Normal and pseudorabies virus infected primary nerve cell cultures in scanning electron microscopy

Primary cell cultures from the central nervous system of the embryonic rat were inoculated with pseudorabies virus. Their morphological changes were studied by phase contrast microscopy and by scanning as well as by transmission electron microscopy. Uninfected cultures display two distinct cell layers in scanning electron microscopy: a flat continuous monolayer supports a heterogeneous population of individual, presumably neural cells, which emit processes of different number and size. The latter cells form contacts by a dense network of fibres.Infectious virus is propagated in these nerve cell cultures with similar effectivity as in other cultures. The infection leads to fusion and death of the cells. By the time the cytopathic effect is visible, nearly all cells, including those of neuronal and those of nonneuronal appearance, are studded with ample amounts of virus-sized particles. The particles represent viruses as demonstrated by transmission electron microscopy or by treatment with a hyperimmune serum directed against pseudorabies virus structural components. Hyperimmune serum leads to clustering of the particles at the cell surface. The amount of virus particles per surface unit was about 10 times higher on neural cells as compared to primary rabbit kidney cells. The concentration of infectious particles in the supernatant, however was approximately the same. The system described appears to be useful for the study of acute virus effects on neural tissue under strictly controlled conditions.Part of this work will be presented in the thesis of U. BijokWork was supported by Sonderforschungsbereich 47 and the Bundesministerium des Inneren.     

Preparation of primary cell cultures from lamprey

The lamprey is an important model for studies of evolution and comparative biology. The ability to culture cells from lamprey tissues makes it possible to employ an in vitro approach to address basic questions in these areas. Methods are described for the initiation of cell cultures derived from tissues of adult and larval sea lamprey (Petromyzon marinus). Primary cultures initiated from gill, muscle, gut, brain, ovary, heart and kidney were viable for up to eight months and several of the cultures were propagated for multiple passages. Most cultures were initiated from tissue explants in basal nutrient medium supplemented with fetal bovine and trout sera on a culture surface treated with fibronectin and collagen. Variations of these culture conditions to meet the specific growth requirements of certain cell types are discussed.     

Development of primary cell cultures from Nephrops norvegicus

This paper reports the first attempt to develop primary cell cultures from tissues (ovary, testes, hepatopancreas, haematopoietic tissue, heart, gut, gill, eye-stalk and nerve tissues) of the Dublin Bay prawn (also known as the Norwegian squat lobster and scampi) Nephrops norvegicus (L.) using Leibovitz's L-15, MEM and M199 media supplemented with 5, 10 or 20 percent (w/v) foetal bovine serum (FBS). The best results were achieved with ovary tissues in 2× Leibovitz's L-15. Also, primary cell cultures were developed with testicular and haematopoietic tissues. With ovary tissues, results with 5 percent (w/v) FBS at an osmolality of 800 mOsm/kg were superior for the maintenance of epithelial-type cells, and enabled longer survival, i.e. more than 3 months. Little success was achieved with M199 and MEM media. One subculture of ovary cells was obtained.     

Galactosamine-induced cell death in primary cultures of rat hepatocytes.

Primary cultures of rat hepatocytes were exposed to 0.5 mM D-galactosamine. After 36 hours, only 10-20% of the original cells were viable, as assessed by trypan blue exclusion. In the absence of galactosamine, there was no loss of viability over this same period. The addition of 3 mM uridine to the culture medium completely prevented the cell death produced by galactosamine. Glucosamine had no effect on the viability of the hepatocytes. The extent of galactosamine-induced cell death was dependent upon the concentration of Ca++ ions in the culture medium. With the only source of Ca++ that added with the fetal calf serum, galactosamine had only a very slight effect on viability. With higher Ca++ than with the fetal calf serum, galactosamine had only a very slight effect on viability. With higher Ca++ concentrations, from 0.9 to 3.6 mM, the viability ranged from 75% to 31% 18 hours after treatment with galactosamine. The addition of 1.4 microM chlorpromazine to culture medium containing 1.8 mM Ca++ decreased the extent of the galactosamine-induced cell death. This protective effect was progressively reduced by raising the Ca++ concentration to 3.6 and 5.4 mM. Chlorpromazine given to intact rats 2 hours after treatment with 400 mg/kg galactosamine prevented the appearance of liver cell necrosis. At the same time, chlorpromazine prevented the increases in liver cell Ca++ content. These results indicate that many of the features of the effect of galactosamine on intact rat liver cells can be reproduced in primary cultures of these same cells. The data also support the hypothesis that a disturbance in intracellular Ca++ homeostasis leading to accumulations of these ions is causally related to the cell death produced by galactosamine.     

Establishment of primary cell cultures: Experiences with 155 cell strains

Summary Cell culture systems allow the examination of cell populations in a functional state. To simulate in vivo conditions as closely as possible freshly established cell strains are superior to permanent cell lines. Different aspects for the establishment of primary cell cultures obtained from various tissues are compared: (1) Disintegration, (2) culture media supplemented with basal additions, (3) special supplements (growth factors, hormones), and (4) attachment factors. The proliferation rates of the attained cell strains were evaluated by determination of cell doubling times. Procedures for how to obtain a relatively high plating efficiency (approx. 70% in our series of 219 attempts) of primary growth in vitro are described: (1) Mechanical disintegration is superior to enzymatic digestion. If mechanical treatment alone did not produce a sufficient number of viable cells, additional digestion with collagenase/dispase revealed a higher number of proliferating primary cultures than with trypsin. (2) Proliferation of cell cultures from normal and tumorous tissues of epithelial origin was superior in Leibovitz L 15 medium (58 of 87 (67%) cases). Cultures from mesenchymal tissues and tumors were found to have shortest cell doubling times in MEM and RPMI 1640 (16 of 23 (70%) cases). The media were supplemented with the basal additions indicated. (3) In approx. 30% of the cases special supplements like growth factors or hormones increased cell replication, although they were almost always not essential for cell growth. (4) Attachment factors only rarely contributed to the initiation of primary monolayer cultures. The application of various culture conditions does not lead to a protocol optimal for all tissues, for all probes of the same type of tumor, or for all tumor specimens of unique differentiation.Abbreviations EGF epidermal growth factor - FGF fibroblast growth factor - GHL glycyl-L-histidyl-L-lysine - PAN pancreozymin - E estrogen - PR progesterone Supported by the Deutsche Forschungsgemeinschaft, grant Di 276/1–2, SFB 232, Hamburger Landesverband zur Krebsbekämpfung und Krebsforschung, and Hamburger Stiftung zur Förderung der Krebsbekämpfung     

Antigenic staining patterns of human glioma cultures: primary cultures, long-term cultures and cell lines

Summary The immunocytochemical staining patterns of cultured glioma cells were investigated. Fifty nine individual cases were stained at differentin vitro ages for glial fibrillary acidic protein, fibronectin, galactocerebroside, HNK-1/Leu 7, A2B5, vimentin, factor VIII and A4. Histologically, the cases were composed of eight low-grade astrocytomas, 11 high-grade astrocytomas, four low-grade oligodendrogliomas, seven high-grade oligodendrogliomas and 29 glioblastomas. The 45 cases were analysed within the first 3 weeks of culture, many of them as primary cultures. In 11 cases stainings were performed repeatedly at intervals of up to 6 months.Glial fibrillary acidic protein staining was positive in most of the early cultures of astrocytomas (low and high grade) and glioblastomas; expression in more than 50% of the cells was found in 1 of 5 low-grade astrocytomas, 5 of 11 high-grade astrocytomas and 14 of 29 glioblastomas. Two of the high-grade astrocytomas were stained once more after 6 weeks in culture and were found to be only 1% positive for glial fibrillary acidic protein but strongly positive for fibronectin. The same was true for five of the glioblastoma cases. Two of these cases remained glial fibrillary acid protein positive and developed into stable permanent cell lines. Only one case started with 1% of glial fibrillary acidic protein positive cells and later developed into a 99% glial fibrillary acidic protein positive cell line. Neither HNK-1/Leu 7 expression nor A2B5 staining appeared to have a relationship to the glial fibrillary acidic protein staining. It was observed that glial fibrillary acidic protein and HNK-1/Leu 7 were both 100% in some cases but that later one of the two antigens disappeared but not the other. The amount of glial fibrillary acidic protein staining does not allow the prediction of A2B5 staining.The study shows that initiation of primary cultures on an extracellular matrix yields more glial fibrillary acidic protein positive cells in primary cultures than have been found in other studies. It is concluded that only a rigid standardization of culture conditions will ensure the validity of comparisons ofin vitro data obtained in primary cultures.     

Ten commandments for preventing contamination of primary cell cultures

Procedures for preventing contamination in primary cell cultures must be carefully defined and strictly followed in order to obtain healthy cells. Protocols have been developed and refined in our laboratory for establishing primary cultures of muscle and fat stem cells without contamination from a variety of animals. Contamination of cell cultures is not only frustrating, but is also very expensive both in time and loss of materials. Through the consistent use of proper aseptic techniques, most instances of contamination may be avoided. We suggest that the basic principles detailed here will find wide applicability in the culturing of primary cells without contamination from many different types of animals and tissues.     

Cytogenetic aberrations in primary cell cultures of the ovarian surface epithelium

Our objective was to determine the presence of chromosomal abnormalities in primary cultures of ovarian surface epithelial cells in women of different ages with no history of cancer. Throughout conventional cytogenetic techniques, we analyzed chromosome spreads of cultured ovarian epithelial cells from 10 donors who were 50 or more years old (B) and 16 controls between 20 and 49 years old (A), belonging to the mestizo population in Bogota DC, Colombia. Of the 26 cultures that were analyzed in passage 1, 61.5% had an abnormal chromosome complement (62.5% in A, and 60% in B). Abnormalities included polyploidies, endoduplications and monosomies. Deletions in chromosomes 3 and 11 were found in just one metaphase. None of the samples showed weaknesses or breakpoints. After transforming and applying the exact student's t-test for variance heterogeneity, we found significant differences in the frequency of metaphases, that were higher in A than in B (p=0.05), and in the frequency of polyploidies, which were higher in B than in A (p=0.044). Through the application of the Mann-Whitney test, we determined that the frequency of endoduplications was higher in A than in B (p=0.126), without reaching significant differences. There were no significant differences in the frequency of monosomies. The level of significance was set at p < or = 0.05. Taking into account that polyploidization is a marker of chromosomal instability and that the risk of cancer arising from the ovarian surface epithelium augments substantially after menopause, the increase in the frequency of age-associated polyploidies could be used as a predictor of ovarian cancer in women from an ethnically homogeneous population as the mestizo one in Bogota DC.     

Cytogenetic studies of primary cultures of esophageal squamous cell carcinoma

Cytogenetic analysis was performed on primary cultures of 21 squamous cell carcinomas of the esophagus (SCCE). Seven cases exhibited mosaic clonal chromosome abnormalities distributed as follows: two contained tetraploid cell populations, one with t(3;7)(p21;q11); two showed loss of the Y chromosome, one with double minutes; single cases demonstrated der(11)t(4;11)(q?27;q23); add(1)(p35) and del(4)(p12); and del(7)(p13), del(7)(q22q34), and der(11)t(7;11)(p?15;p?13). The remaining 14 cases had apparently normal karyotypes, possibly derived from stromal elements. These results demonstrate numerical abnormalities and the multiple occurrence of rearrangements involving chromosomes 7 and 11 in SCCE.     

Peroxidase-labeled primary antibody method for detection of pestivirus contamination in cell cultures

Contaminating bovine viral diarrhea (BVD) virus in cell cultures and in fetal bovine serum (FBS) is a well recognized problem. This study describes a direct peroxidase (DP) labeled primary antibody method for detection of pestivirus antigens in cell culture that is simple and reliable. Using this method, most bovine and porcine cell cultures, a cat lung, four mosquito and two monkey cell cultures were found contaminated with BVD virus. The rodent and human cell cultures tested were negative by this method for BVD virus.     

A method to reduce glutathione peroxidase levels in primary endothelial cell cultures

The purpose of this study was to develop culture conditions that would reduce glutathione peroxidase activity in bovine mammary endothelial cells. Conditions of reduce glutathione peroxidase activity were produced in vitro by culturing cells in selenium-deficient media. Low selenium levels were achieved by reducing serum concentrations; however, levels of essential growth factors also were reduced by this method. Therefore, cell proliferation was promoted by supplementation with combinations of defined serum components including insulin, transferrin, linoleic acid, bovine brain extract, and human epidermal growth factor. Out of seven different formulas tested, F12K medium containing 2% fetal bovine serum, insulin, transferrin, and linoleic acid was found to be conducive for cell proliferation. Upon confluence, endothelial cells cultured under these conditions consistently displayed short passage rates, consistent cell numbers, and classic cobblestone morphology when grown in the presence or absence of supplemental selenium. Additionally, these cells retained typical endothelial cell characteristics such as uptake of 1,1-dioctadecyl-3,3,3,3-tetramethylindo-carb ocyanine perchlorate acetylated low-density lipoprotein and the expression of cell adhesion molecules and von Willebrand Factor.     

Ultrastructural studies of the replication of fowl adenoviruses in primary cell cultures

The replication of four fowl adenovirus strains (FAV-1, strain Phelps; FAV-5, strains 340 and TR-22; and FAV-7, strain YR-36), in primary chick kidney cell cultures, is described. Differences were found in the distribution of virus particles and virus associated inclusions between viruses belonging to different cytopathology subgroups. Thus in cells infected with FAV-1 (Phelps) and FAV-5 (340) (i.e. subgroup 1) virus particles, as they increased in number, tended to become distributed peripherally, close to the nuclear membranes, with the virus associated inclusions in the centre of the nucleus. With FAV-5 (TR-22) and FAV-7 (YR-36) (i.e. subgroup 2) virus particles and associated inclusions became concentrated initially in the central nuclear area later increasing to fill the whole nucleus, with virus particles and inclusions completely intermixed. The virus-associated inclusions were found to be identical to those described in human adenovirus infected cells and the same nomenclature was adopted. Other inclusions found in infected nuclei, included tubular structures and inclusions composed of granular particles.     

Respiratory syncytial virus-specific RNA synthesis in primary monkey kidney cell cultures.

Primary rhesus monkey kidney (MK) cell cultures were inoculated with respiratory syncytial virus and treated or untreated with actinomycin D before pulse labeling with uridine-5-3H. The virus-specific RNA synthesis was noted at its peak in the nucleoplasm and possibly less so in the cytoplasm of infected cells. At 48 and 72 hours post-inoculation (p.i.), small fractions of available cells were synthesising virus-specific RNA with labeling index of 15% and 18% respectively. By 48 hours p.i. syncytia started appearing and a higher grain count in the cytoplasm of actinomycin D-treated infected cells was noted.     

Laser microdissection and primary cell cultures improve pharmacogenetic analysis in pancreatic adenocarcinoma

A key focus of research on pancreatic ductal adenocarcinoma (PDAC) is identifying new techniques to tailor gemcitabine and 5-fluorouracil treatments. Availability of tumor tissue is critical for the accurate assessment of gene expression, and laser microdissection (LMD) and primary cell cultures may be useful tools to separate tumor cells from the stromal reaction. The aim of this study was (1) to address the genetic profile relevant to drug activity and (2) to evaluate differences between microdissected and non-microdissected tumors, normal tissues, and primary cell cultures. Quantitative PCR of seven key genes was performed on mRNA from 113 microdissected and 28 non-microdissected tumors, a pool of normal tissues and four established primary cell lines. Protein expression was evaluated by western blot and immunocytochemistry and cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. LMD allowed the analysis of 110 samples and revealed significant differences in mRNA levels between microdissected tumors and normal tissues, as well as between non-microdissected and microdissected tumors from the same patients. In contrast, primary cell lines showed similar expression profiles with respect to their respective microdissected tumors. In particular, expression levels of human equilibrative nucleoside transporter-1 and thymydilate synthase were significantly related to gemcitabine and 5-fluorouracil cytotoxicity. We conclude that LMD is a reliable technique for mRNA extraction, and allows detection of significant differences in the expression of specific target genes when compared to non-microdissected specimens and normal tissues. Moreover, expression levels in microdissected tumors are similar to those observed in primary tumor cell cultures, both at mRNA and protein level, and are related to drug chemosensitivity. The use of these ex vivo techniques for molecular analysis of tumors therefore appears to be of some value in implementing the clinical management of PDAC.     

Age-dependent changes in the proportion of macrophages in primary thymus non-lymphoid cell cultures

Primary murine thymus cell cultures consist of thymus epithelial cells, macrophages and interdigitating dendritic cells. Results of the present study indicated that the proportion of macrophage-like cells in cultures was directly related to the age of the thymus donors through 7 weeks of age. We standardized procedures to compare primary thymic cultures derived from mice, 1 day to 115 days of age. Cultures were grown 6 to 8 days and then tested for nonspecific esterase, acid phosphatase, phagocytosis of latex beads, Fc receptors, and MAC-1 staining. Less than 10% macrophage-like cells were identified in cultures from mice 1 to 3 days-old, but the proportion increased to 50% macrophage-like cells in cultures from 7 week-old mice. Thereafter the proportion plateaued although there was considerable variation among older mice. Further studies showed that the proportions of macrophage-like cells was not affected by the number of cells initially seeded into culture, nor by the amount of serum added to the culture medium. These data suggest that macrophage/interdigitating cell precursors immigrate into the thymus for 4 to 6 weeks after birth and support the hypothesis that these precursors may be influenced to differentiate into thymus macrophages or interdigitating cells by the thymic microenvironment.     

Na-independent and Na-dependent transport of neutral amino acids in the human red blood cell

Experiments performed at 25°C, pH 7.4, confirmed that the human red blood cell possesses both Na-independent and Na-dependent transport systems for neutral amino acids. Further evidence for the existence of a major Na-independent pathway for the large neutral amino acids (L-leucine, L-isoleucine, L-phenylalanine, L-valine and L-methionine), designated the ‘L-system’, was provided from trans-acceleration experiments of L-leucine efflux. Transport via the L-system with respect to kinetics of substrates and stereospecificity was studied. Experiments on inhibition of transport and on kinetics of Na-dependence of uptake of L-alanine and glycine were consistent with Na-dependent amino acid transport being mediated by two different systems, i. e. an ASC-system for L-alanine, L-serine and L-cysteine and a Gly-system for glycine transport. When compared with a ‘high capacity/low affinity’ pattern of kinetics of the L-system, Na-dependent uptake of L-alanine and glycine was found to exhibit ‘low capacity/high affinity’ kinetics. The Na-dependence of L-alanine uptake conformed to first order interaction, that of glycine uptake to second order. An effect predominantly on the maximum transport capacity (V₁₂) of the saturable Nadependent uptake route of L-alanine and glycine, respectively, by a 50% reduction of the extracellular Na⁺ concentration, was suggested.