Monitoring of autophagy in Chinese hamster ovary cells using flow cytometry

Recently, autophagy, which is a degradative process, has drawn attention as an anti-cell death engineering target in addition to apoptosis in recombinant Chinese hamster ovary (rCHO) cell cultures for enhanced production of therapeutic proteins. Appropriate autophagy monitoring methods, that are suitable for long term CHO cell cultures, are necessary in order to investigate the culture conditions that affect the autophagy pathway and to select appropriate engineering targets for autophagy control. Herein, detailed protocols for autophagy monitoring methods based on flow cytometry are provided using the GFP-LC3-overexpressing CHO DG44 host cell line or MDC-like molecules in rCHO cells grown as an adherent culture with serum-containing medium or suspension culture with serum-free medium. Furthermore, combined with the apoptosis detection based on the Annexin V-PS interaction, the simultaneous detection of autophagy and apoptosis is also described. It is anticipated that the protocols described herein will assist in the fast, high throughput monitoring of autophagy that can support other existing autophagy assays.     

Effect of sodium butyrate on autophagy and apoptosis in Chinese hamster ovary cells

Sodium butyrate (NaBu), which is widely used in recombinant Chinese hamster ovary cell (rCHO) cultures for high-level expression of therapeutic proteins, is known to induce apoptosis in a dose-dependent manner. Lately, the significance of autophagy has increased in the field of CHO cell culture due to the fact that autophagy is related to the programmed cell death mechanism. To determine the effect of NaBu on autophagy as well as apoptosis of rCHO cells, rCHO cells producing erythropoietin were subjected to NaBu treatment. NaBu treatment up to 5 mM increased cleaved forms of PARP, caspase-3, and Annexin V positive population, confirming the previous results that NaBu induces apoptosis. Concurrently, NaBu treatment increased the level of accumulation of the autophagic marker, LC3-II, independently of nutrient depletion, suggesting that NaBu induces autophagy. To elucidate the potential role of autophagy induced by NaBu, a representative autophagy inducer (rapamycin) or an inhibitor (bafilomycin A1) was added to cultures together with NaBu. It was found that autophagy had the potential role of a positive cell survival mechanism under NaBu treatment. Furthermore, gradual reduction in mitochondrial membrane potential/mass and recruitment of a mitophagy protein, Parkin, to the mitochondria were observed under NaBu treatment, suggesting that this positive function of autophagy might be mediated by the autophagic removal of damaged mitochondria. Taken together, autophagy was observed in rCHO cell culture under NaBu treatments and the results obtained here support the positive effects of autophagy induced by NaBu treatments.     

Autophagy and apoptosis in Chinese hamster ovary cell culture

Upon nutrient deprivation during Chinese hamster ovary (CHO) cell culture for foreign protein production, cells are subjected to two types of programmed cell death (PCD), apoptosis and autophagy. However, only apoptosis has drawn attention in the field of CHO cell culture. Numerous studies on engineering genes or supplementing essential nutrients or chemical additives to culture media to overcome cell death induced by various stimuli have been limited to apoptosis. Recently, autophagic morphologies were demonstrated by the processing of LC3 into the 16 kDa LC3-II form, and the accumulation of multiple autophagosomes in CHO cell culture. Therefore, it seems worthwhile to revisit the issue of cell death in CHO cell culture with the concept of autophagy in mind, in order to achieve a maximum production of foreign proteins by protecting cells from both types of PCD.     

Autophagy and apoptosis of recombinant Chinese hamster ovary cells during fed-batch culture: effect of nutrient supplementation

Upon nutrient depletion during recombinant Chinese hamster ovary (rCHO) cell batch culture, cells are subjected to apoptosis, type I programmed cell death (PCD), and autophagy which can be type II PCD or a cell survival mechanism. To investigate the effect of nutrient supplementation on the two PCDs and protein production in rCHO cells, an antibody-producing rCHO cell line was cultivated in batch and fed-batch modes. The feed medium containing glucose, amino acids, and vitamins was determined through flask culture tests and used in bioreactor cultures. In the bioreactor cultures, the nutrient feedings extended the culture longevity and enhanced antibody production. In addition, cells in the fed-batch culture showed delayed onset of both apoptosis and autophagy, compared with those in the batch culture. The inhibition of apoptosis was demonstrated by a decreased amount of cleaved caspase-7 protein and less fragmentation of chromosomal DNA. Concurrently, reduced LC3 conversion, from LC3-I to LC3-II, was observed in cells that received the feeds. Cultivation with pharmacological autophagy inducer (rapamycin) or inhibitor (bafilomycin A1) indicated that autophagy is necessary for the cells to survive under nutrient depletion. Taken together, the delayed and relieved cell death by nutrient supplementation could improve antibody production.     

Effect of Akt overexpression on programmed cell death in antibody-producing Chinese hamster ovary cells

Upon nutrient deprivation, Chinese hamster ovary (CHO) cells are subjected to two types of programmed cell death, apoptosis and autophagy. CHO cell engineering, as a means to improve foreign protein production, has focused mainly on anti-apoptosis. In this study, to determine the effect of Akt, which is known to regulate both apoptosis and autophagy, on cell survival and foreign protein production, constitutively active Akt was overexpressed in antibody-producing cells. Compared with the control cells, Akt overexpressing cells showed delayed onset of apoptosis and autophagy during batch culture. The inhibition of apoptosis was demonstrated by reduced amount of cleaved poly(ADP-ribose) polymerase and caspase 3 proteins and less fragmentation of chromosomal DNA. Moreover, under nutrient-limiting conditions, decreased level of autophagosome accumulation was observed in Akt overexpressing cells by the less accumulation of the 16kDa form of LC3-II and autophagic vacuoles. Taken together, the overexpression of constitutively active Akt in CHO cells could delay the onset of both types of programmed cell death during batch culture.     

Combinatorial engineering of <Emphasis Type="Italic">ldh-a</Emphasis> and <Emphasis Type="Italic">bcl-2</Emphasis> for reducing lactate production and improving cell growth in dihydrofolate reductase-deficient Chinese hamster ovary cells

In Chinese hamster ovary (CHO) cells, rapid glucose metabolism normally leads to inefficient use of glucose, most of which is converted to lactate during cell cultures. Since lactate accumulation during the culture often exerts a negative effect on cell growth and valuable product formation, several genetic engineering approaches have been developed to suppress lactate dehydrogenase-A (LDH-A), the enzyme converting pyruvate into lactate. However, despite the reduced lactate accumulation, such cell cultures are eventually terminated in the late period of the culture, mainly due to apoptosis. Therefore, we developed an apoptosis-resistant, less lactate-producing dhfr ⁻ CHO cell line (CHO-Bcl2-LDHAsi) by overexpressing Bcl-2, one of the most well-known anti-apoptotic proteins, and by downregulating LDH-A in a dhfr ⁻ CHO cell line. When the dhfr ⁻ CHO-Bcl2-LDHAsi cell line was used as a host cell line for the development of recombinant CHO (rCHO) cells producing an Fc-fusion protein, the culture longevity of the rCHO cells was extended without any detrimental effect of genetic engineering on specific protein productivity. Simultaneously, the specific lactate production rate and apparent yield of lactate from glucose were reduced to 21–65% and 37–78% of the control cells, respectively. Taken together, these results show that the use of an apoptosis-resistant, less lactate-producing dhfr ⁻ CHO cell line as a host cell line saves the time and the effort of establishing an apoptosis-resistant, less lactate-producing rCHO cells for producing therapeutic proteins.     

Rapamycin treatment inhibits CHO cell death in a serum‐free suspension culture by autophagy induction

Rapamycin, a specific mTOR inhibitor, has been used as a chemical activator in autophagy research both in vitro and in vivo. Recently, autophagy has received attention as an anti‐cell death engineering target in addition to apoptosis in the Chinese hamster ovary (CHO) cell engineering field. Here, the effect of rapamycin and the subsequent autophagy induction is investigated on two CHO cell lines, DG44 host and an antibody‐producing recombinant CHO (rCHO), in a serum‐free suspension culture. In both cell lines, the rapamycin treatment delayed the viability drop and apoptosis induction. In particular, the improved cell viability of the antibody‐producing rCHO cell line resulting from the rapamycin treatment led to a 21% increase in the maximum antibody concentration. From observations that a rapamycin derivative, everolimus, demonstrated similar positive effects in both cell lines, but not FK‐506, which forms the same complex as rapamycin, but does not inhibit mTOR, it was demonstrated that the positive effects of rapamycin appear to be mTOR‐dependent. In addition, the cultivation with rapamycin and/or an autophagy inhibitor, bafilomycin A1, indicated that the autophagy induction is related to the positive effects of rapamycin. The genetic perturbation of the autophagy pathway through the regulation of the expression level of Beclin‐1, an important autophagy regulator, resulted in a delayed autophagy induction and apoptosis inhibition in response to the rapamycin treatment in the DG44 host cell line. Taken together, the results obtained in this study imply a positive role for autophagy and predict the usefulness of pro‐autophagy engineering in CHO cell cultures. Biotechnol. Bioeng. 2012; 109: 3093–3102. © 2012 Wiley Periodicals, Inc.     

Quantitative immunocytochemical staining for recombinant tissue-type plasminogen activator in transfected Chinese hamster ovary cells

Tissue-type plasminogen activator (tPA) is a serine protease which cleaves plasminogen to its active form, plasmin. tPA plays a physiologic role in hemostasis, wound healing, and embryogenesis. Therapeutically, recombinant human tPA is used as a thrombolytic in myocardial infarction. Although production of therapeutic quantities of tPA in Chinese hamster ovary (CHO) cells transfected with the human gene for tPA is practical, production costs remain high. One important factor which determines the ultimate cost of tPA (or any other recombinant protein expressed in mammalian cells) is its production level on a per cell basis. We have used postembedding immunocytochemical staining with colloidal gold to study the subcellular localization of tPA in CHO cells expressing recombinant tPA (rCHO) in an effort to understand the factor(s) which might limit secretion. Staining for tPA was evaluated visually and by morphometric analysis and was specific and reproducible. Serially passaged rCHO showed no significant change in staining density over 31 serial passages. Staining density was greatest over dilated cisternae of the rough endoplasmic reticulum and nuclear envelope. Golgi stacks and large acid phosphatase-positive vacuoles (probably lysosomes) were also heavily stained. Staining of lysosomal vacuoles suggested that rCHO might be degrading nascent tPA. Incubation of rCHO with 125I-tPA showed that the cells were not internalizing tPA from the media. These results suggest that rCHO fail to secrete a portion of the tPA they synthesize and that it is degraded in lysosomes. This observation may have important implications on the choice of expression systems for efficient production of large quantities of recombinant proteins.     

Bcl-x(L) overexpression delays the onset of autophagy and apoptosis in hyperosmotic recombinant Chinese hamster ovary cell cultures

Hyperosmolality in recombinant Chinese hamster ovary (rCHO) cell cultures induces autophagy and apoptosis. To investigate the effect of Bcl-x_L overexpression on autophagy and apoptosis in hyperosmotic rCHO cell cultures, an erythropoietin (EPO)-producing rCHO cell line with regulated Bcl-x_L overexpression was subjected to hyperosmolality resulting from NaCl addition in a batch culture and nutrient supplementation in a fed-batch culture. In the batch culture, Bcl-x_L overexpression suppressed apoptosis, as evidenced by a decreased amount of cleaved caspase-7 and PARP. Concurrently, Bcl-x_L overexpression also delayed autophagy, as indicated by reduced LC3 conversion, from LC3-I to LC3-II. As a result, the cell viability and EPO production were improved by Bcl-x_L overexpression. In the fed-batch culture, the simultaneous application of Bcl-x_L overexpression and nutrient feeding increased the culture longevity and maximum EPO concentration. Taken together, Bcl-x_L overexpression delayed autophagy and apoptosis in hyperosmotic rCHO cell cultures, resulting in increased EPO production.     

Effect of Bcl‐xL overexpression on apoptosis and autophagy in recombinant Chinese hamster ovary cells under nutrient‐deprived condition

Upon nutrient deprivation during culture, recombinant Chinese hamster ovary (rCHO) cells are subjected to two types of programmed cell death (PCD), apoptosis and autophagy. To investigate the effect of Bcl‐x_L overexpression on apoptosis and autophagy in rCHO cells, an erythropoietin (EPO)‐producing rCHO cell line with regulated Bcl‐x_L overexpression (EPO‐off‐Bcl‐x_L) was established using the Tet‐off system. The expression level of Bcl‐x_L in EPO‐off‐Bcl‐x_L cells was tightly regulated by doxycycline in a dose‐dependent manner. Bcl‐x_L overexpression enhanced cell viability and extended culture longevity in batch culture. Upon nutrient depletion in the later stage of batch culture, Bcl‐x_L overexpression suppressed apoptosis by inhibiting the activation of caspase‐3 and ‐7. Simultaneously, Bcl‐x_L overexpression also delayed autophagy, characterized by LC3‐II accumulation. Immunoprecipitation analysis with a Flag‐tagged Bcl‐x_L revealed that Bcl‐x_L interacts with Bax and Bak, essential mediators of caspase‐dependent apoptosis, as well as with Beclin‐1, an essential mediator of autophagy, and may inhibit their pro‐cell death function. Taken together, it was found that Bcl‐x_L overexpression inhibits both apoptosis and autophagy in rCHO cell culture. Biotechnol. Bioeng. 2009;103: 757–766. © 2009 Wiley Periodicals, Inc.     

RNAi suppression of Bax and Bak enhances viability in fed-batch cultures of CHO cells

Bcl-2 family proteins play a crucial role in the regulation of the mitochondrial pathway that leads to apoptosis. Members of the Bcl-2 family can be divided into the anti-apoptotic proteins such as Bcl-2 and Bcl-X(L), and the pro-apoptotic proteins such as Bax and Bak and the BH3-only proteins. In this study, siRNA constructs to silence the Bax and Bak genes in Chinese hamster ovary (CHO) cells were generated. Stable CHO cell lines in which the expression of Bax and Bak were significantly knocked down were screened by Western blot analysis and confirmed by RT-PCR. CHO cells with both Bax and Bak knocked down showed a clear resistance against cytotoxic lectins and UV irradiation-induced apoptosis. Compared to original CHO-K1 cells, these cells also survived longer when cultured under extreme conditions such as complete nutrient depletion or in high-osmolality medium. CHO cells with both Bax and Bak genes knocked down displayed an extended lifespan as well as higher viable cell densities in fed-batch cultures, both in adherent form on microcarrier beads and in suspension. The IFN-gamma productivity by a rCHO IFN-gamma cell line in which both Bak and Bax were knocked down increased by 35% compared to the control cells. These results indicate that the genetic inactivation of Bax and Bak in recombinant CHO cells can be an effective strategy in delaying the onset of apoptosis in batch and fed-batch cultures.     

Proteomic understanding of intracellular responses of recombinant chinese hamster ovary cells adapted to grow in serum‐free suspension culture

To understand the intracellular responses in recombinant Chinese hamster ovary (rCHO) cells adapted to grow in serum‐free suspension culture, a proteomic approach was employed. After rCHO cells producing erythropoietin were adapted to grow in suspension culture with the two different serum‐free media (SFM4CHO? and SF‐L1), proteome analyses were carried out using 2‐D PAGE and based on spot intensities, 58 high‐intensity protein spots were selected. Of the 58 protein spots, which represented 34 different kinds of proteins, 55 were identified by MALDI‐TOF‐MS, and MS/MS. Compared with the results in serum‐containing medium, six proteins, four de novo synthesis of nucleotides‐related proteins (dihydrolipoamide S‐acetyltransferase, transaldolase, inosine‐5′‐monophosphate dehydrogenase 2, and lymphoid‐restricted membrane protein) and two molecular chaperones (heat shock protein 70 kDa and 60 kDa [HSC70, HSP60]) were significantly increased in SFM4CHO?. From the results of proteomic analysis, HSP60 and HSC70, which were increased in both SFM, were selected as candidate proteins for engineering and rCHO cell lines overexpressing these genes were constructed. Cells overexpressing HSP60 and/or HSC70 showed 10–15% enhanced cell concentration during serum‐free adaptation and 15–33% reduction in adaptation time. Taken together, identification of differentially expressed proteins in rCHO cells by a proteomic study can provide insights into understanding the intracellular events and clues to find candidate genes for cell engineering for improved performance of rCHO cells during adaptation to serum‐free suspension culture. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Proteomic understanding of intracellular responses of recombinant Chinese hamster ovary cells cultivated in serum-free medium supplemented with hydrolysates

In order to understand the intracellular responses in recombinant CHO (rCHO) cells producing antibody in serum-free medium (SFM) supplemented with optimized hydrolysates mixtures, yielding the highest specific growth rate (μ, SFM#S1) or the highest specific antibody productivity (q _Ab, SFM#S2), differentially expressed proteins in rCHO cells are measured by two-dimensional gel electrophoresis combined with nano-LC-ESI-Q-TOF tandem MS. The comparative proteomic analysis with basal SFM without hydrolysates revealed that the addition of hydrolysate mixtures significantly altered the profiles of CHO proteome. In SFM#S1, the expression of metabolism-related proteins, cytoskeleton-associated proteins, and proliferation-related proteins was up-regulated. On the other hand, the expression of anti-proliferative proteins and pro-apoptotic protein was down-regulated. In SFM#S2, the expression of various chaperone proteins and proliferation-linked proteins was altered. 2D-Western blot analysis of differentially expressed proteins confirmed the proteomic results. Taken together, identification of differentially expressed proteins in CHO cells by a proteomic approach can provide insights into understanding the effect of hydrolysates on intracellular events and clues to find candidate genes for cell engineering to maximize the protein production in rCHO cells.     

Application of sodium propionate to the suspension culture of Chinese hamster ovary cells for enhanced production of follicle-stimulating hormone

In this study, we found that adding sodium propionate to batch culture medium resulted in cell growth suppression in a dosedependent manner, while it enhanced follicle-stimulating hormone (FSH) production in Chinese hamster ovary (CHO) cells. In a pseudo-perfusion culture, with the exchange of fresh medium containing 2.5 mM sodium propionate, a final FSH of 532 μg was obtained, which was approximately 1.5-fold higher than the control. Overall, the results demonstrate that the application of sodium proplonate for the commercial production of recombinant proteins in rCHO cells is feasible.     

Development of apoptosis-resistant dihydrofolate reductase-deficient Chinese hamster ovary cell line

Apoptosis-resistant dihydrofolate reductase-deficient CHO cell line (dhfr(-) CHO-bcl2) was developed by introduction of the bcl-2 gene into the dhfr(-) CHO cell line (DUKX-B11, ATCC CRL-9096) and subsequent selection of clones stably overexpressing Bcl-2 in the absence of selection pressure. When the dhfr(-) CHO-bcl2 cell line was used as a host cell line for development of a recombinant CHO (rCHO) cell line expressing a humanized antibody, it displayed stable expression of the bcl-2 gene during rCHO cell line development and no detrimental effect of Bcl-2 overexpression on specific antibody productivity. Taken together, the results obtained demonstrate that the use of an apoptosis-resistant dhfr(-) CHO cell line as the host cell line saves the effort of establishing an apoptosis-resistant rCHO cell line and expedites the development process of apoptosis-resistant rCHO cells producing therapeutic proteins.     

Hyperosmotic stress induces autophagy and apoptosis in recombinant Chinese hamster ovary cell culture

During recombinant Chinese hamster ovary (rCHO) cell culture, various events, such as feeding with concentrated nutrient solutions or the addition of base to maintain an optimal pH, increase the osmolality of the medium. To determine the effect of hyperosmotic stress on two types of programmed cell death (PCD), apoptosis and autophagy, of rCHO cells, two rCHO cell lines, producing antibody and erythropoietin, were subjected to hyperosmotic stress resulting from NaCl addition (310–610 mOsm/kg). For both rCHO cell lines, hyperosmolality up to 610 mOsm/kg increased cleaved forms of PARP, caspase‐3, caspase‐7, and fragmentation of chromosomal DNA, confirming the previous observation that apoptosis was induced by hyperosmotic stress. Concurrently, hyperosmolality increased the level of accumulation of LC3‐II, a widely used autophagic marker, which was determined by Western blot analysis and confocal microscopy. When glucose and glutamine concentrations were measured during the cultures, glucose and glutamine concentrations in the culture medium at various osmolalities (310–610 mOsm/kg) showed no significant differences. This result suggests that induction of PCD by hyperosmotic stress occurred independently of nutrient depletion. Taken together, autophagy as well as apoptosis was observed in rCHO cells subjected to hyperosmolality. Biotechnol. Bioeng. 2010;105: 1187–1192. © 2009 Wiley Periodicals, Inc.     

Inhibition of sodium butyrate-induced apoptosis in recombinant Chinese hamster ovary cells by constitutively expressing antisense RNA of caspase-3

Sodium butyrate (NaBu) can enhance the expression of genes controlled by some of the mammalian promoters, but it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on a foreign protein expression is compromised by its cytotoxic effect on cell growth. To overcome this cytotoxic effect of NaBu, the expression vector of antisense RNA of caspase-3 was constructed and transfected to recombinant Chinese hamster ovary (rCHO) cells producing a humanized antibody. Using this antisense RNA strategy, rCHO cells (B3) producing a low level of caspase-3 proenzyme were established. When batch cultures of both B3 cells and control cells transfected with antisense RNA-deficient plasmid were performed in the absence of NaBu, both cells showed similar profiles of cell growth and antibody production. Compared with control cell culture, under the condition of 5 mM NaBu addition at the exponential growth phase, expression of antisense RNA of caspase-3 significantly suppressed the NaBu-induced apoptosis of B3 cells and extended culture longevity by >2 days if the culture was terminated at cell viability of 50%. However, compared with control cell culture, the final antibody concentration of B3 cell culture was not increased in the presence of NaBu, which may be due to the loss of cellular metabolic capability resulted from the depolarization of mitochondrial membrane. Taken together, this study suggests that, although expression of antisense RNA of caspase-3 does not improve antibody productivity of rCHO cells, it can suppress NaBu-induced apoptotic cell death of rCHO cells and thereby may reduce problems associated with cellular disintegration.     

表达外源rhBMP2的CHO细胞株稳定性分析

骨形态发生蛋白2(BMP2)属于TGF-β超家族,是诱导成骨活性最强的BMPs之一, 具有广泛的临床应用的前景。本实验室已成功构建高效表达rhBMP2的重组CHO细胞株, 现选取其中一株表达量最高的细胞rCHO(hBMP2)-C8进行长期体外培养, 并在培养过程中比较了添加和去除压力筛选中使用的MTX对细胞生长, rhBMP2基因拷贝数及rhBMP2分泌蛋白表达的影响;检测了该细胞株在无血清培养基中可以连续表达rhBMP2的时间以及培养基中rhBMP2的温度敏感性等等。该研究为进一步采用动物细胞规模化培养技术生产rhBMP2奠定了基础。