Different responses to acute or progressive osmolarity increases in the mIMCD3 cell line

Cells from the kidney medulla are able to survive and function when exposed to high concentrations of NaCl and urea. In vitro, cultured epithelial cells from the kidney medulla are able to survive stronger acute hyperosmotic shocks when both solutes are present. However, in vivo, increases in osmolarity are not acute. In this study, we compared the survival of a murine renal epithelial cell line during acute or progressive (two step) adaptation to hypertonic NaCl and/or urea. Increasing osmolarity to 700 mOsm/l with NaCl or urea in a single step led to massive cell death ( 50% in 24 hours). However, genomic DNA of dying cells was not degraded, and electron microscopy revealed weak condensation of chromatin, absence of membrane blebbing, and no nuclear indentation. Pre-adaptation to permissive concentrations of NaCl (200 mOsm/l giving a final osmolarity of 500 mOsm/l) protected cells against subsequent increases in osmolarity, allowing adaptation to final osmolarities as high as 900 mOsm/l. In contrast, pre-adaptation to permissive concentrations of urea (200 mOsm/l) did not lead to enhanced cell survival after a subsequent 200 mOsm/l step. Cell death was as rapid as after an acute shock, but was more typical of apoptosis (genomic DNA laddering, strong chromatin condensation, nuclear indentation, and blebbing of the membrane giving rise to apoptotic bodies). Thus, acute hyperosmolarity induces cell death with essentially similar responses to NaCl and urea. In contrast, progressive adaptation of mIMCD3 cells to NaCl allows cell survival, whereas progressive adaptation to hyperosmotic urea triggers a cell death pathway different from the one triggered by acute hyperosmotic shocks.     

Hypertonic stress response

Mammalian renal inner medullary cells are normally exposed to extremely high NaCl concentrations. Remarkably, under these normal conditions, the high NaCl causes DNA damage and inhibits its repair, yet the cells survive and function both in cell culture and in vivo. The interstitial NaCl concentration in parts of a normal renal medulla can be 500 mM or more, depending on the species. Studies of how the cells survive and function despite this extreme stress have led to the discovery of protective adaptations, including accumulation of large amounts of organic osmolytes, which normalize cell volume and intracellular ionic strength, despite the hypertonicity of the high NaCl. Those adaptations, however, do not prevent DNA damage. High NaCl induces DNA breaks rapidly, and the DNA breaks persist even after the cells become adapted to the high NaCl. The adapted cells proliferate rapidly in cell culture and function adequately in vivo despite the DNA breaks. Both in cell culture and in vivo the breaks are rapidly repaired if the NaCl concentration is lowered. Although acute elevation of NaCl causes transient cell cycle arrest and, when the elevation is too extreme, apoptosis, proliferation of adapted cells is not arrested in culture and apoptosis is not evident either in culture or in vivo. Further, high NaCl impairs activation of several components of the classical DNA damage response such as Mre11, H2AX and Chk1 leading to inhibition of DNA repair. Nevertheless, other regular participants in the DNA damage response, such as Gadd45a, Gadd153, p53, Hsp70, and ATM are still upregulated by high NaCl. How high NaCl causes the DNA breaks and how the cells survive them is conjectural at this point. We discuss possible answers to these questions, based on current knowledge about induction and processing of DNA breaks.     

Renal response of euryhaline toad (Bufo viridis) to acute immersion in tap water, NaCl, or urea solutions

Green toads (Bufo viridis) were acclimated to either tap water, 230 mOsmol NaCl kg-1 H2O (saline), 500 mOsmol NaCl kg-1 H2O (high saline), or 500 mmol L-1 urea. Renal functions for each acclimation group were studied on conscious animals that had one ureter chronically catheterized. Reciprocal immersion of tap-water- and saline-acclimated toads in the opposite solution did not stress the animals osmotically, and plasma osmolality increased or decreased by no more than 15%. However, urine osmolality and ionic composition changed immediately and profoundly on exposure to the other solution. Exposure of tap-water-acclimated toads to saline decreased urine flow by 30%, whereas the reciprocal immersion led to an increase of 30%. Immersion of tap-water-acclimated toads in high saline led to immediate cessation of urine flow, whereas immersion of 500 NaCl- or urea-acclimated toads in tap water led to a large increase in urine flow, with an overshoot that lasted 10 h (as a result of either salt or urea diuresis). Urine flow then stabilized at a level 5-6 times higher than the value attained at high-salt environment. On immersion of 500 urea-acclimated toads in 500 NaCl, urine flow doubled, accompanied by a change in ion composition, without change in the osmolality. In all experimental conditions, plasma potassium concentration was maintained within a narrow range. The results show that the toad's kidneys contributed efficiently both to osmo- and ionoregulation in a wide range of ambient solutions.     

Utilization of osmoprotective compounds by hybridoma cells exposed to hyperosmotic stress

A search was undertaken for osmoprotective compounds for mouse hybridoma cell line 6H11 grown in culture. When the osmolality of the growth medium was increased above the normal osmolality of 330 mOsmol/kg, growth rates were decreased in a dose-dependent fashion, reaching zero when the osmolality of the medium reached approx. 435 mOsmol/kg through the addition of KCl (60 mM), or 510 mOsmol/kg through the addition of NaCl (100 mM), or sucrose (175 mM). For NaCl or sucrose-stressed cultures, the inclusion of glycine betaine, sarcosine, proline, glycine, or asparagine in the growth medium gave a moderate to strong osmoprotective effect, measured as the ability of these compounds to enhance cell growth rates under hyperosmotic conditions. Inclusion of dimethylglycine may also give a strong osmoprotective effect under these stress conditions.In KCl-stressed cell cultures, addition of glycine betaine, sarcosine, or dimethylglycine gave strong osmoprotective effects. Of 38 compounds tested during NaCl stress, 7 gave weak osmoprotective effects and 25 gave no osmoprotective effect. The osmoprotective compounds accumulated inside the stressed cells. Accumulation was completed after 4 to 8 h, reaching intracellular concentrations of approx. 0.27 pmol/cell, or 0.15 M, in NaCl stressed cells (100 mM NaCl added).Glycine betaine, dimethylglycine, and sarcosine accumulation was observed only when these protectants were included in the medium. For all osmoprotectants, a growth medium concentration between 5 and 30 mM gave the maximal protective effect, with the exception of dimethylglycine, for which the optimum concentration was approx. 65 mM. Osmoprotective effects obtained with glycine, sarcosine, dimethylglycine, and glycine betaine, indicate that the more methylated compounds are the most effective protectants.The cellular content of glycine betaine and the glycine betaine uptake rate increased with medium osmolality in a linear fashion. Glycine betaine uptake was described by a model comprising a saturable component obeying Michaelis-Menten kinetics and a nonsaturable component. K(m) and V(max) for glycine betaine uptake were determined at 420 mOsmol/kg (50 mM NaCl added) and 510 mOsmol/kg (100 mM NaCl added). A K(m) value of approx. 2.5 mM was obtained at both medium osmolalities, while V(max) increased from 0.010 pmol/cell . h to 0.018 pmol/cell . h as the osmolality of the growth medium was increased, indicating an effect of medium osmolality on the maximal rate of transport rather than on the affinity of the transporters for glycine betaine. Hybridoma cells were not able to utilize the glycine betaine precursors choline or glycine betaine aldehyde for osmoprotection, suggesting that the cells lack part, or all, of the choline-glycine betaine pathway or the appropriate uptake mechanism.The uptake rate for glycine in NaCl-stressed hybridoma cells was approx. four times higher than the uptake rate for glycine betaine. Furthermore, if equimolar amounts of glycine betaine, glycine, sarcosine, and proline were simultaneously added to NaCl-stressed cell cultures, the intracellular concentrations of glycine, proline, and sarcosine were significantly higher than the concentration of glycine betaine.A 40% increase in hybridoma cell volume was observed when the growth medium osmolality was increased from 300 to 520 mOsmol/kg. (c) 1994 John Wiley & Sons, Inc.     

Hyperosmotic NaCl and urea synergistically regulate the expression of the UT-A2 urea transporter in vitro and in vivo

The UT-A2 urea transporter is involved in the recycling of urea through the kidney, a process required to maintain high osmotic gradients. Dehydration increases UT-A2 expression in vivo. The tissue distribution of UT-A2 suggested that hyperosmolarity, and not vasopressin, might mediate this effect. We have analyzed the regulation of UT-A2 expression by ambiant osmolarity both in vitro (mIMCD3 cell line) and in vivo (rat kidney medulla). The UT-A2 mRNA was found to be synergistically up-regulated by a combination of NaCl and urea. Curiously, the UT-A2 protein was undetectable in this hypertonic culture condition, or after transfection of the UT-A2 cDNA, whereas it could be detected in HEK-293 transfected cells. Treating rats with furosemide, a diuretic which decreases the kidney interstitium osmolarity without affecting vasopressin levels, led to decreased levels of the UT-A2 protein. Our results show that the UT-A2 urea transporter is regulated by hyperosmolarity both in vitro and in vivo.     

Differential response of nucleus pulposus intervertebral disc cells to high salt, sorbitol, and urea

Nucleus pulposus intervertebral disc cells are routinely confronted with high osmolality in their microenvironment and respond to this stress in vitro by regulating cell cycle progression and by activating a DNA repair machinery in order to counteract its genotoxic effect. In the present study, we attempted to identify the origin of this osmo-regulatory response, by using an ionic NaCl/KCl solution, the compatible osmolyte sorbitol, and the readily permeant urea. High salt and sorbitol were found to activate similar molecular pathways, including the p38 MAPK and the p53-p21(WAF1)-pRb axis, that were not stimulated by high urea. On the other hand, only high urea led to the phosphorylation of ERKs and JNKs. Furthermore, salt- and sorbitol-treated cells were able to phosphorylate histone H2A.X on Ser139, in contrast to cells exposed to urea, indicating a common mechanism for DNA repair, which was achieved by a p53-dependent activation of the G1 checkpoint by both solutes. DNA repair, as directly measured by a host cell reactivation assay, occurred under conditions of hyperosmolar salt and sorbitol, although to a lesser extent in sorbitol-treated cells than in cells exposed to high salinity. Taken as a whole, our findings suggest that the hyperosmolality-provoked DNA damage and the responses of nucleus pulposus cells induced by this genotoxic stress most probably originate from cell volume alterations mediated by hypertonicity and not from increased intracellular ionic concentration.     

Fertilization environment of the non-copulating marine sculpin, Hemilepidotus gilberti

To clarify the extracellular environment for external fertilization in the non-copulating marine sculpin Hemilepidotus gilberti, sperm motility was measured in NaCl, KCl, mannitol solutions, seawater, and ovarian fluid. Spermatozoa of H. gilberti actively moved in seminal plasma the moment they were removed from the genital papilla. Spermatozoa showed higher motility in NaCl solution at osmolalities between 300–400 mOsmol kg^-1. In KCl and in mannitol solutions, spermatozoa actively moved at osmolalities between 500 and 800 mOsmol kg^-1, and at osmolality 300 mOsmol kg ^-1, respectively. The ovarian fluid was a transparent and viscous gelatinous material, rich in sodium with an osmolality of 340 mOsmol kg^-1. Sperm motility in the ovarian fluid lasted more than 90 min, which was six times longer than in seawater. This sperm motility under conditions isotonic to body fluid is similar to that of copulating marine sculpins rather than to other non-copulating marine fishes. In addition, eggs of H. gilberti could be fertilized in the ovarian fluid. This suggests that external fertilization takes place under physiological conditions similar to the internal conditions of the ovary provided by the ovarian fluid, which isolates the eggs from sea water for several hours after spawning. This manner of fertilization is thought to be one of the evolutionary pre-adaptations allowing copulation among marine sculpins.     

Urea sensitizes mIMCD3 cells to heat shock-induced apoptosis: protection by NaCl

Urea, with NaCl, constitutes the osmotic gradient that allows water reabsorption in mammalian kidneys. Because NaCl induces heat shock proteins, we tested the responses to heat shock of mIMCD3 cells adapted to permissive urea and/or NaCl concentrations. We found that heat-induced cell death was stronger after adaptation to 250 mM urea. This effect was reversible, dose dependent, and, interestingly, blunted by 125 mM NaCl. Moreover, we have shown that urea-adapted cells engaged in an apoptotic pathway upon heat shock, as shown by DNA laddering. This sensitization is not linked to a defect in the heat shock response, because the induction of HSP70 was similar in isotonic and urea-adapted cells. Moreover, it is not linked to the presence of urea inside cells, because washing urea away did not restore heat resistance and because applying urea and heat shock at the same time did not lead to heat sensitivity. Together, these results suggest that urea modifies the heat shock response, leading to facilitated apoptosis.     

High osmolality activates the G1 and G2 cell cycle checkpoints and affects the DNA integrity of nucleus pulposus intervertebral disc cells triggering an enhanced DNA repair response

Nucleus pulposus intervertebral disc cells experience a broad range of physicochemical stimuli in their native environment including osmotic fluctuations. Here we show that hyperosmotic treatment reduced nucleus pulposus cells’ proliferation by activating the G2 and G1 cell cycle checkpoints. p38 MAPK was found to participate in the manifestation of the G2 arrest under conditions of increased osmolality, since inhibition of its activity by SB203580 released the cells from G2 phase into mitosis. High osmolality resulted in the ATM-mediated phosphorylation of p53 on Ser15, the up-regulation of p21^WAF1 and the hypophosphorylation of the retinoblastoma protein in accordance to the observed G1 arrest. siRNA knocking down of p53 inhibited the expression of p21^WAF1 while maintaining the hyperphosphorylated form of the retinoblastoma protein and thus abrogated the G1 arrest observed under hyperosmotic conditions. Comet assay revealed that high osmolality provoked DNA damage to nucleus pulposus cells. Several previous reports have shown that renal cells become unable to sense and repair DNA damage under conditions of increased osmolality. On the contrary, nucleus pulposus cells residing within a hyperosmotic environment clearly preserved their ability to sense newly introduced DNA damage, as confirmed by the reactivation of p53 by ionizing radiation, retained the MRN complex in the nucleus and phosphorylated H2A.X on Ser139. H2A.X phosphorylation was attenuated in cells persistently experiencing hyperosmotic stress which, combined with the concurrent reduction in comet tails’ length, indicated an active DNA repair machinery. Even more, when the DNA repair efficiency of nucleus pulposus cells was directly measured by a host cell reactivation of luciferase activity assay, it was found to be significantly increased under hyperosmotic pressure. Finally, p53 depletion of nucleus pulposus cells by siRNA enhanced and prolonged H2A.X phosphorylation, attributing to p53 a regulatory role in the DNA repair pathway induced by increased osmolality.     

Inactivation of Cdc7 kinase in mouse ES cells results in S-phase arrest and p53-dependent cell death

Cdc7-related kinases play essential roles in the initiation of yeast DNA replication. We show that mice lacking murine homologs of Cdc7 (muCdc7) genes die between E3.5 and E6.5. We have established a mutant embryonic stem (ES) cell line lacking the muCdc7 genes in the presence of a loxP-flanked transgene expressing muCdc7 cDNA. Upon removal of the transgene by Cre recombinase, mutant ES cells cease DNA synthesis, arresting growth with S-phase DNA content, and generate nuclear Rad51 foci, followed by cell death with concomitant increase in p53 protein levels. Inhibition of p53 leads to partial rescue of muCdc7(-/-) ES cells from cell death. muCdc7(-/-)p53(-/-) embryos survive up to E8.5, and their blastocysts generate inner cell mass of a significant size in vitro, whereas those of the muCdc7(-/-)p53(+/-) embryos undergoes complete degeneration. These results demonstrate that, in contrast to cell cycle arrest at the G(1)/S boundary observed in yeasts, loss of Cdc7 in ES cells results in rapid cessation of DNA synthesis within S phase, triggering checkpoint responses leading to recombinational repair and p53-dependent cell death.     

Effect of osmotic immobilization on refrigerated storage and cryopreservation of sperm from a viviparous fish, the green swordtail Xiphophorus helleri

In this study, refrigerated storage and cryopreservation of sperm from the green swordtail Xiphophorus helleri were investigated. Previous cryopreservation research in this species utilized motile sperm because unlike in most fish species, Xiphophorus sperm can remain continuously motile after collection for a week with refrigerated storage. However, this species reproduces by internal fertilization, and given the significant requirements for motility within the female reproductive tract and potential limitations on sperm energetic capacities, immobilization of sperm prior to insemination could be used to improve fertilization success. Thus, the goal in this study was to use osmotic pressure to inhibit the motility of sperm after collection from X. helleri, and to test the effect of immobilization on refrigerated storage and cryopreservation. The objectives were to: (1) estimate the motility of sperm at different osmotic pressures, and determine an osmotic pressure suitable for immobilization; (2) cryopreserve the immobilized sperm, and estimate the motility after thawing with or without dilution, and (3) compare motility of non-immobilized and immobilized sperm after thawing, centrifugation, and washing to remove cryoprotectant. Motility was determined when sperm were suspended in 11 different osmotic pressures (24-500 mOsmol/kg) of Hanks' balanced salt solution (HBSS). Motility was observed between 116 and 425 mOsmol/kg. Sperm were not motile when the osmolality was lower than 116 or higher than 425 mOsmol/kg. Motility of the immobilized (non-motile) sperm could be activated by changing the osmotic pressure to 291-316 mOsmol/kg, and motility of immobilized sperm from hypertonic HBSS (425 mOsmol/kg) was significantly higher than that from hypotonic HBSS (145 mOsmol/kg) after 48 h of storage. At an osmolality of 500 mOsmol/kg, HBSS was used as extender to maintain immobilized sperm during cryopreservation with glycerol as the cryoprotectant. High motility (approximately 55%) was obtained in sperm after thawing when cryopreserved with 10-15% glycerol, and dilution of thawed sperm in fresh HBSS (1:4; V:V) was found to decrease the motility significantly. No difference was found in the motility of thawed sperm cryopreserved with 14% glycerol and extended in 310 and 500 mOsmol/kg HBSS. Washing by centrifugation prolonged the motility of thawed sperm from 24 to 72 h in HBSS at 310 and 500 mOsmol/kg. This study showed that sperm from X. helleri could be immobilized by use of specific osmotic pressures, and that the immobilization did not affect sperm motility after thawing. The immobilization of sperm by osmotic pressure could minimize reduction of the energetic capacities necessary for insemination, traversal, and residence within the female reproductive tract, and fertilization.     

Proteolytic cleavage of human p53 by calpain: a potential regulator of protein stability.

The p53 tumor suppressor protein is activated in cells in response to DNA damage and prevents the replication of cells sustaining genetic damage by inducing a cell cycle arrest or apoptosis. Activation of p53 is accompanied by stabilization of the protein, resulting in accumulation to high levels within the cell. p53 is normally degraded through the proteasome following ubiquitination, although the mechanisms which regulate this proteolysis in normal cells and how the p53 protein becomes stabilized following DNA damage are not well understood. We show here that p53 can also be a substrate for cleavage by the calcium-activated neutral protease, calpain, and that a preferential site for calpain cleavage exists within the N terminus of the p53 protein. Treatment of cells expressing wild-type p53 with an inhibitor of calpain resulted in the stabilization of the p53 protein. By contrast, in vitro or in vivo degradation mediated by human papillomavirus E6 protein was unaffected by the calpain inhibitor, indicating that the stabilization did not result from inhibition of the proteasome. These results suggest that calpain cleavage plays a role in regulating p53 stability.     

Constitutive mdmx expression during cell growth, differentiation, and DNA damage.

The mdmx gene was shown to possess high homology to the mdm-2 gene and to encode a protein that can bind p53 and block p53 transactivation. Because Mdm-2 protein blocks the growth-suppressive activity of the p53 tumor-suppressor protein through similar activities, we examined the expression patterns of mdmx to determine how MdmX expression correlates with p53 protein levels. In this study, the expression pattern and protein levels of mdmx were examined in a number of cell culture systems. Like mdm-2, mdmx gene expression was constitutive during serum deprivation/restimulation of murine fibroblasts and differentiation of either murine teratocarcinoma or preadipocyte cells. In contrast, whereas mdm-2 gene expression was induced after cisplatin damage to ovarian carcinoma cells, mdmx expression remained constitutive. Because p53 transactivation is critical following a genotoxic stress, we examined p53:MdmX complexes after in vitro DNA-PK phosphorylation, a posttranslational modification that blocks p53 association with Mdm-2. The DNA-PK phosphorylation of p53 was capable of inhibiting p53:MdmX association. Thus, whereas DNA damage does not regulate mdmx mRNA levels, posttranslational modifications induced during DNA damage may block p53:MdmX association in vivo. These results demonstrate that, in the cell lines examined, mdmx gene expression remains constitutive during cell proliferation and differentiation or following DNA damage. Taken together, the data suggest that cells retain a constant level of MdmX. Thus, in undamaged cells, there exists the potential for an MdmX:p53 reservoir.     

Characterization and purification of a mammalian osmoregulatory protein, aldose reductase, induced in renal medullary cells by high extracellular NaCl

GRB-PAP1 is a continuous line of epithelial cells derived from a rabbit renal inner medulla. Elevation of the NaCl concentration in the medium bathing these cells strongly induced the expression of a soluble protein with an apparent molecular mass of 39 kDa. The protein, purified by affinity chromatography with Amicon Matrex Gel Orange A, had enzyme activity characteristic of aldose reductase (alditol:NADPH+ oxidoreductase, EC 1.1.1.21). Goat antiserum against this purified aldose reductase selected the 39-kDa band from immunoblots of cells grown in a medium containing high NaCl. When the osmolality of the medium was increased by adding NaCl, the amount of aldose reductase protein and the aldose reductase activity increased together from very low to sustained high levels over several days. The aldose reductase protein was more than 10% of the soluble cell protein when cells were propagated in medium made hyperosmotic by adding NaCl to increase medium osmolality to 600 mosm.kg-1.     

Effects of changes in plasma hepatic-portal and systemic osmolality on plasma concentrations of arginine vasopressin in conscious newborn calves

Systemic plasma concentrations of arginine vasopressin (AVP) were studied in three groups of 10-15 day-old conscious newborn calves. Animals in the first group (control group) and in the second group (systemic-hypertonic-injected group) received respectively isotonic and hypertonic (8 mmol NaCl/kg body weight) saline injection into the right jugular vein. Animals in the third group were fitted with chronic mesenteric and hepatic-portal catheters and received a 1 h-hypertonic saline infusion (2 mmol NaCl/kg body weight) into the main mesenteric vein. In animals in the second group there were parallel increases in systemic plasma concentration of Na+ (from 148.0 +/- 2.6 to 177 +/- 8 mmol/l; P less than 0.01), osmolality (from 289 +/- 2 to 319 +/- 4 mOsmol/kg H2O; P less than 0.01) and systemic plasma concentrations of AVP (from 4.2 +/- 0.4 to 11.1 +/- 0.6 pmol/l; P less than 0.01) 10 min after the injection. There were no significant changes in control animals. Hypertonic saline infusion into the main mesenteric vein in the third group induced an increase in concentration of Na+ (from 147.3 +/- 2.0 to 165.0 +/- 5.0 mmol/l; P less than 0.01) and osmolality (from 288 +/- 5 to 315 +/- 10 mOsmol/kg H2O; P less than 0.01) in hepatic-portal vein plasma but did not alter systemic plasma osmolality or concentrations of Na+ and AVP. This study demonstrates that the relationship between plasma concentrations of AVP and systemic osmolality is operative in the newborn calf but does not support the hypothesis that hepatic portal osmo-receptors sensitive to hyperosmolality influence AVP release.     

Effects of osmolality and ions on the motility of stripped and testicular sperm of freshwater-and seawater-acclimated tilapia, Oreochromis mossambicus

A significantly higher concentration of testicular spermatozoa was obtained from freshwater Oreochromis mossambicus (9·9×10⁹ spermatozoa ml⁻¹) than seawater O. mossambicus (4·6×10⁹ spermatozoa ml⁻¹). The mean osmolality of the urine of freshwater fish (78·5 mOsmol kg⁻¹) was significantly different from that of seawater fish (304·8 mOsmol kg⁻¹). The mean length of the mid-piece of the spermatozoa together with the tail was more variable in freshwater O. mossambicus (8·80±0·23μm) than in seawater specimens (8·27±0·18 μm). Stripped sperm of freshwater O. mossambicus was highly contaminated by urine which was a good activator of sperm motility in O. mossambicus held in both fresh and sea water. The osmolality for initiation of motility in freshwater O. mossambicus spermatozoa was from 0 to 333 mOsmol kg⁻¹ while for seawater O. mossambicus spermatozoa it was from 0 to 1022 mOsmol kg⁻¹. The optimum osmolality for motility was from 70 to 333 mOsmol kg⁻¹ for freshwater O. mossambicus spermatozoa and from 333 to 645 mOsmol kg⁻¹ for seawater fish. In freshwater O. mossambicus spermatozoa, the presence of 20 mM CaCl₂ increased the permissive osmolality of NaCl from 184 to 645 mOsmol kg⁻¹. For seawater O. mossambicus spermatozoa, solutions of NaCl devoid of CaCl₂ were unable initiate motility, but the addition of 1·5 to 30 mM CaCl₂ to the NaCl solution (0–934 mOsmol kg₁) had a full motility initiating effect.     

Effects of elevated pCO2 and osmolality on growth of CHO cells and production of antibody-fusion protein B1: a case study

Partial pressure of CO2 (pCO2) and osmolality as high as 150 mmHg and 440 mOsm/kg, respectively, were observed in large-scale CHO cell culture producing an antibody-fusion protein, B1. pCO2 and osmolality, when elevated to high levels in bioreactors, can adversely affect cell culture and recombinant protein production. To understand the sole impact of pCO2 or osmolality on CHO cell growth, experiments were performed in bench-scale bioreactors allowing one variable to change while controlling the other. Elevating pCO2 from 50 to 150 mmHg under controlled osmolality (about 350 mOsm/kg) resulted in a 9% reduction in specific cell growth rate. In contrast, increasing osmolality resulted in a linear reduction in specific cell growth rate (0.008 h(-1)/100 mOsm/kg) and led to a 60% decrease at 450 mOsm/kg as compared to the control at 316 mOsm/kg. This osmolality shift from 316 to 445 mOsm/kg resulted in an increase in specific production rates of lactate and ammonia by 43% and 48%, respectively. To elucidate the effect of high osmolality and/or pCO2 on the production phase, experiments were conducted in bench-scale bioreactors to more closely reflect the pCO2 and osmolality levels observed at large scale. Increasing osmolality to 400-450 mOsm/kg did not result in an obvious change in viable cell density and product titer. However, a further increase in osmolality to 460-500 mOsm/kg led to a 5% reduction in viable cell density and a 8% decrease in cell viability as compared to the control. Final titer was not affected as a result of an apparent increase in specific production rate under this increased osmolality. Furthermore, the combined effects from high pCO2 (140-160 mmHg) and osmolality (400-450 mOsm/kg) caused a 20% drop in viable cell density, a more prominent decrease as compared to elevated osmolality alone. Results obtained here illustrate the sole effect of high pCO2 (or osmolality) on CHO cell growth and demonstrate a distinct impact of high osmolality and/or pCO2 on production phase as compared to that on growth phase. These results are useful to understand the response of the CHO cells to elevated pCO2 (and/or osmolality) at a different stage of cultivation in bioreactors and thus are valuable in guiding bioreactor optimization toward improving protein production.