Enhanced expression of various exogenous genes in recombinant Chinese hamster ovary cells in presence of dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) (1% v/v) stimulated stable transformed Chinese hamster ovary (CHO) cells to synthesize different recombinant proteins and repress their proliferation rate. The expressions of a fusion protein and -galactosidase were increased 1.6- and 1.4-fold after adding DMSO. The expression of fusion protein was increased by up to 2.8-fold that of uninduced control by the simultaneous addition of DMSO and pentanoic acid. However, DMSO did not increase the production of the monoclonal antibody (immunoglobulin G) of three hybridoma cell lines (OKM1, OKT4 and HyGPD-YK-1-1), although it could inhibit the growth rates of the hybridoma.     

Expression of the casein kinase 2 subunits in Chinese hamster ovary and 3T3 L1 cells provides information on the role of the enzyme in cell proliferation and the cell cycle.

In order to investigate the in vivo functions of protein kinase CK2 (CK2), the expression of Myc-tagged versions of the subunits, Myc-CK2alpha and Myc-CK2beta, was carried out in Chinese hamster ovary cells (CHO cells) and in 3T3 L1 fibroblasts. Cell proliferation in these cells was examined. CHO cells that transiently overexpressed the Myc-CK2beta subunit exhibited a severe growth defect, as shown by a much lower value of [(3)H]thymidine incorporation than the vector controls, and a rounded shrunken morphology. In contrast, cells overexpressing Myc-tagged CK2alpha showed a slightly but consistently higher value of [(3)H]thymidine incorporation than the controls. The defect in cell growth and changes in morphology caused by Myc-CK2beta overexpression were partially rescued by coexpression of Myc-tagged CK2alpha. In parallel to the studies in CHO cells, the stable transfection of Myc-CK2alpha and Myc-CK2beta subunits was achieved in 3T3 L1 fibroblast cells. Similarly, the ectopic expression of Myc-CK2beta, but not Myc-CK2alpha, caused a growth defect. By measuring [(3)H]thymidine incorporation, it was found that expression of Myc-CK2beta prolonged the G(1) phase and inhibited up-regulation of cyclin D1 expression during G(1). In addition, a lower mitotic index and lower mitotic cyclin-dependent kinase activities were detected in Myc-CK2beta-expressing cells. Detailed analysis of stable cells that were synchronously released into the cell cycle revealed that the expression of Myc-CK2beta inhibited cells entering into mitosis and prevented the activation of mitotic cyclin-dependent kinases. Taken together, results from both transient and stable expression of CK2 subunits strongly suggest that CK2 may be involved in the control of cell growth and progression of the cell cycle.     

p27Kip1-mediated controlled proliferation technology increases constitutive sICAM production in CHO-DUKX adapted for growth in suspension and serum-free media

We have engineered dihydrofolate reductase-deficient (dhfr(-)) Chinese hamster ovary (CHO)-DUKX B11 cells adapted for growth in serum-free suspension cultures for unlinked muristerone-inducible expression of the cyclin-dependent kinase inhibitor p27Kip1 and constitutive expression of the soluble intercellular adhesion molecule-1 (sICAM), a potent common cold therapeutic. Conditional overexpression of p27Kip1 resulted in a sustained G1-specific growth arrest of transgenic CHO-DUKX associated with up to fivefold-increased specific sICAM productivity. Herein we exemplify the implementation of controlled proliferation technology in a major biopharmaceutical production cell line that is compatible with key requirements for large-scale production procedures, including constitutive transgene expression and anchorage-independent growth in serum-free media.     

Tissue polarity genes of Drosophila regulate the subcellular location for prehair initiation in pupal wing cells

The purpose of this study was to explore the functional role of the cytoplasmic domain of the alpha subunit of the alpha 5/beta 1 integrin, a fibronectin receptor. Mutant CHO cells that express very low levels of endogenous hamster alpha 5 subunit (CHO clone B2) were transfected with an expression vector containing full-length or truncated human alpha 5 cDNAs to form chimeric human alpha 5/hamster beta 1 integrins. Three transfectants were examined: B2a27 expresses a full-length human alpha 5 subunit with 27 amino acids in the cytoplasmic domain; B2a10 expresses an alpha 5 with a 17-amino acid cytoplasmic truncation; B2a1 expresses an alpha 5 with a 26-amino acid truncation. Levels of alpha 5/beta 1 surface expression in B2a27 and B2a10 cells were similar to that in wild type CHO cells. The expression of alpha 5/beta 1 in B2a1 cells was less, amounting to 15-20% of WT levels, despite message levels that were three to five times greater than those of B2a27. The transfectants were used to examine the role of the alpha 5 cytoplasmic domain in cell adhesion, cell motility, cytoskeletal organization, and integrin-mediated tyrosine phosphorylation. The adhesion characteristics of B2a27 and B2a10 cells on fibronectin substrata were similar to each other and to wild type CHO cells. B2a1 cells displayed slight reductions in the strength and rate of adhesion to fibronectin. Cell motility in the presence of fibronectin was similar for B2a27, B2a10, and wild type CHO cells, while the B2a1 cells were substantially less motile. Comparable degrees of cell spreading and extensive organization of actin filaments were observed for B2a27, B2a10, and wild type CHO cells on fibronectin substrata. The B2a1 cells spread to a lesser degree, and some organization of actin was observed; the untransfected B2 cells remained round on fibronectin substrata and showed no actin reorganization. Since the reduced motility and cell spreading observed in the B2a1 cells might be due either to reduced surface expression of alpha 5/beta 1 or to the truncation in the alpha 5 cytoplasmic domain, we used flow cytometric cell sorting to select populations of B2a1 and B2a27 cells expressing similar levels of cell surface alpha 5. The deficits in spreading and motility were present in B2a1 cells expressing high levels of alpha 5. Thus the region of the alpha 5 cytoplasmic domain adjacent to the membrane seems to play an important role in cytoskeletal organization and cell motility. We also examined whether alpha subunit truncation would affect integrin- mediated tyrosine phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Recombinant cyclin E expression activates proliferation and obviates surface attachment of chinese hamster ovary (CHO) cells in protein-free medium

Exogenous growth factors normally required in cell culture activate cell proliferation via the molecular controls of cell-cycle progression. Highly differing influences of mitogenic stimulation of Chinese hamster ovary (CHO) cells by insulin and basic fibroblast growth factor(bFGF) have been clearly observed in a defined protein-free medium. CHO K1 cells stimulated only with insulin grow with flattened cell morphology and extensive cell-cell contact, whereas stimulation with only bFGF or bFGF plus insulin results in loss of cell-cell contact and a transformed and rounded-up morphology. Compared with insulin-stimulated cells, bFGF-stimulated cells exhibit a relatively long G1, and short S phase, and contain higher levels of cyclin E. Observation of elevated levels of cyclin E in wild-type CHO K1 cells mitogenically stimulated by basic fibroblast growth factor motivated transfection of these cells by a cyclin E expression vector. These transfectants grew rapidly in protein-free basal medium and had similar cyclin b levels, distributions of nuclear cell-cycle times, and cell morphologies as bFGF-stimutated CHO K1 culture. Metabolic engineering of cell-cycle regulation thus bypasses exogenous growth factor requirements, addressing a priority objective in economical, reproducible, and safe biopharmaceutical manufacturing. (c) 1995 John Wiley & Sons Inc.     

p27 modulates cell cycle progression and chemosensitivity in human malignant glioma.

The cell cycle regulatory protein p27, an inhibitor of cyclin-dependent kinases (CDK), has been attributed a role in (i) prognosis in breast and colon cancer, (ii) induction of apoptosis in cancer cells, and (iii) resistance to cancer chemotherapy. Here we report that p27 is widely expressed in human malignant gliomas in vivo and in glioma cell lines in vitro. Serum deprivation or confluency promotes p27 protein accumulation in vitro. Neither baseline p27 levels nor p27 levels induced by confluency or serum deprivation correlate with p53 status or drug sensitivity of human glioma cell lines. Expression of antisense p27 mRNA increased the doubling times in T98G glioma cells, whereas sense p27 mRNA had no such effect. There was a density-dependent and drug-specific modulation of chemosensitivity by sense or antisense mRNA expression in T98G cells. Taken together, these data define a strong p27 response to altered growth conditions and suggest a role for p27 in modulating response to chemotherapy in human malignant glioma cells.     

Insulin-like growth factor-I and transferrin mediate growth and survival of Chinese hamster ovary cells

The aim of this investigation was to elucidate the roles of insulin-like growth factor-I (IGF-I) and transferrin in the survival and proliferation of Chinese hamster ovary (CHO) cells upon withdrawal of serum. For this purpose, we employed DNA analysis and flow cytometry to compare CHO cell lines expressing either IGF-I alone or IGF-I and transferrin. The ability of cells to cycle and the occurrence of apoptosis were monitored in these cells in serum-free medium. These results indicate that IGF-I alone is able to maintain the viability of CHO cells for an extended length of time in the absence of serum. Transferrin alone does not promote survival or proliferation. Only in the presence of both IGF-I and transferrin do cells survive and proliferate. Therefore, in attached CHO cultures, IGF-I alone does not stimulate cell proliferation but is a requirement for growth in serum-free medium in cooperation with transferrin. We report on the dual role of IGF-I as a survival factor in CHO cells and its interlocking role with transferrin to stimulate cell growth.     

Reversible G1 arrest in the cell cycle of human lymphoid cell lines by dimethyl sulfoxide

Proliferation of human B- and T-lymphoid cell lines including Raji and Akata cells was found to be arrested at the G1 stage in the cell cycle by dimethyl sulfoxide (DMSO). The G1 arrest by DMSO occurred gradually and was completed within 96 h after addition of 1.5% DMSO concomitantly with a decrease in growth rate. Progression of G1-phase cells containing a larger amount of RNA into S-phase began 9-12 h after removal of DMSO. At 24 h, the DNA pattern of the cell cycle was similar to that of nontreated log-phase cells. The expression of six differentiation markers on the lymphoid cells was not appreciably changed by treatment with DMSO. On the other hand, the expression of transferrin receptor (one of the growth-related markers) on G1-phase cells 96 h after addition of DMSO was decreased to one-fourth that on log-phase cells and was completely restored 24 h after removal of DMSO. These results indicate that DMSO, known as an inducer of differentiation in several myeloid cell lines, acts as an agent inducing G1 arrest in the cell cycle of the lymphoid cells.     

A cell cycle and mutational analysis of anchorage-independent growth: cell adhesion and TGF-beta 1 control G1/S transit specifically

We have examined cell cycle control of anchorage-independent growth in nontransformed fibroblasts. In previous studies using G0-synchronized NRK and NIH-3T3 cells, we showed that anchorage-independent growth is regulated by an attachment-dependent transition at G1/S that resembles the START control point in the cell cycle of Saccharomyces cerevisiae. In the studies reported here, we have synchronized NRK and NIH-3T3 fibroblasts immediately after this attachment-dependent transition to determine if other portions of the fibroblast cell cycle are similarly regulated by adhesion. Our results show that S-, G2-, and M-phase progression proceed in the absence of attachment. Thus, we conclude that the adhesion requirement for proliferation of these cells can be explained in terms of the single START-like transition. In related studies, we show that TGF-beta 1 overrides the attachment-dependent transition in NRK and AKR-2B fibroblasts (lines in which TGF-beta 1 induces anchorage-independent growth), but not in NIH-3T3 or Balb/c 3T3 fibroblasts (lines in which TGF-beta 1 fails to induce anchorage- independent growth). These results show that (a) adhesion and TGF-beta 1 can have similar effects in stimulating cell cycle progression from G1 to S and (b) the differential effects of TGF-beta 1 on anchorage- independent growth of various fibroblast lines are directly reflected in the differential effects of the growth factor at G1/S. Finally, we have randomly mutagenized NRK fibroblasts to generate mutant lines that have lost their attachment/TGF-beta 1 requirement for G1/S transit while retaining their normal mitogen requirements for proliferation. These clones, which readily proliferate in mitogen-supplemented soft agar, appear non-transformed in monolayer: they are well spread, nonrefractile, and contact inhibited. The existence of this new fibroblast phenotype demonstrates (a) that the growth factor and adhesion/TGF-beta 1 requirements for cell cycle progression are genetically separable, (b) that the two major control points in the fibroblast cell cycle (G0/G1 and G1/S) are regulated by distinct extracellular signals, and (c) that the genes regulating anchorage- independent growth need not be involved in regulating contact inhibition, focus formation, or growth factor dependence.     

SPRR1B overexpression enhances entry of cells into the G0 phase of the cell cycle

Many studies have established the role of SPRR1B during squamous differentiation of skin and respiratory epithelial cells. However, its role in nonsquamous cells is largely unknown. We reported that expression of SPRR1B in Chinese hamster ovary (CHO) cells is increased as they enter the G0 phase of the cell cycle. The purpose of this study was to further investigate the SPRR1B expression pattern in nonsquamous tumors and to study its role in these cells. Expression of SPRR1B was detected by Northern blotting in a higher percentage of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced compared with beryllium metal-induced rat lung adenocarcinomas. In situ hybridizations confirmed that SPRR1B is expressed in individual or clusters of cells of nonsquamous cells from mouse, rat, and human adenocarcinomas. The same pattern of expression was observed in adenocarcinomas formed in nude mice from cell lines established from adenocarcinomas. SPRR1B expression was downregulated in the cell lines derived from adenocarcinoma when cells were enriched in G0 at low confluence. Tetraploidy was induced in CHO, mouse, and human tumor cell lines by stably overexpressing SPRR1B, whereas control cells showed no change in ploidy. Inducible expression of this protein for shorter periods using the ecdyson system did not affect growth rate or the ploidy of CHO cells but accelerated entry into G0/G1 compared with controls. These findings indicate that SPRR1B is likely coupled primarily to signals responsible for withdrawal from the proliferative state rather than the final stages of cellular quiescence and that its overexpression for prolonged periods may disrupt the normal progression of mitosis.     

Expression of beta 1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility

The integrin subunit beta 1B, a beta 1 isoform with a unique sequence at the cytoplasmic domain, forms heterodimers with integrin alpha chains and binds fibronectin, but it does not localize to focal adhesion sites (Balzac, F., A. Belkin, V. Koteliansky, Y. Balabanow, F. Altruda, L. Silengo, and G. Tarone. 1993. J. Cell Biol. 121:171-178). Here we analyze the functional properties of human beta 1B by expressing it in hamster CHO cells. When stimulated by specific antibodies, beta 1B does not trigger tyrosine phosphorylation of a 125- kD cytosolic protein, an intracellular signalling pathway that is activated both by the endogenous hamster or the transfected human beta 1A. Moreover, expression of beta 1B results in reduced spreading on fibronectin and laminin, but not on vitronectin. Expression of beta 1B also results in severe reduction of cell motility in the Boyden chamber assay. Reduced cell spreading and motility could not be accounted for by preferential association of beta 1B with a given integrin alpha subunit. These data, together with our previous results, indicate that beta 1B interferes with beta 1A function when expressed in CHO cells resulting in a dominant negative effect on cell adhesion and migration.     

Cell cycle variation associated with feeder effects in cultures of Chinese hamster fibroblasts

Sub-confluent monolayer cultures of an established line of Chinese hamster fibroblast (Don) are shown to exhibit a density-dependent stimulation of growth. Evidence is presented that both long and short range ‘feeder effects’ are involved. Using the technique of autoradiography, cell cycle parameters have been studied in sub-confluent cultures seeded at different densities to identify the source of this density-dependent variation in growth rate. The durations of S phase, G2, and mitosis are constant as indicated by “percentage labelled mitoses” curves. A simple procedure has been developed for measurement of the fraction of a cell population in the G1 state, and this fraction is shown to be inversely related to the density of the culture. It is concluded that regulation of cell growth associated with feeder effects in cultured Don cells occurs within the G1 state. The data obtained from “percentage labelled mitoses” curves are shown to be highly consistent with the predictions of the Transition Probability model for cell cycle regulation.     

Glucose-6-phosphate dehydrogenase and the oxidative pentose phosphate cycle protect cells against apoptosis induced by low doses of ionizing radiation

The initial and rate-limiting enzyme of the oxidative pentose phosphate shunt, glucose-6-phosphate dehydrogenase (G6PD), is inhibited by NADPH and stimulated by NADP(+). Hence, under normal growth conditions, where NADPH levels exceed NADP(+) levels by as much as 100-fold, the activity of the pentose phosphate cycle is extremely low. However, during oxidant stress, pentose phosphate cycle activity can increase by as much as 200-fold over basal levels, to maintain the cytosolic reducing environment. G6PD-deficient (G6PD(-)) cell lines are sensitive to toxicity induced by chemical oxidants and ionizing radiation. Compared to wild-type CHO cells, enhanced sensitivity to ionizing radiation was observed for G6PD(-) cells exposed to single-dose or fractionated radiation. Fitting the single-dose radiation response data to the linear-quadratic model of radiation-induced cytotoxicity, we found that the G6PD(-) cells exhibited a significant enhancement in the alpha component of radiation-induced cell killing, while the values obtained for the beta component were similar in both the G6PD(-) and wild-type CHO cell lines. Here we report that the enhanced alpha component of radiation-induced cell killing is associated with a significant increase in the incidence of ionizing radiation-induced apoptosis in the G6PD(-) cells. These data suggest that G6PD and the oxidative pentose phosphate shunt protect cells from ionizing radiation-induced cell killing by limiting the incidence of radiation-induced apoptosis. The sensitivity to radiation-induced apoptosis was lost when the cDNA for wild-type G6PD was transfected into the G6PD(-) cell lines. Depleting GSH with l-BSO enhanced apoptosis of K1 cells while having no effect in the G6PD(-) cell line     

Integrin Signaling at the M/G1 Transition Induces Expression of Cyclin E

The activities of the mammalian G1 cyclins, cyclin D and cyclin E, during cell cycle progression (G1/S) are believed to be regulated by cell attachment and the presence of growth factors. In order to study the importance of cell attachment and concomitant integrin signaling on the expression of G1 cyclins during the natural adhesion process from mitosis to interphase, protein expression was monitored in cells that were synchronized by mitotic shake off. Here we show that in Chinese hamster ovary (CHO) and neuroblastoma (N2A) cells, expression of cyclin E at the M/G1 transition is regulated by both growth factors and cell attachment, while expression of cyclin D seems to be entirely dependent on the presence of serum. Expression of cyclin E appears to be correlated with the phosphorylation of the retinoblastoma protein, suggesting a link with the activity of the cyclin D/cdk4 complex. Expression of the cdk inhibitors p21^cip1/Waf1 and p27^Kip1 is not changed upon serum depletion or detachment of cells during early G1, suggesting no direct role for these CKIs in the regulation of cyclin activity. Although inhibition of cyclin E/cdk2 kinase activity has been reported previously, this is the first time that cyclin E expression is shown to be dependent on cell attachment.     

DIMETHYL SULFOXIDE-INDUCED REVERSION OF SEVERAL FEATURES OF POLYOMA TRANSFORMED BABY HAMSTER KIDNEY CELLS (BHK-21) : Alterations in Growth and Morphology

Cells of a polyoma virus transformed clonal line (Cl-I) of baby hamster kidney fibroblasts (BHK-21) were grown in medium containing 2 percent dimethylsulfoxide (DMSO). Unlike the untransformed BHK-21 cells, Cl-I cells adapted to replication in the presence of DMSO, and they exhibited a rapidly reversible phenotypic reversion of a number of properties characteristic of the transformed state. Restoration of density dependent growth inhibition with accumulation of cells in the G₁ phase of the cell cycle occurred and was associated with restoration of contact dependent behavior and with reversion of histological and ultrastructural features towards those which characterize untransformed cells. Concomitantly, Cl-I cells grown in 2 percent DMSO lost the ability to form colonies in semisolid medium. The data presented suggest that DMSO alters the expression of cellular functions which were altered as a result of viral transformation and which may be involved in cell tumorigenicity.     

Epidermal growth factor induces cell cycle arrest and apoptosis of squamous carcinoma cells through reduction of cell adhesion

Most squamous epithelial cells are strictly anchorage-dependent cell types. We observed that epidermal growth factor (EGF) promoted the growth of A431 squamous carcinoma cells in suspension cultures but suppressed cell growth and induced apoptosis in monolayer cultures, suggesting that loss of adhesion is responsible for the effects observed in monolayer culture, before cell death. Consistent with this finding, we demonstrated that EGF reduced cell attachment, cell-cell interaction, and cell spreading. Treatment with EGF increased cell adhesion-regulated expression of p21 but suppressed expressions of cyclin A, D1, cdk2, and retinoblastoma protein (pRb), leading to cell cycle arrest and adhesion-regulated programmed cell death. To test directly whether promoting cell adhesion could reduce the effects of EGF, we grew cultures on plates coated with type II collagen. On these plates, cell adhesion was enhanced and EGF treatment had little effect on cell adhesion and apoptosis when cells were attached to the collagen. The collagen effects were dose dependent, and cell cycle and cell cycle-associated proteins were altered accordingly. Finally, when cultures were plated on bacterial Petri dishes, which completely disrupted cell attachment to substratum, the level of apoptosis was greatly higher and cell cycle was arrested as compared with monolayer cultures. Taken together, our results strongly suggest that the EGF-induced cell cycle arrest and apoptosis in monolayer cultures was the result of a decline in cell adhesion.     

Nutritional folate deficiency in Chinese hamster ovary cells. I. Characterization of the pleiotropic response and its modulation by nucleic acid precursors

Nutritional folate deficiency in Chinese hamster ovary (CHO)-K1 cells inhibited population growth rate and caused growth arrest within 3 days of culture in Fol- medium [without folate, hypoxanthine (Hx), and thymidine (TdR)]. Coincident with impaired population growth was a transient delay in cell cycle progression through S phase and an increase in cell size. The growth-arrested population of predominantly G1 phase cells exhibited an increased adhesion to the culture substratum. There was a time-dependent loss of cell reproductive capacity. All these various perturbations of cellular phenotype induced by folate deficiency were prevented by the addition of folate or a combination of TdR and Hx to the Fol- medium. However, the singular presence of each nucleotide precursor differentially affected the pleiotropic response. The addition of Hx to Fol- medium exacerbated the aforementioned abnormalities, producing a threefold increase in mean cell volume, a 72 hr accumulation of cells in the S phase of the cell cycle, and a rapid demise in cell clonogenicity. Unexpectedly, we found reduced cell adhesion in these cultures. In contrast, folate-deficient cells supplemented with TdR exhibited a general amelioration of cell perturbations with respect to cell size, cell cycle distribution, and reproductive viability. Notably, such populations were not released from growth inhibition or subsequent growth arrest, and the cells became elongated and highly adherent with time. When cell populations from each of the three conditions of folate-deficient culture were released from growth arrest by addition of complete medium, the respective profiles of synchronous cell cycle progression were distinctive.     

Changes in p21(Cip1) and p27(Kip1) expression are not required for cell cycle entry and progression to S phase in Swiss 3T3 cells

The cyclin-dependent kinase inhibitors, p21(Cip1) and p27(Kip1), play an important role in the regulation of progression through G(1) to S phase in mammalian cells. Here we report that confluent 3T3 cells expressed p21(Cip1) and p27(Kip1) predominantly in the nucleus, and the level of both proteins declined as the cells entered the cell cycle and progressed through G(1) in response to serum growth factors. However, when confluent cells were serum starved prior to treatment, no downregulation of p21(Cip1) or p27(Kip1) expression was observed. Notably, serum starvation did not significantly influence the capacity of the cells to progress to the S phase. It was observed that serum starvation reduced cell density. Further, when cells were plated at a range of different densities, starved of serum to render them quiescent and then subsequently treated with serum, a reduction in p21(Cip1) and p27(Kip1) expression was observed in cells plated at high density but not in those at low density. Again, the extent and timing of progression to S phase was not influenced by cell density. To establish the potential role of cell:cell contact in the observed density-dependent regulation of p21(Cip1) and p27(Kip1) expression, cells were plated onto micorarrays of adhesive islands that prevented individual cells from making any contact with other cells. Under these conditions serum growth factors induced p21(Cip1) and p27(Kip1) downregulation, and hence, there is no requirement for cell:cell contact. Together, these data indicate that there are conditions under which 3T3 cells can progress to the S phase without downregulation of p21(Cip1) and p27(Kip1). The significance of these observations and mechanisms by which density-dependent regulation of p21(Cip1) and p27(Kip1) expression may occur are discussed.     

Growth behavior of Chinese hamster ovary cells in a compact loop bioreactor: 1. Effects of physical and chemical environments.

Chinese hamster ovary (CHO) cells were cultivated in a compact loop bioreactor using MEM-alpha medium supplemented with 10% fetal calf serum. Effects of physical and chemical environments, i.e., pH in the medium, stirring speed of impellers, temperature and partial pressure of oxygen (pO2) upon growth of suspended cells in the bioreactor were determined in batch cultures. Growth behavior was characterized by specific rates of growth (mu), glucose consumption (qG) and lactate production (qL), and the yield coefficients (cell yield from glucose, YX/G, and lactate yield from glucose, YL/G). An effect of medium osmolality was also evaluated with T-flask monolayer cultivation. The best growth was observed at pH 7.6, 37 degrees C, 400 rpm, 50-100% saturation with oxygen and 320 mOsmol kg-1. Corresponding to the previous work with a human melanoma cell line, the sophisticated cultivation and process control systems have been improved for CHO cells.