Changes in bleb formation following hyperthermia treatment of Chinese hamster ovary cells

Chinese hamster ovary cells in suspension cultures were heated for various times at 41.5, 43.5, and 45.5 degrees C, and quantitative determinations of microblebbing and macroblebbing of the cell membrane were performed for cells maintained at 4, 25, and 37 degrees C after hyperthermia. The percentage of cells with blebs following heating at 45.5 degrees C was dependent upon the duration of heating with increases from 40% for 5 min to 90% for 30 min. Cells exposed to lower temperatures exhibited less blebbing which was not quantifiable. The changes in bleb formation following 45.5 degrees C were dependent upon the posthyperthermia temperature: a slight decrease of macroblebbing at 25 degrees C, a decrease to 50% by 2 h at 37 degrees C, and a sharp decrease of macroblebbing to less than 10% by 1 h at 4 degrees C. Microblebbing increased slightly at 37 degrees C. When cells were transferred rapidly from the 4 degrees C posthyperthermia incubation to 37 degrees C, the bleb formation percentages returned rapidly to the higher levels which existed before posthyperthermia incubation at the lower temperatures. Gamma irradiation of 20 and 50 Gy produced only a small increase in microblebbing at longer periods (5 to 6 h) but no increase in macroblebbing. The survival of cells heated for 20 min at 45.5 degrees C was decreased 40% for suspension cells maintained at 4 degrees C for 2 to 3 h before incubation at 37 degrees C for colony formation compared to cells immediately incubated at 37 degrees C after heating. The survival of cells maintained at 25 degrees C after heating was not altered in comparison.     

The chronic effects of db-cAMP on cell cycle parameters and cell size in asynchronous culture of Chinese hamster ovary cells

N⁶,O^2′-dibutyryl adenosine 3′,5′-cyclic-phosphate (db-cAMP) has been shown to convert Chinese hamster cells of ovarian origin (CHO-K₁) from compact, randomly oriented cells growing in multilayers to elongated fibroblast-like cells which grow in monolayers. This compound also has been reported to have a variety of effects on the cell cycle. Most such studies have employed synchronized cells to determine cell cycle effects, and consequently have been limited to the short-term effects of the compound. We have looked for chronic effects on the cell cycle in cultures exposed continuously to db-cAMP from the initiation of the cultures until they had reached or approached the plateau phase. This was done by combined autoradiography and Feulgen microspectrophotometry plus measurements of the protein content of mitotic cells to detect any influence on cell size. The overall results were that continuous exposure to db-cAMP had at most only minor effects on the cell cycle and cell size when the culture medium was renewed daily. Somewhat greater effects were found on plateau-phase cells in cultures in which the medium was not renewed. In this case fewer cells appeared to remain in the cell cycle in the cultures with db-cAMP. Comparison with our earlier results with Chinese hamster V79 cells led to the conclusions that cell cycle parameters and cell size at mitosis were less altered during culture growth in CHO cells, but that CHO cells seemed to be less able to maintain cells in the cell cycle in crowded cultures.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

Chromosome-mediated gene transfer with the Chinese hamster ovary cell line

Using an improved method of chromosome-mediated gene transfer, we have investigated transfer of the codominantly expressed methotrexate-resistant dihydrofolate reductase (MtxRIIIdhfr) gene into Chinese hamster ovary (CHO) cell recipients. The frequency of dhfr gene transfer with CHO cells varied considerably from clone to clone, ranging from 4 X 10(-7) to 5 X 10(-5). Using appropriate cell recipients we were able to test for linkage of several genetic markers available in the CHO cell line. For example, the mutation resulting in the auxotrophic glyB-CHO cell line has been reported by others to be linked to the dhfr gene. However, we could not demonstrate cotransfer of these two markers when glyB- recipient cells were treated with MtxRIII chromosomes and transformant clones were selected for either methotrexate-resistance (MtxR) or glycine prototrophy. We conclude that these two genes are not closely linked in the hamster genome. However, the genes for thymidine kinase (tk) and galactokinase (gk), which are known to be linked in mammalian genomes, were found to cotransfer into CHO recipients with a frequency of about 50%.     

The survival of cytochalasin-induced multinucleation following irradiation of Chinese hamster ovary cells

Chinese hamster ovary cells were cultured for up to 280 hr in medium containing 1.75 mcg/ml cytochalasin B. The distribution of the number of nuclei per cell in unirradiated cultures on the 6th day was unimodal with some cells containing 27 or more nuclei. The DNA content distribution was in contrast polymodal with the means of the two terminal major peaks occurring at approximately 40 and 80 units of DNA content (antimodes at 29 and 58 units), where 1 unit is the content of untreated G1 cells. Irradiation (gamma, 137-Cs) at doses up to 10 Gy caused an exponential reduction in the proportion of plated cells able to reach high nucleus- or DNA-contents. The reduction due to 5 Gy was stable at least up to 280 hr in culture. The accumulation of total DNA in the culture was well-fitted by a Gompertz function, with little further increase after 230 hr when the average DNA content per cell reached about 90 units.     

A correlation between membrane glycopeptide composition and losses in concanavalin a agglutinability induced by db-cAMP in Chinese hamster ovary cells

A double-label method, employing [¹⁴C] - and [³H] -fucose, has been used to compare the carbohydrate components of surface glycoproteins from four different sub-clones of Chinese hamster ovary cells grown in the presence or absence of either 3′: 5′-dibutyryl cyclic AMP (db-cAMP), 3′: 5′-cyclic AMP (cAMP) or a phosphodiesterase inhibitor SQ 20009. Following growth in one or more of these drugs, a number of these sub-clones showed a fairly small, but consistent reduction in the amount of the more rapidly eluting fucopeptides that could be isolated from the plasma membrane and a corresponding increase in lower molecular weight components as determined by Sephadex G-50 chromatography. This apparent decrease in the size of surface fucopeptides was related to a reduced sialic acid content of a class of surface glycopeptides isolated from the treated cells. This surface change was always correlated with a loss of concanavalin A (ConA)-mediated agglutinability. However, this surface change was not invariably associated with the drug-induced morphological transition towards a more fibroblast-like form. More-over, the sialic acid-rich glycopeptides bound only poorly to ConA affinity columns and were probably not therefore the lectin receptors. Double-label experiments have shown that upon addition of db-cAMP to the cells, existing glycopeptides are apparently unmodified but rather new components reaching the cell surface have a reduced amount of sialic acid associated with them. We propose that the loss in lectin-induced agglutinability and the reduction in glycopeptide size are related phenomena resulting from a primary change in cell surface chemistry.     

Characterization of a Chinese hamster ovary cell line resistant to uncouplers

The chemiosmotic theory of oxidative phosphorylation and the action of uncouplers was examined by characterizing a clone, UH5, of Chinese hamster ovary (CHO TK-) cells resistant to 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S-13), a potent uncoupler of oxidative phosphorylation. About 9-times and 4-times more S-13 was required to effect growth and respiration respectively of UH5 cells compared to the parental CHO TK- cells. UH5 cells were cross-resistant to the uncouplers SF-6847 (3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile), carbonylcyanide p-trifluoromethoxyphenylhydrazone and 2,4-dinitrophenol but not to oligomycin, venturicidin or Tevenel. Size, chromosome number and DNA content indicated that the UH5 cell line was probably pseudotetraploid compared to the parental pseudodiploid CHO TK- cells. Hybrid and cybrid cells formed from crosses of UH5 cells and cytoplasts, respectively, with an uncoupler-sensitive cell line were sensitive to S-13 indicating that resistance is probably nuclear-determined. UH5 cell mitochondria had increased cytochrome oxidase and decreased H+-ATPase activities. A fivefold resistance of oxidative phosphorylation to uncouplers was found at the mitochondrial level with respiration driven by either succinate or ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine. In contrast, no difference in sensitivity was found to valinomycin between mitochondria from UH5 and CHO TK- cells. The oligomycin-sensitive H+-ATPase activity of UH5 and CHO TK- cell mitochondria was equally stimulated by the uncoupler S-13. Uncoupler-resistant mitochondria would not be expected on the basis of the chemiosmotic theory, and the relation of the results to other modes of coupling is considered.     

Transformation of UV-hypersensitive Chinese hamster ovary cell mutants with UV-irradiated plasmids

Transfection of UV-hypersensitive, DNA repair-deficient Chinese hamster ovary (CHO) cell lines and parental, repair-proficient CHO cells with UV-irradiated pHaprt-1 or pSV2gpt plasmids resulted in different responses by recipient cell lines to UV damage in transfected DNA. Unlike results that have been reported for human cells, UV irradiation of transfecting DNA did not stimulate the genetic transformation of CHO recipient cells. In repair-deficient CHO cells, proportionally fewer transformants were produced with increasing UV damage than in repair-proficient cells in transfections with the UV-irradiated hamster adenine phosphoribosyltransferase (APRT) gene contained in plasmid pHaprt-1. However, transfection of CHO cells with UV-irradiated pSV2gpt resulted in neither decline in transformation frequencies in repair-deficient cell lines relative to repair-proficient cells nor stimulation of genetic transformation by UV damage in the plasmid. Blot hybridization analysis of DNA samples isolated from transformed cells showed no dramatic changes in copy number or arrangement of transfected plasmid DNA with increasing UV dose. We conclude that the responses of recipient cells to UV-damaged transfecting plasmids depend both on the type of recipient cell and the characteristics of the genetic sequence used for transfection.     

Improved product formation in high density Chinese hamster ovary cell cultures transfected at confluency

Cell-specific productivity was compared in Chinese hamster ovary cell clones transfected at confluency versus subconfluency. Following lipofection of a pCDNA1-derived vector encoding a proteinA-fucosyltransferaseIV fusion protein, selection and single cell sorting by flow cytometry, clones were expanded to tissue flasks and assessed for cell specific productivity. The probability of obtaining high producers at confluency was significantly higher from cells transfected at confluency than at subconfluency (P=0.05).     

Acetate accumulation and regulation by process parameters control in Chinese hamster ovary cell culture

Chinese hamster ovary (CHO) cells represent a group of predominantly used mammalian hosts for producing recombinant therapeutic proteins. Known for their rapid proliferation rates, CHO cells undergo aerobic glycolysis that is characterized by fast glucose consumption, that ultimately gives rise to a group of small-molecule organic acids. However, only the function of lactate has been extensively studied in CHO cell culture. In this study, we observed the accumulation of acetate from the late exponential phase to harvest day, potentially contributing to the pH decline in late culture stage regardless of lactate consumption. In addition, we evaluated the acidification of the fresh media and the cell culture suspension, and the data revealed that acetate presented a lower acidification capacity compared to lactate and exhibited limited inhibitory effect on cells with less than 20 mM supplemented in the media. This study also explored the ways to control acetate accumulation in CHO cell culture by manipulating the process parameters such as temperature, glucose, and pH control. The positive correlation between the specific glucose consumption rate and acetate generation rate provides evidence of the endogenous acetate generation from overflow metabolism. Reducing these parameters (temperature, glucose consumption) and HCl-controlled low pH ultimately suppress acetate build-up. In addition, the specific acetate generation rate and relevant glucose consumption rate are found to be a metabolic trait associated with specific cell lines. Taken together, the results presented in these experiments provide a means to advance industrial CHO cell culture process control and development.     

鼠抗人纤维蛋白单链抗体-尿激酶杂合基因的构建及其在中国仓鼠卵巢细胞中的表达

采用 DNA重组技术构建了表达鼠抗人纤维蛋白单链抗体与低分子量尿激酶融合基因的真核表达载体。通过磷酸钙共沉淀法 ,将该表达载体转染到中国仓鼠卵巢细胞二氢叶酸还原酶基因缺陷株 ( CHO- dhfr-)中 ,利用选择培养基筛选出稳定表达的细胞株 ,溶解圈法测定融合蛋白的表达水平为每 1 0 6细胞每天 5 8IU。该融合蛋白保留了与纤维蛋白的结合活性和溶解纤维蛋白的溶纤活性。SDS- PAGE,Western印迹法分析证明融合蛋白的相对分子质量约为 70× 1 0 3     

Inositol metabolism and cell growth in a Chinese hamster ovary cell myo-inositol auxotroph

The intracellular concentrations of polyphosphoinositides and inositol phosphates were determined, and their role in growth factor-initiated cell division was investigated in a Chinese hamster ovary cell inositol auxotroph (CHO-K1-Ins). Metabolic labeling experiments during inositol starvation of CHO-K1-Ins cells showed that 1) the lipid-linked inositol component was maintained at the expense of the soluble inositol pool, 2) the decreasing cellular content of phosphatidylinositol was replaced by phosphatidylglycerol, and 3) the concentrations of inositol polyphosphates and polyphosphoinositides were conserved at the expense of inositol and phosphatidylinositol. These data show that homeostatic mechanisms exist for the maintenance of the polyphosphoinositide and inositol phosphate pools at the expense of inositol and phosphatidylinositol. The addition of alpha-thrombin to growth-arrested (serum-starved) CHO-K1-Ins cells stimulated the incorporation of [3H]thymidine into DNA to the same extent as that observed following serum readdition. gamma-Thrombin was also an effective mitogen, but active site-inhibited alpha-thrombin was not. Both alpha- and gamma-thrombin, but not catalytic site-inhibited alpha-thrombin, initiated phosphatidylinositol turnover in vivo and increased phosphatidylinositol 4,5-bisphosphate phospholipase C activity in vitro. Serum and insulin were potent CHO-K1-Ins cell mitogens, but neither triggered phosphatidylinositol turnover in vivo nor activated phospholipase C in vitro. The activation of phospholipase C plays a determinant role in thrombin-initiated cell cycle progression in Chinese hamster ovary cells, although other growth factor-signaling pathways exist that are independent of polyphosphoinositide catabolism.     

Decoupling cell growth and product formation in Chinese hamster ovary cells through metabolic control.

The development of a strategy for the culture of Chinese hamster ovary (CHO) cells producing tissue plasminogen activator (t-PA) is investigated. This strategy is based on the replacement of the main carbon source, glucose, by another compound that is slowly metabolizable, particularly galactose. The introduction of this change allows for acute change in cell behavior at various levels. Cell growth is stopped after this nutrient shift, and the cells can be kept in long-duration culture at a low growth rate and high viability as compared with a culture strategy based solely on glucose utilization. Moreover, the capability of cells to produce recombinant proteins (t-PA in this work) can be maintained over the entire period of galactose feeding. From the metabolic point of view, use of a slowly metabolizable carbon source (galactose) introduces important changes in the production of lactate, ammonia, and some amino acids. The use of this metabolic shift enables the generation of biphasic processes, with a first phase with cell growth on glucose and a second stationary phase on galactose, which is particularly suited to perfusion systems.     

Genetic analysis of mitomycin-C-sensitive mutants of a Chinese hamster ovary cell line

5 mutants of a Chinese hamster ovary (CHO) cell line, which exhibit similar levels of sensitivity to killing by mitomycin C, have been analysed genetically to determine whether they represent one or more genetic complementation groups. Hybrids were constructed by fusing cells carrying either the neo or the Ecogpt marker and selecting in medium containing G418 and mycophenolic acid. Selectable markers were introduced into the cells by DNA transfection using pSV5-neo or pSV5-gpt, which represents a quick and convenient method for generating resistant derivatives. Hybrids generated by crosses between any one mutant and the parental cell line exhibited near wild-type resistance to mitomycin C, indicating that the mutants are phenotypically recessive. Self-cross hybrids for all 5 mutants had D37 values for killing by mitomycin C of between 20 and 30 ng/ml. The values obtained for crosses between different mutants were 60-105 ng/ml, with the exception of 1 pairing which gave a value of 33 ng/ml. These results indicate that that the mutants represent at least 4 different genetic complementation groups, suggesting that cellular resistance to mitomycin C is mediated via a number of different mechanisms.     

Comparative metabolite analysis to understand lactate metabolism shift in Chinese hamster ovary cell culture process

A metabolic shift from lactate production (LP) to net lactate consumption (LC) phenotype was observed in certain Chinese hamster ovary (CHO) cell lines during the implementation of a new chemically defined medium (CDM) formulation for antibody production. In addition, this metabolic shift typically leads to process performance improvements in cell growth, productivity, process robustness, and scalability. In our previous studies, a correlation between a key media component, copper, and this lactate metabolism shift was observed. To further investigate this phenomenon, two complementary studies were conducted. In the first study, a single cell line was cultivated in two media that only differed in their copper concentrations, yet were known to generate an LP or LC phenotype with that cell line. In the second study, two different cell lines, which were known to possess inherently different lactate metabolic characteristics, were cultivated in the same medium with a high level of copper; one cell line produced lactate throughout the duration of the culture, and the other consumed lactate after an initial period of LP. Cell pellet and supernatant samples from both studies were collected at regular time intervals, and their metabolite profiles were investigated. The primary finding from the metabolic analysis was that the cells in LP conditions exhibited a less efficient energy metabolism, with glucose primarily being converted into pyruvate, sorbitol, lactate, and other glycolytic intermediates. This decrease in energy efficiency may be due to an inability of pyruvate and acetyl-CoA to progress into the TCA cycle. The lack of progression into the TCA cycle or overflow metabolism in the LP phenotype resulted in the inadequate supply of ATP for the cells. As a consequence, the glycolysis pathway remained the major source of ATP, which in turn, resulted in continuous LP throughout the culture. In addition, the accumulation of free fatty acids was observed; this was thought to be a result of phospholipid catabolism that was being used to supplement the energy produced through glycolysis in order to meet the needs of LP cells. A thorough review of the metabolic profiles indicated that the lactate metabolic shift could be related to the oxidative metabolic capacity of cells.     

Conserved microRNA function as a basis for Chinese hamster ovary cell engineering

The use of microRNAs (miRNAs) for improving the efficiency of recombinant protein production by CHO cells is gaining considerable interest for their ability to regulate entire molecular networks. Differential miRNA expression profiling and large-scale transient screening have been the prerequisite for the selection of miRNA candidates for stable manipulation, reported in CHO cells expressing a range of recombinant products. We selected a potent and well characterised tumour suppressor miRNA, miR-34a, as a high priority candidate for CHO cell engineering based on the conservation of both its sequence and function across species and cell type. Ectopic expression of miR-34a retained its functional conservation in CHO-SEAP cells by inhibiting growth by 90 % in addition to decreasing the viable cell population by 30 % when compared to controls. When the miR-34 family was stably depleted using a miRNA sponge decoy vector, the overall product yield was enhanced by ~2-fold in both fed-batch and small scale clonal batch cultures, despite having a negative impact on cell growth. These findings further strengthen the utility of miRNAs as engineering tools to modify and improve CHO cell performance.     

Diversity in host clone performance within a Chinese hamster ovary cell line

Much effort has been expended to improve the capabilities of individual Chinese hamster ovary (CHO) host cell lines to synthesize recombinant therapeutic proteins (rPs). However, given the increasing variety in rP molecular types and formats it may be advantageous to employ a toolbox of CHO host cell lines in biomanufacturing. Such a toolbox would contain a panel of hosts with specific capabilities to synthesize certain molecular types at high volumetric concentrations and with the correct product quality (PQ). In this work, we examine a panel of clonally derived host cell lines isolated from CHOK1SV for the ability to manufacture two model proteins, an IgG4 monoclonal antibody (Mab) and an Fc‐fusion protein (etanercept). We show that these host cell lines vary in their relative ability to synthesize these proteins in transient and stable pool production format. Furthermore, we examined the PQ attributes of the stable pool‐produced Mab and etanercept (by N‐glycan ultra performance liquid chromatography (UPLC) and liquid chromatography ‐ tandem mass spectrometry (LC‐MS/MS), respectively), and uncovered substantial variation between the host cell lines in Mab N‐glycan micro‐heterogeneity and etanercept N and O‐linked macro‐heterogeneity. To further investigate the capabilities of these hosts to act as cell factories, we examined the glycosylation pathway gene expression profiles as well as the levels of endoplasmic reticulum (ER) and mitochondria in the untransfected hosts. We uncovered a moderate correlation between ER mass and the volumetric product concentration in transient and stable pool Mab production. This work demonstrates the utility of leveraging diversity within the CHOK1SV pool to identify new host cell lines with different performance characteristics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1187–1200, 2015