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PubMed2005

Hyperhydricity in shoot cultures of Scrophularia yoshimurae can be effectively reduced by ventilation of culture vessels.

An effective procedure for obtaining healthy shoots from nodal segments of Scrophularia yoshimurae is described. Nodal segments cultured on Murashige and Skoog's (MS) basal medium supplemented with 1.0 mg L(-1) benzyladenine (BA) and 0.2 mg L(-1) alpha-naphthaleneacetic acid (NAA) induced multiple shoots in conical flasks that differed in the way they were closed and sealed. Hermitically sealed culture vessels resulted in high hyperhydricity/vitrification. High concentrations of ethylene and CO2 were found to accumulate in these vessels. The hyperhydricity of the shoot cultures could be decreased by progressively ventilating the vessels. Exchange of gases was achieved by removing the Parafilm sealing without affecting sterility. This reduced the hyperhydricity rate and gave a good recovery of vitrified shoots, but resulted in decreased proliferation and a dehydration of proliferating nodal segments and the culture medium. The best number of normal shoots was observed when the parafilm was removed for gaseous exchange after four weeks of culture incubation. The results show that hyperhydricity in shoot cultures of S. yoshimurae could be prevented by sufficient gas exchange during culture.

Aerobiosis↗
PubMed1989

Growth rate of human fibroblasts is repressed by the culture within reconstituted collagen matrix but not by the culture on the matrix.

Effect of reconstituted type I collagen gel on in vitro growth of fibroblasts was examined. A prolonged lag period was observed by culture either on the surface of three-dimensional collagen gel ("on-gel") or within the gels ("in-gel") as compared with the culture on plastic dishes. The rate of cell proliferation in logarithmic phase growth was repressed by the culture "in-gel" but not by the culture "on-gel". The differential growth rates between "in-gel" culture and "on-gel" culture should be ascribed to difference in distributions of interacting sites between cell and collagen fibrils. The repression of cell growth was more marked in a contracting collagen gel which contains higher density of collagen fibrils. The cell density in contracted gel per unit cross-section area was found to be much lower than that of confluent monolayer culture. These results suggest that the repression of cell growth by collagen fibrils in the three-dimensional gels is not due to direct cell-cell contact, but due to the distribution and number of contact sites between a cell and collagen fibrils.

Cell Count↗
PubMed2001

Angiotensin II-induced growth effects in vascular smooth muscle in cell culture and in the aortic tunica media in organ culture.

Several different studies have investigated the growth effects of angiotensin II on vascular smooth muscle cells in culture. However, smooth muscle cells change their phenotype when placed in culture. The objective of the present study was to investigate the effects of angiotensin II on (3)H-thymidine and (3)H-proline incorporation in vascular smooth muscle cells in culture and in the tunica media of blood vessels perfused at normal physiological pressures in organ culture, thus avoiding the phenotypic changes observed in cell culture. The perfusion system consisted of a peristaltic pump and a closed circuit of plastic tubing connected to a culture media bottle where thoracic rat aortae were placed. Angiotensin II induced an increase in (3)H-thymidine and (3)H-proline incorporation in both culture systems. The results suggest that angiotensin II may play a role in mediating cell growth in vascular smooth muscle cells in their 'contractile' as well as in their 'synthetic' phenotype.

Angiotensin II↗
PubMed1999

Comparison of human blastulation rates and total cell number in sequential culture media with and without co-culture.

Recent interest in delayed embryo transfers necessitated the evaluation of two improved in-vitro systems that could generate viable blastocysts. A total of 178 two-pronucleated embryos (entire cohorts) from 19 patients was cultured in IVF50 medium (100 microl) under oil for 24 h until day 2. Each patient's day 2 embryos were then equally allotted to two in-vitro systems. Embryos in system A were grown until the morning of day 3 on Vero cells covered with IVF50 medium (100 microl) under oil. The medium was then replaced on day 3 with a 1:1 mixture (100 microl) of IVF50:S2 medium and on day 4 with S2 medium only. The same culture protocol was used for system B without Vero cells. Throughout the 5 days all dishes were housed in sealed humidified modular chambers containing a triple gas atmosphere. Separately, 175 spare embryos from 80 patients were grown in system A and B up to days 6 and 7 for total cell number (TCN) analysis. Blastulation rates were not significantly different between system A and B (67.4 versus 68.5%; P > 0.01) although co-cultured embryos cleaved slightly faster by day 4. The overall pregnancy and implantation rates were 52.0% and 32.1% for the 19 patients each of whom received a mixed cohort of three day 5 embryos from both systems. TCN values for the day 6 and 7 blastocysts from both systems were high and increased steadily from days 6-7 and from expanded to hatching stages. There were no significant differences in TCN for day 6 expanded blastocysts between the two systems although day 6 hatching and hatched co-cultured blastocysts had greater values than non-co-cultured blastocysts (246.0 +/- 18.5 and 236.7 +/- 17.8 versus 173.0 +/- 13.5 and 166.5 +/- 16.0; P < 0.01). The results demonstrated that the culture protocol using the sequential IVF50-S2 media combination was a good substitute for Vero cell co-culture for the transfer of viable day 3-6 embryos.

Animals↗
PubMed2002

Sensitivity and rapidity of blood culture bottles in the detection of cornea organ culture media contamination by bacteria and fungi.

AIMS: To test the bactericidal activity of standard organ culture medium, and to compare the sensitivity and rapidity of blood culture bottles with conventional microbiological methods for detection of bacteria and fungi inoculated in a standard cornea organ culture medium. METHODS: The bactericidal activity of contaminated standard organ culture medium containing 100 IU/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 micro g/ml amphotericin B was evaluated after 48 hours of incubation at 31 degrees C with five inocula of 14 bacteria. Two yeasts (Candida spp) and one Aspergillus were also tested. Contaminated media were then inoculated in three blood bottles (aerobic, anaerobic, fungal) placed in a Bactec 9240 automat; three conventional microbiological broths were the control. Changes in colour of organ culture medium and growth on conventional broth were screened daily by visual inspection. The sensitivity and rapidity of detection of contamination were compared between the three methods: blood bottle, conventional, and visual. RESULTS: Organ culture medium eradicated five bacteria irrespective of the starting inoculums: Streptococcus pneumoniae, Branhamella catarrhalis, Escherichia coli, Propionibacterium acnes, and Haemophilus influenzae. For micro-organisms where the medium was ineffective or bactericidal only (methicillin resistant Staphylococcus aureus, methicillin sensitive Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Pseudomonas aeruginosa, Acinetobacter baumannii, Bacillus subtilis, Klebsiella pneumoniae, Enterococcus faecalis, Candida albicans, Candida kruzei, Aspergillus fumigatus), the blood bottle, conventional, and visual methods detected microbial growth in 100%, 76.5%, and 70% of cases respectively. Mean detection time using blood bottles was 15.1 hours (SD 13.8, range 2-52). In cases of detection by the blood bottle method and the conventional method, the former was always faster: 95.5% against 65.2% detection within 24 hours (p=0.022) respectively. CONCLUSIONS: Blood bottles detect more efficiently and more rapidly a wider range of bacteria and fungi than the conventional microbiological method and the visual inspection of organ culture media.

Bacteria↗
PubMed1997

Thyrotropin-releasing hormone gene expression by anterior pituitary cells in long-term cultures is influenced by the culture conditions and cell-to-cell interactions.

It has been suggested that TRH, synthesized by anterior pituitary (AP) cells in long-term monolayer cultures, may act as a paracrine or autocrine regulator. Because local control through messenger molecules depends on the cellular microenvironment, we were interested in studying the synthesis of TRH by AP cells in different culture systems and under various conditions. When AP cells were cultured as monolayers in medium containing 10% FCS for long periods of time (up to 3 weeks), a considerable increase in TRH content and prepro-TRHmessengerRNA (preproTRHmRNA) levels could be demonstrated by RIA and Northern blot analysis, whereas the cellular content of the TRH-like peptide pyroGlu-Glu-Pro-NH2 decreased with time in culture to undetectable levels. The release of TRH could be stimulated by depolarizing concentrations of K+ (55 mM), by the Ca++ ionophore A23187, and by GnRH, but not by CRH or GRF, indicating that TRH is stored in gonadotropes. Moreover, a combined in situ hybridization and immunocytochemical analysis demonstrated colocalization of LH in preproTRHmRNA-positive AP cells. When AP cells were cultured as reaggregates in the same (FCS-containing) medium, only a marginal increase in TRH content and preproTRHmRNA levels was observed. Irrespective of the culture systems and the culture conditions used, TRH gene expression was not observed when FCS was omitted. These results indicate that TRH gene expression more likely reflects derepression, rather than induction, of the TRH gene.

Animals↗
PubMed2002

Biphasic culture strategy based on hyperosmotic pressure for improved humanized antibody production in Chinese hamster ovary cell culture.

Hyperosmotic pressure increased specific antibody productivity (q(Ab)) of recombinant Chinese hamster ovary (rCHO) cells (SH2-0.32) and it depressed cell growth. Thus, the use of hyperosmolar medium did not increase the maximum antibody concentration substantially. To overcome this drawback, the feasibility of biphasic culture strategy was investigated. In the biphasic culture, cells were first cultivated in the standard medium with physiological osmolality (294 mOsm/kg) for cell growth. When cells reached the late exponential growth phase, the spent standard medium was replaced with the fresh hyperosmolar medium (522 mOsm/kg) for antibody production. The q(Ab) in growth phase with the standard medium was 2.1 microg per 10(6) cells/d, whereas the q(Ab) in antibody production phase with the hyperosmolar medium was 11.1 microg per 10(6) cells/d. Northern blot analysis showed a positive relationship between the relative contents of intracellular immunoglobulin messenger ribonucleic acid and q(Ab). Because of the enhanced q(Ab) and the increased cell concentration in biphasic culture, the maximum antibody concentration obtained in biphasic culture with 522 mOsm/kg medium exchange was 161% higher than that obtained in batch culture with the standard medium. Taken together, the simple biphasic culture strategy based on hyperosmotic culture is effective in improving antibody production of rCHO cells.

Animals↗
PubMedDate not supplied

Dilute culture media as an environmental or physiological simulant in cultured gill epithelia from freshwater rainbow trout.

The electrophysiological and ion-transporting properties of cultured gill epithelia from freshwater (FW) rainbow trout were examined in the presence of dilute cell culture media as an environmental or physiological simulant. Gill epithelia were cultured on cell culture inserts under symmetrical conditions (L15 apical-L15 basolateral) for 6-7 d. The following experiments were then conducted. (1) To mimic a gradual lowering of environmental salinity, apical L15 medium was progressively diluted with FW (first to 2/3 L15 for 8 h and then to 1/3 L15 for 6 h) before the introduction of apical FW (FW apical-L15 basolateral, analogous to a fish in a natural FW environment). Dilute apical media had no significant effect on the electrophysiological properties of preparations compared with symmetrical culture conditions, and no evidence for active Na(+) or Cl(-) transport was observed. Preparations subsequently exposed to apical FW exhibited a negative transepithelial potential and evidence of active Cl(-) uptake and slight Na(+) extrusion. (2) To mimic the extracellular fluid dilution that occurs in euryhaline fish after abrupt transfer from saline to FW, the osmolality or ionic strength (or both) of basolateral media was reduced by 20-40% (using either FW or FW + mannitol) while simultaneously replacing apical media with FW. Under these conditions, Na(+) and Cl(-) influx rates were low compared with efflux rates, while the Ussing flux ratio analysis generally indicated active Cl(-) uptake and Na(+) extrusion. The Na(+)-K(+) adenosine triphosphatase activity was not affected by alterations in basolateral osmolality. Our studies indicate that cultured trout gill epithelia are tolerant of media dilution from both the apical and the basolateral direction; however, neither treatment alone appeared to increase ion influx rates or stimulate active Na(+) uptake in cultured trout gill epithelia.

Animals↗
PubMed1996

Effect of medium renewal during culture in two different culture systems on development to blastocysts from in vitro produced early bovine embryos.

Three experiments investigated the effects of culture systems and renewal of medium (synthetic oviduct fluid medium (SOFM) supplemented with glutamine and amino acids) during in vitro development of in vitro-produced 2- to 4-cell bovine embryos. In Exp. 1, the early cleaved embryos were cultured under three different conditions for 186 h using 24-well dishes. The proportions of blastocysts (P < .05) and hatched blastocysts (P < .001) were lower for culture in SOFM + .8% BSA with renewal of medium at 48-h intervals (26.7 and 7.9%) than for cultures in SOFM + 10% human serum without renewal of medium (32.6 and 20.6%) and in SOFM + BSA without renewal of medium (32.2 and 23.0%). Two culture systems (well and microdrop) with or without renewal of medium (SOFM + BSA) were compared in Exp. 2 (186-h culture) and 3 (138-h culture). In Exp. 2, the microdrop system was superior (P < .05) to the well system for development of hatched blastocysts (19.1 and 11.7%), and the highest development to blastocysts (39.6%) and hatched blastocysts (27.8%) was observed in the microdrop system without renewal of medium. Experiment 3 also showed that the microdrop system was superior (P < .05) to the well system for development to blastocysts (32.7 and 27.2%), and the highest development rate to blastocysts (37.6%) occurred without renewal of medium. When the medium was not renewed in Exp. 2 and 3, the ammonium concentrations increased in both the well and microdrop systems. The results indicate that despite the reduction in ammonium concentrations resulting from medium renewal, renewal resulted in reduced development of embryos to hatched blastocysts in both the well and microdrop systems.

Amino Acids↗
PubMed1981

A comparison of various laboratory scale culture configurations for microcarrier culture of animal cells.

When developing procedures for microcarrier culture it is important to use equipment which results in even suspension of microcarriers with minimal shear forces and which permits maximum growth of the culture. A variety of different laboratory scale culture systems were investigated including traditional magnetic spinner flasks, modified spinner flasks, cultures stirred with bulb-shaped rods and a fluid lift culture system. All systems were compared with respect to growth of either MRC-5 or Vero cells on Cytodex microcarriers. Although most of the earlier work with microcarriers has been performed with traditional magnetic spinner flasks, recently modified equipment resulted in better plating efficiencies and greater cell yields. Cultures stirred with a bulb-shaped rod and contained in flasks with contoured bases were particularly useful for cells having a low plating efficiency and provided thorough stirring of the medium with minimum shear forces. A fluid lift system based on upward perfusion of medium through a culture contained in a chromatographic column is described. This technique could provide a useful alternative for suspending microcarriers and provides for accurate in-line control of gas tensions.

Animals↗
PubMed1998

[Culture of human keratocytes. Influence of culture conditions and ultrastructural aspects].

BACKGROUND: To investigate the influence of fetal calf serum (FCS) and fibroblast growth factor (FGF) on human keratocyte growth in vitro and cell differentiation, and to describe cultured human keratocyte ultra-structure. METHODS: Human keratocytes were cultured in TC 199/Ham F12 media, supplemented or not with 10% FCS, aFGF, and bFGF. Keratocyte growth was studied. Cultured keratocytes were analyzed by means of immunochemistry and transmission electron microscopy. RESULTS: Without fetal calf serum, cell population doubling occurred after 7 days of culture and no alpha smooth muscle-actin cell expression was observed. With serum, cell population increased by 1 log after 7 days of culture and all of the cells were alpha SM-actin + bFGF or aFGF-addition to the serum-containing medium resulted in a dramatic decrease in this alpha SM-actin expression. Nuclei were found to be oval and regular in cross-sections, and round and indented in frontal sections. Numerous cytoplasmic organelles were observed, as were cell expansions, gap junctions, omega-shaped structures, and fenestrations. Cultured keratocytes synthesized collagen fibers and filaments. CONCLUSION: Fetal calf serum allows human keratocytes to grow with a myofibroblast cell phenotype, whereas addition of FGF results in a fibroblast cell phenotype. Ultrastructure of cultured keratocyte is similar to that observed in situ.

Actin Cytoskeleton↗
PubMed1999

Decrease in culture temperature releases monolayer endothelial cell sheets together with deposited fibronectin matrix from temperature-responsive culture surfaces.

Bovine aortic endothelial cells were cultured on surfaces grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), in the presence of serum. Cells adhered, spread, proliferated, and reached confluency as observed on ungrafted tissue culture polystyrene dishes. A decrease in culture temperature released cells only from the grafted surfaces without enzymatic or ethylenediaminetetraacetic acid treatment. Upon lowering temperature, the culture surfaces changed from hydrophobic to hydrophilic owing to the hydration of grafted PIPAAm and thus weakened the cell attachment to the dishes. Released cells maintained cell-cell junctions composing monolayer cell sheets. Immunoblotting and immunofluorescence microscopy revealed that fibronectin (FN) was deposited and accumulated on the grafted surfaces during the culture. Furthermore, the deposited FN matrix adhering to cell sheets was also recovered from temperature-responsive surfaces by low-temperature treatment, while trypsin treatment destroyed the matrix. The recovery of FN by low-temperature treatment was as high as by physical scraping with a rubber blade. Temperature-responsive surfaces can provide a novel method to use cultured confluent cell sheets for tissue engineering, and also to elucidate structure and function of deposited extracellular matrix during cell culture.

Acrylamides↗
PubMed1987

Osteoclast-like cell formation in fetal and newborn long-term baboon marrow cultures is more sensitive to 1,25-dihydroxyvitamin D3 than adult long-term marrow cultures.

It is unknown if osteoclasts derived from animals at different developmental stages differ. To study this question, we used a long-term baboon marrow culture system in which osteoclast-like multinucleated cells (MNC) are formed. Fetal, newborn, or adult baboon marrow cultures were tested to determine if they differ in their responsiveness to osteotropic hormones. In fetal and newborn marrow cultures 1,25-dihydroxyvitamin D3 (1,25D3) at 10(-10) M significantly increased MNC formation with a maximal effect seen at 10(-9) M. Higher concentrations of 1,25D3 progressively decreased MNC formation. In contrast, in adult baboon marrow cultures, 10(-9) M 1,25D3 was required to significantly increase MNC formation, with a maximal affect at 10(-8) M 1,25D3. Calcitonin (25-200 ng/ml) inhibited MNC formation in fetal, newborn, or adult baboon marrow cultures treated with 1,25D3 in an identical manner. The effects of 1,25D3 on granulocyte-macrophage progenitor cells (CFU-GM), the probable precursors of MNC, were identical in fetal and adult baboon marrow cultures, with a significant inhibition of CFU-GM colony formation at 10(-8) M 1,25D3. These results suggest that 1) osteoclast precursors are more sensitive to some osteotropic hormones during the fetal and newborn periods, and 2) differences in the 1,25D3 sensitivity of osteoclast-like MNC formation in fetal, newborn, and adult baboon marrow cultures are not due to effects on early proliferating precursors but may result from effects of 1,25D3 on fusion of later precursors for MNC.

Age Factors↗
PubMed2003

Detection of Staphylococcus aureus in peripheral blood stem cell cultures after sterilization of standard blood cultures.

We report central venous catheter (CVC)-associated Staphylococcus aureus bacteremia detected by apheresis product culture after sterilization of standard blood cultures. A 64-year-old man with non-Hodgkin's lymphoma underwent peripheral blood stem cell (PBSC) collection. Five days after placement of a CVC, inflammation was evident at the insertion site. The CVC was removed and cephalexin was begun. Discharge at the site contained neutrophils and gram-positive cocci in pairs and clusters. Cultures of the discharge, of blood drawn via the CVC, of the CVC tip and of the apheresis product collected that day grew methicillin-susceptible S. aureus (MSSA). Cephalexin was discontinued in favor of oxacillin. Three days after removal of the CVC, PBSC collections were resumed via a contralateral CVC. Three sets of standard blood cultures drawn the day the new CVC was placed and the following day were negative, yet apheresis product cultures from each of these days grew MSSA. PBSC collections were halted, the CVC was removed, and oxacillin was continued via a peripherally inserted central catheter. Transesophageal echocardiography after two weeks of therapy revealed thickened mitral leaflets and damage to the posterior leaflet. Transthoracic echocardiography 11 weeks preceding this study had demonstrated normal mitral valve anatomy and function. Oxacillin was continued for six weeks, after which PBSC collections were resumed. Pulsed-field gel electrophoresis of the MSSA isolates revealed a clonal pattern. Cultures of apheresis product may be more sensitive to the presence of bacteremia than standard blood cultures, and they should guide clinical decisions when the bacteria isolated are potential pathogens or suggest clinical infection.

Blood Cells↗
PubMed1995

Adhesive substrates influence acid-productive activities of cultured rabbit osteoclasts: cultured osteoclasts with large vacuoles have enhanced acid-productive activities.

Since acid secretion of polarized osteoclasts on the bone surface has an important role in bone resorption, we examined the acid-productive activities of osteoclasts by measuring the pH of acidic organelles such as endosomes, lysosomes, and vacuoles in cultured rabbit osteoclasts with FITC-dextran as a fluorescent pH probe. The average pH value of acidic organelles of osteoclasts cultured on plain glass coverslips was 5.3 +/- 0.2. The pH of the acidic organelles correlated well with the size and amount of the vacuoles in the cells. Using this FITC-dextran method, we also examined the effects of adhesive substrates, such as type I collagen, vitronectin, and dentine, on the acid-productive activities of osteoclasts and found that the pH value of acidic organelles of osteoclasts cultured on dentine slices was significantly lower than that of osteoclasts cultured on plain glass coverslips. Likewise, in the case of the osteoclasts cultured on type I collagen- or vitronectin-coated glass coverslips, the pH values of acidic organelles were slightly lower and the proportion of osteoclasts having large vacuoles was increased compared with the cells cultured on the plain glass coverslips. These results indicate that osteoclasts containing large vacuoles have high acid-productive activities, and adhesive substrates such as type I collagen and vitronectin influence the formation of large vacuoles in cultured osteoclasts.

Acids↗
PubMed1990

The advantage of the aggregate culture of isolated ovarian cell types over the monolayer culture.

Ovarian cells such as theca interna, granulosa and corpus luteum cells were isolated from pig ovaries and cultured in Erlenmayer flasks (25 ml) containing 3.5 ml of culture medium. The media were replaced every second day and frozen to -20 degrees C for later steroid analysis. The reaggregation of cells was completed within 2-3 days and this was then followed by a period of cell migration. During the subsequent 5-6 day period the reaggregates became larger. The best results were obtained in cultures of isolated theca alone and in combination with granulosa cells, as well as of early corpus luteum cells. Granulosa cells did not aggregate as easily or as completely as the corpus luteum cells. All types of cells investigated were able to secrete progesterone into the culture medium. They secreted more progesterone and for a longer time than cells cultured as monolayers. The aggregate culture seems to be a good model to study the secretion of ovarian cells, by creating the tri-dimensional, and thus more physiological, culture system.

Animals↗
PubMed1979

Tapping culture--an improved method for cell suspension culture.

A new culture vessel was designed for cell suspension culture. A silicone-covered magnet bar fixed by one end to the side wall of the bottle was held horizontally a short distance from the bottom. A standard type magnetic stirrer was used. In contrast to the conventional horizontal movement of "stirring" in cultures the bar moves vertically with a "tapping" motion. This improvement resulted in less cell injury, higher rate of cell proliferation and formation of fewer bubbles than in the conventional type. Nine cell types were simultaneously cultivated in tapping, stirring and stationary culture. All cell types proliferated more luxuriously in tapping cultures then in stirring cultures. Serial cultivation of cells in tapping cultures was also successful.

Animals↗
PubMed1990

High oxygen atmosphere for neuronal cell culture with nerve growth factor. I. Primary culture of basal forebrain cholinergic neurons from fetal and postnatal rats.

Cholinergic neurons cultured from postnatal days 11-13 (P11-P13) rat basal forebrain showed better survival in the culture condition using a 50% O2 atmosphere with and without nerve growth factor (NGF) than in a low (10 or 20%) O2 atmosphere. Except for the culture at a low cell density, the beneficial effect of the highly oxidized culture condition was found in the culture from P3 neurons, but not from embryonic day 18 neurons. The survival of microtubule-associated protein 2 (MAP2)-positive neurons in culture from P3 basal forebrain regions was more enhanced in a 50% O2 atmosphere than in 20% and also 10% O2 atmosphere. The viable number of the MAP2-positive neurons in a 10% O2 condition was about half of that in a 20% condition. These results suggest that the response of the cultured neurons to an incubator O2 concentration changes during the neuronal development in CNS from fetal to postnatal stages.

Acetylcholine↗