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At least 19 recordsLinked to original sources
PubMed1999

Impact of culture conditions, culture media volumes, and glucose content on metabolic properties of renal epithelial cell cultures. Are renal cells in tissue culture hypoxic?

When renal proximal tubular cells are brought into tissue culture, they revert from oxidative metabolism and gluconeogenesis to high rates of glycolysis. Among the factors possibly responsible for this metabolic conversion, limited oxygen availability and/or substrate supply are discussed. In order to study the role of these factors on long-term cultures, the impact of growth conditions, culture media volume, and glucose content on carbohydrate metabolism of the continuous renal cell lines LLC-PK(1) (porcine kidney) and OK (opossum kidney) was investigated. The impact of culture media volumes and glucose content, respectively, was determined by overlaying confluent monolayer cultures of LLC-PK(1) and OK cells (i) with increasing volumes of culture medium and thus increasing amounts of glucose, and (ii) with increasing culture medium volumes at constant absolute amounts of glucose by adding glucose-free medium, in order to increase volume at a constant glucose supply. Alternatively, and in order to improve cell oxygenation, LLC-PK(1) cells were also cultured in roller bottles. Cell carbohydrate metabolism was assessed by measuring rates of glucose consumption and lactate production, respectively, and by determination of specific activities of the key glycolytic enzymes hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase (LDH). Mitochondrial phosphate-dependent glutaminase (PDG) was assayed as marker enzyme of oxidative metabolism of glutamine. In LLC-PK(1) and OK cells, rates of glucose consumption were independent of the initial glucose concentrations and/or the culture media volumes used. Glucose was quantitatively converted to lactate, which accumulated in a 1:2 molar ratio. Lactate in culture media reached a maximum content after 24 h, and was reutilized by the cell lines thereafter. Interestingly, the rates of lactate reuptake strictly depended on culture medium volume, indicating a volume-induced stimulation of oxidative lactate metabolism. Marked changes were found for the specific activities of glycolytic enzymes. In LLC-PK(1) cells, increased glucose supply caused increases in HK, PFK, PK and LDH activities, which were superimposed to the stimulatory effects of increased media volumes. Enzyme activity showed a biphasic response, indicating that both glucose supply and culture media volumes covering the cell monolayer are factors determining glycolytic rates of LLC-PK(1) renal cells. Conversely, in OK cells glycolytic enzyme activities decreased with increasing culture media volumes at constant glucose levels. As expected, under conditions of enhanced oxygenation of LLC-PK(1) cells in roller bottle culture, glycolytic enzyme activities decreased, whereas PDG activity increased, which was paralleled by increased rates of ammonia generation. Thus, changes in nutrient supply and oxygenation of renal epithelial cell cultures by altered culture media volumes dramatically influence metabolic rates and levels of enzyme activities, respectively.

Animals↗
PubMed2003

Confronting "culture" in medicine's "culture of no culture".

The author presents reflections from medical anthropology on the institutional culture of medicine and medical education, which sees itself as a "culture of no culture" and which systematically tends to foster static and essentialist conceptions of "culture" as applied to patients. Even though requirements designed to address cultural competence are increasingly incorporated into medical school curricula, medical students as a group may be forgiven for failing to take these very seriously as long as they perceive that they are quite distinct from the real competence that they need to acquire. To change this situation will require challenging the tendency to assume that "real" and "cultural" must be mutually exclusive terms. Physicians' medical knowledge is no less cultural for being real, just as patients' lived experiences and perspectives are no less real for being cultural. Whether this lesson can be effectively conveyed within existing curricular frameworks remains an open question. Cultural competence curricula will, perhaps, achieve their greatest success if and when they put themselves out of business-if and when, that is, medical competence itself is transformed to such a degree that it is no longer possible to imagine it as not also being "cultural."

Attitude of Health Personnel↗
PubMed1994

Viability of one-cell bovine embryos cultured in vitro: comparison of cell-free culture with co-culture.

In this study, the quality (number of cells) and pregnancy rates of bovine blastocysts produced by in vitro maturation/in vitro fertilization (IVM/IVF) following cultivation in either cell-free culture or co-culture were compared. Bovine one-cell IVM/IVF embryos obtained 6 h after insemination were stripped of cumulus cells and assigned to either cell-free culture or co-culture with granulosa cell monolayers for 9 days (Expt 1) or 10 days (Expts 2 and 3). In Expt 3, day-7 (day 0 = day of insemination) blastocysts, day-8 expanded blastocysts and day-9 hatched blastocysts were air-dried, fixed and stained to determine the number of cells. Expanded blastocysts obtained in Expt 1 were cryopreserved using propylene glycol as a cryoprotectant and were used later for embryo transfer. There were no significant differences between cell-free culture and co-culture in the percentage of one-cell embryos that developed to 2- to 16-cells (66.7% versus 72.4% for Expt 1, 71.0% versus 78.2% for Expt 2). However, significantly more (P < 0.05) of the one-cell embryos co-cultured with granulosa cell monolayers developed to morula, blastocyst and expanded blastocyst stages compared with those in cell-free culture (35.0 versus 27.1%, 25.1 versus 14.2%, 15.6 versus 5.4% for Expt 1; 37.6 versus 24.0%, 25.7 versus 11.0%, 16.8 versus 3.0% for Expt 2). Only embryos co-cultured with granulosa cell monolayers hatched (Expt 2). Embryos co-cultured with granulosa cell monolayers also had higher (P < 0.01) numbers of cells (92 +/- 42 versus 56 +/- 21 for blastocysts, 149 +/- 53 versus 81 +/- 29 for expanded blastocysts).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

Primary socialization theory: culture, ethnicity, and cultural identification. The links between culture and substance use. IV.

Ethnicity, perceived membership in a cultural group, and cultural identification, the strength of one's affiliation with a group, develop primarily through interactions with the primary socialization sources, the family, the school, and peer clusters. Cultural norms for substance use are also transmitted as part of these interactions. Substance use differs across cultures; in different cultures some forms of substance use are culturally required, others are tolerated, and others are sanctioned. Ethnicity and cultural identification, therefore, should relate to substance use. However, primary socialization theory indicates that simple relationships are not likely to be found for a number of reasons: 1) All members of an ethnic group do not have the same level of cultural identification and may not, therefore, have the same conformance to substance use norms. 2) Primary socialization,sources are embedded in subcultures, and subcultures have norms that may differ from those of the larger ethnic group. 3) The individual may experience and report differing levels of cultural identification and different substance use norms in different social contexts. 4) For an individual, ethnicity and cultural identification may derive from different primary socialization sources than drug use norms.

Adolescent↗
PubMed1989

Long-term survival of murine erythroid progenitors in long-term bone marrow culture and stromal cell culture: differentiation in peritoneal diffusion chamber culture.

Bone marrow cells of normal and cytosine-arabinoside (Ara-C) treated C57B1 mice were cultured in primary long-term culture (LTBMC) for a period of eight weeks. Non-adherent cells collected at weekly culture feedings consisted of neutrophils, macrophages and megakaryocytes. These were transferred into a) secondary peritoneal diffusion chamber cultures (DC) and b) secondary stromal cell cultures (SCC) first, and then into tertiary DC cultures. While in LTBMC and SCC there was no evidence of erythropoiesis, many erythroid colonies developed in DC cultures. It appears that undifferentiated erythroid progenitors may have a long survival in LTBMC and SCC devoid of erythropoietin and then differentiate in vivo in DC cultures in host mice without specific erythropoietic stimuli. Terminal differentiation and maturation of erythroid progenitors occurs to a limited extent in conventional DC cultures. The large number of erythroid colonies in DC observed in the present study could be due to increased sensitivity of undifferentiated erythroid progenitors from LTBMC to physiological levels of Epo in host mice of DC.

Animals↗
PubMed1990

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

The immunocytochemical staining patterns of cultured glioma cells were investigated. Fifty nine individual cases were stained at different in 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 of in vitro data obtained in primary cultures.

Antigens, Neoplasm↗
PubMed2000

Comparison of gen-probe AccuProbe group B streptococcus culture identification test with conventional culture for the detection of group B streptococci in broth cultures of vaginal-anorectal specimens from pregnant women.

The performance of the AccuProbe Group B Streptococcus Culture Identification Test (Gen-Probe Incorporated, San Diego, CA, USA) for the detection of group B streptococci (GBS) directly from LIM broth cultures of vaginal-anorectal swab specimens from pregnant women (two swabs per patient in most cases) was evaluated by comparing results to those of conventional GBS culture. Of 411 specimens analyzed, 82 were positive and 312 were negative for GBS by both methods. After initial testing, the percent agreement was 95.9%. The initial sensitivity, specificity, and positive and negative predictive values for the AccuProbe test were 90.1%, 97.5%, 91.1%, and 97.2%, respectively. Results were discrepant for 17 specimens: eight were GBS positive by probe and negative by culture; nine were negative by probe and positive by culture. To resolve discrepancies, culture plates were re-examined for GBS colonies, AccuProbe testing was repeated on the initial LIM broth cultures, and the second swab (if received) was inoculated to LIM broth for AccuProbe testing after overnight incubation. After discrepant resolution testing, the percent agreement between the two test methods was 97.8%. The final sensitivity, specificity, and positive and negative predictive values for the AccuProbe test were 95.6%, 98.4%, 94.6%, and 98.7%, respectively. These data suggest that the AccuProbe test is a reliable method for detecting GBS in vaginal-anorectal specimens, providing results more rapidly than conventional culture. However, strict adherence to the manufacturer's test protocol is necessary to limit technical errors.

Anal Canal↗
PubMed1984

Production of somatomedin-like activity by human adult tumor-derived, transformed, and normal cell cultures and by cultured rat hepatocytes: effects of culture conditions and of epidermal growth factor (urogastrone).

We have measured the production of a basic-somatomedin-like activity (SLA) by a variety of human tumor-derived, transformed, and normal postnatal cell cultures; and we have compared the production of SLA by these cell types with the production of SLA by adult rat hepatocytes cultured in serum-free medium. Cells derived from a human epidermoid carcinoma (KB), a pancreatic carcinoma (Panc-1), a Simian virus 40 transformed adult human skin-derived cell line (SV40 fibroblasts), and a normal adult human skin-derived fibroblast line released SLA when cultured in a serum-free growth medium. No SLA was recovered from the culture medium of human choriocarcinoma-derived cells (BeWo) or of a human lymphoblastoid cell line (IM-9). The production of SLA by rat hepatocytes cultured in serum-free medium appeared to exceed the production of SLA by the other cell cultures. In cultures of KB cells, SV40 fibroblasts, and rat hepatocytes, the production of SLA depended on the frequency with which the growth medium was renewed; in general, the highest rates of SLA production were observed when the medium was renewed every 48-72 h. The presence of mouse epidermal growth factor (urogastrone) (EGF-URO) in the serum-free culture medium stimulated the production of SLA by KB cells and by rat hepatocytes, but did not increase SLA production by normal or by SV40-transformed human skin-derived fibroblasts. We conclude that tumor-derived cells are capable of producing somatomedin-like activity and that the production of SLA by such cells can be subject to controls (nutrient availability, EGF-URO stimulation) that regulate SLA production, either by normal adult tissues, like liver, or by a variety of normal embryonic tissues.

Animals↗
PubMed1983

Difference in growth factor requirements of rat 3Y1 cells among growth in mass culture, clonal growth in low density culture, and stimulation to enter S phase in resting culture.

A semiserum-free medium was developed for monolayer culture of rat 3Y1 fibroblastic cells. The main components of the developed medium added to Dulbecco's modified Eagle's medium (DMEM) were insulin, transferrin, epidermal growth factor, poly-D-lysine, bovine albumin, oleic acid, and bovine alpha-globulin. In this medium, 3Y1 cells grew in mass culture at much the same rate as in DMEM supplemented with 10% fetal bovine serum (FBS), and colonies, albeit of smaller sizes, did form. Virally transformed derivatives of 3Y1 (simian virus 40-3Y1, polyoma virus-3Y1 and adenovirus type 12-3Y1) also formed colonies in the semiserum-free medium. When trypsinized 3Y1 cells were seeded with the medium lacking alpha-globulin, neither growth in the mass culture nor clonal growth in the low density culture (clonal growth) occurred. In this case, cell spreading was inhibited by albumin, and this inhibition was overcome by adding alpha-globulin or treating dishes with serum. When albumin was excluded from the semiserum-free medium, clonal growth did not occur, whereas growth in mass culture and stimulation of DNA synthesis in the resting mass culture (stimulation of DNA synthesis) were not so drastically affected. When oleic acid was removed, growth in mass culture was inhibited considerably, but no considerable effect was seen on clonal growth or on stimulation of DNA synthesis. In the absence of insulin, stimulation of DNA synthesis was inhibited more markedly than when other components were removed, but such was not the case with growth in mass culture and clonal growth.

Albumins↗
PubMed1992

[Purification and culture of mouse gallbladder epithelial cells in secondary culture using microexplant culture on collagen gel].

We established a new method of purification and culture of mouse gallbladder epithelial cells. In primary culture, mouse gallbladder tissue was cultured on collagen gel using microexplant culture method; the epithelial cells spread in a single sheet from the microexplant toward the periphery on the collagen gel. Tiny fragment of the collagen gel containing the border of epithelial cell layer was retransplanted onto the another collagen gel for secondary culture. This retransplantation allowed the proliferation of the epithelial cells without mesenchymal contamination, which spread on the collagen gel at the rate of 0.29 +/- 0.06 mm per day for more than two weeks. The cultured epithelial cells showed cuboid or columnar shape with focal mucus production, which is morphologically and functionally similar to the in vivo epithelial cells. This suggests that this in vitro culture method can be used to pathophysiological studies of biliary epithelial cells as well as for the method of purification of the epithelial cells.

Animals↗
PubMed1980

Chinese hamster lymphocyte cultures. Relationship between lymphocyte proliferation, cell concentration, culture time and culture area.

Different numbers of Chinese hamster lymphocytes were cultured in microtiter plates with flat-, round- and V-bottomed wells for different culture times. The smallest number of cells could be stimulated in plates with V-bottomed wells. At least 25,000-50,000 cells and a longer culture time than for round- or V-bottomed plates were required for maximal stimulation in flat-bottomed plates. For a given well conformation the optimal day of culture is earlier with higher and later with lower cell concentrations. The optimal culture time for a given number of cells is shortest in V- and longest in flat-bottomed plates. The amount of PHA producing the highest thymidine incorporation for a given number of cells depends upon the well conformation. It decreases with lower cell concentration and increases with longer culture time.

Animals↗
PubMed1983

Decreased insulin binding to cultured cells from a patient with the Rabson-Mendenhall syndrome: dichotomy between studies with cultured lymphocytes and cultured fibroblasts.

[125I]Insulin binding has been studied with cultured cells from an insulin-resistant patient with the Rabson-Mendenhall syndrome. Previously, this patient has been demonstrated to have decreased insulin binding to circulating blood cells. Similarly, [125I]insulin binding to cultured lymphocytes and cultured fibroblasts was markedly decreased. Whereas the decrease in [125I]insulin binding to cultured lymphocytes appeared to result from a decrease in receptor number in cultured lymphocytes, there appeared to be a reduction in the affinity of [125I]insulin binding to cultured fibroblasts. This discrepancy between the observations with the two cell types is a general phenomenon which has been described in patients with a variety of syndromes of extreme insulin resistance. It is possible that the interpretation of the studies with the fibroblasts is complicated by the confounding influence of receptors for insulin-like growth factors on the surface of those cells. If the number of insulin receptors is sufficiently reduced on fibroblasts from the patient with extreme insulin resistance, then the low level of residual [125I]insulin binding may involve primarily receptors for insulin-like growth factors, which bind insulin with relatively low affinity.

Adolescent↗
PubMed1991

Two types of Na(+)-currents in cultured rat optic nerve astrocytes: changes with time in culture and with age of culture derivation.

Na+ channel expression was studied in cultures of rat optic nerve astrocytes using whole-cell voltage-clamp recordings. Astrocytes from postnatal day 7 rat optic nerve (RON) expressed two distinct types of Na+ currents, which had significantly different h infinity curves. Stellate, GFAP+/A2B5+ astrocytes showed currents with h infinity curve midpoints close to -65 mV, similar to Na+ currents in most neurons. In contrast, flat fibroblast-like GFAP+/A2B5- astrocytes showed Na+ currents with h infinity midpoints around -85 mV, almost 20 mV more hyperpolarized than in neurons or A2B5+ astrocytes. Interestingly, Na+ current expression was maintained in A2B5+ astrocytes but began to decrease in A2B5- astrocytes after 6 days in vitro (DIV) and fell to or below the level of detection (i.e., 1 pA/pF) at 12 DIV. Astrocytes cultured from neonatal rats (P0) are almost exclusively GFAP+/A2B5-. These cells did not display measurable Na+ currents when studied at 2 DIV; however, Na+ current was observed after 5 DIV in A2B5- astrocytes from these neonatal (P0) cultures. These findings were substantiated by immunocytochemical experiments using 7493, an antibody raised against purified rat brain Na+ channels; in P0-derived astrocyte cultures 7493 antibody staining was initially lacking (up to 3 DIV), but it was prominent in cultures after 5 DIV, suggesting that Na+ current expression in RON astrocytes occurs postnatally.

Animals↗
PubMed1994

A novel two-compartment culture dish allows microscopic evaluation of two different treatments in one cell culture simultaneously. Influence of external pH on Na+/Ca2+ exchanger activity in cultured rat cardiomyocytes.

A new type of culture dish containing two separate compartments is described, that can be used in high-magnification microscopy. Using the dish, two halves of a single-cell culture, grown on a standard coverslip, can be exposed to different treatments simultaneously, allowing the effect of one treatment to be compared with that of the other treatment in the same culture. This way, the natural variability that might exist between different individual cultures is circumvented. In addition, by simultaneously conducting two experiments per dish, the number of experiments needed can be decreased. This both reduces the time to complete a series of experiments and allows the optimal use of specimens that are difficult to obtain, such as human material. We found there is an excellent barrier between the two compartments for lipophilic and hydrophilic compounds, and for low-molecular-mass cations. To illustrate the use of the dish we describe the influence of external pH on the activity of the Na+/Ca2+ exchanger in intact cultured neonatal rat ventricular cardiomyocytes. The intracellular free calcium concentration ([Ca2+]i) in the cardiomyocytes, measured using fura-2 and imaging fluorescence microscopy, was studied during sodium-free incubation. The resulting rise in [Ca2+]i at pH 7.4 in one compartment was compared with that in the other compartment in which the pH was either 6.0, 7.0, 7.4 or 8.0. It was found that below pH 7.4, Na+/Ca2+ exchanger activity was diminished, whereas at pH higher than 7.4 the Na+/Ca2+ exchanger activity was increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Removal of endotoxin from culture media by a polymyxin B sepharose column. The activity of contaminating endotoxin in culture media measured by the interleukin 1 inducing effect on human monocyte cultures and by the Limulus test.

The in vitro study of monocytes (Mo) poses several problems. Minor contamination with endotoxin (ET) of media and utensils as well as adherence to glass or plastic surfaces may activate the cells and cause pronounced production of monokines. Many commercially liquid culture media were found to contain ET in concentrations above 25 X 10(-12) g/ml. A simple system for the removal of ET from media and solutions was established by use of a commercially available Polymyxin B Sepharose gel. To measure the lipopolysaccharide (LPS) binding capacity of the gel, known concentrations of LPS were added to culture media, which were passed through a column consisting of the Polymyxin B Sepharose gel. The content of ET and added LPS in media was measured by the Limulus amoebocyte lysate (LAL) test before and after passage of the column. The LPS-binding capacity of the gel was approximately 2.4 X 10(-6) g/10 ml. The biological activity of contaminating ET and added LPS in media, before and after passage of the column, was also characterized by the capacity of the media to induce interleukin 1 (IL-1) secretion in human Mo cultures. The content of IL-1 in Mo culture supernatants was determined by the mouse thymocyte costimulatory (LAF) assay. By comparison of the activity of ET in these different biological systems, it was demonstrated that 15-20 X 10(-12) g/ml of ET stimulate human Mo cultures to IL-1 secretion.

Adult↗