Evaluation of attachment and growth of anchorage-dependent cells on culture surfaces with type I collagen coating

The effects of coating the culture surface with bovine type I collagen on the culture properties of anchorage-dependent cells were investigated. When human fibroblasts were cultured on a surface coated with collagen at 5.8 x 10(-3) mg/cm2, cell attachment and subsequent cell growth were both enhanced compared to the culture on an uncoated surface. The degrees of cell attachment and growth enhancement were numerically characterized using the time constant of cell adhesion (tau) and doubling time (t(d)) as kinetic parameters. These parameters applied to cultures of human keratinocytes and rabbit chondrocytes allowed the effects of collagen coating on the respective culture properties of both types of cells to be evaluated. In addition, the relative parameters R(tau) and R(t(d)) (defined as the ratios of the tau and t(d) values at a given collagen concentration against those without collagen coating, respectively) were employed to estimate the effects of collagen based on a standardized criterion. Similar R(tau) and R(t(d)) profiles were obtained for collagen concentrations ranging from 5.8 x 10(-13) to 5.8 x 10(-3) mg/cm2, whether the cells were fibroblasts, keratinocytes or chondrocytes. It was also revealed that coating the surface with collagen at a concentration over 5.8 x 10(-7) mg/cm2 led to reductions in both the R(tau) and R(t(d)) values, i.e. the promotion of cell attachment and growth, in the culture of each type of cells examined.     

Compare the effects of chondrogenesis by culture of human mesenchymal stem cells with various type of the chondroitin sulfate C

Chondroitin sulfate C (CSC) is a kind of glycosaminoglycans (GAGs) with molecular weights of 10,000 to 50,000 Da and a high charge density. GAGs are major components in extracellular matrix (ECM), which play important role in the regulation of cell proliferation, migration, and differentiation. In this study, we studied the effects of chondroitin sulfate C (CSC) on the differentiation of human mesenchymal stem cells (MSCs) toward the chondrocyte lineage. The MSCs were either cultured on type II collagen (COL II) scaffolds with high molecular weight CSC addition in the medium (free CSC) or with free oligosaccharide CSC. Special attention was given to the effects of MSCs cultured on CSC cross-linked type II scaffolds (cross-linked CSC). According to the analysis of histology stain, gene expression, and ECM secretion, our results showed that MSCs cultured with free CSC, free oligosaccharides CSC, and on the cross-linked CSC scaffolds all would be induced into chondrocytes. Moreover, free oligosaccharide CSC present in the microenvironment could significantly up-regulate MSC chondrogenesis gene expression and stimulate cartilage ECM accumulation more than free CSC with high molecular weight after 3-week induction. Importantly, cross-linked CSC had the most excellent effects on the MSC chondrogenesis. Thus, we believed that cross-linked CSC in the scaffold would play the similar roles with free oligosaccharide CSC in the medium. Cross-linked CSC would be a potential candidate for cartilage repair in the cell therapy and tissue engineering.     

Growth and adhesion of osteoblast-like cells derived from neonatal rat calvaria on calcium phosphate ceramics

The effects of biocompatible ceramics on the growth and adhesion of osteoblast-rich rat calvarial cell cultures were investigated. Osteoblast-like cells and mouse fibroblast-like L-929 cells were cultured on composite sinters of hydroxyapatite (HAP) and beta-tricalcium phosphate (TCP) culture carriers, whose Ca/P molar ratios were adjusted to values of 1.50, 1.55, 1.60, 1.64 and 1.67. The growth rates of both cell types were accelerated on the TCP-HAP ceramics as compared to those on polystyrene plastic (LUX) or bioinert zirconia ceramics. The population of osteoblast-like cells reached a density of 2.28 x 10(5) cells/cm2 on 100% HAP (Ca/P ratio 1.67) at 9 d of culture, while the corresponding cell density was 1.66 x 10(5) cells/cm2 on LUX and 1.26 x 10(5) cells/cm2 on zirconia. Adhesion of the osteoblast-like cells on TCP-HAP ceramics was similarly increased as compared with that on LUX or zirconia ceramics. The adhesion of L-929 cells on TCP-HAP ceramics was found to be weaker than that on cultures on LUX or zirconia ceramics. The time-dependent variations in the alkaline phosphatase activity of the osteoblast-like cells showed that the osteoblastic phenotype was potentiated by culturing the cells in calcium-rich media. The surface analyses of the Ca/P ratio and the microstructure by XRD and FTIR suggest that the Ca-rich surface was newly formed by recombination on the surface layer in the culture medium containing fetal bovine serum. These results suggest that the surface of TCP-HAP ceramics, especially that of 100% HAP ceramics, are effective for accelerating growth and differentiation of osteoblast-like cells. This is most probably due to the chemical and physical instability and composition of 100% HAP, which promote the formation of a Ca-rich layer at the cell-material interface and provision of Ca ions to the osteoblast-like cells.     

Production of recombinant protein by baculovirus-infected insect cells in immobilized culture using porous biomass support particles

Immobilization of insect cells using porous biomass support particles (BSPs) and production of a recombinant protein by the immobilized cells after infection with a baculovirus were investigated in a shake-flask culture. Sf9 cells were passively immobilized in reticulated polyvinyl formal (PVF) resin BSPs (2 x 2 x 2 mm cubes) with matrices of 60 mum mean pore diameter in situ in shake-flasks. The cell density in the BSPs was over 5 x 10(7) cells/cm3-BSP in cultures with regular replacement of the culture medium, as estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After infection with a recombinant baculovirus carrying the beta-galactosidase gene, immobilized cells within the BSPs showed a high specific productivity, comparable to the maximum productivity in shake-flask cultures of non-immobilized cells, as long as nutrients in the medium were not depleted. Even when immobilized cells at a high density of 5 x 10(7) cells/cm3-BSP were infected with the baculovirus, efficient beta-galactosidase production with a high specific productivity was possible by replacing the medium at appropriate intervals to avoid nutrient depletion.     

Hepatic differentiation of mouse embryonic stem cells in a three-dimensional culture system using polyurethane foam

Embryonic stem (ES) cells are a type of pluripotent stem cell line isolated from the inner cell mass of blastocysts and characterized by an almost unlimited self-renewal capacity and differentiation potential in vitro into multiple cell lineages. Therefore the use of ES cells has recently received much attention as a novel cell source for various hybrid artificial organs. To use ES cells, it is necessary to be able to produce functional matured cells from ES cells in large quantities. In this study, we applied polyurethane foam (PUF)/spheroid culture, which enables spontaneous spheroid formation and mass cultivation of cultured cells, to mouse ES cells for hepatic differentiation. Mouse ES cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of the PUF within 1 d. To induce hepatic differentiation, specific growth factors were added to the culture medium. Mouse ES cells proliferated by day 20, and high cell density (about 1.0 x 10(8) cells/cm(3)-PUF) was achieved. Differentiating ES cells expressed endodermal-specific genes, such as alpha-fetoprotein, albumin and tryptophan 2,3-dioxygenase. The activity of ammonia removal of mouse ES cells per unit volume of the module was detected by day 21 and increased with culture time. Maximum expression levels were comparable to those of primary mouse hepatocytes. Mouse ES cells could express liver-specific functions at high level because of the high cell density culture and hepatic differentiation. These results suggest that the PUF/spheroid culture method could be useful to develop mass differentiation cultures.     

Optimal production of recombinant protein by the baculovirus-insect cell system in shake-flask culture with medium replacement

Sf9 insect cells infected with a recombinant baculovirus expressing beta-galactosidase and suspended in fresh medium (TNM-FH supplemented with 10% fetal bovine serum) at the time of infection were cultured in shake flasks at various combinations of initial cell density and multiplicity of infection (MOI). The effects of cell density and MOI on beta-galactosidase production were quantitatively analyzed by plotting the beta-galactosidase yield against the time integral of the viable cell density from the time of infection to the time when the beta-galactosidase production reached a plateau. The beta-galactosidase yield had a maximum value at a viable cell density time integral of approximately 8 x 10(6) cells.d/cm3 for each MOI used in a range from 0.01 to 10 plaque-forming units per cell (pfu/cell). Since glucose and fructose were exhausted when the culture reached 8 x 10(6) cells.d/cm3, it was concluded that protein production in a high-cell-density culture was limited by nutrient depletion in the culture medium, and hence the nutritional capacity of the medium was able to be determined as the viable cell density time integral at which the maximum product yield was attained. In cultures infected at a low MOI (< or =1 pfu/cell), the specific productivity, and thereby the yield, of beta-galactosidase declined with decreasing MOI due to the reduction in the proportion of initially infected cells. These results indicate that production of a recombinant protein in a culture with medium replacement at the time of infection can be optimized if the cells are infected at a high MOI (> or = 1 pfu/cell) and at a cell density such that the viable cell density time integral reaches the nutritional capacity just as the protein production is completed.     

Influence of fetal calf serum on differentiation of mesenchymal stem cells to chondrocytes during expansion

Mesenchymal stem cells (MSCs) should be expanded in vitro while maintaining their multilineage potential before differentiation to one mesenchymal lineage for application to regeneration therapy. The effect of fetal calf serum (FCS) on undesirable differentiation during subcultivations for the expansion was investigated. The expression level of the aggrecan gene, which is a marker of chondrogenic differentiation, gradually and markedly increased during the subcultivations of MSCs with the addition of 10% FCS and without additional cytokines. The percentage of cells positive for CD90 and CD166, which are markers of MSCs, decreased, and the percentage of large polygonal cells and the average cell adhesion area increased during the expansion. There was a marked difference in the increase in the aggrecan expression level between the two expansion cultures employing different FCS lots, although their proliferation rates were almost the same. The decrease in FCS concentration resulted in a higher percentage of CD90(+)CD166(+) cells, a lower percentage of large polygonal cells, and a lower level of aggrecan expression. Consequently, FCS components could stimulate MSC differentiation to chondrocytes and a lower concentration could decrease this differentiation.     

Cultivation of recombinant Chinese hamster ovary cells grown as suspended aggregates in stirred vessels

Recombinant Chinese hamster ovary (rCHO) cells capable of producing a prourokinase mutant (mPro-uk) grown as suspended aggregates in stirred vessels were described and characterized. The addition of chitosan to a mixture of DMEM and Ham's F12 (D-MEM/F-12) medium promoted cell aggregation and spheroid formation efficiently. Multicellular aggregates formed immediately after the rCHO cells were inoculated into the chitosan-added medium, and the mean diameter of the cell aggregates reflecting the aggregate size increased with culture time, shifting from 65 to 163 mum after 2 and 9 d of culture in spinner flasks. No significant difference in the metabolism performance of the rCHO cells was observed between suspended aggregates and anchored monolayers. However, the cells cultured as suspended aggregates showed a marked decrease in growth rate as evaluated from specific growth rate (mu). Replacing D-MEM/F-12 medium with CD 293 medium caused compact spherical cell aggregates to dissociate into small irregular aggregates and single cells without apparent effects on cell performance in subcultures. The perfusion culture of the rCHO cells grown as suspended aggregates in a 2-l stirred tank bioreactor for 15 d resulted in a maximum viable cell density of 5.6 x 10(6) cells ml(-1) and an mPro-uk concentration of about 2.6 x 10(3) IU ml(-1), and cell viability was remained at roughly 90% during the entire run.     

CoCl(2) inhibits neural differentiation of retinoic acid-treated embryoid bodies

The effects of CoCl(2) on retinoic acid (RA)-treated embryoid bodies (EBs) were investigated. Four-day EBs were treated with 5x10(-6) M of RA for 4 d, then subjected to attached culturing for 7 d in the presence of CoCl(2) at 0, 20, and 100 microM. Differentiation into MAP2- and GFAP-immunopositive cells was inhibited by CoCl(2) in a dose-dependent manner. Next, RA-treated EBs were dissociated into single cells and cultured for 7 d at an initial cell density of 1x10(3)/cm(2). The number of cells increased in a CoCl(2)-dose dependent fashion. In cultures with 100 microM of CoCl(2), more than 90% of the cells were immunopositive for nestin and nestin-immunopositive cells formed clusters, while there were few cells immunopositive for MAP2 or GFAP. These results suggest that CoCl(2) inhibits neural differentiation of RA-treated EB cells and promotes the proliferation of nestin-immunopositive cells, i.e., embryonic stem (ES)-derived neural stem-like cells.     

Aggregate formation of rCHO cells and its maintenance in repeated batch culture in the absence of cell adhesion materials

Aggregate formation of recombinant Chinese hamster ovary (rCHO) cells capable of producing granulocyte colony-stimulating factor (G-CSF), using medium lacking cell adhesion materials in a repeated batch culture, was examined together with cell growth, cell viability and G-CSF production. The rCHO culture was conducted in a rotary shaker and the medium was changed every five days. The formation of stable cell aggregates with high reproducibility was observed after the first medium change. The size of the cell aggregates (consisting of several 10s to 40,000 cells) formed during the repeated batch culture ranged from 30 to 600 microm. The cell density of the aggregates reached as high as 2 x 10(6) cells/ml and the viability was maintained at more than 80% for 19 d. Changing the medium to avoid glucose exhaustion effectively maintained the cell density, cell viability and G-CSF productivity at high levels.     

Effect of ascorbic acid on bone marrow-derived mesenchymal stem cell proliferation and differentiation

Mesenchymal stem cells (MSCs) derived from bone marrow are an important tool in tissue engineering and cell-based therapies because of their multipotent capacity. Majority of studies on MSCs have investigated the roles of growth factors, cytokines, and hormones. Antioxidants such as ascorbic acid can be used to expand MSCs while preserving their differentiation ability. Moreover, ascorbic acid can also stimulate MSC proliferation without reciprocal loss of phenotype and differentiation potency. In this study, we evaluated the effects of ascorbic acid on the proliferation, differentiation, extracellular matrix (ECM) secretion of MSCs. The MSCs were cultured in media containing various concentrations (0-500 microM) of L-ascorbate-2-phosphate (Asc-2-P) for 2 weeks, following which they were differentiated into adipocytes and osteoblasts. Ascorbic acid stimulated ECM secretion (collagen and glycosaminoglycan) and cell proliferation. Moreover, the phenotypes of the experimental groups as well as the differentiation potential of MSCs remained unchanged. The apparent absence of decreased cell density or morphologic change is consistent with the toxicity observed with 5-250 microM concentrations of Asc-2-P. The results demonstrate that MSC proliferation or differentiation depends on ascorbic acid concentration.     

Reconstruction of cartilage tissue using scaffold-free organoid culture technique

We applied the scaffold-free culture method to chondrocytes and attempted to reconstitute articular cartilage grafts. Primary rat costal chondrocytes were immobilized into hollow fibers by centrifugation at a density of 3 x 10(8) cells/cm(3) to induce the formation of cylindrical-shaped multicellular aggregates (organoids) and cultured for one month. The organoids were evaluated by histological and gene expression analyses. Chondrocytes formed cylindrical organoids in hollow fibers (HFs). Histochemical analysis revealed the accumulation of a cartilage extracellular matrix (collagen and proteoglycan) around cells in the lumen of HFs with culture time, forming a low-cellular-density tissue similar to native cartilage by day 28. Furthermore, in contrast to that in traditional monolayer culture, the organoid maintained the gene expression of the cartilage extracellular matrix (type II collagen, aggrecan) for one month of culture. In conclusion, our organoid formation method was effective in producing a cartilage-like tissue. This result suggests that the technique may be applicable to the development of an articular cartilage graft.     

Proliferation and chondrogenic differentiation of human adipose-derived mesenchymal stem cells in porous hyaluronic acid scaffold

Human adipose-derived mesenchymal stem cells (AD-MSCs) attracted much interest as a promising alternative to autologous chondrocytes and bone marrow-derived mesenchymal stem cells for cartilage regeneration. Developing a suitable culture technique to direct AD-MSCs into the chondrogenic lineage could be a crucial prerequisite for the cartilage defect repair application of AD-MSCs. Herein, we prepared the PEGDG-crosslinked porous three-dimensional (3D) hyaluronic acid (HA) scaffold and evaluated for its feasibility to induce proliferation and chondrogenic differentiation of the AD-MSCs. In addition, the effect of bone-morphogenetic protein-2 (BMP-2) and platelet-derived growth factor (PDGF) on chondrogenic differentiation was further investigated. Proliferation and chondrogenic differentiation were evaluated by cell morphology, DNA contents, s-GAG contents, and level of mRNA expression of relevant marker genes. When cultured with reference chondrogenic medium (RCM; serum-free DMEM-HG supplemented with 10 ng/mL of transforming growth factor-β1 (TGF-β1), 50 nM ascorbate, 100 nM dexamethasone, and 5 μg/mL of ITS), better proliferation and chondrogenic differentiation of AD-MSCs were obtained in the 3D HA scaffold culture as compared to the micromass culture, a standard 3D culture system. Moreover, the level of chondrogenic differentiation of AD-MSCs in the HA scaffold-RCM culture system was further increased by BMP-2, and decreased by PDGF. These results suggested that the HA scaffold with RCM was a promising chondrogenic culture system of AD-MSCs, and that BMP-2 could potentially serve as a chondrogenic supplement for AD-MSCs. However, PDGF was determined to be an inappropriate supplement based on its inhibition of the chondrogenic differentiation of AD-MSCs.     

Combination of cytomegalovirus enhancer with human cellular promoters for gene-induced chondrogenesis of human bone marrow mesenchymal stem cells

High-expression plasmid vectors for human mesenchymal stem cells (MSCs) were constructed by combination of cytomegalovirus immediate-early enhancer with cellular promoters. MSCs transfected with the vector showed higher transgene production of a cytokine, which increased the differentiation level to chondrocytes.     

Ex vivo cartilage defect model for the evaluation of cartilage regeneration using mesenchymal stem cells

An ex vivo cartilage defect model for the evaluation of cartilage regeneration using mesenchymal stem cells (MSCs) was developed. Porcine chondrocytes and human MSCs were transplanted into cartilage defects created on the porcine osteochondral and chondral discs and cultivated for 3 weeks. Although the regeneration of cartilage-like tissues was observed after the transplantation of chondrocytes to defects on both of the osteochondral and chondral discs, the transplanted MSCs formed cartilage-like tissues only in the defect on the chondral disc, in which an in vivo cartilage-like structure was partly observed, and a degraded tissue was observed in the defect on the osteochondral disc. The effects of medium additives such as serum, transforming growth factor-β3 (TGF-β3), and fibroblast growth factor-2, and the transfection of TGF-β3 gene to MSCs could be clearly estimated using the cartilage defect model. In conclusion, a chondral disc with defects is useful for evaluating cartilage regeneration using MSCs.     

Release of embryogenic carrot cells with high regeneration potency from immobilized alginate beads

For the mass-production of regenerated carrot plantlets, embryogenic carrot callus immobilized in calcium alginate gel beads was cultivated in a growth medium and the regeneration frequency of cells released from alginate gel beads was compared with that in a suspension culture. Cells released in the immobilized culture were regenerated at a frequency which was about 1.5 times higher than that obtained in the suspension culture. When CaCl2 was added to the growth medium at 5 mM, repeated batch culture for plantlet production continued for 245 d with no significant decrease in the productivity (1.6 x 10(5) plantlets/l-medium/d).     

Saccharification of chitin using solid-state culture of Aspergillus sp. S1-13 with shellfish waste as a substrate

Saccharification of chitin was performed in a suspension (mash) of a solid-state culture of chitinase-producing Aspergillus sp. Sl-13 with acid-treated shellfish waste as a substrate. The conditions for the saccharifying reaction and the solid-state cultivation were examined from the viewpoint of saccharification in the mash. Optimum cultivation conditions were defined: a solid-state medium consisting of 5 g of 10% lactic acid-treated crab shells (0.50-2.36 mm in size) and 3 ml of a basal medium (0.028% KH2PO4 0.007% CaCl2.2H2O, and 0.025% MgSO4.7H2O) supplemented with 0.3% peptone was inoculated with 4 ml of spore suspension (1 x 10(7) spores/ml), and the water content of the medium was adjusted to 75%; static cultivation at 37 degrees C for 7 d. When a culture obtained under the optimum conditions was suspended in 70 ml of 50 mM sodium phosphate-citrate buffer (pH 4.0) and incubated at 45 degrees C for 11-13 d, 55 mM N-acetylglucosamine (GlcNAc) was formed in the solid-state culture mash, indicating that at least 33% of the initial chitin in the solid material was hydrolyzed. Through the experiments, the amounts of G1cNAc formed in the solid-state culture mash varied in a way similar to that of the water-extractable pnitrophenyl beta-D-N-acetylglucosaminide-hydrolyzing enzyme in the culture, but not to that of the colloidal chitin-hydrolyzing enzyme. G1cNAc-assimilating lactic acid bacteria, which were inoculated into the mash after or at the start of the saccharification, formed lactic acid with decreasing GlcNAc.     

Shrinkage-free preparation of scaffold-free cartilage-like disk-shaped cell sheet using human bone marrow mesenchymal stem cells

Aiming for the clinical application of cartilage regeneration, a culture method for mesenchymal stem cells (MSCs) derived from human bone marrow to obtain scaffold-free cartilage-like disk-shaped sheet of uniform sizes without the shrinkage was investigated. A disk-shaped cell sheet having the same diameter as that of the membrane without the shrinkage was formed after the cultivation of MSCs (18.6 × 10(5)cells/well) for 3 weeks in a cell culture insert (CCI) containing a flat membrane whose porosity was 12%, while 6.2 and 31.0 × 10(5)MSCs/well, respectively, resulted in the shrinkage of the aggregate and the hole formation in the center part of the sheet. Cell aggregates shrunk also in a 96-well plate and CCIs having lower porosity. The disk-shaped cell sheet showed the comparable thickness (1.2mm) and sulfated glycosaminoglycan (sGAG) density to those of the pellet formed in a pellet culture. The gene expression levels of aggrecan and type II collagen in the disk-shaped cell sheet were not lower than those in the pellet. In conclusion, the usage of CCI having 12% porosity and 18.6 × 10(5)MSCs/well could avoid the shrinkage from the formation of the scaffold-free cartilage-like disk-shaped cell sheet.     

Role of gap junctions in regulation of progesterone secretion by ovine luteal cells in vitro

To evaluate the role of gap junctions in the regulation of progesterone secretion, two experiments were conducted. In Experiment 1, luteal cells obtained on days 5, 10, and 15 were cultured overnight at densities of 50 x 10(3), 100 x 10(3), 300 x 10(3), and 600 x 10(3) cells/dish in medium containing: (1) no treatment (control), (2) LH, or (3) dbcAMP. In Experiment 2, luteal cells from days 5 and 10 of the estrous cycle were transfected with siRNA, which targeted the connexin (Cx) 43 gene. In Experiment 1, progesterone secretion, Cx43 mRNA expression, and the rates of gap junctional intercellular communication (GJIC), were affected by the day of the estrous cycle, cell density, and treatments (LH or dbcAMP). The changes in progesterone secretion were positively correlated with the changes in Cx43 mRNA expression and the rates of GJIC. Cx43 was detected on the luteal cell borders in every culture, and luteal cells expressed 3beta-hydroxysteroid dehydrogenase. In Experiment 2, two Cx43 gene-targeted sequences decreased Cx43 mRNA expression and progesterone production by luteal cells. The changes in Cx43 mRNA expression were positively correlated with changes in progesterone concentration in media. Thus, our data demonstrate a relationship between gap junctions and progesterone secretion that was supported by (1) the positive correlations between progesterone secretion and Cx43 mRNA expression and GJIC of luteal cells and (2) the inhibition of Cx43 mRNA expression by siRNA that resulted in decreased production of progesterone by luteal cells. This suggests that gap junctions may be involved in the regulation of steroidogenesis in the ovine corpus luteum.     

Effect of antioxidants on the apoptosis of CHO cells and production of tissue plasminogen activator in suspension culture

The effects of antioxidants on the apoptosis of Chinese hamster ovary (CHO) 1-15,500 cells and production of tissue plasminogen activator (tPA) in suspension culture were investigated. After cell growth to 2 x 10(5) cells/ml in Ham's F12 medium containing 10% newborn bovine serum (NBS) in a spinner bottle, CHO cells were maintained for 6 d in Ham's F12 medium containing 0 or 0.4% NBS and 10 mM antioxidants, namely, l-ascorbic acid 2-phosphate (VCP) or the reduced form of glutathione (GSH). The viable cell concentrations at day 6 in the serum-free culture with GSH and in the low-serum culture wiht VCP or GSH were 0.57, 1.04 and 1.69 x 10(5) cells/ml, respectively, while those in the serum-free and low-serum cultures without the antioxidants were only 2.33 and 1.17 x 10(3) cells/ml, respectively. The percentages of apoptotic cells in the serum-free and low-serum cultures with VCP (73.2, 44.6%) and GSH (76.9, 38.6%) measured using a flowcytometer after annexin V-FITC/propidium iodide staining were markedly lower than those in the cultures without antioxidants (96.3, 92.5%). The percentage of cells having a high mitochondrial membrane potential among the viable cells in the cultures with antioxidants determined using a flowcytometer after 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide staining was clearly higher than those in the cultures without the antioxidants. The production of tPA in the serum-free and low-serum media with VCP (0.282, 2.92 mg/l) or GSH (1.01, 1.61 mg/l) was markedly higher than that in the cultures without the antioxidants (0.275, 0.689 mg/l). Consequently, the suppression of apoptosis through the maintenance of the membrane potential of mitochondria by VCP or GSH resulted in a marked increase in tPA production by CHO cells in the serum-free and low-serum cultures.