Influence of hyperosmolar basal media on hybridoma cell growth and antibody production

To investigate the influence of hyperosmolar basal media on hybridoma response, S3H5/γ2bA2 and DB9G8 hybridomas were cultivated in a batch mode using hyperosmolar basal media resulting from additional sodium chloride supplementation. The basal media used in this study were IMDM, DMEM, and RPMI 1640, all of which are widely used for hybridoma cell culture. In IMDM, two hybridomas showed different responses to hyperosmotic stress regarding specific MAb productivity (q _MAb), though they showed similar depression of cell growth in hyperosmolar media. Unlike S3H5/γ2bA2 hybridoma, the q _MAb of DB9G8 hybridoma was not enhanced significantly around 390 mOsm kg^?1. The variation of basal media influenced DB9G8 hybridoma response to hyperosmotic stress regarding q _MAb. In IMDM, the q _MAb of DB9G8 hybridoma was increased by more than 200% when the osmolality increased from 281 to 440 mOsm/kg. However, in RPMI 1640 and DMEM, similar amplitude of osmolality increase resulted in less than 100% increase in q _MAb. The variation of basal media also influenced the cell growth in hyperosmolar medium. Both hybridomas were more tolerant against hyperosmotic stress in DMEM than in IMDM, which was found to be due to the high osmolality of standard DMEM. The osmolalities of standard IMDM and DMEM used for inocula preparation were 281 and 316 mOsm kg^?1, respectively. Thus, when the cells were cultivated at 440 mOsm kg^?1, the cells in IMDM experienced higher osmotic shock than in DMEM. By using the inoculum prepared at 317 mOsm kg^?1 in IMDM, S3H5/γ2bA2 cell growth at 440 mOsm kg^?1 in IMDM was comparable to that in DMEM. Taken together, the results obtained from this study show that the selection of basal media is an important factor for MAb production by employing hyperosmotic stress.     

Effect of Bcl‐xL overexpression on lactate metabolism in chinese hamster ovary cells producing antibody

Previously, overexpression of anti‐apoptotic proteins, such as E1B‐19K and Aven, was reported to alter lactate metabolism of CHO cells in culture. To investigate the effect of Bcl‐x_L, a well‐known anti‐apoptotic protein, on lactate metabolism of recombinant CHO (rCHO) cells, two antibody‐producing rCHO cell lines with regulated Bcl‐x_L overexpression (CS13*‐0.02‐off‐Bcl‐x_L and CS13*‐1.00‐off‐Bcl‐x_L) were established using the Tet‐off system. When cells were cultivated without Bcl‐x_L overexpression, the specific lactate production rate (q_Lac) of CS13*‐0.02‐off‐Bcl‐x_L and CS13*‐1.00‐off‐Bcl‐x_L were 7.32 ± 0.37 and 6.78 ± 0.56 pmol/cell/day, respectively. Bcl‐x_L overexpression, in the absence of doxycycline, did not affect the q_Lac of either cell line, though it enhanced the viability during cultures. Furthermore, activities of the enzymes related to glucose and lactate metabolism, such as hexokinase, glucose‐6‐phosphate dehydrogenase, lactate dehydrogenases, and alanine aminotransferase, were not affected by Bcl‐x_L overexpression either. Taken together, Bcl‐x_L overexpression showed no significant effect on the lactate metabolism of rCHO cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1594–1598, 2013     

Effects of anisosmotic medium on cell volume,transmembrane potential and intracellular K+ activity in mouse hepatocytes

Summary Mouse hepatocytes in primary monolayer culture (4 hr) were exposed for 10 min at 37°C to anisosmotic medium of altered NaCl concentration. Hepatocytes maintained constant relative cell volume (experimental volume/control volume) as a function of external medium relative osmolality (control mOsm/experimental mOsm), ranging from 0.8 to 1.5. In contrast, the relative cell volume fit a predicted Boyle-Van't Hoff plot when the experiment was done at 4°C. Mouse liver slices were used for electrophysiologic studies, in which hepatocyte transmembrane potential (V _m ) and intracellular K⁺ activity (a _K ⁱ ) were recorded continuously by open-tip and liquid ion-exchanger ion-sensitive glass microelectrodes, respectively. Liver slices were superfused with control and then with anisosmotic medium of altered NaCl concentration.V _m increased (hyperpolarized) with hypoosmotic medium and decreased (depolarized) with hyperosmotic medium, and ln [10(experimentalV _m /controlV _m )] was a linear function of relative osmolality (control mOsm/experimental mOsm) in the range 0.8–1.5. Thea _K ⁱ did not change when medium osmolality was decreased 40–70 mOsm from control of 280 mOsm. Similar hypoosmotic stress in the presence of either 60mm K⁺ or 1mm quinine HCl or at 27°C resulted in no change inV _m compared with a 20-mV increase inV _m without the added agents or at 37°C. We conclude that mouse hepatocytes maintain their volume anda _K ⁱ in response to anisosmotic medium; however,V _m behaves as an osmometer under these conditions. Also, increases inV _m by hypoosmotic stress were abolished by conditions or agents that inhibit K⁺ conductance.     

Use of NaCl prevents aggregation of recombinant COMP–Angiopoietin-1 in Chinese hamster ovary cells

To investigate the effect of hyperosmotic medium on production and aggregation of the variant of Angiopoietin-1 (Ang1), cartilage oligomeric matrix protein (COMP)–Ang1, in recombinant Chinese hamster ovary (CHO) cells, CHO cells were cultivated in shaking flasks. NaCl and/or sorbitol were used to raise medium osmolality in the range of 300–450 mOsm/kg. The specific productivity of COMP–Ang1, q_COMP–Ang1, increased as medium osmolality increased. At NaCl-450 mOsm/kg, the q_COMP–Ang1 was 7.7-fold higher than that at NaCl-300 mOsm/kg, while, at sorbitol-450 mOsm/kg, it was 2.9-fold higher than that at sorbitol-300 mOsm/kg. This can be attributed to the increased relative mRNA level of COMP–Ang1 at NaCl-450 mOsm/kg which was approximately 2.4-fold higher than that at sorbitol-450 mOsm/kg. Western blot analysis showed that COMP–Ang1 aggregates started to occur in the late-exponential phase of cell growth. When sorbitol was used to raise the medium osmolality, a severe aggregation of COMP–Ang1 was observed. On the other hand, when NaCl was used, the aggregation of COMP–Ang1 was drastically reduced at NaCl-400 mOsm/kg. At NaCl-450 mOsm/kg, the aggregation of COMP–Ang1 was hardly observed. This suggests that environmental conditions are critical for the aggregation of COMP–Ang1. Taken together, the use of NaCl-induced hyperosmotic medium to cell culture process turns out to be an efficient strategy for enhancing COMP–Ang1 production and reducing COMP–Ang1 aggregation.     

Calnexin overexpression sensitizes recombinant CHO cells to apoptosis induced by sodium butyrate treatment

Sodium butyrate (NaBu) can enhance the expression of foreign genes in recombinant Chinese hamster ovary (rCHO) cells, but it can also inhibit cell growth and induce cellular apoptosis. In this study, the potential role of calnexin (Cnx) expression in rCHO cells treated with 5 mM NaBu was investigated for rCHO cells producing tumor necrosis factor receptor FC. To regulate the Cnx expression level, a tetracycline-inducible system was used. Clones with different Cnx expression levels were selected and investigated. With regard to productivity per cell (q_p), NaBu enhanced the q_p by over twofold. Under NaBu treatment, Cnx overexpression further enhanced the q_p by about 1.7-fold. However, under NaBu stress, the cells overexpressing Cnx showed a poorer viability profile with a consistent difference of over 25% in the viability when compared to the Cnx-repressed condition. This drop in the viability was attributed to increased apoptosis seen in these cells as evidenced by enhanced poly (ADP-ribose) polymerase cleavage and cytochrome C release. Ca²⁺ localization staining and subsequent confocal imaging revealed elevated cytosolic Ca²⁺ ([Ca²⁺]_c) in the Cnx-overexpressing cells when compared to the Cnx-repressed condition, thus endorsing the increased apoptosis observed in these cells. Taken together, Cnx overexpression not only improved the q_p of cells treated with NaBu, but it also sensitized cells to apoptosis.     

New cell line development for antibody-producing Chinese hamster ovary cells using split green fluorescent protein

Background

The establishment of high producer is an important issue in Chinese hamster ovary (CHO) cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS)-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production.

Results

An internal ribosome entry site (IRES) was introduced for using two green fluorescence protein (GFP) fragments as a reporter to both antibody chains, the heavy chain and the light chain. The cells co-transfected with two GFP fragments showed the emission of green fluorescence by the reconstitution of split GFP. The FACS-sorted pool with GFP expression had a higher specific antibody productivity (q _Ab) than that of the unsorted pool. The q _Ab was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and q _Ab in individual selected clones.

Conclusions

This study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of q _Ab with reconstitution of GFP. Taken together, we developed an efficient FACS-based screening method for high antibody-producing CHO cells with the benefits of the split GFP system.     

Effect of hypoosmotic stress on hybridoma cell growth and antibody production

To investigate the response of hybridoma cells to hypoosmotic stress, S3H5/gamma2bA2 and DB9G8 hybridomas were cultivated in the hypoosmolar medium [Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% serum] resulting from sodium chloride subtraction. Both hybridomas showed similar responses to hypoosmotic stress in regard to cell growth and antibody production. The cell growth and antibody production at 276 mOsm/kg were comparable to those at 329 mOsm/kg (standard DMEM). Both cells grew well at 219 mOsm/kg, though their growth and antibody production were slightly decreased. When the osmolality was further decreased to 168 mOsm/kg, the cell growth did not occur. When subjected to hyperosmotic stress, both cells displayed significantly enhanced specific antibody productivity (q(Ab)). However, the cells subjected to hypoosmotic stress did not display enhanced q(Ab). Taken together, both hyperosmotic and hypoosmotic stresses depressed the growth of S3H5/gamma2bA2 and DB9G8 hybridomas. However, their response to hypoosmotic stress in regard to q(Ab) was different from that to hyperosmotic stress. (c) 1997 John Wiley & Sons, Inc. Biotechnol Biong 55: 565-570, 1997.     

Bcl‐xL overexpression does not enhance specific erythropoietin productivity of recombinant CHO cells grown at 33°C and 37°C

Overexpression of bcl‐xL in recombinant Chinese hamster ovary (rCHO) cells has been known to suppress apoptotic cell death and thereby extend culture longevity during batch culture. However, its effect on specific productivity (q) of rCHO cells is controversial. This study attempts to investigate the effect of bcl‐xL overexpression on q of rCHO cells producing erythropoietin (EPO). To regulate the bcl‐xL expression level, the Tet‐off system was introduced in rCHO cells producing EPO (EPO‐off‐bcl‐xL). The bcl‐xL expression level was tightly controlled by doxycycline concentration. To evaluate the effect of bcl‐xL overexpression on specific EPO productivity (q_EPO) at different levels, EPO‐off‐bcl‐xL cells were cultivated at the two different culture temperatures, 33°C and 37°C. The q_EPO at 33°C and 37°C in the presence of 100 ng/mL doxycycline (without bcl‐xL overexpression) were 4.89 ± 0.21 and 3.18 ± 0.06 μg/10⁶cells/day, respectively. In the absence of doxycycline, bcl‐xL overexpression did not affect q_EPO significantly, regardless of the culture temperature, though it extended the culture longevity. Taken together, bcl‐xL overexpression showed no significant effect on the q_EPO of rCHO cells grown at 33°C and 37°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

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.     

Response of recombinant Chinese hamster ovary cells to hyperosmotic pressure: effect of Bcl-2 overexpression

In an attempt to use the hyperosmotic pressure for improved foreign protein production in recombinant Chinese hamster ovary (rCHO) cells, the response of rCHO cells producing a humanized antibody (SH2-0.32-(Delta)bcl-2 cells) to hyperosmotic pressure was determined in regard to cell growth and death, and antibody production. Further, the feasibility of Bcl-2 overexpression in improving rCHO cell viability under hyperosmotic pressure was also determined by comparing control cells (SH2-0.32-(Delta)bcl-2) with Bcl-2 overexpressing cells (14C6-bcl-2). After 3 days of cultivation in the standard medium (294 mOsm x kg(-1)), the spent medium was exchanged with the fresh media with various osmolalities (294-640 mOsm x kg(-1)). The results obtained show that hyperosmotic pressure inhibited cell growth in a dose-dependent manner, though 14C6-bcl-2 cells were less susceptible to hyperosmotic pressure than SH2-0.32-(Delta)bcl-2 cells. At 522 mOsm x kg(-1), SH2-0.32-(Delta)bcl-2 cells underwent a gradual cell death mainly through apoptosis due to the cytotoxic effect of hyperosmotic pressure. In contrast, Bcl-2 overexpression in 14C6-bcl-2 cells could delay the apoptosis induced by 522 mOsm x kg(-1) by inhibiting caspase-3 activation. Bcl-2 overexpression could also improve the cellular membrane integrity of 14C6-bcl-2 cells. When subjected to hyperosmotic pressure, the specific antibody productivity of SH2-0.32-(Delta)bcl-2 cells and 14C6-bcl-2 cells was increased in a similar extent. As a result, the final antibody concentration achieved in 14C6-bcl-2 cells at 522 mOsm x kg(-1) was 2.5-fold higher than that at 294 mOsm x kg(-1). At 580 mOsm x kg(-1), acute hyperosmotic pressure induced the rapid loss of viability in both SH2-0.32-(Delta)bcl-2 and 14C6-bcl-2 cells through necrosis rather than through apoptosis. Taken together, Bcl-2 overexpression and optimized hyperosmotic pressure could improve the antibody production of rCHO cells.     

Biphasic culture strategy based on hyperosmotic pressure for improved humanized antibody production in Chinese hamster ovary cell culture

Hyperosmotic pressure increased specific antibody productivity (q(Ab)) of recombinant Chinese hamster ovary (rCHO) cells (SH2-0.32) and it depressed cell growth. Thus, the use of hyperosmolar medium did not increase the maximum antibody concentration substantially. To overcome this drawback, the feasibility of biphasic culture strategy was investigated. In the biphasic culture, cells were first cultivated in the standard medium with physiological osmolality (294 mOsm/kg) for cell growth. When cells reached the late exponential growth phase, the spent standard medium was replaced with the fresh hyperosmolar medium (522 mOsm/kg) for antibody production. The q(Ab) in growth phase with the standard medium was 2.1 microg per 10(6) cells/d, whereas the q(Ab) in antibody production phase with the hyperosmolar medium was 11.1 microg per 10(6) cells/d. Northern blot analysis showed a positive relationship between the relative contents of intracellular immunoglobulin messenger ribonucleic acid and q(Ab). Because of the enhanced q(Ab) and the increased cell concentration in biphasic culture, the maximum antibody concentration obtained in biphasic culture with 522 mOsm/kg medium exchange was 161% higher than that obtained in batch culture with the standard medium. Taken together, the simple biphasic culture strategy based on hyperosmotic culture is effective in improving antibody production of rCHO cells.     

Linkage between low-density lipoprotein and igk light-chain genes in rabbits

The rabbit geneLpq, which codes for a low-density serum lipoprotein², is linked (34.6 ± 5.3 centimorgans) to the Ig kappa light-chain gene (Ab). There is no evidence thatLpq is linked to another gene,Prt, that was previously found to be linked to theAb gene. This suggests that the gene order for the three genes isPrt- Ab- Lpq. Abbreviations used in this paper Ig immunoglobulin - a the heavy-chain variable-region geneAa - b the kappa light-chain geneAb - q the low-density serum lipoprotein geneLpq     

Hypotonically induced changes in the plasma membrane of cultured mammalian cells

Summary Cells from three cell lines were electrorotated in media of osmotic strengths from 330 mOsm to 60 mOsm. From the field-frequency dependence of the rotation speed, the passive electrical properties of the surfaces were deduced. In all cases, the area-specific membrane capacitance (C _m) decreased with osmolality. At 280 mOsm (iso-osmotic), SP2 (mouse myeloma) and G8 (hybridoma) cells had C _mvalues of 1.01 ± 0.04 F/cm² and 1.09 ± 0.03 F/cm², respectively, whereas dispase-treated L-cells (sarcoma fibroblasts) exhibited C _m=2.18±0.10/F/cm². As the osmolality was reduced, the C _mreached a well-defined minimum at 150 mOsm (SP2) or 180 mOsm (G8). Further reduction in osmolality gave a 7% increase in C _m, after which a plateau close to 0.80F/cm2²was reached. However, the whole-cell capacities increased about twofold from 200 mOsm to 60 mOsm. L-cells showed very little change in C _mbetween 280 mOsm and 150 mOsm, but below 150 mOsm the C _mdecreased rapidly. The changes in C _mcorrelate well with the swelling of the cells assessed by means of van't Hoff plots. The apparent membrane conductance (including the effect of surface conductance) decreased with C _m, but then increased again instead of exhibiting a plateau. The rotation speed of the cells increased as the osmolality was lowered, and eventually attained almost the theoretical value. All measurements indicate that hypo-osmotically stressed cells obtain the necessary membrane area by using material from microvilli. However, below about 200 mOsm the whole-cell capacities indicate the progressive incorporation of extra membrane into the cell surface.We thank Mr. B.G. Klarmann for his help with the measurements. This work was supported by grants of the DFG (SFB 176 B5 to U.Z. and W.M.A.) and of the BMFT (DARA 50 WB 9212 to U.Z.). We also thank the Umweltbundesamt, Berlin, for support enabling the construction of some of the rotation generators used in this work.     

BiP Inducer X: An ER Stress Inhibitor for Enhancing Recombinant Antibody Production in CHO Cell Culture

Prolonged endoplasmic reticulum (ER) stress reduces protein synthesis and induces apoptosis in mammalian cells. When dimethyl sulfoxide (DMSO), a specific monoclonal antibody productivity (q_mAb)‐enhancing reagent, is added to recombinant Chinese hamster ovary (rCHO) cell cultures (GSR cell line), it induces ER stress and apoptosis in a dose‐dependent manner. To determine an effective ER stress inhibitor, three ER stress inhibitors (BiP inducer X [BIX], tauroursodeoxycholic acid, and carbazole) are examined and BIX shows the best production performance. Coaddition of BIX (50 μm ) with DMSO extends the culture longevity and enhances q_mAb. As a result, the maximum mAb concentration is significantly increased with improved galactosylation. Coaddition of BIX significantly increases the expression level of binding immunoglobulin protein (BiP) followed by increased expression of chaperones (calnexin and GRP94) and galactosyltransferase. Furthermore, the expression levels of CHOP, a well‐known ER stress marker, and cleaved caspase‐3 are significantly reduced, suggesting that BIX addition reduces ER stress‐induced cell death by relieving ER stress. The beneficial effect of BIX on mAb production is also demonstrated with another q_mAb‐enhancing reagent (sodium butyrate) and a different rCHO cell line (CS13‐1.00). Taken together, BIX is an effective ER stress inhibitor that can be used to increase mAb production in rCHO cells.     

Plasmolysis and cell wall deposition in wheat root hairs under osmotic stress

We analysed cell wall formation in rapidly growing root hairs of Triticum aestivum under reduced turgor pressure by application of iso- and hypertonic mannitol solutions. Our experimental series revealed an osmotic value of wheat root hairs of 150 mOsm. In higher concentrations (200–650 mOsm), exocytosis of wall material and its deposition, as well as callose synthesis, still occurred, but the elongation of root hairs was stopped. Even after strong plasmolysis when the protoplast retreated from the cell wall, deposits of wall components were observed. Labelling with DiOC₆(3) and FM1-43 revealed numerous Hechtian strands that spanned the plasmolytic space. Interestingly, the Hechtian strands also led towards the very tip of the root hair suggesting strong anchoring sites that are readily incorporated into the new cell wall. Long-term treatments of over 24 h in mannitol solutions (150–450 mOsm) resulted in reduced growth and concentration-dependent shortening of root hairs. However, the formation of new root hairs does occur in all concentrations used. This reflects the extraordinary potential of wheat root cells to adapt to environmental stress situations.     

A rapid procedure for plant regeneration from protoplasts isolated from suspension cultures and leaf mesophyll cells of wild Solanum species and Lycopersicon pennellii

A rapid procedure for protoplast isolation, culture and plant regeneration has been developed for two Solanum species (S. lycoperisicoides and S. verrucosum) and Lycopersicon pennellii. Freshly isolated protoplasts were initially cultured in liquid Solanum Culture Medium (SCM), containing 2,4-dichlorophenoxy acetic acid (2,4-D). Subsequent dilution with fresh culture medium without auxins appeared to be essential to obtain rapid regeneration medium later on. The resulting micro calli were first grown in a culture medium containing 0.5 mg/l 6-BAP and 0.05 mg/l NAA and 0.2 M mannitol and 7.3 mM sucrose to induce greening, at a lower osmolarity (300 mOsm · kg⁻¹). Then, the green micro calli were transferred to shoot induction medium, containing 2 mg/l zeatin, 0.1 mg/l IAA and 2% sucrose (150 mOsm · kg⁻¹). In this way plants could be regenerated from leaf mesophyll protoplasts and suspension cell-derived protoplasts of L. pennellii and S. lycopersicoides within 2 months. Shoot regeneration from leaf mesophyll protoplasts of the two lines of S. verrucosum could be obtained 3 months after protoplast isolation.     

Effect of salinity on the osmotic,chloride, total protein and calcium concentrations in the hemolymph of the prawn Peneaus monodon (fabricius)

• 1.1. Osmolality and chloride concentrations in the hemolymph of Penaeus monodon became stable 1 day after molting in 32 ppt, while total protein and calcium concentrations remained stable throughout the molting cycle. When intermolt (≥ 36 hr postmolt) animals were transferred from control (32 ppt) to experimental (8–40 ppt) salinities, osmolality, chloride and total protein, but not calcium, concentrations in the hemolymph achieved steady state values 24–48 hr after transfer.
• 2.2. The hemolymph osmolality was a linear function (slope = 0.28) of medium osmolality at salinities between 8 and 40 ppt. It was isosmotic to seawater at 698 mOsm (10 g prawns) and 752 mOsm (30 g), and was hyperosmotic to the medium below isosmotic concentrations, and hypoosmotic to those above.
• 3.3. Hemolymph chloride concentration was isoionic to seawater at 334 mM, and was hyperregulated below isoionic concentrations, and hyporegulated to those above.
• 4.4. P. monodon maintained its hemolymph calcium concentration between 6.4 and 10 mM when medium salinities increased from 8 to 40 ppt.
• 5.5. Total protein concentration in the hemolymph was independent of medium salinity (8–40 ppt) and hemolymph osmolality (540–850 mOsm).