Rotary Culture System Research Articles

In vitro characterization of 3D culture-based differentiation of human liver stem cells.

Introduction: The lack of functional hepatocytes poses a significant challenge for drug safety testing and therapeutic applications due to the inability of mature hepatocytes to expand and their tendency to lose functionality in vitro. Previous studies have demonstrated the potential of Human Liver Stem Cells (HLSCs) to differentiate into hepatocyte-like cells within an in vitro rotary cell culture system, guided by a combination of growth factors and molecules known to regulate hepatocyte maturation. In this study, we employed a matrix multi-assay approach to comprehensively characterize HLSC differentiation. Methods: We evaluated the expression of hepatic markers using qRT-PCR, immunofluorescence, and Western blot analysis. Additionally, we measured urea and FVIII secretion into the supernatant and developed an updated indocyanine green in vitro assay to assess hepatocyte functionality. Results: Molecular analyses of differentiated HLSC aggregates revealed significant upregulation of hepatic genes, including CYP450, urea cycle enzymes, and uptake transporters exclusively expressed on the sinusoidal side of mature hepatocytes, evident as early as 1day post-differentiation. Interestingly, HLSCs transiently upregulated stem cell markers during differentiation, followed by downregulation after 7days. Furthermore, differentiated aggregates demonstrated the ability to release urea and FVIII into the supernatant as early as the first 24h, with accumulation over time. Discussion: These findings suggest that a 3D rotation culture system may facilitate rapid hepatic differentiation of HLSCs. Despite the limitations of this rotary culture system, its unique advantages hold promise for characterizing HLSC GMP batches for clinical applications.

A Morphological Post-Processing Approach for Overlapped Segmentation of Bacterial Cell Images

Scanning electron microscopy (SEM) techniques have been extensively performed to image and study bacterial cells with high-resolution images. Bacterial image segmentation in SEM images is an essential task to distinguish an object of interest and its specific region. These segmentation results can then be used to retrieve quantitative measures (e.g., cell length, area, cell density) for the accurate decision-making process of obtaining cellular objects. However, the complexity of the bacterial segmentation task is a barrier, as the intensity and texture of foreground and background are similar, and also, most clustered bacterial cells in images are partially overlapping with each other. The traditional approaches for identifying cell regions in microscopy images are labor intensive and heavily dependent on the professional knowledge of researchers. To mitigate the aforementioned challenges, in this study, we tested a U-Net-based semantic segmentation architecture followed by a post-processing step of morphological over-segmentation resolution to achieve accurate cell segmentation of SEM-acquired images of bacterial cells grown in a rotary culture system. The approach showed an 89.52% Dice similarity score on bacterial cell segmentation with lower segmentation error rates, validated over several cell overlapping object segmentation approaches with significant performance improvement.

Simulated Microgravity Using a Rotary Culture System Compromises the In Vitro Development of Mouse Preantral Follicles.

BackgroundGrowing cells in simulated weightlessness condition might be a highly promising new technique to maintain or generate tissue constructs in a scaffold-free manner. There is limited evidence that microgravity condition may affect development of ovarian follicles. The objective of the present study was to investigate the effects of simulated microgravity on the in vitro development of mouse preantral follicles.Methods and ResultsOvarian tissue from 14-day-old mice, or preantral follicles mechanically isolated from 14-day-old mouse ovaries were cultured at a simulated microgravity condition generated using a rotating wall vessel apparatus. Follicle survival was assessed quantitatively using H&E staining. Follicle diameter and oocyte diameter were measured under an inverted microscope. Ultrastructure of oocytes was evaluated using transmission electron microscopy. We observed that simulated microgravity compromised follicle survival in vitro, downregulated PCNA and GDF-9 expressions, and caused ultrastructural abnormalities in oocytes.ConclusionThis study showed for the first time that three-dimensional culture condition generated by simulated microgravity is detrimental to the initial stage development of mouse preantral follicles in vitro. The experimental setup provides a model to further investigate the mechanisms involved in the in vitro developmental processes of oocytes/granulosa cells under the microgravity condition.

Integrated Biophysical and Biochemical Signals Augment Megakaryopoiesis and Thrombopoiesis in a Three-Dimensional Rotary Culture System.

Platelet transfusion has been widely used in patients undergoing chemotherapy or radiotherapy; however, the shortage of the platelet supply limits the care of patients. Although derivation of clinical-scale platelets in vitro could provide a new source for transfusion, the devices and procedures for deriving scalable platelets for clinical applications have not been established. In the present study, we found that a rotary cell culture system (RCCS) can potentiate megakaryopoiesis and significantly improve the efficiency of platelet generation. When used with chemical compounds and growth factors identified via small-scale screening, the RCCS improved platelet generation efficiency by as much as ∼3.7-fold compared with static conditions. Shear force, simulated microgravity, and better diffusion of nutrients and oxygen from the RCCS, altogether, might account for the improved efficient platelet generation. The cost-effective and highly controllable strategy and methodology represent an important step toward large-scale platelet production for future biomedical and clinical applications. Significance: Platelet transfusion has been widely used in patients undergoing chemotherapy or radiotherapy; however, the shortage of platelet supply limits the care of patients. Thus, derivation of clinical-scale platelets in vitro would provide a new source for transfusion. The present study evaluated a rotary suspension cell culture system that was able to potentiate megakaryopoiesis and significantly improved the efficiency of platelet generation. When used with chemical compounds and growth factors identified via small-scale screening, the three-dimensional system improved platelet generation efficiency compared with the static condition. The three-dimensional device and the strategy developed in the present study should markedly improve the generation of large-scale platelets for use in future biomedical and clinical settings.

EFFECTS OF SIMULATED MICROGRAVITY ON PROLIFERATION AND CHEMOSENSITIVITY IN MALIGNANT GLIOMA CELLS

BACKGROUND: Everything on the earth can be affected by gravity of the earth. So, microgravity, as small as 10 minus 3 times G, can make some effects to the growth or maturation of cells. Under this hypothesis we examined cell proliferation and chemosensitivity after exposure to simulated microgravity using the malignant glioma cell lines. We exposed cells to simulated microgravity using a 3D-clinostat which is a type of rotating culture system that has two independent axes of rotation to disperse the gravity vector throughout the whole steric angle. METHODS: D54MG, U251MG and T98G cell lines were used in this study. All samples were identically treated before the start of the experiment and randomized for clinorotation (CL group) or culturing at 1G (C group). The Opticell cell culture system, mounted in a 3D-clinostat, was used for culture under simulated microgravity. Cells were cultured under a 1G environment for 24 h before using the 3D-clinostat to achieve adhesion. Morphological changes in glioblastoma cells during culture were examined using an inverted phase-contrast microscope. We analyzed the viability of the CL group and the C group cells by Trypan-blue exclusion method. The mitochondrial membrane potential was measured using the fluorescent dye Rhodamine123. Cell cycle distribution was measured with a FACS Caliber flow cytometer. Immunoblotting and sensitivity to CDDP was examined. RESULTS: Proliferation was significantly lower in all cell lines in the CL group compared to the C group on day three. The percentage of living cells in the CL group showed no significant differences compared to the C group. Cell cycles indicated by flow cytometry showed no differences between the CL and C groups. In both groups, FACS analysis also showed there was no significant proportion of apoptotic cells in any of the cell lines. In the CL group, significant deterioration of mitochondrial activity was shown in all malignant glioma cell lines compared to the C group. CONCLUSIONS: This is the first study to examine the effect of simulated microgravity on the proliferation, mitochondrial activity, and chemosensitivity of human malignant glioma cells. Our data suggests that simulated microgravity induces growth inhibition through reduction of mitochondrial activity, and enhances the chemosensitivity of malignant glioma cells to CDDP. Our findings using 3D-clinorotation provide new insight into the responses to simulated microgravity of malignant glioma cells. SECONDARY CATEGORY: Tumor Biology.

Human ovarian cancer multicellular spheroids: a model for testing antiproliferation activity of Devil's club (Oplopanax horridus) and anticancer agents.

This study was conducted to employ an ovarian cancer Ovcar 10 three-dimensional model to assess the antiproliferation activity of the medicinal plant Devil's club, Oplopanax horridus, and its active compound, alone and in combination, with chemotherapeutic agents compared to Ovcar 10 two-dimensional cells grown as monolayer cells. Ovcar 10 three-dimensional spheroids were prepared with a rotary cell culture system. Cell counting kit-8 assessed the antiproliferation activity. Apoptosis-related gene expression in three-dimensional spheroids and two- dimensional cells was analyzed with an apoptosis antibody array. Flow cytometry was used to analyze the cell cycle. Ovcar 10 cells formed compact three-dimensional spheroids after 5 days of culture in a rotary culture system. Ovcar 10 three-dimensional spheroids were significantly more resistant to killing by Devil's club extract, its active compound alone, gemcitabine, and paclitaxel, but not cisplatin compared to two-dimensional cells, with IC50 levels closer to that observed in vivo. Devil's club extract and its active compound alone significantly enhanced the antiproliferation activity of cisplatin and gemcitabine at some concentrations, but did not affect the activity of paclitaxel. A number of apoptosis-related genes were differentially expressed in three-dimensional spheroids, two-dimensional cells, and cells treated with Devil's club extract compared to untreated controls. In three-dimensional spheroids, the proportion of cells in the G2/M phase was slightly increased and the S phase was slightly decreased compared to two-dimensional cells. Ovcar 10 cells in three-dimensional spheroids altered the expression of multiple apoptosis-related genes, which may have contributed to the increased resistance of the cells to some drugs.

Novel alginate three‐dimensional static and rotating culture systems for effective ex vivo amplification of human cord blood hematopoietic stem cells and in vivo functional analysis of amplified cells in NOD/SCID mice

Autologous and allogeneic hematopoietic stem cell (HSC) transplantations serve as effective therapy for a variety of hematologic and other diseases. Umbilical cord blood (UCB) is an important source of HSCs. However, it is difficult to obtain a sufficient number of HSCs with complete self-renewal capability derived from a single unit of UCB for use in adult transplantation. In this study, we investigated two novel three-dimensional (3D) culture systems (static and rotating) for ex vivo expansion of HSCs from UCB. We encapsulated the human cord blood mononuclear cells (CBMCs) in alginate 3D static and rotating culture systems, compared the cell number amplification, the proportion of CD34+ cells, and the colony-forming capacity of these systems to those of the conventional two-dimensional (2D) system. The amplified cells were transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice to confirm the hematopoiesis reconstruction capacity of the cells. The increase in the cell number and the proportion of CD34+ cells in the CBMCs was more effective in these 3D alginate culture systems than in the conventional 2D culture system under the same conditions (p < 0.05). The stem cell maintenance capability was confirmed by flow cytometry and colony-forming assay ex vivo and NOD/SCID mice xenogeneic transplantation model in vivo. Our results demonstrated that these 3D alginate culture systems are an efficient way to amplify cord blood HSCs for extended periods without having them lose their self-renewal capacity in vivo. These novel 3D alginate culture systems are promising for the amplification of UCB-derived HSCs for clinical application in the future.

&lt;i&gt;In Vitro&lt;/i&gt; Fertilization and Development of Porcine Oocytes Matured in Follicular Fluid

This study was conducted to assess the fertilization and development of porcine oocytes matured in a solo follicular fluid (pFF) using different in vitro culture systems and insemination periods. Cumulus-oocyte complexes (COCs), follicular cells (FCs), and pFF were collected from the follicles of ovaries. The pFF was used as a maturation medium (MpFF) after supplementation with follicle stimulating hormone (FSH) and antibiotics. The COCs were matured in a 15 ml test tube containing 3.5 ml of MpFF with FCs (5.2 × 106 cells/ml; rotating culture system) or 2 ml of MpFF without FCs in a 35-mm petri dish (static culture system) for 44 to 48 h. After maturation culture, oocytes were co-incubated with frozen-thawed spermatozoa for 5 h and then cultured for 7 days. The total mean rates of sperm penetration, normal fertilization, male pronucleus (MPN) formation, cleavage, and development to the blastocyst stage of oocytes after insemination were significantly higher (P<0.01) in the rotating culture system than in the static culture system. In conclusion, compared with the static culture system, the rotating culture system is adequate for the production of developmentally competent porcine oocytes when MpFF is used as a maturation medium.

Differentiation and selection of hepatocyte precursors in suspension spheroid culture of transgenic murine embryonic stem cells.

Embryonic stem cell-derived hepatocyte precursor cells represent a promising model for clinical transplantations to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology investigations. This study aimed to establish an in vitro culture system for scalable generation of hepatic progenitor cells. We used stable transgenic clones of murine embryonic stem cells possessing a reporter/selection vector, in which the enhanced green fluorescent protein- and puromycin N-acetyltransferase-coding genes are driven by a common alpha-fetoprotein gene promoter. This allowed for “live” monitoring and puromycin selection of the desired differentiating cell type possessing the activated alpha-fetoprotein gene. A rotary culture system was established, sequentially yielding initially partially selected hepatocyte lineage-committed cells, and finally, a highly purified cell population maintained as a dynamic suspension spheroid culture, which progressively developed the hepatic gene expression phenotype. The latter was confirmed by quantitative RT-PCR analysis, which showed a progressive up-regulation of hepatic genes during spheroid culture, indicating development of a mixed hepatocyte precursor-/fetal hepatocyte-like cell population. Adherent spheroids gave rise to advanced differentiated hepatocyte-like cells expressing hepatic proteins such as albumin, alpha-1-antitrypsin, cytokeratin 18, E-cadherin, and liver-specific organic anion transporter 1, as demonstrated by fluorescent immunostaining. A fraction of adherent cells was capable of glycogen storage and of reversible up-take of indocyanine green, demonstrating their hepatocyte-like functionality. Moreover, after transplantation of spheroids into the mouse liver, the spheroid-derived cells integrated into recipient. These results demonstrate that large-scale hepatocyte precursor-/hepatocyte-like cultures can be established for use in clinical trials, as well as in in vitro screening assays.

The Effect of the Microgravity Rotating Culture System on the Chondrogenic Differentiation of Bone Marrow Mesenchymal Stem Cells

We investigated the influence of the microgravity rotating culture system on the chondrogenic differentiation of bone marrow mesenchymal stem cells (MSCs). During chondrogenic induction, MSCs combined with polyglycolic acid (PGA) were cultured by static culture or microgravity rotating culture and chondrocyte formation was confirmed by toluidine blue staining. Furthermore, the mRNA and protein expressions of a specific cartilage extracellular matrix protein (collagen type II and Aggrecan) were evaluated by real-time RT-PCR and western blot, respectively. Toluidine blue staining indicated the OD values of proteoglycans semi-determination were higher in the microgravity rotating culture group than the static culture group. Following chondrogenic induction, mRNA and proteins of collagen type II and Aggrecan were more significantly expressed in cells of the microgravity rotating culture group compared with the controls. Compared with routine three-dimensional static culture, the microgravity rotating culture system was more effective for the construction of tissue-engineered cartilage in vitro.

Effects of oxygen tension and follicle cells on maturation and fertilization of porcine oocytes during in vitro culture in follicular fluid

The objective was to investigate the effects of oxygen tension and follicle cells (FCs) during in vitro maturation of porcine oocytes in only porcine ( Sus scrofa domesticus) follicular fluid (pFF), using static and non-static (rotating) culture systems, on the nuclear maturation and subsequent in vitro fertilization of the oocytes. In the first experiment, cumulus-oocyte complexes (COCs) were matured for 48 h in pFF supplemented with (+) or without (−) FCs (5.2 × 10 6 cells/mL), using the static (S) and rotating (R) culture systems (+FC/S, −FC/S, +FC/R, and −FC/R) under 5% or 20% O 2. Co-culture with FCs in the static culture system (+FC/S) had a detrimental effect on the meiotic competence of oocytes, whereas co-culture with FCs in the rotating culture system (+FC/R) increased maturation rates. In both culture systems, oxygen tension had no apparent effects on meiotic competence of oocytes, irrespective of culture system and FC addition. In the second experiment, COCs were matured under 5% or 20% O 2 using the −FC/S or +FC/R culture systems and then fertilized. Oxygen tension had no significant effects on fertilization parameters, irrespective of the culture system. The rotating culture system increased rates of sperm penetration and male pronuclear formation and decreased polyspermic fertilization compared with the static culture system (P < 0.05). In conclusion, both −FC/S and +FC/R culture systems supported meiotic competence, irrespective of oxygen tension. However, the +FC/R culture system may be superior to the −FC/S culture system for promoting fertilization.

Optimization of Allograft Implantation Using Scaffold-Free Chondrocyte Plates

If a tissue-engineered cartilage transplant is to succeed, it needs to integrate with the host tissue, to endure physiological loading, and to acquire the phenotype of the articular cartilage. Although there are many reported treatments for osteochondral defects of articular cartilage, problems remain with the use of artificial matrices (scaffolds) and the stage of implantation. We constructed scaffold-free three-dimensional tissue-engineered cartilage allografts using a rotational culture system and investigated the optimal stage of implantation and repair of the remodeling site. We evaluated the amounts of extracellular matrix and gene expression levels in scaffold-free constructs and transplanted the constructs for osteochondral defects using a rabbit model. Allografted 2-week constructs expressed high levels of proteoglycan and collagen per DNA content, integrated with the host cartilage successfully, and were able to counter physiological loads, and the chondrocyte plate contributed reparative mesenchymal stem cells to the final phenotype of the articular cartilage.

Characteristics of a Scaffold-Free Articular Chondrocyte Plate Grown in Rotational Culture

We investigated whether articular chondrocytes could form three-dimensional tissue-engineered cartilage in a rotational culture system without a scaffold. A suspension of chondrocytes derived from Japanese white rabbits was inoculated into a mold. Eight hours later, the cell suspension in the mold showed cell aggregation, forming a chondrocyte plate. The mold was removed, and the plate was cultured under static conditions. After 7 days of primary static culture, the plate was cultured under dynamic conditions, using rotational culture. After 2-3 weeks of rotational culture, the chondrocyte plate maintained a constant form and was considered stable enough to be handled with surgical pincers. Conversely, after 3 weeks of static culture, the plate gradually changed into an arch over that time. Histological and immunohistochemical evaluations indicated that the plate had cartilaginous qualities in terms of cell distribution and organization and the production of glycosaminoglycans and type II collagen in rotational cultures. Chondron units were detected with scanning electron microscopy. In contrast, a plate cultivated in static culture for 3 weeks was irregular in shape, and histological analysis indicated irregularly accumulated glycosaminoglycans. TUNEL-positive cells had increased significantly in the central region in 3-week static cultures, compared with those in 3-week rotational cultures. In this study, cartilaginous tissue in a scaffold-free environment has been produced. Significantly rotational cultures produce a construct, which is stable enough to be handled with surgical forceps after only 2 weeks of rotational culture. This system should be useful for implantation in the future.

Recent technological advancements related to articular cartilage regeneration

Some treatments for full thickness defects of the articular cartilage, such as the transplantation of cultured chondrocytes have already been performed. However, in order to overcome osteoarthritis, we must further study the partial thickness defects of articular cartilage. It is much more difficult to repair a partial thickness defect because few repair cells can address such injured sites. We herein show that bioengineered and layered chondrocyte sheets using temperature-responsive culture dishes may be a potentially useful treatment for the repair of partial thickness defects. We also show that a chondrocyte-plate using a rotational culture system without the use of a scaffold may also be useful as a core cartilage of an articular cartilageous defect. We evaluated the properties of these sheets and plates using histological findings, scanning electrical microscopy, and photoacoustic measurement methods, which we developed to evaluate the biomechanical properties of tissue-engineered cartilage. In conclusion, the layered chondrocyte sheets and chondrocyte-plates were able to maintain the cartilageous phenotype, thus suggesting that they could be a new and potentially effective therapeutic product when attached to the sites of cartilage defects.

Characteristics of a Scaffold-Free Articular Chondrocyte Plate Grown in Rotational Culture

We investigated whether articular chondrocytes could form three-dimensional tissue-engineered cartilage in a rotational culture system without a scaffold. A suspension of chondrocytes derived from Japanese white rabbits was inoculated into a mold. Eight hours later, the cell suspension in the mold showed cell aggregation, forming a chondrocyte plate. The mold was removed, and the plate was cultured under static conditions. After 7 days of primary static culture, the plate was cultured under dynamic conditions, using rotational culture. After 2-3 weeks of rotational culture, the chondrocyte plate maintained a constant form and was considered stable enough to be handled with surgical pincers. Conversely, after 3 weeks of static culture, the plate gradually changed into an arch over that time. Histological and immunohistochemical evaluations indicated that the plate had cartilaginous qualities in terms of cell distribution and organization and the production of glycosaminoglycans and type II collagen in rotational cultures. Chondron units were detected with scanning electron microscopy. In contrast, a plate cultivated in static culture for 3 weeks was irregular in shape, and histological analysis indicated irregularly accumulated glycosaminoglycans. TUNEL-positive cells had increased significantly in the central region in 3-week static cultures, compared with those in 3-week rotational cultures. In this study, cartilaginous tissue in a scaffold-free environment has been produced. Significantly rotational cultures produce a construct, which is stable enough to be handled with surgical forceps after only 2 weeks of rotational culture. This system should be useful for implantation in the future.

Cleavage and survival of Xenopus embryos exposed to 8 T static magnetic fields in a rotating clinostat

In this study, we examined cleavage and survival of fertilized Xenopus embryos exposed to 8 T static magnetic fields (SMFs). We investigated fertilized Xenopus embryos exposed to magnetic field either in static chamber or in a rotating culture system. Our results showed that the exposure to the strong magnetic field of 8 T changed the third cleavage furrow from the usual horizontal one to a perpendicular one; however, when the direction of gravity was randomized by exposing embryos to magnetic field in a rotating culture system, the third cleavage furrow were formed horizontally, a finding which suggests that the observed distortion of the third cleavage furrow in magnetism-exposed embryos was accomplished by altering gravity effects which were elicited by diamagnetic force due to high gradient magnetic field. Our results also showed that the exposure to the strong magnetic field did not damage survival. These results demonstrate that SMF and altering gravity cause distortion of the third cleavage furrow and show that effects of exposing cleavage embryos to magnetic field were transient and did not affect the post-cleavage development. We also showed that strong magnetic field is not hazardous to the cleavage and blastula-gastrula transition of developing embryonic cells.

286 EFFECTS OF PORCINE FOLLICULAR FLUID DURING IN VITRO MATURATION OF OOCYTES ON IN VITRO FERTILIZATION AND EMBRYONIC DEVELOPMENT

When used as a solo maturation medium for oocytes, porcine follicular fluid (pFF) promoted male pronucleus formation (MPF) of oocytes after in vitro maturation (IVM), using a static system, and in vitro fertilization (IVF) in pigs (Naito et al. 1988 Gamete Res. 21, 289–295). However, the developmental competence of oocytes matured in pFF after IVM/IVF has not been reported. This study was conducted to assess the ability of pFF as a maturation medium to support IVM/IVF of porcine oocytes and their subsequent in vitro development. pFF, including cumulus–oocyte complexes (COCs), was aspirated from follicles (2–5 mm in diameter) of prepubertal crossbred gilt ovaries, and large clusters of follicular cells (FC) were removed from pFF by filtration through 212 �m of mesh. All of the COCs in filtered pFF were collected, and COCs with compact cumulus cells were selected for IVM. Also, small clusters of FC were collected by centrifugation of the filtered pFF, and pFF without any cells was prepared by centrifugation and used as a maturation medium (MpFF) after supplementation with FSH and antibiotics. COCs were transferred to 3.5 mL (in a 15-mL test tube) of MpFF with FC (5.2 � 106 cells mL-1) and cultured for 44–48 h at 38.5�C in 5% O2 and 5% CO2 using the rotating culture system. As a control group, COCs were cultured in 2 mL of MpFF without FC in a 35-mm Petri dish by the standard static culture system. After maturation, culture oocytes were co-incubated (IVF) for 5 h with frozen–thawed sperm in vitro, as reported previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041), and then some of them were fixed 10 h after IVF to assess the fertilization status; the rest of them were cultured in PZM (Yoshioka et al. 2002 Biol. Reprod. 60, 112–119) for 7 days to assess their early embryonic development. All of the data were analyzed by ANOVA. Oocytes cultured with FC in the rotating system (R group) showed significantly higher sperm penetration (71.0%), MPF formation (70.5%), and normal fertilization (monospermic fertilization with female and male pronuclei; 31.5%) rates than those in the control group (56.0%, 56.9%, and 17.6%, respectively; P &amp;lt; 0.05). Also, the R group showed significantly higher rates of 8-cell embryos at 2 days after IVF and blastocyst formation at 7 days after IVF than those of the control group (17.2% vs. 8.3% and 10.9% vs. 4.5%, respectively; P &amp;lt; 0.05). These results indicate that porcine oocytes matured in pFF supplemented with FC using the rotating system have the ability to be penetrated by sperm and form MPF, and to develop to the blastocyst stage at higher rates, than oocytes cultured in the standard static maturation culture system. In conclusion, the pFF can be a sole and simple maturation culture medium useful for the in vitro production of blastocysts in pigs.

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&amp;ndash;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 &amp;micro;m 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 &amp;times; 106 cells mL&amp;minus;1) added and were cultured for 48 h at 38.5&amp;deg;C in 5&amp;percnt; CO2 and 20&amp;percnt; O2 (in air) or 5&amp;percnt; O2 using the S or R culture systems (5O2/FC&amp;minus;/S, 5O2/FC&amp;plus;/S, 20O2/FC&amp;minus;/S, 20O2/FC&amp;plus;/S, 5O2/FC&amp;minus;/R, 5O2/FC&amp;plus;/R, 20O2/FC&amp;minus;/R, and 20O2/FC&amp;plus;/R groups). The oxygen tension had no effect on nuclear maturation except for the FC&amp;minus;/R groups. In the FC&amp;minus;/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&amp;plus;/R (67.7&amp;percnt;) and 20O2/FC&amp;plus;/R (65.9&amp;percnt;) than those in 5O2/FC&amp;plus;/S (17.5&amp;percnt;) and 20O2/FC&amp;plus;/S (35.7&amp;percnt;) (chi-squared test; P &amp;lt; 0.05). In contrast, when cultured without FC, oocytes matured to MII at higher rates in 5O2/FC&amp;minus;/S (78.3&amp;percnt;) and 20O2/FC&amp;minus;/S (76.5&amp;percnt;) than those in 5O2/FC&amp;minus;/R (32.7&amp;percnt;) and 20O2/FC&amp;minus;/R (11.4&amp;percnt;) (P &amp;lt; 0.05). In Experiment 2, oocytes were cultured in 5O2/FC&amp;minus;/S, 20O2/FC&amp;minus;/S, 5O2/FC&amp;plus;/R, or 20O2/FC&amp;plus;/R; fertilized in vitro, as reported previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033&amp;ndash;1041), and fixed 10 h after IVF. Oocytes cultured in 20O2/FC&amp;plus;/R showed a significantly higher sperm penetration rate (95.9&amp;percnt;) than those cultured in 5O2/FC&amp;minus;/S and 20O2/FC&amp;minus;/S (88.0&amp;percnt; and 88.4&amp;percnt;, respectively), but were similar to those cultured in 5O2/FC&amp;plus;/R (90.9&amp;percnt;) (P &amp;lt; 0.05). Oocytes cultured in 5O2/FC&amp;plus;/R formed a male pronucleus at a higher rate (77.1&amp;percnt;) than those cultured in 5O2/FC&amp;minus;/S, 20O2/FC&amp;minus;/S, and 20O2/FC&amp;plus;/R (52.8, 51.6, and 63.3&amp;percnt;, respectively) (P &amp;lt; 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&amp;plus;/R than in 20O2/FC&amp;plus;/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&amp;percnt; 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.

Proliferation Rates of HepG2 Cells Encapsulated in Alginate Are Increased in a Microgravity Environment Compared With Static Cultures

This study investigates the effect of rotary culture compared with static culture on the proliferation, cell viability, synthetic function and detoxificatory capacity of HepG2 cells encapsulated in 1% alginate. Cell viability and alginate bead morphology were maintained in the rotary culture system at day 10, while cell number showed a 4.5-fold increase compared with static culture. Protein production was increased in rotary cultures with a 4.1-fold increase in total albumin and a 4.4-fold increase in alpha1 antitrypsin levels in rotary compared with static culture at day 10. CYP4501A1/2 activity was maintained between the two culture systems. In conclusion, rotary culture increases proliferation rates leading to improved bead packing and a concomitant increase in total protein synthesis, along with maintenance of detoxificatory capacity. This allows a greater level of hepatic function to be expressed in a given volume, offering clear advantages for the design of liver support systems.

Rotation culture with a newly developed holder enables long-term liver slice culture for study of liver fibrosis

Background/aims: Conventional slice rotation culture has employed fibrin that is produced from fibrinogen by thrombin, to fix tissue slices on a slide glass. However, thrombin transmits various stimuli to cells through the thrombin receptor and affects experimental results. To exclude this disadvantage of thrombin, we developed a new holder and studied long-term liver rotation culture without using thrombin. Methods: Liver slices about [Formula: see text] were produced from the liver of 8-day-old Wister rats. The slice was fixed to a newly development holder on a slide glass and cultured in tube on rotary culture system. To evaluate whether slice survives and maintains functions, morphological structure, LDH leakage into the medium, ATP synthesis, glutathione-S-transferase activity and glycogen synthesis were examined. We also studied collagen synthesis after treatment with TGF-beta or thrombin. Results: Slice tissue survived and maintained its functions for over 10 days. After treatment with TGF-beta or thrombin, the tissue became shrunken and showed increased collagen synthesis. On the other hand, no stimulation of collagen synthesis was found in cultured tissue without treatment. Conclusions: Our data indicate that rotation culture with the new holder is suitable for long-term culture for study of liver fibrosis.