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Results for “Primary Cell Culture”

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At least 19 recordsLinked to original sources

PubMed1980

A study of irrigation fluids for neurosurgery on brain primary cell cultures.

Primary cell cultures from newborn rat brain hemispheres were exposed to different irrigation fluids used in neurosurgery. The cells died after incubation for 5 min with hydrogen peroxide, and the number of cells was drastically decreased after 10 sec of incubation. They shrank after incubation in Elliott's artificial cerebrospinal fluid for 3 h, but the viability as determined by trypan blue exclusion test was not affected. Physiological sodium chloride, Ringer's solution and the culture medium 199 with Hank's salt had no noticeable effect on the viability or morphology of the cells.

Animals↗

PubMed1996

Cytotoxicity, zinc protection, and stress protein induction in rat proximal tubule cells exposed to cadmium chloride in primary cell culture.

Primary cell culture was utilized to study the relationships between stress protein induction by zinc in vivo and cadmium toxicity in vitro. Effects of cadmium on cell viability were evaluated by the alamar blue assay, in conjunction with the ultrastructural morphology of cells by transmission electron microscopy. The expression of stress protein gene products was evaluated by 35S two-dimensional gel electrophoresis. The results showed cytotoxicity of CdCl2 at and above 129 microM (14.55 micrograms cadmium/mL medium) following 4 h of exposure. Prior zinc administration (20 mg zinc/kg, s.c., two daily doses) in vivo significantly protected the cells in vitro as demonstrated by improved cell viability. The 35S labeling of proteins induced by CdCl2 exposure clearly demonstrated for the first time that gene product of the 70-kDa family was induced in cultured rat proximal tubule cells which are the target cells for cadmium toxicity in vivo. Zinc in vivo pretreatment of animals induced proteins in the 90-, 70-, and 38-kDa families, which may act together with metallothionein to protect cells against cadmium toxicity. The results also indicate that the protective effect of zinc remains after the cells have been put in culture and thus provides a system in which we can study the changes that occur as a result of zinc exposure that decreases cadmium toxicity.

Animals↗

PubMed1988

Differentiated properties of rabbit tracheal epithelial cells in primary culture.

The purpose of this study was to establish whether rabbit tracheal epithelial cells, grown in primary cell culture, retain neurohormonal receptor and mediator activity. Epithelial cells were isolated by enzymatic treatment and cultured on a collagen matrix. The culture medium consisted of a 1:1 mixture of Dulbecco's modified Eagle medium and Ham's F12 supplemented with 5% fetal calf serum, epidermal growth factor, insulin, transferrin, fibronectin, hydrocortisone, and trace elements. Cultures had a population doubling time of 48 h. Mucus-containing cells and cilia were not observed after 7 days of incubation. Positive immunofluorescent staining with monoclonal antibodies to keratins established the epithelial nature of the cell cultures. Primary cell cultures responded to beta-adrenergic agonists with a dose- and time-dependent increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels. Propranolol, a beta-adrenergic antagonist, blocked the effects of the beta-adrenergic agonist. Adrenergic agonists also mediated a dose-dependent increase in the generation and release of prostaglandin E2 (PGE2). PGE2 caused an increase in cAMP levels. The results demonstrate that primary cultures of rabbit tracheal epithelial cells retain hormonal responses of the isolated epithelium and tracheal mucosa-submucosa.

Animals↗

PubMed1987

Inhibition of human natural killer activity by monolayers of primary cell cultures.

Some primary and continuous cell cultures were tested for their capacity to regulate human natural killer (NK) activity. Primary cultures of endothelial cells, fetal fibroblasts, adult fibroblasts, amnion epithelial cells, renal parenchymal cells, and ovarian carcinoma cells inhibited NK activity when peripheral blood lymphocytes were preincubated on target cell monolayers for 18 h before testing the cytotoxicity against K-562. The supernatants of the inhibiting cell cultures were not suppressive. Prostaglandins or suppressive lymphocytes were not involved in the phenomenon. The binding capacity of the effector cells was not changed, suggesting that the suppressive signal was targeted at the cytolytic machinery of NK cells. The down-regulating capacity of the cell cultures weakened significantly during subculturing in vitro, and continuous cell lines were not inhibitory. The inactivation of NK cells may be one of the mechanisms by which target cells are protected from NK activity.

Cell Communication↗

PubMed1998

Colloidal silica-coated tissue culture dishes for primary cell cultures: growth of rabbit renal proximal tubule cells.

The use of colloidal silica as a substratum for primary cultures of differentiated cells has significant advantages over classic tissue culture polystyrene. In this report, the growth and the level of expression of differentiated function of primary rabbit renal proximal tubule (RPT) cell cultures on colloidal silica is examined, using hormonally defined serum-free medium. Primary RPT cells grew to confluence more rapidly on colloidal silica than on tissue culture polystyrene (TC+). Moreover, following three passages, the RPT cells increased in number threefold more than parallel cultures on TC+. The morphology of primary RPT cells on colloidal silica were found by means of transmission electron microscopy to possess a polarized morphology with a brush border, and differentiated markers were retained even after passaging, including the Na+/glucose cotransport system and Glut 7.

Animals↗

PubMed1996

Prognostic value of monolayer culture patterning in primary cell culture of oesophageal cancer.

The growth of primary cell cultures of oesophageal cancer was compared with the clinical outcome of patients from whom the cancers were taken. Ninety-three patients underwent curative resection, with no operative deaths, and were divided into three groups according to the monolayer culture pattern of the primary cell culture: culture from 43 patients (46 per cent) grew no malignant cells (group 1), 21 (23 per cent) produced monolayer epithelial growth (group 2) and the rest (from 29 patients) established cell lines (group 3). The 5-year survival rate of patients in group 2 (29 per cent) and group 3 (23 per cent) was significantly lower (P < 0.005) than that of those in group 1 (51 per cent). Monolayer epithelial growth potential is a significant prognostic factor in patients with oesophageal cancer.

Adult↗

PubMed2000

Ultrastructure of white spot syndrome virus development in primary lymphoid organ cell cultures.

Primary cell cultures from the lymphoid organ of Penaeus monodon were used to investigate in vitro propagation and morphogenesis of white spot syndrome virus (WSSV). Double-strength Leibovitz's L15 supplemented with 20% fetal bovine serum, pH 7.5, with a final osmolarity of 530 +/- 5 mOsm kg-1 was identified as the most suitable culture medium. In this medium, the lymphoid cells remained viable for more than 1 wk. Migrating cells were inoculated with WSSV, and the consequent cytopathic effects documented by light and electron microscopy. WSSV appears capable of following 2 alternative assembly sequences, one similar to the morphogenesis of the Oryctes rhinocerus virus and another which is more typical of baculoviral assembly. Possible relationships between WSSV, Oryctes virus, and baculoviruses are discussed.

Animals↗

PubMed2006

Molecular detection of measles virus in primary cell cultures of otosclerotic tissue.

CONCLUSION: Primary cell cultures were established from otosclerotic/otospongiotic footplate bone particles. Although this procedure is time-consuming, the quality and quantity of RNA isolated from these cells were much higher in comparison with the direct isolation of RNA from footplate bone samples and the preparation was more suitable for the detection of measles virus (MeV) RNA. OBJECTIVE: Morphological and biochemical investigations suggest that persistent MeV infection participates in the development of otosclerotic foci. However, this hypothesis is controversial because the detection of MeV in otosclerotic foci is inconsistent since the results are dependent on the presence and stage of foci in the investigated bone particles. Unfortunately, this cannot be confirmed before investigation. To study the presence of the MeV by different techniques in otosclerotic foci, stapes footplate fragments were collected during stapedectomy from patients suffering from clinical otosclerosis. MATERIALS AND METHODS: MeV-specific RT-PCR was performed on total RNA isolated directly from four fresh frozen footplate bone fragments and from the cells of 16 primary cultures of otosclerotic tissue samples. In order to rescue persisting MeV, the primary footplate cells were cocultured with MeV permissive B95a cells. RESULTS: MeV was not detected in RNA from fresh frozen otosclerotic materials, but analysis of the RNA from 5 of the 16 primary cell cultures showed MeV-positive results. Nucleotide sequencing of a 317 bp MeV-specific RT-PCR fragment confirmed the presence of the MeV RNA genome. Here, we report the first determination of MeV sequences in total RNA isolated from primary cells cultured from otosclerotic tissue. Persisting MeV in primary footplate cells could not be recovered by coculturing with B95a cells.

Adult↗

PubMed1992

Dissociated neurons of the pupal honeybee brain in cell culture.

Primary cell cultures were prepared from specific regions of the pupal honeybee brain which are involved in proboscis extension learning. Defined areas could be dissociated purely by mechanical treatment. We show that cultured neurons regenerate new neurites and remain viable for up to three weeks in a serum-free, chemically-defined medium. Several labelling techniques were employed to identify subpopulations of cultured neurons. For example, acetylcholinesterase staining; fluorescent beads to distinguish identified cell populations of co-cultured brain areas; various markers for surface antigens such as a monoclonal antibody to olfactory projection neurons of the antennoglomerular tracts and monopolar cells of the optic lobes, as well as anti-HRP immunoreactivity and alpha-bungarotoxin binding; and various antisera for detecting transmitter phenotype. The appearance of transmitter-immunoreactive cells agreed closely with that expected from their known distribution in situ. Our results suggest that cultured cells retain surface properties and transmitter phenotype of their in vivo counterparts, despite differences in basic morphology. Thus our culture system provides the important initial step for future in vitro investigations of the cellular and electrophysiological properties of neurons mediating proboscis extension learning.

Acetylcholinesterase↗

PubMed1977

Estrogen action in vitro: regulation of thyroid stimulating and other pituitary hormones in cell cultures.

Primary cell cultures of ovine pituitaries can maintain production of thyroid stimulating hormone (TSH) for as long as 24 days. These cultures responded in a normal fashion to thyroxine by decreasing TSH secretion. Addition of thyrotropin releasing hormone increased TSH secretion. Physiologic levels of estradiol-17 beta (10(-11)-10(-9)M) produced a five-fold increase in secretion of TSH and a two-fold increase in intracellular TSH concentration in cell cultures. Common estrogens, but not common progestins, androgens and glucocorticoids affected TSH production. Markedly different effects of estrogen in the pituitary on follicle stimulating, luteinizing and thyroid stimulating hormones and prolactin are discussed in terms of current models of estrogen action.

Animals↗

PubMed1994

[Intracellular calcium and pH as sensitive parameters of toxicity in neural cell culture]

Primary cell cultures and synaptosomes of brain serve as tools to investigate neurotoxic compounds. Changes of intracellular calcium and pH indicate cell injury. In the present study, mesencephalic cell cultures were prepared from embryonal C57/BL6 mouse brain and intact synaptosomes from dog brain. In both model systems, the effect of MPTP, MPP+ and paraquat on intracellular calcium homeostasis and pH were studied. Direct addition of these neurotoxins (10 µM) to fura-2-labeled cells did not change intracellular calcium concentration. Mesencephalic neurons were exposed to 10 µM MPP+ for 24 hours led to a 36% calcium increase. Thus both in vitro models of changes of calcium homeostasis and pH may serve to study prolonged toxic effects of substances and also means of their prevention.

Journal Article↗

PubMed1990

Immunocytochemical and biochemical evidence of renin in human lactotrophic cell cultures.

Primary cell cultures from human prolactin (PRL)-secreting adenomas were used to test the ability of human lactotrophs to synthesize renin in vitro. The renin content of the culture medium and of cellular extracts was measured by enzyme-linked immunosorbent assay. The level of PRL release in the culture medium and the amount of PRL in a cellular extract were determined by radioimmunoassay. Morphologic studies included indirect immunofluorescence, pre-embedding immunoelectron microscopy using a three-layer peroxidase-antiperoxidase method and postembedding immunoelectron microscopy using protein A-gold complexes. Renin was detected in cellular extracts and was found to be absent in the culture medium, whereas PRL was extracellularly secreted. PRL and renin immunoreactivity was observed in all the cultures studied by immunofluorescence. The subcellular localization of renin was found to be similar to that of PRL and was observed in the rough endoplasmic reticulum, the Golgi apparatus, and cytoplasmic secretory granules. The results suggest that, in vitro, renin may be synthesized and intracellularly metabolized in human adenomatous lactotrophic cells rather than secreted. Cell cultures may be a useful model to further the understanding of the role of a local renin-angiotensin system in PRL secretion.

Female↗

PubMed2004

A new culturing strategy optimises Drosophila primary cell cultures for structural and functional analyses.

Neurons in primary cell cultures provide important experimental possibilities complementing or substituting those in the nervous system. However, Drosophila primary cell cultures have unfortunate limitations: they lack either a range of naturally occurring cell types, or of mature physiological properties. Here, we demonstrate a strategy which supports both aspects integrated in one culture: Initial culturing in conventional serum-supplemented Schneider's medium (SM(20K)) guarantees acquisition of all properties known from 30 years of work on cell type-specific differentiation in this medium. Through subsequent shift to newly developed active Schneider's medium (SM(active)), neurons adopt additional mature properties like the ability to carry out plastic morphological changes, neurotransmitter expression and electrical activity. We introduce long-term FM-dye measurements as a tool for Drosophila primary cell cultures demonstrating the presence of increased, action potential-dependent synaptic activity in SM(active). This is confirmed by patch-clamp recordings, which in addition show that SM(active)-cultured neurons display different spiking patterns. Furthermore, we demonstrate that transmission can be evoked in SM(active) cultures, revealing the existence of synaptic plasticity. Thus, these culture conditions support developmental, structural and physiological properties known or expected from the nervous system, enhancing possibilities for future experiments complementing or substituting those in nervous systems of Drosophila.

Action Potentials↗

PubMed1990

Influence of experimental conditions on osteoblast activity in human primary bone cell cultures.

Primary bone cell cultures were derived from human bone explants. Cellular activity was characterized by the alkaline phosphatase (AP) activity, osteocalcin, and type I and III collagen secretions in the supernatant. The determination of bone cell activity was performed in three different wells. No significant difference was noted between wells: the coefficient of variation was 8.0 +/- 2.9% for AP activity, 18.3 +/- 1.9% for osteocalcin secretion, and 22.5 +/- 14.3% for collagen. The AP activity and osteocalcin secretion significantly decreased with the number of passages: they were the highest after the first passage. Between each subject, the coefficient of variation was 85% for AP activity and osteocalcin secretion and 63 and 57% for type I and III collagen secretion, respectively. The AP activity did not differ with the age or sex of the donor. In contrast, osteocalcin secretion was significantly lower in females than in males. In males, osteocalcin significantly decreased with the age of the donor (r = -0.61; p less than 0.05). Cellular activity did not depend on the site of the biopsy. When bone explants from one donor were cultured in two different petri dishes, the activity of cells was similar in both dishes, except in one case. Primary cell cultures derived from human bone explants are the only model providing untransformed osteoblastlike cells of human origin. Because of the experimental conditions, some factors may have influenced the cellular activity and they must be taken into account to validate further in vitro studies.

Adolescent↗

PubMed1970

Use of cell cultures in vaccine production: primary cell cultures.

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Animals↗

PubMed1992

Hormonal regulation of endometriotic cell growth in primary cell culture system.

A primary cell culture of endometriotic cells was established to examine the hormonal regulation of endometriosis. The addition of estradiol had no effect on the DNA synthesis of cultured endometriotic cells. Progesterone at 10(-7) M caused a significant reduction in the amount of the DNA synthesis. An inhibitory effect of progesterone was completely eliminated by the concomitant addition of RU486, an antiprogesterone. The addition of epidermal growth factor (EGF) at 1 ng/ml significantly increased the DNA synthesis. Other growth factors, such as fibroblast growth factor and insulin-like growth factor also produced a modest but significant increase in DNA synthesis. These results seem to provide clues for clarifying the pathophysiology of endometriosis.

Cell Division↗

PubMed1985

Chicken egg white genes: multihormonal regulation in a primary cell culture system.

A primary cell culture from estrogen-withdrawn chicken oviduct has been developed in which the genes for the major egg white proteins, ovalbumin and conalbumin, are induced by steroid hormones. The responsiveness of the isolated cells depends on the combination of enzymes employed to dissociate the oviduct; trypsin and pronase are essential. Administration of estradiol to cultured cells is insufficient to induce ovalbumin mRNA (mRNAov) to levels achieved in vivo. Both insulin and a second steroid (a glucocorticoid, progestin, or androgen) are required for the induction of the ovalbumin gene by estradiol to the extent reached in vivo. Although low levels of a progestin or androgen can synergize with estradiol to obtain an induction of mRNAov, we have demonstrated that a physiological concentration of corticosterone (10(-8) M) potentiates a large response to estradiol without causing an induction when added alone. When estradiol, corticosterone, and insulin are present in the culture medium, mRNAov increases from about 20 to 6500 molecules/cell by 55 h. With the same culture conditions, conalbumin mRNA increases from about 120 to 4300 molecules/cell by 52 h. After about 55 h, the level of egg white mRNAs decreases, and this reduction in mRNAov correlates with a diminished transcription of that gene. The data obtained with the cultured cells demonstrate that the induction of the ovalbumin gene requires the permissive effects of insulin and a second steroid (probably a glucocorticoid in vivo) to facilitate the response to estradiol.

Animals↗

PubMed1967

Studies on the mode of action of diphtheria toxin. II. Protein synthesis in primary heart cell cultures.

Primary heart cell cultures of embryonic guinea pigs and the neonatal rat were established and incubated with purified diphtheria toxin. The rat heart cell cultures were refractory to the effects of the toxin; protein synthesis proceeded normally as measured by the incorporation of tritiated leucine into cell proteins; beating heart cells continued to contract; and the cell monolayers remained intact after exposure to the toxin for periods as long as 72 hr. These findings are compatible with the species resistance of the rat to diphtheria toxin. The guinea pig heart cell cultures were found to be extremely sensitive to the toxin. Protein synthesis was inhibited by approximately 50% after incubation with small quantities of toxin for 3 hr. Increasing the concentration of the length of exposure to the toxin did not increase this inhibition significantly. In addition, diphtheria toxin exerted a true cytopathic effect on the guinea pig heart cells. Monolayers were destroyed when incubated with the toxin for 2 to 3 days. The results show that the heart cells reflect species resistance or sensitivity to diphtheria toxin in the absence of neural or endocrine influences and suggest further that the toxin exerts a direct toxicity to muscle cells of the heart. It is not yet possible to explain in biochemical terms why the toxin seems to act specifically on cardiac tissues.

Animals↗