Increasing Time In Culture Research Articles

Production of phenolic compounds in hairy root culture of tartary buckwheat (Fagopyrum tataricum Gaertn)

Fagopyrum tataricum Gaertn (tartary buckwheat) is an excellent medicinal and nutrient-rich crop. It has a high content of rutin and other phenolic compounds. An experiment was conducted to investigate in vitro production of phenolic compounds from hairy root culture of tartary buckwheat. Hairy root growth was promoted by increasing culture time in MS medium. The highest hairy root growth reached up to 11.2 g/l dry weight at 18 d after placement. Transformation was confirmed by PCR using rol genes, rol A (304 bp), B (797 bp), C (550 bp), and D (1035 bp) genes which is transferred into hairy roots from the Ri-plasmid in Agrobacterium rhizogenes and is responsible for the induction of hairy root from plant species. Rutin, quercetin, (−) epicatechin, (−) catechin hydrate, gallic acid, ferulic acid, chlorogenic acid, and caffeic acid were identified both in hairy and wild type roots of tartary buckwheat. The main compound found in the both types of root was epicatechin followed by rutin. The concentration of phenolic compounds in the hairy roots of tartary buckwheat was several-fold higher compared with wild type roots of same species. Our results indicate that hairy root culture of F. tataricum is a valuable alternative approach for the production of phenolic compounds.

The repair of osteochondral defects using baculovirus-mediated gene transfer with de-differentiated chondrocytes in bioreactor culture

Baculovirus has emerged as a promising gene delivery vector. Hereby de-differentiated rabbit chondrocytes were transduced ex vivo with a recombinant baculovirus expressing BMP-2 (Bac-CB), seeded to scaffolds and cultured statically for 1 day (Bac-w0 group) or in a rotating-shaft bioreactor (RSB) for 1 week (Bac-w1 group) or 3 weeks (Bac-w3 group). Mock-transduced constructs were cultured statically for 1 day to serve as the control (Mock-w0 group). We unraveled that Bac-CB transduction and increasing culture time in the RSB yielded more mature cartilaginous constructs in vitro. Eight weeks after implanting into the rabbit osteochondral defects, Mock-w0 constructs failed to repair the lesion while Bac-w0 constructs resulted in augmented, yet incomplete, repair. Bac-w1 constructs yielded neocartilage layers rich in glycosaminoglycans and collagen II, but the integration between the graft and host cartilages was not complete. In contrast, Bac-w3 constructs led to the regeneration of hyaline cartilages as characterized by cartilage-like appearance, improved integration, chondrocytes clustered in lacunae, smooth and homogeneous matrix rich in collagen II and glycosaminoglycans but deficient in collagen I. In conclusion, combining baculovirus-modified de-differentiated chondrocytes and RSB culture creates constructs that repair osteochondral defects, and in vitro culture time dictates the construct maturation and subsequent in vivo repair.

Cytocompatibility of Poly(L-lactide-co-glycolide) Porous Scaffold Materials for Tissue Engineering

The development of a suitable three-dimensional scaffold for the maintenance of cellular viability and differentiation is critical for applications in periodontal tissue engineering. Poly(L-lactide-co-glycolide) (PLGA) porous scaffold materials were fabricated by using NaHCO3 particulates as progen, and the compatibility test in vitro was carried out. The scaffold shows an interconnected structure with large pore sizes ranging from 100 to 450 μm. Cells did attach to the culture plate when cultured in PLGA extract, and the number of cells increased with the increase of culture time. Von Kossa-positive mineralization nodules were first observed after 21 days of culture time. The percent of viable cells cultured in PLGA extract was higher than that in PLLA extract at the same culture period. Nodule formation and multilayer structures were observed on the scaffold surface.

The Effect of Serum on the PEG-Based Crosslinker-Induced Spheroid Formation of Pancreatic β-Cell

A polymeric crosslinker poly(ethylene glycol) derivative with dioleyl groups as hydrophobic group at both ends was developed for promoting cellular spheroid formation. Our approach to bridge cells was based on the crosslinking of cell membrane using a crosslinker via the hydrophobic interaction. Using the crosslinker, spheroid formation of pancreatic β-cell line RIN in a round bottom 96-well plate was evaluated, especially in effect of serum on spheroid formation. In the presence or absence of serum, the size of prepared spheroid was found to decrease with increasing culture time and with increasing crosslinker concentration. However, the medium without serum proved to be a favorable condition for promoting cell aggregation because in this case spheroid with smaller size could be obtained. It was clarified that spheroid formation and insulin secretion of the spheroid prepared by the crosslinker were enhanced, especially in the medium without serum.

Enhanced Cell Survival and Yield of Rat Small Hepatocytes by Honeycomb-Patterned Films

Surface designing of substrate to regulate cell adhesion and function in nano and micro scale is a critical issue in biomaterial science. In this study, we describe the fabrication of highly regular patterned porous films (honeycomb-patterned film) formed by a simply casting technique, and the culture of mature hepatocytes and small hepatocytes on the films. The pore size of the honeycomb-patterned films used was 6, 12, and 16 µm. We evaluated the effect of the honeycomb-patterned films on the morphology, cell yield, survival and the differentiated hepatic function (albumin production) of the both hepatocytes. Both hepatocytes attached on the flat films appeared to spread well, showing a typical monolayer morphology. They peeled off from the films at 7 days in culture on the flat films. On the other hand, spreading of the each hepatocytes was restricted on the honeycomb-patterned films at 3 and 7 days in culture. The cell yield and survival of the each hepatocytes increased with increasing culture time. Small hepatocyte on the pore sizes of 16 µm showed the highest cell yield (approximately 3 times). Albumin production of mature hepatocyte on the pore sizes of 16 µm (224.1.3 ±157 ng ml-1 well-1 at 1 day in culture, 369.5 ±222 ng ml-1 well-1 at 3 days in culture) was higher than that of the hepatocytes on the flat films (119.3 ±9.3 ng ml-1 well-1 at 1 day in culture, 262.8 ±47.3 ng ml-1 well-1 at 3 days in culture), although that of small hepatocytes on the honeycomb-patterned films (pore size: 16 µm) was similar on the flat film. These results indicated that both the surface topography and the pore size of the honeycomb-patterned film affected the hepatic metabolic function.

Studies on Saccharification from Alginate using Stenotrophomonas maltophilia

Various environmental factors affecting saccharification from alginate using Stenotrophomonas maltophilia were investigated in flask cultures. The cell concentrations increased from 0.6 to 0.92 optical density (OD) at 660 nm when the agitation rate increased from 15 to 90 rpm. On the other hand, the maximum concentration of sugar was obtained at 3.8 g/l after 4 days of culture at 15 rpm. After 3 days of preculture at 33 °C, the sugar concentration peaked at 5.0 g/l after 5 days of culture. When 10 g/l of NaCl was used, the maximum concentration of sugar, 5.3 g/l, was obtained after 5 days of culture. Yeast extract and peptone were the best nitrogen source for effective saccharification. Especially, the sugar concentration was 6.1 g/l after 5 days of culture using a mixture of 1.0 g/l of yeast extract and 1.0 g/l of peptone. Under optimum conditions of culture and media, scale-up for effective saccharification from alginate was carried out in 5 l flasks. The cell concentration after 2 days of culture was 0.61 OD at 660 nm and showed no further increase after 3 days of culture. The sugar concentrations from alginate were increased with increasing culture time to 7.9 g/l after 9 days of culture.

Low intensity ultrasound-induced apoptosis in human gastric carcinoma cells

To investigate the low intensity ultrasound (US)-induced apoptosis in human gastric carcinoma cells and its potential mechanism and to suggest a new therapeutic approach to gastric carcinoma. Human SGC-7901 gastric carcinoma cells were cultured in vitro and irradiated by low intensity US for 10 min at different intensities with different incubation times after irradiation. Morphologic changes were examined under microscope with trypan blue staining and then the percentage of early apoptotic cells was detected by flow cytometry (FCM) with double staining of fluorescein isothiocyanate (FITC)-Annexin V/propidium iodide (PI). Two-dimensional electrophoresis (2DE) was used to get the protein profile and some proteins differently expressed after US irradiation were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Functional analysis was performed to investigate the mechanism of US-induced cell apoptosis. The percentage of apoptotic cells increased about 10% after US irradiation (12.0 W/cm(2), 12 h culture). The percentage of early apoptosis and secondary necrosis in the US-irradiated cells increased with the increased US intensity. Moreover, apoptotic cells increased with the increased culture time after US irradiation and reached its maximum at about 12 h. Several new proteins appeared after US irradiation and were up or down regulated more than 2 times. Some heat shock proteins (HSPs) were found to be associated with the signal process simulating the apoptosis of cells. Low intensity US could induce apoptosis in human gastric carcinoma cells. US-induced apoptosis is related to US intensity/culture time. US-induced apoptosis may be caspases-dependent and endoplasmic reticulum (ER) stress-triggered apoptosis may also contribute to it. Proteomic experimental system is useful in finding the protein alteration in carcinoma cells after US irradiation, helping to develop a new cancer therapy.

Effect of Culture Conditions on the Protein Fibril Expression of Candida albicans

Candida albicans is an important human pathogen that causes systemic infections, predominantly among population with weakened immune system. Cell wall structures of C. albicans are important to adhere to host tissue and evade to host immune system. Among cell wall structure, the outermost fibrillar layer of C. albicans is of interest since it may play important roles in antigenicity, phagocytosis, and adherence. The expression of virulent factors could be affected by the growth conditions. The dynamic nature of the cell surface alters the physical properties of the fungal interface with host cells and thereby influences adhesion to the host and recognition by components of the host immune system. In this study, we investigated the effects of culture conditions on cell surface fibril expression of C. albicans by a transmitting electron microscopy and SDS-PAGE. The protein fibril of C. albicans was expressed in the presence of whole serum, however, the fibril expression was decreased in 25% serum and serum containing 1% glucose. Also, germ tube can be induced by serum, RMPI medium, N-acetyl glucosamine, and 39℃ culture condition, hence, the fibrillar structure of C. albicans was detected only in serum-induced germ tube. The expression of fibril layer and the major fibril proteins of 66, 47, 30 kDa were reduced as increasing cell concentration of intial inoculum from 2×107 cells/㎖ to 8×107 cells/㎖. The fibrillar layer of C. albicans was expressed in serum early within 10 min, and the thickness of fibril layer was increased according to the increase of culture time. When the fibrillar proteins were analysed by SDS-PAGE, major protein of 30 kDa was maintained continuously during over night culture although expression of the other proteins were various. These results suggest that expression of serum induced protein fibril is influenced by culture conditions and is not related to hyphal transition of C. albicans.

A novel cultured tissue model of rat aorta: VSMC proliferation mechanism in relationship to atherosclerosis

Development of a cultured tissue experimental model of rat aorta was explored in order to study mechanism of vascular smooth muscle (VSMC) proliferation. This particular model has potential with regard to amelioration of atherosclerosis and other vascular diseases in comparison to whole animal and cell culture models. The aorta segments of rats were divided into 4 experimental groups: the injured endothelium, injured endothelium plus BQ123, without injured endothelium and without injured endothelium plus BQ123. Each of group was subdivided into a further 2 subgroups and cultured with 20% serum and with serum-free DMEM. Each group cultured in vitro for 5, 8 and 13 days respectively. The control group was not cultured in vitro. Bromodeoxyuridine (BrDU 8 × 10 − 4 mol/l) was added into the cultured medium of all groups, 24 h prior to harvesting. These segments were fixed in 4% paraformaldehyde for paraffin slice used to HE and immunocytochemical staining and other aorta segments were used to detect the expressions of hypertension-related gene-1 (HRG-1) and smooth muscle 22 alpha (SM22α) by RT-PCR. ET-1 content in the supernatant was detected with radioimmunology. Proliferous VSMC can be observed on artery segments cultured in vitro, and conspicuous plaques were developed on model vascular wall cultured for 13 days. Labeled cells increased with an increase in culture time but were not seen in the control group. A greater number of labeled cells were observed in injured endothelium group cultured in 20% serum DMEM. Hyperplasia was inhibited after BQ123 was added into the medium, suggesting that serum and ET-1 are important factors that lead to VSMC proliferation. Expressions of HRG-1 and SM22α were decreased while the aorta segments were cultured in vitro, minimum or even absent mRNA expressions of HRG-1 and SM22α were detected in injured endothelium cultured in 20% serum DMEM and increased in injured endothelium plus BQ123 group cultured. ET-1 content in the supernatant increased in injured endothelium cultured in 20% serum DMEM. These results show that the phenotypic transform and VSMC proliferation on cultured artery segments were related not only to serum culture, but also to ET-1 secreting. ET-1 and serum may be the main factors of contributing to the proliferation and phenotypic transform. This model provides a favorable experimental platform for research into the mechanism of vascular proliferous diseases as well as its prevention and treatment.

A new and practical application of JH antagonist KK-42 to promoting growth of shrimp Penaeus schmitti

The shrimps ( Penaeus schmitti), 0.6–0.8 cm long, were soaked for 1 min in juvenile hormone antagonist KK-42 solution at a concentration of 1.95 × 10 − 4 mol/L, and then cultured in normal way. The body weight of animals rose with increasing culture time in all groups, but was higher in KK-42 treatment. The increase rate of body weight of treatment group was 167% and 68% higher than control group at 30th day and 60th day, respectively; the body weight rose by 42.5% at 90th day and 22.1% at 120th day. Histological observations showed that the number of acini of mandibular organ increased gradually as animals grew, and the diameter of acinus-forming cells was statistically bigger at 120th day than at other times. After KK-42 treatment, both the number of acini and the diameter of gland cells decreased in the entire experiment observed. The results indicated the inhibition of anti-juvenile hormone KK-42 on the development of mandibular organ in P. schmitti.

The Influence of Pore Size of a Honeycomb Film on Early Spreading Process of Endothelial Cells

The mechanism that cells use to recognize micro-patterned topographies was clarified. First, 3D double-layer poly (e-caprolactone) scaffolds equipped with honeycomb-patterned micro-pores (“honeycomb films”) were prepared. Then, porcine aortic endothelial cells (PAECs) were cultured on these scaffolds for 1-6 h in serum-free medium. Finally, their initial spreading process was investigated by using AFM and confocal laser scanning microscopy. The attachment and spreading of PAECs on honeycomb films having either 6-or 16-μm pore diameters resulted in voids within the cell cytoplasm, which correspond with the size and location of the honeycomb micropores. The number of cells with this unique morphology decreased with increasing culture time. This dependence of morphology on film pore size and culture time suggests a spreading process of PAECs in which the cells spread trying to sense suitable sites to adhere. Using thick filopodia, the cells spread along the rim of the film and produced pores by close contact between two spreading filopodia. Evidently, these pores became filled in during culture, presumably as the cells began to reorganize their cytoplasma.

An Improved, Non-Viral Approach for Production of hCD40L+ B-CLL Cells for Cancer Immunotherapy.

Previously we reported (Li LH et al, 2006, Cancer Gene Therapy 13:215–224) rapid and efficient production of human CD40L+ (hCD40L) B-Chronic lymphocytic leukemia (B-CLL) tumor vaccine by electrotransfecting the cells with a DNA plasmid encoding hCD40L. The hCD40L-transfected B-CLL cells cryopreserved at 3 hrs post transfection showed cell viability ≥50%, and CD40L expression level ≥50% (N=10). Although costimulatory molecule upregulation was not detected at 3 hrs, we hypothesized the vaccine would upregulate costimulatory molecules in vivo, emulating levels seen in vitro among viable cells after 12–24h culture. The suboptimal immunologic and clinical results of our previous vaccine preparation reported last year (Fratantoni JC et al, 2005, Blood 106:136a) suggested that costimulatory molecule upregulation in vivo was insufficient. Simply increasing culture time of hCD40L-transfected B-CLL cells is limited by the resulting low cell viability caused by DNA uptake-mediated toxicity. In this study, we report a simple modified vaccine manufacturing method that yields a vaccine with good cell viability and expression of co-stimulatory molecules prior to injection. During vaccine production, a portion of the B-CLL cells were first transfected with pCMV-hCD40L via electroporation (provider cells) and then mixed with non-transfected autologous B-CLL cells (naïve recipients) followed by coculture for 12 to 24hrs. Our data show that hCD40L expression levels on the transfected provider cells and the ratio of provider cells to naïve recipient cells directly correlate with hCD40L molecule expression on the naïve recipient cells. The naïve recipient cells in the mixture maintained high cell viability, 80%–90%, when normalized by the input naïve cell number, while cell viability of the provider cells declined to 19 ± 9% at 1d and further down to 2 ± 1% at 7ds post transfection (n=4). The percentage of cells expressing hCD40L depended on the mixing ratio. When a 10:1 ratio (provider: naïve) was used, the hCD40L expression level in naïve cells was up to 80%. In order to make an hCD40L+ B-CLL vaccine with high cell viability, a 1:1 ratio was applied. The viability of the final tumor vaccine product including both provider and recipient cells was 56 ± 6%, while hCD40L was detected among 34%±12% of the cells at 12–24h post mixing (n=10). Expression of CD80, CD86 and CD54 in the mixed cells were increased by 16 ± 8, 10 ± 5 and 24 ± 17 folds respectively, when compared to those of the naïve B-CLL cells (3 patients). Furthermore, we examined the capacity of the vaccine product to present antigen using an allo MLR, and monitored IFN-g secretion and proliferation of CFSE-labeled allo PBL. Data from 3 CLL patients' samples showed that vaccine prepared by the mixing process could induce 6.8 ± 0.01, 2.1 ± 0.35 and 2.5 ± 1 fold more allo PBL proliferation and ≥25 folds higher IFN-g production than the control B-CLL cells (p<0.05). In summary, we could produce viable functional hCD40L+ CLL tumor vaccine with upregulated costimulatory molecules using autologous B-CLL cells. The process can be scaled up to produce >2x1010 modified cancer cells. This simple, non-viral vaccine manufacturing process is practical and currently under evaluation in Phase I/II clinical study.

In Vitro Tendon Engineering with Avian Tenocytes and Polyglycolic Acids: A Preliminary Report

Although there are many reports of in vivo tendon engineering using different animal models, only a few studies involve the short-term investigation of in vitro tendon engineering. Our previous study demonstrated that functional tendon tissue could be engineered in vivo in a hen model using tenocytes and polyglycolic acid (PGA) fibers. This current study explored the feasibility of in vitro tendon engineering using the same type of cells and scaffold material. Tenocytes were extracted from the tendons of a hen's foot with enzyme digestion and cultured in DMEM plus 10% FBS. Unwoven PGA fibers were arranged into a cord-like construct and fixed on a U-shape spring, and tenocytes were then seeded on PGA fibers to generate a cell-PGA construct. In experimental group 1, 22 cell-scaffold constructs were fixed on the spring with no tension and collected at weeks 4 (n = 7), 6 (n = 7) and 10 (n = 8); in experimental group 2, five cell-scaffold constructs were fixed on the spring with a constant strain and collected after 6 weeks of culture. In the control group, three cell-free scaffolds were fixed on the spring without tension. The collected engineered tendons were subjected to gross and histological examinations and biomechanical analysis. The results showed that tendon tissue could be generated during in vitro culture. In addition, the tissue structure and mechanical property became more mature and stronger with the increase of culture time. Furthermore, application of constant strain could enhance tissue maturation and improve mechanical property of the in vitro engineered tendon (1.302 +/- 0.404 Mpa with tension vs 0.406 +/- 0.030 Mpa without tension at 6 weeks). Nevertheless, tendon engineered with constant strain appeared much thinner in its diameter than tendon engineered without mechanical loading. Additionally, its collagen fibers were highly compacted when compared to natural tendon structure, suggesting that constant strain may not be the optimal means of mechanical load. Thus, application of dynamic mechanical load with a bioreactor to the construction of tendon tissue will be our next goal in this series of in vitro tendon engineering study.

Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates

Liposuction aspirates (primarily saline solution, blood, and adipose tissue fragments) separate into fatty and fluid portions. Cells isolated from the fatty portion are termed processed lipoaspirate (PLA) cells and contain adipose-derived adherent stromal cells (ASCs). Here we define cells isolated from the fluid portion of liposuction aspirates as liposuction aspirate fluid (LAF) cells. Stromal vascular fractions (SVF) were isolated separately from both portions and characterized under cultured and non-cultured conditions. A comparable number of LAF and PLA cells were freshly isolated, but fewer LAF cells were adherent. CD34+ CD45- cells from fresh LAF isolates were expanded by adherent culture, suggesting that LAF cells contain ASCs. Although freshly isolated PLA and LAF cells have distinct cell surface marker profiles, adherent PLA and LAF cells have quite similar characteristics with regard to growth kinetics, morphology, capacity for differentiation, and surface marker profiles. After plating, both PLA and LAF cells showed significant increased expression of CD29, CD44, CD49d, CD73, CD90, CD105, and CD151 and decreased expression of CD31 and CD45. Multicolor FACS analysis revealed that SVF are composed of heterogeneous cell populations including blood-derived cells (CD45+), ASCs (CD31- CD34+ CD45- CD90+ CD105- CD146-), endothelial (progenitor) cells (CD31+ CD34+ CD45- CD90+ CD105low CD146+), pericytes (CD31- CD34- CD45- CD90+ CD105- CD146+), and other cells. After plating, ASCs showed a dramatic increase in CD105 expression. Although some adherent ASCs lost CD34 expression with increasing culture time, our culture method maintained CD34 expression in ASCs for at least 10-20 weeks. These results suggest that liposuction-derived cells may be useful and valuable for cell-based therapies.

Effect of stocking density on growth, settlement and survival of clam larvae, Meretrix meretrix

To determine the optimal larval density for hatchery culture of the clam Meretrix meretrix, experiments with stocking densities of 5, 10, 20, 40 and 60 larvae ml − 1 were designed, which included the developmental stages from D-veliger to 8 days post-settlement. Shell length, settlement time and survival rate of the larvae were recorded. Results showed that, at each sampling time, larvae reared at the highest density had the smallest mean size, whereas larvae reared at the lowest density had the largest mean size. Statistical differences in mean shell length at different stocking densities appeared from day 2, and greater differences occurred with increased culture time. Specific growth rate (SGR) in the rapid growing stage (day 0–3) was negatively correlated with density; however, no correlation was found between SGR and density in the slow growing stage (days 3–7). Settlement time was prolonged and shell length of settled larvae decreased as density increased. However, larval survival rate (74.8–79.1%) was independent of stocking density. Results showed that a high stocking density, in the designated range, is feasible for larval culture of the clam M. meretrix. However, for large-scale culture, in the interest of costs and safety, a stocking density of 10–20 larvae ml − 1 is recommended.

Hepatocyte spheroid formation on a titanium dioxide gel surface and hepatocyte long-term culture

The cell morphology and expression of differentiated functions of primary rat hepatocytes on a titanium dioxide (TiO(2)) gel surface were investigated. Polystyrene culture dishes were coated with TiO(2) gel by spin-coating an ethanol solution of titanium n-butoxide, hydrolyzing in a humidity chamber and drying with nitrogen gas. The TiO(2) gel layer formed on the polystyrene dishes was transparent and robust, and its surface was quite flat. Rat hepatocytes inoculated on the TiO(2) gel-coated polystyrene dishes gradually accumulated with increasing culture time, and then spontaneously formed many hepatocyte spheroids at 90 +/- 21 microm diameter from about 3 days of culture. The diameter of the spheroids increased during the culture, and was 151 +/- 43 microm at 14 days of culture. Ammonia removal and albumin secretion by hepatocytes on the TiO(2) gel-coated polystyrene dishes were maintained at a high level for at least 14 days of culture compared with on a type I collagen-coated dish and a non-coated polystyrene dish. These results indicate that TiO(2) gel is an adequate material for hepatocyte spheroid formation and long-term culture of spheroids.

Down-regulation of tight junction mRNAs in human endothelial cells co-cultured with Plasmodium falciparum-infected erythrocytes

To understand the mechanism of sequestration in the microvasculature of patients with falciparum malaria, we examined the patterns of expression of mRNAs for adhesion molecules (ICAM-1, VCAM-1, and E-selectin) and tight junction molecules (occludin, vinculin, and ZO-1) in human umbilical vein endothelial cells (HUVECs) co-cultured with Plasmodium falciparum-parasitized red blood cells (PRBCs) in vitro. The PRBCs were collected from patients with uncomplicated, severe, or cerebral malaria (CM). Patterns of mRNA expression in HUVECs co-cultured with PRBCs were examined by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Levels of mRNAs for all the three adhesion molecules increased with increased culture time within 3 h, regardless of the source of the PRBCs. In contrast, the patterns of mRNA expression for the tight junction molecules varied between the different co-cultures. When HUVECs were cultured with PRBCs from uncomplicated malaria patients, levels of mRNAs for tight junction molecules increased according to the culture time. HUVECs co-cultured with PRBCs from severe malaria patients showed no change in the mRNAs levels during 3 h of observation. When HUVECs were cultured with PRBCs from CM patients, levels of mRNAs for tight junction proteins decreased according to the culture time. Although the mechanisms underlying these phenomena are not clear, our results suggest that PRBCs can alter expression of tight junction proteins in endothelial cells at the site of sequestration and thereby influence disease severity.

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P &lt; 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

Development of a fed-batch culture process for enhanced production of recombinant human antithrombin by Chinese hamster ovary cells

Antithrombin is a serine protease inhibitor that inactivates several coagulation proteases, primarily thrombin and factor Xa. The Chinese hamster ovary (CHO) cell line transfected with a vector expressing recombinant human antithrombin (rAT) and a selectable marker, glutamine synthetase (GS), was cultivated in a 2-l fed-batch culture process using serum-free, glutamine-free medium. To maximize the rAT yield, effects of culture pH, balanced amino acid feeding, and an increased glutamate concentration on cell metabolism and rAT production were investigated. When cells were grown at pH values of 6.6, 6.8, 7.0, and 7.2, the maximum cell density and maximum lactate concentration decreased with decreasing pH. The highest production level of rAT was obtained at culture pH 6.8 due to the extended culture lifetime. Compared to the imbalanced amino acid feeding at culture pH 6.8, the balanced amino acid feeding increased the amount of rAT activity by 30% as a result of an increased viable cell number. A decrease in the specific glucose consumption rate (q(Glc)) with increasing culture time was observed in all the above-mentioned experiments, while the glucose concentration was maintained above 0.7 g l(-1). In addition, a decrease in the specific rAT production rate (q(rAT)) was observed after the depletion of lactate in the late cultivation stage. Taken together, these results suggest that the reduced availability of cellular energy caused by the decrease in q(Glc) and depletion of lactate led to the decrease in q(rAT). This decrease in q(rAT) was partially prevented by increasing the residual glutamate concentration from 1 mM to 7 mM, thus resulting in an additional 30% increase in the amount of rAT activity. The optimized fed-batch culture process yielded 1.0 g l(-1) rAT at 287 h of cultivation.

Characteristics of a thyroid hormone responsive reporter gene transduced into a Xenopus laevis cell line using lentivirus vector

We introduced a self-inactivation (SIN) lentivirus vector (LV) into Xenopus laevis cell lines and established a permanent cell line expressing a reporter gene in a 3,5,3′- l-triiodothyronine (T 3) dependent manner. The SIN LV contained the luciferase gene downstream from the X. laevis T 3-response elements (TREs) and the SV40 promoter, and the enhanced green fluorescent protein (EGFP) gene downstream from the cytomegalovirus (CMV) promoter. It was integrated into the genome of X. laevis XL58, XTC2, and KR cells. The SIN LV transduced the X. laevis cells as efficiently as mammalian cells; however, the expression of EGFP in the transgene decreased with increasing culture time. A cell clone exhibiting the highest TH-dependent luciferase gene expression (XL58-TRE-Luc clone) was isolated from the EGFP-positive XL58 cell pool and characterized. The minimum effective concentration of T 3 that significantly induced the luciferase gene expression was 10 −11 M in the XL58-TRE-Luc clone. The application of the luciferase gene assay using the permanent XL58-TRE-Luc clone for the screening of thyroid-disrupting chemicals revealed that tetrachlorobisphenol A, at 10 −6 M, had a weak T 3-agonist activity, whereas trichlorobisphenol A, at 10 −8–10 −6 M had a weak T 3-antagonist activity. Our results indicated that the permanent X. laevis cell line containing a T 3-response transgene could be used as a bioassay, with small intra-assay variation, for the rapid screening, identification, and characterization of the thyroid-disrupting chemicals.