CHO Cultures Research Articles

Pluronic Enhances the Robustness and Reduces the Cell Attachment of Mammalian Cells

The addition of the non-ionic surfactant, Pluronic F-68, to serum-free CHO cultures causes multi-functional effects that enhance cell yield in agitated cultures and reduce cell adhesion in stationary cultures. Three independent CHO cell lines were subjected to high liquid shear in assay systems that either included or excluded a liquid-gas interface. In the absence of Pluronic, there was a loss in cell viability in either assay system, although there was an intrinsic variability in sensitivity of the cell lines to shear damage. Supplementation with Pluronic prevented loss of cell viability, indicating protection in either a gas sparged or bubble-free environment. However, we found no evidence of long-term protection of cells once Pluronic was removed. Pluronic was capable of repairing trypsin-damaged cells as evidenced by enhanced growth, reduced membrane porosity, and improved robustness under liquid shear. The proportion of adherent cells was reduced to a minimal level by the presence of Pluronic although its effect was rapidly reversible with a high proportion (70%) of adherent cells observed within a few culture passages of its removal. The observed effects of Pluronic on these cultures are compatible with a mechanism in which the polymer forms a protective layer on the cell membrane, which has a significantly lower hydrophobicity.

Process parameter shifting: Part I. Effect of DOT, pH, and temperature on the performance of Epo‐Fc expressing CHO cells cultivated in controlled batch bioreactors

The impact of process environment changes on process performance is one of the most crucial process safety issues when cultivating mammalian cells in a bioreactor. In contrast, directed shifting of process parameters can also be used as an optimization tool providing higher cell and product yields. Compared to other strategies that also aim on the regulation of cell growth and protein expression process parameter shifts can be easily performed without reagent addition or even genetic modification of the host cell line. However, a successful application of changing process conditions implies a profound understanding of the provoked physiological changes within the cells. In a systematic approach we varied the dissolved oxygen tension (DOT), pH, and temperature of CHO cultures in controlled bioreactors and investigated the impact on growth, productivity, metabolism, product quality and cell cycle distribution using a recombinant CHO cell line expressing the highly glycosylated fusion protein Epo-Fc. We found the reduction of cultivation temperature and the reduction of (external) pH to exert the most significant effects on process performance by mainly reducing cell growth and metabolism. With respect to the cell line used we identified a set of parameters capable of affecting cell proliferation in favor of an increased specific productivity and total product yield. The well directed alteration of the process environment has emerged as a tool adequate for further process optimization applying a biphasic cultivation strategy.

Modulation of streptonigrin's clastogenic effects in CHO cells by the metal-chelating agent 1,10-phenanthroline.

The effect of the metal-chelating agent 1,10-phenanthroline (PNT) on the clastogenesis induced by streptonigrin (SN) in CHO cells was investigated. When CHO cells were exposed to SN, chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) were formed in a dose-dependent manner (P < 0.05). When PNT was present in the culture medium, the production of CAs by SN was strongly inhibited (inhibition range = 54.9-80.8%). Similarly, the induction of SCEs by SN was significantly decreased by the addition of PNT to CHO cultures (P < 0.05), although the effect was minor. This finding suggests that intracellular transition metals are implicated in the clastogenesis by SN, and that the Fenton reaction (Fe(2+) + H2O2 --> OH* + OH(-) + Fe(3+)) may be responsible for the production of CAs by this compound. Moreover, the fact that PNT did not completely inhibit the induction of SCEs by SN suggests that this phenomenon might be attributable to a different mechanism, in which transition metals and free radicals play a minor role.

Metabolism of peptide amino acids by Chinese hamster ovary cells grown in a complex medium

Metabolic flux analysis is a useful tool for unraveling relationships between metabolism and cell function. Material balancing can be used to provide estimates of major metabolic pathway fluxes, provided all significant metabolite uptake and production rates are measured. Potential sources of metabolizable material in many serum-free media formulations are low molecular weight digests of biological material such as yeast extracts and plant or animal tissue hydrolysates. These digests typically contain large amounts of peptides, which may be utilized as amino acids. This article demonstrates the need for accounting for amino acids liberated from peptides in order to accurately estimate pathway fluxes in Chinese hamster ovary cells grown in a complex (hydrolysate containing) medium. A simplified model of central carbon metabolism provides the framework for analyzing external metabolite measurements. Redundant measurements are included to ensure the consistency of data and assumed biochemistry by comparing redundant measurements with their predicted values from a minimum data set, and by expressing the degree of agreement using a statistical "consistency index." The consistency index tests whether redundancies are satisfied within expected experimental error. For chemostat steady states of CHO cultures grown in a hydrolysate-supplemented medium, consistent data were obtained only when amino acids liberated from peptides were taken into account.

Metabolism of peptide amino acids by Chinese hamster ovary cells grown in a complex medium

Metabolic flux analysis is a useful tool for unraveling relationships between metabolism and cell function. Material balancing can be used to provide estimates of major metabolic pathway fluxes, provided all significant metabolite uptake and production rates are measured. Potential sources of metabolizable material in many serum-free media formulations are low molecular weight digests of biological material such as yeast extracts and plant or animal tissue hydrolysates. These digests typically contain large amounts of peptides, which may be utilized as amino acids. This article demonstrates the need for accounting for amino acids liberated from peptides in order to accurately estimate pathway fluxes in Chinese hamster ovary cells grown in a complex (hydrolysate containing) medium. A simplified model of central carbon metabolism provides the framework for analyzing external metabolite measurements. Redundant measurements are included to ensure the consistency of data and assumed biochemistry by comparing redundant measurements with their predicted values from a minimum data set, and by expressing the degree of agreement using a statistical “consistency index.” The consistency index tests whether redundancies are satisfied within expected experimental error. For chemostat steady states of CHO cultures grown in a hydrolysate-supplemented medium, consistent data were obtained only when amino acids liberated from peptides were taken into account. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 324–335, 1999.

Latent transforming growth factor-beta binding protein domains involved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) is secreted by many cell types as part of a large latent complex composed of three subunits: TGF-beta, the TGF-beta propeptide, and the latent TGF-beta binding protein (LTBP). To interact with its cell surface receptors, TGF-beta must be released from the latent complex by disrupting noncovalent interactions between mature TGF-beta and its propeptide. Previously, we identified LTBP-1 and transglutaminase, a cross-linking enzyme, as reactants involved in the formation of TGF-beta. In this study, we demonstrate that LTBP-1 and large latent complex are substrates for transglutaminase. Furthermore, we show that the covalent association between LTBP-1 and the extracellular matrix is transglutaminase dependent, as little LTBP-1 is recovered from matrix digests prepared from cultures treated with transglutaminase inhibitors. Three polyclonal antisera to glutathione S-transferase fusion proteins containing amino, middle, or carboxyl regions of LTBP-1S were used to identify domains of LTBP-1 involved in cross-linking and formation of TGF-beta by transglutaminase. Antibodies to the amino and carboxyl regions of LTBP-1S abrogate TGF-beta generation by vascular cell cocultures or macrophages. However, only antibodies to the amino-terminal region of LTBP-1 block transglutaminase-dependent cross-linking of large latent complex or LTBP-1. To further identify transglutaminase-reactive domains within the amino-terminal region of LTBP-1S, mutants of LTBP-1S with deletions of either the amino-terminal 293 (deltaN293) or 441 (deltaN441) amino acids were expressed transiently in CHO cells. Analysis of the LTBP-1S content in matrices of transfected CHO cultures revealed that deltaN293 LTBP-1S was matrix associated via a transglutaminase-dependent reaction, whereas deltaN441 LTBP-1S was not. This suggests that residues 294-441 are critical to the transglutaminase reactivity of LTBP-1S.

Induction of structural chromosome aberrations in human lymphocyte cultures and CHO cells by permethrin

The pyrethroid insecticide permethrin was tested for its ability to induce structural chromosome aberrations (CA) in human lymphocyte cultures and CHO cells, in order to confirm the clastogenic effect of itself and to compare the response of the two different cell types. Permethrin was tested in the range of 50-200 micrograms/ml in human lymphocyte cultures and in the range of 20-100 micrograms/ml in CHO cells. In both lymphocyte and CHO cultures, assays were performed in the absence and in the presence of a rat liver activation system (S9 mix). In the absence of S9 mix, two experiments with different duration of the treatment were carried out. Permethrin induced CA in both cultures when it was evaluated in the absence of a metabolic activation system. The activity of a given concentration of permethrin seemed to be decreased more by the reduction of the time of exposure than by the presence of S9 mix. Aberrations induced by permethrin were mainly chromosome-type aberrations in both cultures. Thus, permethrin can be characterised as an S-phase independent clastogenic agent. The response of both lymphocyte and CHO cultures was similar, indicating that both systems showed the same sensitivity for detecting the clastogenicity in vitro of permethrin.

Origin and evolution of binucleated cells and binucleated cells with micronuclei in cisplatin-treated CHO cultures

It has recently been described that cisplatin is an agent able to induce binucleated cells (BC) in cultured CHO cells. Both the origin and the significance of those cells within a population are unknown although several hypothesis have been suggested such as blocking of cytokinesis or cell fusion. Using interval photography we have found that at least two mechanisms are involved in the production of BC. These cells can arise in a culture as a result of an incomplete process of cell division, i.e. karyokinesis with incomplete cytokinesis or as a result of the mitotic division of a pre-existent BC. The mitotic division of a BC can give rise to different types of daughter cells. These BC sometimes enter mitosis but fail to divide and as a consequence they remain BC. When the process of division is successful (in the vast majority of cases), the results that have been found are either two mononucleated cells or one mononucleated and one binucleated cell. The possible implications and significance of BC and BC with micronuclei in a given population are discussed.

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories, IV. Chromosome aberration and sister-chromatid exchange in Chinese hamster ovary, V79 Chinese hamster lung and human lymphocyte cultures

A recommended protocol has been developed for chromosomal aberration and sister-chromatid exchange assays in CHO, V79 and human lymphocyte cultures. The protocol was based on the responses to a detailed questionnaire completed by North-American and European governmental, university, and contract laboratories using these tests. This report identifies those modifications to previously described methods that are used on a regular basis and clarifies confusing or inconsistent practices. These protocols can be modified for use in other types of cells.

Correlated Measurements of DNA, RNA, and Protein in Individual Cells by Flow Cytometry

A cytochemical method was developed to differentially stain cellular DNA, RNA, and proteins with fluorochromes Hoechst 33342, pyronin Y, and fluorescein isothiocyanate, respectively. The fluorescence intensities, reflecting the DNA, RNA, and protein content of individual cells, were measured in a flow cytometer after sequential excitation by three lasers tuned to different excitation wavelengths. The method offers rapid analysis of changes in the cellular content of RNA and protein as well as in the RNA-protein, RNA-DNA, and protein-DNA ratios in relation to cell cycle position for large cell populations. An analysis of cycling cell populations (exponentially growing CHO cultures) and noncycling CHO cells arrested in the G1 phase by growth in isoleucine-free medium demonstrated the potential of the technique.

DNA fork displacement rates in synchronous aneuploid and diploid mammalian cells

DNA fork displacement rates were measured in Chinese hamster ovary cells (CHO), human HeLa cells and human diploid fibroblasts. For CHO cells two independent techniques were used: one based on CsCl equilibrium density gradients and the other on 313 nm photolysis of incorporated bromodeoxyuridine (BrdUrd). Both methods indicated that there was no significant variation in fork displacement rates in CHO cells as they progressed through S phase. Asynchronous CHO cultures displayed the same average value (1.0 μm/min) and range of values as found in synchronous cells. In contrast, the rate of DNA fork displacement in HeLa cells, measured by the BrdUrd-313 nm method, increased continuously from 0.8 μm/min in early S to 2.5 μm/min in late S. For human diploid fibroblasts, in early S, the rate was approximately 0.7 μm/min and decreased to a minimum of 0.5 μm/min in mid S. The replication fork displacement rate then increased to a maximum of 0.9 μm/min in late S and declined again before the end of S phase. This pattern of DNA fork displacement rates roughly paralleled the overall thymidine incorporation rate and appears quite different from the patterns found for HeLa and CHO cells.

Alteration in morphology of Chinese hamster ovary cells by Ni 3S 2 and dibutyryl cAMP

Rounded Chinese hamster ovary cells (CHO) (axial ratio, 1:1) elongated (axial ratio, >;1:3) when exposed to Ni 3S 2. Other metal compounds which were tested had only minor effects on CHO morphology over a wide range of concentrations. These cells were elongated following exposure to Ni 3S 2 at concentrations which did not affect cell proliferation as judged by cell doubling and colony formation. The elongation occurred when agents that elevate intracellular cAMP concentrations were added to cultures. The cells treated with either dibutyryl cAMP or Ni 3S 2 had very similar fibroblastic morphology. Upon removal of media containing dibutyryl cAMP, elongated CHO cells reverted to control morphology within 4 hr. In contrast, the cultures previously treated with Ni 3S 2 did not rapidly revert to normal morphology. Nickel subsulfide activated cAMP-dependent protein kinase within 3 hr after its addition to cultures while NiS or NiCl 2 did not significantly activate protein kinase. Dibutyryl cAMP addition to CHO cultures completely activated this enzyme. High doses of actinomycin D, or cycloheximide did not affect the dibutyryl cAMP-induced elongation of CHO cells while addition of colcemid did. All three of these agents inhibited the Ni 3S 2 elongation of CHO cells. These data demonstrate that Ni 3S 2 induced morphological changes in CHO cells are similar to the morphological changes mediated by cAMP.

Cytoplasmic and nuclear protein kinases during the cell cycle

Nuclear and cytoplasmic protein kinases were measured during the traverse of synchronous CHO cultures through G 1 into S phase. Cells were synchronized by selective detachment of cells blocked in metaphase using colcemid. Nuclei were isolated and the protein kinases extracted from the nuclear preparation with 0.6 M NaCl. This procedure solubilized greater than 90% of the total protein kinase activity present in the nuclear preparation. DEAE chromatography of this extract showed 5 apparently different ionic forms of nuclear protein kinases. The nuclear protein kinases preferred casein and phosvitin to histone as substrates and were cyclic AMP-independent. Nuclear protein kinase activities increased greater than two-fold, when expressed as units of activity per cell nucleus, during G 1 phase traverse, concomitant with a 70% increase in nuclear non-histone proteins (those soluble in 0.6 M NaCl). This resulted in only a 40% increase in the specific activities (units/μg protein in 0.6 M NaCl extractable nuclear fraction) of these enzymes as cells progressed through G 1 into S phase. This was in contrast to cytoplasmic cyclic AMP-dependent protein kinase activities which also increased two-fold during progression through G 1 phase while total cellular protein increased less than 20%. Activation of, as well as synthesis of, cyclic AMP-dependent cytoplasmic protein kinases during G 1 phase suggests a regulatory mechanism for precise temporal phosphorylation, whereas the constant specific activity in nuclear kinases during cell cycle is more compatible with the maintenance of bulk phosphorylation processes in the nucleus.

DNA Replication Kinetics in X-Irradiated Chinese Hamster Ovary Cells

The kinetics of semiconservative DNA replication have been studied in both asynchronous and synchronized Chinese hamster ovary cells (CHO) irradiated with x-ray doses up to 3000 rad. Amounts of DNA replicated were determined by isopycnic gradient centrifugation of DNA from cells which were incubated after irradiation in medium containing 5-bromodeoxyuridine (50 ..mu..g/ml) and 5-fluorodeoxyuridine (0.1 ..mu..g/ml). The results confirm that cells irradiated in early G/sub 1/ phase experience a delay in their entry into S phase. This G/sub 1/ block is dose independent in the range from 300 to 3000 rad and is 0.5 to 0.7 hr in length. Cells at the G/sub 1//S boundary are insensitive to x-ray induced perturbations of bulk DNA synthetic rates when exposed to doses less than 1000 rad. At doses in excess of 1000 rad, these cells are inhibited from replicating their DNA for a time, but ultimately replicate near-control levels of their DNA. Cells irradiated in S phase again show no effects of x-ray doses below 1000 rad on their ability to replicate bulk DNA. After a 3000-rad exposure, however, the rate of DNA replication in these S-phase cells is markedly reduced compared to that of controls. Irradiation of asynchronous cells with dosesmore » from 150 to 3000 rad does reduce the rate of semiconservative DNA replication in these cultures in a dose-dependent manner. These results confirm that x-ray doses greater than 1000 rad reduce the rate of DNA synthesis in irradiated S-phase cells, thus prolonging the length of S phase. The combined data from asynchronous or synchronized cultures, irradiated with x-ray doses less than 1000 rad, indicate that at least a portion of the reduction in DNA replication rates in irradiated asynchronous CHO cultures is due to the x-ray induced G/sub 1/ block, which reduces the overall number of cells in S phase after irradiation.« less

S phase synchrony in monolayer CHO cultures

Parameters are described for reproducible S phase synchrony of Chinese hamster ovary cells growing in monolayer, adapting a method described by Tobey & Crissman [1] for CHO cells growing in suspension culture. Cells are collected at the G1/S boundary in hydroxyurea after reversal of an early G1 block induced by isoleucine deprivation. The entire population enters the S period within 60 min after removal of hydroxyurea and proceeds through the S period with minimal decay of synchrony, as evidenced by autoradiographic and rate studies on [ 3H]TdR uptake. In addition, a method is described for obtaining cells synchronized during two successive S periods. The presence of hydroxyurea during G1 does not measurably affect the rate of uptake of [ 3H]uridine or [ 3H]leucine into TCA-insoluble material; however, cultures released from the hydroxyurea block at 10 h incorporate slightly more [ 3H]uridine (but not [ 3H]leucine) in the next 6 h than cultures maintained in hydroxyurea over this interval. Delaying entry into S with hydroxyurea for as long as 15 h does not significantly change the initial rate or duration of DNA synthesis upon removal of hydroxyurea, arguing against the build-up of substances responsible for initiation of replicons. Furthermore, if DNA synthesis is delayed with hydroxyurea in one cell cycle, a constant minimal interval of 15 h elapses before the population enters into the next S phase, suggesting that the timing of the S period is coupled to the timing of the previous S.