Static Culture System Research Articles

320 EFFECT OF OXYGEN TENSION AND FOLLICLE CELLS DURING IN VITRO CULTURE OF PORCINE OOCYTES IN FOLLICULAR FLUID ON THEIR MATURATION AND FERTILIZATION

If we could use porcine follicular fluid (pFF) as a solo in vitro maturation (IVM) medium, the preparation of complicated medium wouldn't be necessary. In this study, we investigated the effects on nuclear maturation and subsequent IVF of oxygen tension and follicle cells (FC) during IVM of porcine oocytes in pFF using static (S) and rotating (R) systems. In Experiment 1, all of the cumulus–oocyte complexes (COCs) in pFF were collected, and COCs with compact cumulus cells were selected for IVM. Also, small clusters of FC were collected by centrifugation of the pFF after filtration through 212 µm mesh, and pFF without any cells was prepared by centrifugation and used as a maturation medium (MpFF) after supplementation with FSH and antibiotics. The COCs were transferred to 2 mL (in a 35-mm petri dish) or 3.5 mL (in a 15-mL test tube) of MpFF with or without FC (5.2 × 106 cells mL−1) added and were cultured for 48 h at 38.5°C in 5% CO2 and 20% O2 (in air) or 5% O2 using the S or R culture systems (5O2/FC−/S, 5O2/FC+/S, 20O2/FC−/S, 20O2/FC+/S, 5O2/FC−/R, 5O2/FC+/R, 20O2/FC−/R, and 20O2/FC+/R groups). The oxygen tension had no effect on nuclear maturation except for the FC−/R groups. In the FC−/R groups, cumulus cells were detached from oocytes and their maturation rates were so low that it was difficult to discuss the effect of oxygen tension. When cultured with FC, oocytes were surrounded with expanded cumulus cells and matured to metaphase II (MII) at higher rates in 5O2/FC+/R (67.7%) and 20O2/FC+/R (65.9%) than those in 5O2/FC+/S (17.5%) and 20O2/FC+/S (35.7%) (chi-squared test; P < 0.05). In contrast, when cultured without FC, oocytes matured to MII at higher rates in 5O2/FC−/S (78.3%) and 20O2/FC−/S (76.5%) than those in 5O2/FC−/R (32.7%) and 20O2/FC−/R (11.4%) (P < 0.05). In Experiment 2, oocytes were cultured in 5O2/FC−/S, 20O2/FC−/S, 5O2/FC+/R, or 20O2/FC+/R; fertilized in vitro, as reported previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041), and fixed 10 h after IVF. Oocytes cultured in 20O2/FC+/R showed a significantly higher sperm penetration rate (95.9%) than those cultured in 5O2/FC−/S and 20O2/FC−/S (88.0% and 88.4%, respectively), but were similar to those cultured in 5O2/FC+/R (90.9%) (P < 0.05). Oocytes cultured in 5O2/FC+/R formed a male pronucleus at a higher rate (77.1%) than those cultured in 5O2/FC−/S, 20O2/FC−/S, and 20O2/FC+/R (52.8, 51.6, and 63.3%, respectively) (P < 0.05). These results indicate that oxygen tension had no effect on IVM and IVF, except for a higher male pronucleus formation rate in 5O2/FC+/R than in 20O2/FC+/R, for porcine oocytes cultured in pFF, and that the addition of FC to pFF in the rotating culture system promoted nuclear maturation and a high male pronucleus formation rate after IVF, especially under 5% O2. In contrast, when the static culture system was used for IVM, the addition of FC was found to be detrimental to oocyte maturation in pFF.

Propagation kinetics of retrovirus transgene vector and replication-competent retrovirus in static and microcarrier cell culture systems using different medium exchange strategies

While retrovirus vector (RV) is the main virus vector used in human gene therapy trials, the biosafety issues that surround currently used RVs have become a matter of concern. Similar to the insertional mutagenesis in the therapeutic target cells, the generation of replication-competent retrovirus (RCR) must be minimized during the manufacture of the virus vector. This work investigated the kinetics of RV and RCR production in PA317-RCM1 producer cells in static and microcarrier cell culture systems. RCR in the progeny of transduced Mus dunni cells was detected by the PCR method and the titer of RCR was quantified by cell-based S+/L− assay. The specific rates of RCR production in microcarrier cultures were 271–462% higher than those in the static well-plate cultures. Increased medium exchange operations yielded higher specific rates of RCR production in both static and microcarrier cultures. The optimal medium exchange strategy was on an every 2-day schedule, yielding the highest RV/RCR ratio in static culture but not microcarrier culture. Results of this study presented the difficulty for gene therapy processes that together with the product RV also unwanted RCR produced in two different cell culture systems.

Flow modelling within a scaffold under the influence of uni-axial and bi-axial bioreactor rotation

The problem of donor scarcity has led to the recent development of tissue engineering technologies, which aim to create implantable tissue equivalents for clinical transplantation. These replacement tissues are being realised through the use of biodegradable polymer scaffolds; temporary/permanent substrates, which facilitate cell attachment, proliferation, retention and differentiated tissue function. To optimise gas transfer and nutrient delivery, as well as to mimic the fluid dynamic environment present within the body, a dynamic system might be chosen. Experiments have shown that dynamic systems enhance tissue growth, with the aid of scaffolds, as compared to static culture systems. Very often, tissue growth within scaffolds is only seen to occur at the periphery. The present study utilises the Computational Fluid Dynamics package FLUENT, to provide a better understanding of the flow phenomena in scaffolds, within our novel bioreactor system. The uni-axial and bi-axial rotational schemes are studied and compared, based on a vessel rotating speed of 35 rpm. The wall shear stresses within and without the constructs are also studied. Findings show that bi-axial rotation of the vessel results in manifold increases of fluid velocity within the constructs, relative to uni-axial rotation about the X- and Z-axes, respectively.

Enhanced Biological and Mechanical Properties of Artificial Dermis Prepared Using a Perfusion Culture System

A culture system that is capable of providing even and uniform distribution and deposition of cells and extracellular matrix (ECM) is desired to enhance biological functions of the tissue-engineered artificial dermis (TEADs). For this purpose, we have developed a perfusion culture system that offers uniform exchange of nutrients and gases along the scaffold. Viability and effectiveness of the system were investigated by comparing biological and mechanical properties of TEADs. Results showed that the TEADs constructed by the perfusion culture system revealed significantly increased cell growth, ECM synthesis, and elastic modulus compared to those by the conventional static culture system. In addition, histological findings indicated that cells were more evenly distributed and ECM deposition increased in TEADs with the perfusion culture system. Therefore, it can be suggested that the perfusion culture system can constitute a more promising approach for constructing the TEADs.

Rat Ghrelin Stimulates GH Release and GH mRNA Expressionin the Pituitary of Orange-spotted Grouper, Epinephelus Coioides

Ghrelin was recently demonstrated as an endogenous ligand of the growth hormone (GH) secretagogue receptor (GHS-R), which could promote the release of GH in mammal significantly. The present study conducted to determine whether ghrelin stimulate the release and synthesis of GH in orange-spotted grouper (Epinephelus coioides). Rat ghrelin was incubated with the pituitary fragments of grouper in static culture system. The culture medium was collected at 1, 6, 12, 18 and 24 h after incubation to detect the contents of GH by homologous radioimmunoassay. The level of GH mRNA in the pituitary fragments was measured by a sensitive chemiluminescent ribonuclease protection assay. The results showed that rat ghrelin not only stimulated the release of GH but also augmented the GH mRNA level in grouper. It suggested that the ghrelin-like peptide and the GHS-R involved in the regulation of GH synthesis and release in grouper. The present study would provide a better understanding of the regulatory mechanism of GH release in marine fish.

Control of gas phase for enhanced cellulase production by Penicillium decumbens in solid-state culture

The control of gas phase, by periodic pressure oscillation coupled with forced aeration, was developed for improvement of cellulase production by Penicillium decumbens in solid-state culture (SSC). The effects of pressure oscillation amplitude, frequency, and airflow rate on cellulase production were investigated in detail. The technique was shown to be effective in the control of temperature in the substrate bed. The maximal temperature variation in the substrate bed was +1.5 °C of the set value (30 °C) under the operational strategy described above, while only +6.8 °C in static tray systems. At 30 °C, 4 atm, and an airflow rate of 7 l min −1, the maximal FPA and β-glucosidase activities reached 17.7 IU g −1 substrate dry matter (SDM) and 52.8 IU g −1 substrate dry matter, respectively. Only 9.7 and 23.7 IU g −1 substrate dry matter were obtained in static tray culture systems.

Dynamic in vivo imaging of postimplantation mammalian embryos using whole embryo culture

Due to the internal nature of mammalian development, much of the research performed is of a static nature and depends on interpolation between stages of development. This approach cannot explore the dynamic interactions that are essential for normal development. While roller culture overcomes the problem of inaccessibility of the embryo, the constant motion of the medium and embryos makes it impossible to observe and record development. We have developed a static mammalian culture system for imaging development of the mouse embryo. Using this technique, it is possible to sustain normal development for periods of 18-24 h. The success of the culture was evaluated based on the rate of embryo turning, heart rate, somite addition, and several gross morphological features. When this technique is combined with fluorescent markers, it is possible to follow the development of specific tissues or the movement of cells. To highlight some of the strengths of this approach, we present time-lapse movies of embryonic turning, somite addition, closure of the neural tube, and fluorescent imaging of blood circulation in the yolk sac and embryo.

A beneficial coculture: charm abalone Haliotis discus hannai and sea cucumber Stichopus japonicus

Juvenile charm abalone Haliotis discus hannai (0.35±0.12 g) and sea cumber Stichopus japonicus (5.0±1.2 g) were cocultured in a static culture system for 90 days during the overwintering period. The levels of ammonium nitrogen and nitrite in the water of coculture groups were lower than those in control group (abalone alone) throughout the experiment. No mass mortality occurred in any groups, but abalone in coculture groups showed significantly better growth performance and higher survival, suggesting that coculture of juvenile abalone with sea cucumber, a deposit-feeding species, can reduce the levels of inorganic nitrogen in the water and consequently enhance growth. This coculture appears to be beneficial and could be developed as an alternative culture method in abalone farms during the heating-aided overwintering period.

Effect of GnRH and its antagonist (Antarelix) on LH release from cultured bovine anterior pituitary cells.

In the following investigations, the LH secretion of cells from pituitaries in heifers on days 16-18 of their oestrous cycle (n = 14) was analysed. Cells were dissociated with trypsin and collagenase and maintained in a static culture system. For the estimation of LH release, the cells were incubated with various concentrations of mammalian GnRH (Lutrelef) for 6 h. To determine the action of Antarelix (GnRH antagonist), the cells were preincubated for 1 h with concentrations of 10(-5) or 10(-4) M Antarelix followed by 10(-6) M GnRH coincubation for a further 6 h. At the end of each incubation, the medium was collected for LH analysis. Parallel, intracellular LH was qualitatively detected by immunocytochemistry. Changes in the intensity of LH staining within the cells in dependence of different GnRH concentrations were not observed, but a significant increase LH secretion in pituitary cells was measured at 10(-6) M GnRH. Antarelix had no effect on basal LH secretion at concentrations of 10(-4) and 10(-5) M. After coincubation of pituitary cells with Antarelix and GnRH, Antarelix blocked the GnRH-stimulated LH secretion with a maximal effect of 10(-4) M, but the staining of immunoreactive intracellular LH was detected at approximately the same level compared to the pituitary cells treated with exogenous GnRH alone. These data demonstrate that Antarelix is effective in influencing the GnRH-stimulated LH secretion of pituitary cells in vitro. After administration of Antarelix in vivo, the GnRH-stimulated LH secretion of cultured pituitary cells was not inhibited.

Bioprocess development for the cultivation of human T-lymphocytes in a clinical scale.

Adoptive transfer of large numbers of donor-derived T-lymphocytesmay offer a promising treatment of a variety of viral and malignant diseases. The key step in this approach is the ex vivo generation of sufficient quantities of these cells in a short time.We have investigated the influence of several important cultivation parameters on the proliferation of human T-lymphocytes to develop a large-scale fermentation process usingdifferent types of stirred bioreactors. Such systems offer manypotential advantages over the static culture systems commonlyused today.Peripheral blood mononuclear cells of healthy but CMV positive donors were stimulated with monoclonal antibodies (anti-CD3 and anti-CD28) and Interleukin-2. The influence of osmolality, Interleukin-2 concentration, pH, oxygen tension, feeding strategyand temperature on T-cell proliferation was investigated and theoptimised conditions were transferred to a novel stirred suspension bioreactor with an especially designed magnetic stirrbar to minimize the shear force (working volume 550 ml) and a standard stirred vessel (working volume 1000 ml).Preferable conditions for the cultivation of primary T-lymphocytes were an osmolality of 276-330 mOsmol kg(-1),an Interleukin-2 concentration of 100 U ml(-1), a pH rangeof 7.0 to 7.3, an oxygen tension of 5-50% and a temperature of 38.5 degrees C. After 238 h of cultivation 2.8 x 10(9) cells in the stirred vesseland 1.5 x 10(9) cells in the suspension bioreactor were obtained with a percentage of T-cells >94%. The specificity of the cells wasmaintained during cultivation as proven by IFN-gamma secretionafter exposure to a hCMV protein.

Culture of human T cells in stirred bioreactors for cellular immunotherapy applications: shear, proliferation, and the IL-2 receptor.

Ex vivo expansion of T cells is a key step of many cellular immunotherapy protocols, which require large numbers of immune cells to eradicate malignant or virally infected cells. The use of stirred culture systems for T cell expansion offers many potential advantages over the static culture systems commonly used today, including homogeneity of culture conditions, ease of sampling, and implementation of control systems. Primary human T cells as well as the transformed TALL103/2 T cell line were cultured in 100-mL spinner flasks as well as 2-L bioreactors to investigate the effects of shear forces produced by agitation and sparging-based aeration on the expansion of T cells. Primary T cells could be successfully grown at agitation rates of up to 120 rpm in the spinner flasks and to 180 rpm in the bioreactors with no immediate detrimental effects on proliferation. Exposure to agitation and sparging did, however, cause a significantly increased rate of downregulation of the interleukin-2 receptor (IL-2R), resulting in lower overall expansion potential from a single stimulation as compared to static controls, with faster IL-2R downregulation occurring at higher agitation rates. For the primary T cells, no significant effects of agitation were found on expression levels of other key surface receptors (CD3, CD28, or CD62L) examined. No significant effects of agitation were observed on primary T cell metabolism or levels of cellular apoptosis in the cultures. The TALL103/2 T cell line was found to be extremely sensitive to agitation, showing severely reduced growth at speeds above 30 rpm in 100-mL spinner flasks. This unexpected increased fragility in the transformed T cell line as compared to primary T cells points out the importance of carefully selecting a model cell line which will accurately represent the characteristics of the cell system of interest.

Primary porcine hepatocytes with portal vein serum cultured on microcarriers or in spheroidal aggregates

AIM:To develop a culture mode providing durable biomaterials with high yields and activities used in bioartificial liver.METHODS:Hepatocytes were isolated from a whole pig liver by Seglen s method of orthotopic perfusion with collagenase. In culture on microcarriers, primary porcine hepatocytes were inoculated at a concentration of 5center dot10(7)/mL into the static culture systems containing 2g/L Cytodex-3, then supplemented with 100mL/L fetal calf serum (FCS) or 100mL/L porcine portal vein serum (PPVS) respectively. In spheroidal aggregate culture hepatocytes were inoculated into 100mL siliconized flasks at a concentration of 5.0center dot10(6)/mL.RESULTS:In culture on microcarriers hepatocytes tended to aggregate on Cytodex-3 obviously after being inoculated. Typical multi-cellular aggregated spheroids could be found in the two systems 24h-48h after hepatocytes were cultured. The morphological charact-eristics and synthetic functions were maintained for 5wk in FCS culture system and 8wk in PPVS culture system. In spheroidal aggregate culture about 80%-90% isolated hepatocytes became aggregated spheroids 24h after cultured in suspension and mean diameter of the spheroids was 100&mgr;m. The relationship among the hepatocytes resembled that in the liver in vivo. Synthetic functions of albumin and urea of the spheroids were twice those of hepatocytes cultured on monolayers.CONCLUSION:As high-yields and high-activity modes of culture on microcarriers or in spheroidal aggregate culture with portal vein serum are promising to provide biomaterials for bioartificial liver (BAL) efficiently.

Steps in the development of a Vibrio cholerae El Tor biofilm.

We report that, in a simple, static culture system, wild-type Vibrio cholerae El Tor forms a three-dimensional biofilm with characteristic water channels and pillars of bacteria. Furthermore, we have isolated and characterized transposon insertion mutants of V. cholerae that are defective in biofilm development. The transposons were localized to genes involved in (i) the biosynthesis and secretion of the mannose-sensitive haemagglutinin type IV pilus (MSHA); (ii) the synthesis of exopolysaccharide; and (iii) flagellar motility. The phenotypes of these three groups suggest that the type IV pilus and flagellum accelerate attachment to the abiotic surface, the flagellum mediates spread along the abiotic surface, and exopolysaccharide is involved in the formation of three-dimensional biofilm architecture.

Effect of variations in daily feeding frequency and ration size on growth of shrimp, Litopenaeus vannamei (Boone), in zero-water exchange culture tanks

It is generally believed that increasing feeding frequency has immediate benefits, including reduced nutrient leaching and increased shrimp growth. In this study, static-water ecoassays were conducted to determine the effect of feeding frequency and percentage of daily ration on growth of Litopenaeus vannamei and accumulation of inorganic nitrogen and phosphorus in the culture water. Shrimp were fed a semi-purified diet containing 19.5% crude protein. Experiment 1 tested five feeding frequencies (3, 5, 8, 11 and 15 feedings day −1) evenly spaced in time during 24 h. Daily ration was divided in similar amounts for each feeding frequency. Mean shrimp survival was 98.6% for all treatments. No significant differences were found either on shrimp growth or accumulation of total inorganic nitrogen (TIN) or total reactive phosphorus (TRP) regardless of feeding frequency. In experiment 2 daily rations were offered manually 1 (100% at 0800 h), 2 (50% each at 0800 and 2000 h), 3 (33% each at 0800, 1400 and 2000 h), 3 + (45, 20 and 35% at 0800, 1400 and 2000 h, respectively), 4 (25% each at 0800, 1200, 1600 and 2000 h), 4 + (40, 15, 15 and 30% at 0800, 1200, 1600 and 2000 h, respectively) and 6 (16.6% each at 0800, 1030, 1300, 1600, 1800 and 2000 h) times day −1; and with automatic feeders 15 times day −1 (every 96 min). Shrimp survival was 100% for all treatments. Although mean shrimp growth was highest at 2 feedings day −1 no significant differences were found among feeding treatments. Accumulation of TIN in the culture water was significantly lower for tanks receiving feed 2 times day −1 compared to 1 and 3 times day −1. Levels of TIN were not significantly different among the other feeding treatments. Accumulation of TRP was not significantly different regardless of feeding treatment. These data indicated that under these culture conditions increasing feeding frequency or manipulating ration size did not significantly improve shrimp survival or growth. The only significant environmental effect detected in the culture water was a reduction of TIN at a feeding frequency of 2 compared to 1 and 3 times day −1 in experiment 2. The authors suggest that multiple feedings may not be advantageous for some static culture systems, and that the cost-effectiveness of multiple feedings be evaluated for different feeds and culture systems.

Factors affecting proliferation and dedifferentiation of primary bovine oviduct epithelial cells in vitro

The oviduct is the physiological site for key events in reproduction, such as capacitation of spermatozoa, fertilization and early embryonic development. Interactions between oviduct epithelial cells and gametes or embryos cannot sufficiently be studied in vivo. Therefore, model systems are needed which mimic in vivo conditions most closely. In this study we optimised the method for isolating bovine oviduct cells and compared different cell support materials as well as two culture systems (perfusion vs static culture) for their ability to maintain characteristic morphological and functional features of oviduct cells. Out of nine different cell support materials tested, cellulose nitrate (0.45 micron pore size) was the most suitable to maintain cells in a manner similar to freshly isolated oviduct epithelial cells. Comparing static vs perfusion culture by electron microscopy, morphological differences of the cells were insignificant in the first days of culture, while they became more evident after 8 days. The cells in the static system lost typical characteristics such as columnar shape, cilia and secretory protrusions, while these features were still present in perfusion culture. In addition, intense ciliogenesis and cytoplasmic organelles for protein synthesis were found under perfusion conditions. These findings were underlined by differences in expression of the oviduct-specific oestrus-associated glycoprotein 85-97 kDa (GP 85-97) gene as revealed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The RNA levels of this specific gene were significantly higher in perfusion compared to the static culture system. Our data show clear advantages of perfusion vs static culture for primary bovine oviduct epithelial cells.

Effects of mixing conditions on the production of microbial cellulose byAcetobacter xylinum

Microbial cellulose has many potential applications due to its excellent physical properties. The production of cellulose fromAcetobacter xylinum in submerged culture is, however, beset with numerous problems. The most difficult one has been the appearance of negative mutants under shaking culture conditions, which is deficient of cellulose producing ability. Thus genetic instability ofAcetobacter xylinum under shaking culture condition made developing a stable mutant major research interest in recent years. To find a proper type of bioreactor for the production of microbial cellulose, several production systems were developed. Using a reactor system with planar type impeller with bottom sparging system, it was possible to produce 5 g/L microbial cellulose without generating cellulose minus mutants, which is comparable to that of static culture system.

Evaluation of a partial flow-through culture technique for pearl oyster (Pinctada margaritifera L.) larvae

This study evaluated a partial flow-through culture system for larvae of the blacklip pearl oyster (Pinctada margaritifera, L.). Water flowed through the 500-l larval rearing tanks for 12 h day−1 and provided a 100% water exchange during this period. Larvae were prevented from leaving the culture tanks by a conical mesh screen placed over a central stand-pipe. Tanks were completely drained every 7 days to prevent build-up of debris on the tank bottom. First `eyed' larvae were seen on day 23 and, by day 28, approximately 5% of the larvae initially stocked had reached the `eyed' stage and were removed into settlement tanks. Larval growth and survival compared favourably with previous studies in which P. margaritifera larvae were cultured using conventional static culture methods. However, when compared to static culture systems, the partial flow-through system described in this study requires less labour and, as such, is likely to be more appropriate for hatchery culture in developing Pacific nations.

Ex vivo culture of peripheral blood CD34+ cells: effects of hematopoietic growth factors on production of neutrophilic precursors.

A major potential application for ex vivo culture of hematopoietic progenitor cells is the treatment of cytopenia following high-dose chemotherapy and hematopoietic transplantation. We have previously postulated that infusion of a sufficient number of neutrophil postprogenitor cells generated by ex vivo culture of CD34+ cells may be able to abrogate neutropenia. In this article, we describe further development of an efficient stromal-free, cytokine-dependent, static culture system for generation of these cells. Our previous studies indicated that maximal production of nucleated cells and myeloid progenitor cells from PB CD34+ cells occurred with multiple hematopoietic growth factor (HGF), notably the 6-HGF combination of interleukin (IL)-1, IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), and stem cell factor (SCF). In the present study, we determine the contribution of each of these 6 HGF in generation of neutrophilic precursors. SCF, G-CSF, and IL-3 were found to be the most important HGF for production of neutrophilic cells. The 4-HGF combination of IL-3, IL-6, G-CSF, and SCF was optimized by performing dose-response experiments and shown to be as potent as 6 HGF for production of nascent CFU-GM and neutrophilic precursors.

Leptin Stimulates Insulin Secretion and Synthesis in HIT-T 15 Cells

Shimizu, H., K.-I. Ohtani, T. Tsuchiya, H. Takahashi, Y. Uehara, N. Sato, M. Mori. Leptin stimulates insulin secretion and synthesis in HIT-T 15 cells. Peptides 18(8) 1263–1266, 1997.—Leptin, an ob gene product, corrects hyperinsulinemia in ob/ob mice. The leptin receptor may exist in pancreatic islets. The present studies were undertaken to determine the direct effect of 1–100 ng/ml recombinant leptin on insulin secretion and synthesis in HIT-T 15 cells by using static culture system. The addition of recombinant leptin significantly increased insulin secretion for 20 min at the highest concentration (100 ng/ml). The addition of recombinant leptin dose-dependently increased insulin secretion for 24 h in the 7 m M glucose-containing F-12 K medium. The incubation with recombinant leptin for 24 h increased preproinsulin mRNA expression, assessed with reverse transcription-polymerase chain reaction (RT-PCR) method. It was furthermore demonstrated that HIT-T 15 cells possessed the specific binding site for [ 125I]-labeled leptin. The present study demonstrated the existence of the leptin-specific binding sites that mediate its stimulatory effect on insulin secretion and synthesis in HIT-T 15 cells.

A continuous flow, perifusion culture system for 8- to 16-cell bovine embryos derived from in vitro culture

Eight- to 16-cell stage bovine embryos derived from in vitro maturation and in vitro fertilization were obtained after static culture until 80 to 88 h post insemination. The embryos were cultured in a dynamic (continuous flow) perifusion culture system for 24 h and were further co-cultured on a cumulus cell monolayer in a static CO 2 incubator for an additional 80 to 88 h. Based on preliminary experiments, a serum-free bovine embryo culture medium (BECM) supplemented with 25 mM HEPES was used as the perifusion culture medium (PCM), and the flow rate was fixed within the range of 30 to 38 μl/min in subsequent experiments. In Experiment 1, the combined effects of tubing (silicone vs Tygon ™) and/or culture chamber size (0.2 vs 1.5 ml) on prehatching development of the embryos following 24 h perifusion culture were evaluated in an experiment with a 2 × 2 factorial arrangement. More (P < 0.005) embryos developed to the blastocyst stage with Tygon ™ than with silicone tubing, regardless of chamber size. A higher percentage of blastocysts (29%) was obtained following the perifusion culture by the combination of Tygon ™ tubing and the 1.5-ml culture chamber than by any other combination. In addition, a significantly (P < 0.001) lower mean pH of the perifusate was obtained with Tygon ™ (7.57 to 7.58) than with silicone (7.78 to 7.79) tubing, regardless of chamber size. In Experiment 2, embryo development was compared after culture in the perifusion system and in a static culture system using either serum-free medium (control) or serum-free medium plus a cumulus cell monolayer (co-culture). Percentages of morulae (49 vs 57%) and blastocysts (31 vs 33%) obtained from the control system were not significantly (P > 0.4) higher than the percentages obtained from the perifusion system. However, these proportions were significantly (P < 0.01) increased by co-culturing the embryos on a cumulus cell monolayer in the static incubator for the same intervals (71% morulae and 44% in blastocysts). In conclusion, 8- to 16-cell bovine embryos can develop to the blastocyst stage following 24 h perifusion in the dynamic culture system using a serum-free medium. Physical stress due to flow rate within the range of 30 to 38 μl/min in an air atmosphere has little effect on the development of perifused embryos cultured under this system.