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.     

Preservation at -195 degrees C of trypsinized organs for primary cultures

Trypsinized cells from rabbit kidney can be kept at -195, 8 degrees C indefinitely. When necessary, propagation in monolayer cultures is readily obtained as primary cultures, provided that some conditions be respected: 1) only glass culture flask must be used. Falcon plastic vessels do not allow cell attachment in this type of primary culture 2) the lag period is particularly long: it taken about 20 days before the beginning of cell multiplication 3) fetal calf serum is toxic for the cells in the 2nd and 3rd change of the culture medium, during the lag period.     

Astrocytoma derived short-term cell cultures retain molecular signatures characteristic of the tumour in situ

The heterogeneity of tumours and uncertainties surrounding derived short-term cell cultures and established cell lines fundamentally challenge the research and understanding of tumour growth and development. When tumour cells are cultured, changes are inevitably induced due to the artificial growth conditions. Several recent studies have questioned how representative established cell lines or derived short-term cell cultures are of the tumour in situ. We have characterised gene expression changes induced by short-term culture in astrocytoma in order to determine whether derived short-term cell cultures are representative of the tumour in situ. In comparison to the majority of studies, paired biopsies and derived short-term cultures were investigated to reduce the effects of long-term culture and inter-tumour variability when comparing biopsies and derived cultures from tumours with the same histology from different individuals. We have used the Affymetrix GeneChip^® U133A to generate gene expression profiles of 6 paediatric pilocytic astrocytoma (PA) biopsies and derived short-term cell cultures and 3 adult glioblastoma multiforme (GBM) biopsies and derived short-term cultures. Significant differential gene expression is induced by short-term culture. However, when the biopsy and derived short-term cell culture samples were grouped according to tumour type (PA and GBM) a molecular signature of 608 genes showed significant differential expression between the groups. This gene cohort can distinguish PA and GBM tumours, regardless of the sample source, suggesting that astrocytoma derived short-term cultures do retain key aspects of the global tumour expression profile and are representative of the tumour in situ. Furthermore, these genes are involved in pathways and functions characteristic of adult GBM including VEGF signalling, hypoxia and TP53 signalling.     

Effect of culture conditions on the obtention of boar epididymal epithelial cell monolayers

The goal of this study was to investigate the effect of the collagenase digestion time, the initial density of fragments and the culture temperature on the obtention of a boar epididymal epithelial cell culture, which is a useful methodology for the study of epididymal functions. A confluent monolayer of caput, corpus and cauda epididymal epithelial cells was only obtained when an adequate enzymatic digestion of the connective tissue surrounding the epididymal tubule was performed. For the correct digestion of caput and corpus fragments two collagenase digestions of 2 and 1h, respectively, were enough. Cauda fragments, however, needed two collagenase digestions of 3h each. A confluent monolayer of caput, corpus and cauda epididymal epithelial cells was obtained regardless of the initial density tested (15, 30, 60 and 90fragments/well). However, cultures originated from 15 and 30fragments/well showed higher cell concentration during the first 2 weeks of culture than cultures originated from 60 and 90fragments/well. A confluent monolayer of caput, corpus and cauda epididymal epithelial cells was obtained at both 32 and 37 degrees Celsius, but at 32 degrees Celsius cells grew very slowly and confluence was not reached until a week later than it was with cells growing at 37 degrees Celsius. In conclusion, we have observed that the time of digestion with collagenase is an important factor for the successful establishment of boar epididymal cell monolayers, and that the initial density of fragments and the culture temperature should be taken into account.     

Survival of captive and free-ranging Harlequin Ducks (Histrionicus histrionicus) following surgical liver biopsy

We measured intra- and postoperative mortality rates of captive and free-ranging Harlequin Ducks (Histrionicus histrionicus) undergoing surgical liver biopsy sampling for determination of the induction of cytochrome P4501A, a biomarker of oil exposure. Liver biopsies were taken from and radio transmitters were implanted into 157 free-ranging Harlequin Ducks over three winters (55 in 2000, 55 in 2001, and 47 in 2002). No birds died during surgery, but seven (4.5%) died during recovery from anesthesia (three in 2001 and four in 2002). None of the deaths could be attributed directly to the liver biopsy. Four of the 150 (2.7%) birds that were released died in the 2 wk period after surgery. All post-release deaths occurred in 2001; no birds died after release in 2000 or 2002. No mortalities of 36 captive birds occurred during surgery or recovery or in the 2 wk period following surgery. Hemorrhage was a minor problem with one captive bird. Surgical liver biopsies appear to be a safe procedure, but anesthetic complications may occur with overwintering ducks.     

A specific collagenase from rabbit fibroblasts in monolayer culture

1. Explants of rabbit skin and synovium in tissue culture secreted a specific collagenase into their culture media. Primary cultures of fibroblast-like cells, which were obtained from these tissues and maintained in culture for up to 14 subculture passages, also secreted high activities of a specific collagenase into serum-free culture medium. Secretion of enzyme activity from the cell monolayer was at constant rate for over 100h and continued for up to 8 days in serum-free culture medium. The enzymic activity released was proportional to the number of cells in the monolayer. 2. The fibroblast collagenase was maximally active between pH7 and 8. At 24 degrees C the collagenase decreased the viscosity of collagen in solution by 60%. The collagen molecule was cleaved into three-quarters and one-quarter length fragments as demonstrated by electron microscopy of segment-long-spacing crystallites (measured as native collagen molecules aligned with N-termini together along the long axis), and by polyacrylamide-gel electrophoresis of the denatured products. The collagenase hydrolysed insoluble collagen, reconstituted collagen fibrils and gelatin, but had no effect on haemoglobin or Pz-Pro-Leu-Gly-Pro-d-Arg (where Pz=4-phenylazobenzyloxycarbonyl). 3. The fibroblast collagenase was partially purified by gel filtration and the molecular weight was estimated as 38000. The activity of the partially purified enzyme was stimulated by 4-chloromercuribenzoate, inhibited by EDTA, cysteine, 1,10-phenanthroline and serum, but was unaffected by di-isopropyl phosphorofluoridate, Tos-LysCH(2)Cl and pepstatin. 4. Long-term cell cultures originating from rabbit skin or synovium from rabbits with experimentally induced arthritis also secreted specific collagenase. Human fibroblasts released only very small amounts of collagenase.     

A standardized laboratory and surgical method for in vitro culture isolation and expansion of primary human Tenon’s fibroblasts

Good manufacturing practices guidelines require safer and standardized cell substrates especially for those cell therapy products to treat ocular diseases where fibroblasts are used as feeder layers. However, if these are unavailable for stem cells culturing, murine fibroblasts are regularly used, raising critical issues as accidentally transplanting xenogenous graft and adversely affecting stem cell clinical trials. Moreover, human fibroblasts play a significant role in testing novel ophthalmologic drugs. Accordingly, we developed a standardized laboratory and surgical approach to isolate normal and undamaged Tenon’s fibroblasts to implement the setting up of banks for both stem cells-based ocular cell therapy and in vitro drug testing. A 2–3 cm² undamaged Tenon’s biopsy was surgically obtained from 28 patients without mutually correlated ocular diseases. Nineteen dermal biopsies were used as control. Fibroblasts were isolated with or without collagenase, cultured in autologous, fetal bovine or AB serum, tested for viability by trypan blue, vimentin expression and standardized until passage 6. Successful Tenon’s fibroblasts isolation was age dependent (P = 0.001) but not sex, pathology or surgery related. A significant rate of successful cultures were obtained when biopsies were not digested by collagenase (P = 0.013). Moreover, cultures in autologous or fetal bovine serum had comparable proliferative properties (P = 0.77; P = 0.82). Through our surgical and laboratory standardized procedure, we elucidated for the first time key points of this human primary culture system, the role of the autologous serum, comparing Tenon’s and dermal fibroblasts. Our protocol may be clinically useful to reduce the risk above mentioned and may be potentially more effective for ophthalmological clinical purposes.     

Long-lived plasma cells from human small intestine biopsies secrete immunoglobulins for many weeks in vitro

To understand the biology of Ab-secreting cells in the human small intestine, we examined Ab production of intestinal biopsies kept in culture. We found sustained IgA and IgM secretion as well as viable IgA- or IgM-secreting cells after >4 wk of culture. The Ab-secreting cells were nonproliferating and expressing CD27 and CD138, thus having a typical plasma cell phenotype. Culturing of biopsies without tissue disruption gave the highest Ab production and plasma cell survival suggesting that the environment regulates plasma cell longevity. Cytokine profiling of the biopsy cultures demonstrated a sustained presence of IL-6 and APRIL. Blocking of the activity of endogenous APRIL and IL-6 with BCMA-Fc and anti-human IL-6 Ab demonstrated that both these factors were essential for plasma cell survival and Ab secretion in the biopsy cultures. This study demonstrates that the human small intestine harbors a population of nonproliferating plasma cells that are instructed by the microenvironment for prolonged survival and Ab secretion.     

Transfection of "naked" siRNA results in endosomal uptake and metabolic impairment in cultured neurons

RNA interference is rapidly becoming a powerful tool for gene silencing in mammalian cells. Introduction of siRNA into primary cells, however, remains one of the major difficulties of this novel technique. Using cationic lipid-based transfection reagents satisfactory transfection results are observed in cell lines, but low transfection efficiency and cytotoxicity limit applications in primary cells, especially primary neurons. The application of "naked" siRNA has been previously used successfully in nematodes and mammals in vivo. We therefore evaluated the effects of non-cationic-lipid-based siRNA application to primary hippocampal neuron cultures. "Naked" siRNA was bound to the cell surface and was taken up into endosomes. No significant silencing effect of endogenous or reporter genes was observed, rather application of "naked" siRNA was accompanied by a moderate downregulation of metabolic activity in culture. We postulate that endosomal degradation of "naked" siRNA in neurons prevents the induction of significant RNAi-mediated mRNA-downregulation and is accompanied by a global impairment of the cell metabolism. Transfection methods circumventing the endosomal pathway therefore might prove useful for siRNA transduction of primary neurons.     

A METHOD TO COLLECT AND PROCESS SKIN BIOPSIES FOR CELL CULTURE FROM FREE-RANGING GRAY WHALES (ESCHRICHTIUS ROBUSTUS)

For researchers studying mysticere whales few methods for determining gender or for collecting biochemical and genetic information from unrestrained animals are available. The objective of this study was to develop a reliable method for collecting viable tissue samples for establishing continuous cell cultures from skin biopsies of free-ranging whales. A method to collect and process these samples is presented. Six of seven skin biopsies from gray whales were established in cell culture. Our results suggest that the viability of the samples is improved by (1) sterile processing in the field, (2) minimizing the time between collection and delivery to the cell culture facility, (3) reducing the concentration of antifungal agent, and (4) placing tissue explants under a coverslip. While the results reported in this paper are based on a small sample size, we believe that if the procedures are followed, they will increase the probability of successfully culturing cetacean tissue. Established cell lines can supply replenishable material from identified whales still living in the wild. These cultures can then be used for determination of sex from karyotypes, and for assessing genetic relationships of cetaceans from inherited protein, chromosomal and DNA polymorphisms. These much needed analytical tools can be used to determine familial and populational relationships, leading to a better understanding of mating systems, stock identification and effective population sizes of wild cetaceans.     

Long-term storage of tissue samples for cell culture

The establishment of cultured cell lines from skin biopsies stored at -196 degrees C for periods up to 1 year has been investigated. Attempts to initiate cell cultures from the frozen tissue samples were uniformly successful. There was no alteration in chromosome constitution, morphological appearance, or specific activities of lysosomal enzymes in cells cultured from the stored samples. This process can safeguard against failure of the initial tissue culture and provide an alternate means of storing viable cells when it is impossible or impractical to initiate a cell culture immediately.     

The role of energy availability in Mammalian hibernation: an experimental test in free-ranging eastern chipmunks

Reduced torpor expression by hibernating mammals is often attributed to physiological constraints that limit their hibernation ability but may instead reflect adaptive, plastic responses to surplus energy availability. We evaluated this hypothesis by supplementing the food hoards of free-ranging eastern chipmunks (Tamias striatus) before hibernation and then documenting their use of torpor during the subsequent winter. In both years of study, chipmunks that received additional food were euthermic more than twice as frequently as nonsupplemented individuals. Furthermore, when food-supplemented individuals did express torpor, their minimum collar temperature was 5 degrees -10 degrees C warmer than nonsupplemented animals. These results indicate that reduced torpor expression by hibernators can result from an absence of energetic necessity rather than a lack of physiological capability and suggest that even endotherms sequestered in a hibernaculum may benefit from maintaining an elevated body temperature whenever possible.     

In vivo DNA damage in gastric epithelial cells

A number of risk factors have been linked epidemiologically with gastric cancer, but studies of DNA damage in gastric epithelial cells are limited. The comet assay is a simple technique for determining levels of DNA damage in individual cells. In this study, we have validated the comet assay for use in epithelial cells derived directly from human gastric biopsies, determined optimal conditions for biopsy digestion and investigated the effects of oxidative stress and digestion time on DNA damage. Biopsies taken at endoscopy were digested using combinations of pronase and collagenase, ethylenediaminetetra-acetic acid (EDTA) and vigorous shaking. The resultant cell suspension was assessed for cell concentration and epithelial cell and leukocyte content. A score for DNA damage, the comet %, was derived from the cell suspension, and the effect of various digestion conditions was studied. Cells were incubated with H(2)O(2) and DNA damage was assessed. Pronase and collagenase provided optimum digestion conditions, releasing 1. 12x10(5) cells per biopsy, predominantly epithelial. Of the 23 suspensions examined, all but three had leukocyte concentrations of less than 20%. The comet assay had high inter-observer (6.1%) and inter-assay (4.5%) reproducibility. Overnight storage of the biopsy at 4 degrees C had no significant effect on DNA migration. Comet % increased from a median of 46% in untreated cells to 88% in cells incubated for 45 min in H(2)O(2) (p=0.005). Serial 25-min digestions were performed on biopsies from 13 patients to release cells from successively deeper levels in the crypt. Levels of DNA migration were significantly lower with each digestion (r=-0.94, p<0.001), suggesting that DNA damage is lower in younger cells released from low in the gastric crypt. The comet assay is a reproducible measure of DNA damage in gastric epithelial cells. Damage accumulates in older, more superficial cells, and can be induced by oxidative stress.     

Regulation of connective tissue collagenase production: stimulators from adult and fetal epidermal cells

We have examined the ability of primary adult rabbit skin cells to regulate collagenase production in vitro. Dermal cells constitutively produce collagenase in culture, and enzyme production by these cells can be influenced by epithelial cells. Co-culture with skin epidermal cells resulted in more enzyme production by dermal cells, whereas co- culture with corneal epithelial cells yielded less enzyme activity. Connective tissue cells from a different source, cornea, also produced collagenase when co-cultured with skin epidermal cells, although the stromal cells alone made no enzyme. The drug cytochalasin B had very little influence on collagenase production by dermal cells, either alone or in co-culture with epidermal cells, but did significantly potentiate enzyme production by corneal stromal cells responding to epidermal effector molecules. Epidermal-cell-conditioned medium from both fetal and adult rabbit skin was a potent source of stimulators (apparent mol wt 20,500 and 55,000) of connective-tissue-cell collagenase production. Stimulator production by epidermal cultures was cell density dependent. Optimal production of stimulators occurred in adult cultures containing 10(6) epidermal cells/ml of medium, and in fetal cultures containing 10(5) cells/ml. Inhibitors of connective tissue cell enzyme production were not detected in conditioned medium from either adult or fetal epidermal cells.     

Heat shock of rabbit synovial fibroblasts increases expression of mRNAs for two metalloproteinases, collagenase and stromelysin

Two metalloproteinases, collagenase and stromelysin, are produced in large quantities by synovial fibroblasts in individuals with rheumatoid arthritis. These enzymes play a major role in the extensive destruction of connective tissue seen in this disease. In this study, we show that heat shock of monolayer cultures of rabbit synovial fibroblasts increases expression of mRNA for heat shock protein 70 (HSP-70), and for collagenase and stromelysin. We found that after heat shock for 1 h at 45 degrees C, the mRNA expression for HSP-70 peaks at 1 h and returns to control levels by 3 h. Collagenase and stromelysin mRNA expression is coordinate, reaching peak levels at 3 h and returning to control levels by 10 h. The increase in mRNA is paralleled by an increase in the corresponding protein in the culture medium. 3 h of heat shock at a lower temperature (42 degrees C) is also effective in inducing collagenase and stromelysin mRNAs. Concomitant treatment with phorbol myristate acetate (PMA; 10(-8) or 10(-9) M) and heat shock is not additive or synergistic. In addition, all-trans-retinoic acid, added just before heat shock, prevents the increase in mRNAs for collagenase and stromelysin. Our data suggest that heat shock may be an additional mechanism whereby collagenase and stromelysin are increased during rheumatoid arthritis and perhaps in other chronic inflammatory stress conditions.     

Identification of Campylobacter pylori in gastric biopsy smears

The use of gastric biopsy imprint smears to diagnose Campylobacter pylori was compared with the use of tissue sections and cultures. Multiple gastric biopsies were taken from the mucosa of 42 patients during endoscopy. Imprint smears were prepared from the samples used to make tissue sections; other samples were used for microbiologic culture. There was a good concordance (93%) between the morphologic diagnosis of C pylori in the air-dried, Giemsa-stained smears and the tissue sections; the cytologic preparations were clearly positive in six cases (14%) whose sections contained low numbers of the organisms. There was a concordance of 83% between the combined morphologic techniques and the bacteriologic culture. Six positive cases were detected only by the morphologic techniques while one positive case was detected only by bacteriologic culture. C pylori was identified in one or more preparations of the antral biopsy specimens in 23 (55%) of the 42 cases, including 23 (74%) of the 31 cases with a final diagnosis of gastritis or ulcer. These results show the usefulness of the cytologic study of gastric biopsy smears in diagnosing C pylori infections.     

Mammalian growth and development parameters, cell proliferation in culture and the maximal life span]

The maximum life span of mammals is known to be proportional to the pregnancy duration and to the age at puberty. We found that the maximum life span of mammals was also proportional to the number of cell doublings, and inversely proportional to the rate of duplication of these cells, during embryogenesis or for the time from zygote formation to growth termination. We found also that the life span of "stationary phase aging" transformed Chinese hamster cells (time from subcultivation until culture "death", i.e., until the moment when the number of live cells is less than 10% of their number at saturation density) was proportional to the duration of their growth and number of cell doublings during the period from subcultivation to saturation density, and inversely proportional to the rate of cell culture duplication during the same period. The dependencies for cell cultures and mammals proved to be analogous to each other. An approximately twofold decrease in the cell duplication rate, as a result of a decrease of the growth medium temperature from 37 to 27 degrees C or the introduction of ethanol to a final concentration 2%, increased the life span of "stationary phase aging" cultures more than twofold. The data obtained suggest that influences resulting in optimized delay of the rate of cell duplication, and correspondingly the mean rate of proliferation during the period of growth in mammals, may increase their maximum life span.     

SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes

Primary skeletal muscle myoblasts have a limited proliferative capacity in cell culture and cease to proliferate after several passages. We examined the effects of several oncogenes on the immortalization and differentiation of primary cultures of rat skeletal muscle myoblasts. Retroviruses containing a SV40 large T antigen (LT) gene very efficiently immortalize myogenic cells. The immortalized cell lines retain a very high differentiation capacity and form, in the appropriate culture conditions, a very dense network of muscle fibers. As in primary culture, cell fusion is associated with the synthesis of large amounts of muscle-specific proteins. However, unlike normal myoblasts (and previously established myogenic cell lines), nuclei in the multinucleated fibers of SV40-immortalized cells synthesize DNA and enter mitosis. Thus, withdrawal from DNA synthesis is not obligatory for cell fusion and biochemical differentiation. Using a retrovirus coding for a temperature-sensitive SV40 LT, myogenic cell lines were produced in which the SV40 LT could be inactivated by a shift from 33 degrees C to 39 degrees C. The inactivation of LT induced massive cell fusion and synthesis of muscle proteins. The nuclei in those fibers did not synthesize DNA, nor did they undergo mitosis. This approach enabled the reproducible establishment of myogenic cell lines from very small populations of myoblasts or single primary myogenic clones. Activated p53 also readily immortalized cells in primary muscle cultures, however the cells of eight out of the nine cell lines isolated had a fibroblastic morphology and could not be induced to form multinucleated fibers.     

Isolation, culture and characteristics of epididymal epithelial cells from adult cats

A tissue-culture system in which cells retain defined ultrastructural and functional characteristics was established to provide a basis for functional investigations of the epididymal duct in the cat. A widely used culture protocol for rat epididymal epithelium was used as a starting point and subsequently modified. The cellular population of the cat's epididymal epithelium was isolated by successive collagenase and trypsin digestion. A high yield of isolated cells obtained with good viability, were cultured in DMEM/F12 medium supplemented with foetal bovine serum, in absence or in presence of additional dihydrotestosterone (1 nM). The plated primary cultures reached confluence within 5-8 days, producing a monolayer of cohesive cells. Samples taken after 6 days in culture were processed for transmission and scanning electron microscopies. Immunocytochemical staining was used to estimate the purity of the epithelial cell population in the monolayers. The cell cultures displayed several functional traits of in vivo epithelia, including [35S] hypotaurine and [35S] taurine production. These results demonstrate that primary cultures of epididymal epithelial cells isolated from sexually mature cats maintain several differentiated characteristics of the intact organ and therefore provide a valuable system for the study of epididymal epithelial cell functions, metabolic activities and their regulation in cats.     

Comparative analysis and physiological impact of different tissue biopsy methodologies used for the genotyping of laboratory mice

Genotyping of genetically modified mice and control of authenticity of the genetic background of congenic or coisogenic strains by polymerase chain reaction (PCR) is a routine procedure that can be performed with different tissue biopsies causing variable grades of trauma. In this study, some invasive and non-invasive sampling methods were compared, with the main focus on the impact on animal physiology. We compared ear punch, tail biopsy, hair plugging, mouth and rectum swabs and the simple restraint of the animals, scoring for the impact on heart rate (HR), core body temperature (BT) and motor activity by telemetry, during biopsy and for the following 6 h. Furthermore, in order to correlate the physiological impact with the practicability and reliability of the genotyping results, we performed a PCR analysis of the biopsy samples obtained by using the same collection procedures analysed by telemetry. All sampling methods and restraint induced significant increase in HR and BT and a slight increase in motor activity for 1 h, independent of the invasiveness of the method used. Genotyping of all biopsies allowed the proper identification of transgenic animals, tail biopsies, ear punches and hair follicles giving clear signals, the last method being fast, but also susceptible to cross contaminations during sampling by large numbers of animals. Restraint and all biopsy methods provoked similar physiological changes, indicating that the handling of the animals is of major importance and that the sampling procedure does not strongly influence the physiological parameters.