.     

Survival of human lymphoblastoid cells after DNA damage measured by growth in microtiter wells

Survival of cells in suspension culture after treatment with damaging agents is usually measured by extrapolation from growth curves or by growth of colonies in soft agar. We have developed a survival assay which measures the ability of small numbers of cells to initiate microscopic cultures in wells of microtiter plates without agar or feeder layers. Suitable human lymphoblastoid lines were obtained by selection of rapidly growing cultures from microtiter wells in which <200 cells were inoculated in 0.2 ml RPMI 1640 medium and incubated at 37° with 5% CO₂ at 95% relative humidity. Survival after damage was measured by inoculating groups of 24 microtiter wells with appropriate serial dilutions of cells. The wells were examined microscopically at intervals and scored for evidence of cell proliferation. Survival was calculated with the Poisson formula on the basis of the fraction of wells in which cells were not proliferating. Survival did not change appeciably after 2–3 weeks incubation. Survival measured by the microtiter-well assay was found to be similar to survival measured by extrapolation from growth curves after damaging the cells with bleomycin or with 8-methoxypsoralen plus long-wavelength ultraviolet radiation. The microtiter-well assay affords a simple, accurate measure of cell survival in human lymphoblastoid cells with suitable growth ability.     

Hormone-defined cell system for studying G-protein coupled receptor agonist-activated growth modulation: δ-Opioid and serotonin-5HT2C receptor activation show opposite mitogenic effects

G-protein-coupled receptor (GPCR) agonist-activated transformation of NIH/3T3 fibroblast cells has been documented by many workers. Our present interest is in the growth control exerted by these agonists. The mechanisms involved in GPCR agonist-activated growth regulation are not known and investigations using existing cell lines are complicated by the endogenous expression of numerous different GPCRs as well as by the fact that these cell lines are cultured in serum that contains naturally occurring agonists for these receptors. To study the agonist induced growth response of cells transfected with either δ-opioid or serotonin-5HT_2C neurotransmitter receptor genes, we have developed new clonal cell lines derived from NIH/3T3 mouse fibroblast cells. These new cell lines, designated with the suffix 3T3DA, can be cultured stably in serum-free, hormone-defined medium: insulin is the only exogenous growth factor added to the culture medium of proliferating 3T3DA cell lines, and their proliferation can be stopped and started by the respective removal or addition of insulin. Micromolar concentrations of agonists were used to activate the corresponding opioid and serotonin receptors over periods extending to 6 days. We observed distinct patterns of GPCR-specific, agonist-activated growth regulation in serum-free cultures, but not in serum-supplemented cultures. At concentrations > 10 μM, morphine inhibits growth of δ-opioid receptor-expressing cells by 40% with respect to normal 3T3DA cells. Opioid agonist induced inhibition of cyclic AMP (cAMP) production as well as growth down-regulation are pertussis toxin sensitive indicating that the exogenously expressed δ-opioid receptors demonstrate classical opioid receptor signaling. The presence of 1 μM serotonin stimulates growth of serotonin-5HT_2C receptor-expressing cells by approximately 100% with respect to normal 3T3DA cells. Neither the untreated nor the agonist-treated cells form colonies in soft agar, indicating that they retain anchorage-dependent growth control. These cell lines provide a simple system that could be used as a tool for probing the complex molecular mechanisms associated with GPCR agonist-activated growth control. J. Cell. Physiol. 171:61–74, 1997. © 1997 Wiley-Liss, Inc.     

Phenotypic diversity in cultured cerebral microvascular endothelial cells

Summary Diversity exists in both the structure and function of the endothelial cells (EC) that comprise the microvasculature of different organs. Studies of EC have been aided by our ability to first isolate and subsequently establish cultures from microvascularized tissue. After the isolation of microvessel endothelial cells (MEC) derived from rat cerebrum, we observed morphologic differences in colonies of cells that grew in primary cultures. The morphologies ranged from a cobblestone phenotype considered typical of EC in culture to elongated and stellate cell appearances. Serially passaged cell lines were established based on two parameters: initially by growth and, second, on differences in primary colony morphology using selective weeding techniques. Each culture was examined for the presence of EC-characteristic markers which include Factor-VIII-related antigen, angiotensin-I-converting enzyme activity, collagen type IV synthesis, and PGI₂ production. Variable expression of each of these characteristics among the established EC lines was observed. Growth curves established for each of the EC cultures demonstrated differences in both population doubling rates and cell densities at confluence. The endocytic capacity of each EC line was also evaluated. Our ability to isolate and establish a number of morphologically distinct EC cultures indicates that diversity exists within the EC that comprise the cerebral microvasculature. Diversity in the established cell lines suggests either the EC that line the brain microvasculature exist as a mosaic or that morphologically distinct cultures may originate from different microanatomical origins (arteriolar, true capillary, or venular) or may have resulted from cells at different points in their in vitro life spans at the time of isolation. This research was supported by grants HLO3227 and HLO1514 from the National Institutes of Health, Bethesda, MD.     

A serum-free medium that supports the growth of piscine cell cultures

Summary An undefined, serum-free medium was developed for use with fish cell cultures. Lactalbumin hydrolyzate, trypticase-soy broth, Bacto-peptone, dextrose, yeastolate, and polyvinylpyrrolidone were initially combined in 100 ml of distilled H₂O, autoclaved, and added to 5% of the final volume of Medium 199. In addition, filter sterilized bovine pancreatic insulin, glutamine, and nonessential amino acids were added to the medium. The addition of insulin was observed to be unnecessary. Five fish cell lines [goldfish-derived CAR cells, fathead minnow (FHM) cells, epithelioma papillosum cyprini (EPC) cells, chinook salmon embryo (CHSE-214) cells, and a new cell line from goldfish air bladders (ABIII)] were all capable of growth in the serum-free medium at rates equivalent to cells grown in fetal bovine serum (FBS). The morphology of all cell lines, except CHSE-214 cells, was identical to cells grown in FBS. All cell lines were capable of long-term growth in the serum-free medium. The CAR, ABIII, EPC, and CHSE-214 cells in the serum-free medium supported the replication of goldfish virus-2 at levels equivalent to cells grown in FBS.