Effects of elevated pCO(2) and/or osmolality on the growth and recombinant tPA production of CHO cells

Carbon dioxide is a by-product of mammalian cell metabolism that will build up in culture if it is not removed from the medium. Increased carbon dioxide levels are generally not a problem in bench-top bioreactors, but inhibitory levels can easily be reached in large-scale vessels, especially if the aeration gas is enriched in oxygen. Due to the equilibrium attained between dissolved CO(2) and bicarbonate, increased pCO(2) is associated with increased osmolality in bioreactors with pH control. While a few preliminary reports indicate that elevated pCO(2) levels can inhibit cell growth and/or recombinant protein production, no comprehensive analysis of the interrelated effects of elevated pCO(2) and osmolality has been published. We have examined the effects of 140, 195, and 250 mm Hg (187, 260, and 333 mbar, respectively) pCO(2) (with and without osmolality control) on the growth of and recombinant tPA production by MT2-1-8 Chinese hamster ovary (CHO) cells. The effects of elevated osmolality were also investigated at the control pCO(2) of 36 mm Hg. Elevated pCO(2) at 310 mOsm/kg osmolality inhibited cell growth in a dose-dependent fashion, with a maximum decrease of 30% in the specific growth rate (mu) at 250 mm Hg. Osmolality alone had no effect on mu, but the combination of elevated pCO(2) and osmolality increased the maximal reduction in mu to 45%. Elevated pCO(2) at 310 mOsm/kg osmolality decreased the specific tPA production rate (q(tPA)) by up to 40% at 250 mm Hg. Interestingly, while increased osmolality decreased q(tPA) significantly at 140 mm Hg pCO(2), it had no effect or even increased q(tPA) at 195 and 250 mm Hg. (c) 1996 John Wiley & Sons, Inc.     

Hyperosmotic stress and elevated pCO2 alter monoclonal antibody charge distribution and monosaccharide content

Medium osmolality increases with pCO2 at constant pH. Elevated pCO2 and osmolality inhibit hybridoma growth to similar extents in both serum-containing and serum-free media. The combination of osmolality and elevated pCO2 synergizes to negatively impact cell growth. IgG2a glycosylation by hybridoma cells was evaluated under elevated pCO2 (to 250 mmHg pCO2) and/or osmolality (to 476 mOsm/kg). IgG2a site occupancy did not change significantly under any of the conditions studied, which is consistent with the robust glycosylation of other antibodies produced under various environmental stresses. However, changes were observed in the IgG2a charge distribution. Changes in the isoelectric point (pI) were greater under hyperosmotic stress, increasing by 0.32 and 0.41 pH units at 435 mOsm/kg in serum-containing and serum-free medium, respectively. Hyperosmotic stress also resulted in a concomitant increase in the heterogeneity of the charge distribution. The mean pI in serum-containing medium decreased by 0.16 pH units at 250 mmHg pCO2 when osmolality was controlled at 320 mOsm/kg but increased by 0.20 pH units when the osmolality increased with pCO2 (195 mmHg pCO2-435 mOsm/kg). In serum-free medium, elevated pCO2 did not alter pI, regardless of medium osmolality. In contrast to elevated osmolality at control pCO2, elevated pCO2 did not significantly alter the IgG2a charge heterogeneity under any of the conditions studied. The IgG2a was not sialylated, so sialylation changes were not responsible for changes in the charge distribution. IgG2a galactose content decreased with elevated osmolality, as a result of either elevated NaHCO3 or NaCl. However, when osmolality was controlled at elevated pCO2, the galactose content tended to increase. The mannose content decreased with increasing stress, while the fucose content remained relatively unchanged. It is likely that the observed increases in the pI of murine IgG2a were due to increased organellar pH, which is reflected by increased specific beta-galactosidase activity in the supernatant.     

Effects of NHE1 expression level on CHO cell responses to environmental stress

Ammonia, lactate and CO(2) inhibit animal cell growth. Accumulation of these metabolic byproducts also causes a decrease in intracellular pH (pH(i)). Transport systems regulate pH(i) in eukaryotic cells. Ion transporters have been cloned and overexpressed in cells but have not been examined for protection against the buildup of ammonia, lactate or CO(2). The Na(+)/H(+) exchangers (NHE) transport H(+) ions from cells during acidification to increase pH(i). We examined whether overexpression of NHE1 would provide CHO cells with greater protection from elevated ammonia, lactate or CO(2). NHE1 CHO cells were compared to MT2-1-8 ("normal" levels of NHE) and AP-1 (devoid of any NHE activity) CHO cell lines. Expression of at least "normal" levels of NHE1 is necessary for CHO cell survival during exposure to 30 mM lactic acid without pH adjustment or to 20 mM NH(4)Cl with pH adjustment. Resistance to an acute acid-load increased when NHE1 was overexpressed in CHO cells. Surprisingly, the inhibitory effect on cell growth at 195 mmHg pCO(2)/435 mOsm/kg (normal levels are 40 mmHg pCO(2)/ 320 mOsm/kg) was not affected by the NHE1 level. Also, there was no further decrease in CHO cell growth in the absence of NHE1 expression during elevated osmolality alone (up to 575 mOsm/kg).     

Dynamics of protein uptake within the adsorbent particle during packed bed chromatography

Elevated osmolality and pCO(2) have been shown to alter sialylation in a protein-specific manner. In Chinese hamster ovary (CHO)MT2-l-8 cells, tPA sialylation changed only slightly from 40 to 250 mm Hg pCO(2), whereas neural cell adhesion molecule polysialic acid (NCAM PSA) content decreased by up to 70% at 250 mm Hg pCO(2), pH 7.2. NCAM PSA content also decreased with increasing NaCl or NH(4)Cl concentration. This suggests that PSA content is a sensitive indicator of conditions that may alter glycosylation. Amino acids and their derivatives have been used to protect hybridoma and CHO cell growth under hyperosmotic stress. We examined the impact of osmoprotectants on NCAM PSA content in CHO MT2-1-8 cells under hyperosmolality (up to 545 mOsm/kg) and at 195 and 250 mm Hg pCO(2). NCAM PSA content at 545 mOsm/kg was at least two-fold greater in the presence of glycine betaine or L-proline compared to that without osmoprotectant. Surprisingly, in the presence of 20 mM glycine betaine, PSA levels were 50-60% of the control level for osmolalities ranging from 320 to 545 mOsm/kg. Thus, glycine betaine inhibits NCAM polysialylation at osmolalities below 435 mOsm/kg and is beneficial at higher osmolalities. In contrast to glycine betaine, L-proline increased PSA content by 25-120% relative to the unprotected culture at < or =545 mOsm/kg. The decrease in NCAM PSA levels of CHO MT2-1-8 cells cultured at 195 mm Hg pCO(2)-435 mOsm/kg was not mitigated by the presence of 25 mM glycine betaine, glycine, or L-threonine, even though all of these compounds enhanced cell growth. At 250 mm Hg pCO(2), all osmoprotectants tested (20 mM L-threonine, L-proline, glycine, or glycine betaine) increased NCAM polysialylation, with 20 mM glycine betaine restoring NCAM PSA to near control levels. Thus, osmoprotectants may (partially) offset changes in glycosylation, as well as the inhibition of growth, in cells under environmental stress. Supernatant beta-galactosidase levels, which increase upon alkalization of acidic organelles, did not differ significantly under elevated pCO(2) and hyperosmolality from that at control conditions.     

Effects of CO2 and osmolality on hybridoma cells: growth, metabolism and monoclonal antibody production

CO2 partial pressure (pCO2) in industrial cell culture reactors may reach 150–200 mm Hg, which can significantly inhibit cell growth and recombinant protein production. Due to equilibrium with bicarbonate, increased pCO2 at constant pH results in a proportional increase in osmolality. Hybridoma AB2-143.2 cell growth rate decreased with increasing pCO2 in well-plate culture, with a 45% decrease at 195 mm Hg with partial osmolality compensation (to 361 mOsm kg- 1). Inhibition was more extensive without osmolality compensation, with a 63% decrease in growth rate at 195 mm Hg and 415 mOsm kg-1. Also, the hybridoma death rate increased with increasing pCO2, with 31- and 64-fold increases at 250 mm Hg pCO2 for 401 and 469 mOsm kg- 1, respectively. The specific glucose consumption and lactate production rates were 40–50% lower at 140 mm Hg pCO2. However, there was little further inhibition of glycolysis at higher pCO2. The specific antibody production rate was not significantly affected by pCO2 or osmolality within the range tested. Hybridomas were also exposed to elevated pCO2 in continuous culture. The viable cell density decreased by 25–40% at 140 mm Hg. In contrast to the well-plate cultures, the death rate was lower at the new steady state at 140 mm Hg. This was probably due to higher residual nutrient and lower byproduct levels at the lower cell density (at the same dilution rate), and was associated with increased cell-specific glucose and oxygen uptake. Thus, the apparent effects of pCO2 may vary with the culture system. VMdZ and RK contributed equally to the results in this article. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of hybridoma cell responses to elevated pCO(2) and osmolality: intracellular pH, cell size, apoptosis, and metabolism.

CO(2) partial pressure (pCO(2)) in industrial cell culture reactors may reach 150-200 mm Hg, which can significantly inhibit cell growth and recombinant protein production. The inhibitory effects of elevated pCO(2) at constant pH are due to a combination of the increases in pCO(2) and [HCO(-) (3)], per se, and the associated increase in osmolality. To decouple the effects of pCO(2) and osmolality, low-salt basal media have been used to compensate for this associated increase in osmolality. Under control conditions (40 mm Hg-320 mOsm/kg), hybridoma cell growth and metabolism was similar in DMEM:F12 with 2% fetal bovine serum and serum-free HB GRO. In both media, pCO(2) and osmolality made dose-dependent contributions to the inhibition of hybridoma cell growth and synergized to more extensively inhibit growth when combined. Elevated osmolality was associated with increased apoptosis. In contrast, elevated pCO(2) did not increase apoptotic cell death. Specific antibody production also increased with osmolality although not with pCO(2). In an effort to understand the mechanisms through which elevated pCO(2) and osmolality affect hybridoma cells, glucose metabolism, glutamine metabolism, intracellular pH (pHi), and cell size were monitored in batch cultures. Elevated pCO(2) (with or without osmolality compensation) inhibited glycolysis in a dose-dependent fashion in both media. Osmolality had little effect on glycolysis. On the other hand, elevated pCO(2) alone had no effect on glutamine metabolism, whereas elevated osmolality increased glutamine uptake. Hybridoma mean pHi was approximately 0.2 pH units lower than control at 140 mm Hg pCO(2) (with or without osmolality compensation) but further increases in pCO(2) did not further decrease pHi. Osmolality had little effect on pHi. Cell size was smaller than control at elevated pCO(2) at 320 mOsm/kg, and greater than control in hyperosmotic conditions at 40 mm Hg.     

Selected amino acids protect hybridoma and CHO cells from elevated carbon dioxide and osmolality

Elevated pCO(2) inhibits cell growth. This growth inhibition is accompanied by a decrease in intracellular pH (pHi), as well as a decrease in glycolysis. Elevated concentrations (mM) of some amino acids have been shown by others to protect cells exposed to two very different environmental stresses: nutrient starvation and hyperosmolality. The fact that many of the amino acids shown to have protective effects against other stresses are transported into the cell through a pHi-sensitive transporter led us to study the possibility of using these amino acids as protective agents under elevated pCO(2). Screening experiments using 5, 15, and 25 mM of each amino acid showed that not all amino acids that protect cells from hyperosmolality protect them from elevated pCO(2). Glycine betaine and glycine were chosen for further characterization in both hybridoma and CHO cells. Asparagine and threonine were also tested in hybridoma and CHO cells, respectively. All amino acids tested under 195 mm Hg pCO(2)/435 mOsm/kg (50% growth inhibition) restored the specific growth rate (mu) in hybridoma cells to that observed under control conditions (40 mm Hg/320 mOsm/kg). Addition of each amino acid resulted in an increase in the consumption rate and intracellular accumulation of that amino acid. In CHO cells, glycine betaine also restored mu to control values, while glycine and threonine partially restored mu. In hybridoma cells, the higher specific antibody productivity obtained at elevated pCO(2) was maintained with the lowest amino acid concentration (5 mM). Productivity decreased toward control values with increasing amino acid concentrations. Elevated pCO(2) decreased the specific tPA productivity in the CHO cell line studied. Only glycine betaine resulted in a 20% increase in productivity at 195 mm Hg/435 mOsm/kg. With the exception of glycine betaine in hybridoma cells, amino acids did not mitigate the associated pHi decrease of at least 0.2 pH units at 195 mm Hg/435 mOsm/kg. pHi in hybridoma cells under elevated pCO(2) in the presence of glycine betaine was about 0.1 pH units below that of control. Amino acids had no effect on the cell size response of hybridoma cells, while they partially offset the increase in CHO cell size at elevated pCO(2). Glycine betaine, asparagine, and glycine increased the specific glucose consumption rate observed at 195 mm Hg/435 mOsm/kg (50% of control) to values greater than 70% of control in hybridoma cells. In CHO cells, only glycine betaine increased q(glc) (by 20%) under elevated pCO(2). All amino acids tested improved the cell yield from glutamine at 195 mm Hg/435 mOsm/kg in both cell lines.     

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.     

Identifying inhibitory threshold values of repressing metabolites in CHO cell culture using multivariate analysis methods

Elevation of lactate, ammonia, osmolality, and carbon dioxide to inhibitory levels was reported to have adverse effects on cell growth and protein productivity in mammalian cell culture. Multivariate analysis methods were used to investigate the roles of these repressing metabolites in a fed-batch CHO cell culture for antibody fusion protein B1 (B1) production. Principal Factor Analysis methodology was applied to manufacturing-scale data of 112 cell culture runs, which identified threshold values of four repressing metabolites as follows: (1) ammonium levels above 5.1 mM inhibit cell growth; (2) both lactate and osmolality levels above 58 mM and 382 mOsm/kg affect cell viability; and (3) carbon dioxide levels at or above 111 mmHg reduce protein quality. These threshold values were then verified by simulations using Monod-type equations and Canonical Correlation. These results suggest that adverse effects on cell growth, productivity, and product quality may be minimized under the ideal cell culture condition, in which the peak values of all four repressing metabolites are maintained below the threshold values. This strategy was evaluated in 45 cell culture runs in 50-L bioreactors. Eight out of 45 runs were operated under the ideal condition, while the remaining 37 runs had at least one repressing metabolite with peak value at or above the threshold. In comparison to the remaining runs, the eight cell culture runs under the ideal condition had 17%, 40%, and 11% higher values in peak viable cell density, final B1 titer, and quality attribute, respectively. The unique methodology used in this study may be generally applicable in characterizing cell culture processes.     

Dependence on glucose limitation of the pCO2 influences on CHO cell growth, metabolism and IgG production

The culture levels of glucose and CO(2) have been reported to independently have important influences on mammalian cell processes. In this work the combined effects of glucose limitation and CO(2) partial pressure (pCO(2)) on monoclonal antibody (IgG) producing Chinese Hamster Ovary cells were investigated in a perfusion reactor operated with controlled cell specific medium feed rate, pH and osmolality. Under high glucose conditions (14.3 +/- 0.8 mM), the apparent growth rate decreased (from 0.021 to 0.009 h(-1)) as the pCO(2) increased to approximately 220 mmHg, while the cell specific IgG productivity was almost unchanged. The lactate yield from glucose was not affected by pCO(2) up to approximately 220 mmHg and glucose was mainly converted to lactate. A feed medium modification from high (33 mM) to low (6 mM) glucose resulted in <0.1 mM glucose in the culture. As a result of apparently shifting metabolism towards the conversion of pyruvate to CO(2), both the ratio of lactate to glucose and the alanine production rate were lowered (1.51-1.14 and 17.7-0.56 nmol/10(6) cells h, respectively). Interestingly, when the pCO(2) was increased to approximately 140 mmHg, limiting glucose resulted in 1.7-fold higher growth rates, compared to high glucose conditions. However, at approximately 220 mmHg pCO(2) this beneficial effect of glucose limitation on these CHO cells was lost as the growth rate dropped dramatically to 0.008 h(-1) and the IgG productivity was lowered by 15% (P < 0.01) relative to the high glucose condition. The IgG galactosylation increased under glucose- limited compared to high-glucose conditions.     

Increased CHO cell fed-batch monoclonal antibody production using the autophagy inhibitor 3-MA or gradually increasing osmolality

Modulating autophagy provides a new method to increase CHO cell protein production. A fed-batch protocol using the autophagy inhibitor 3-methyl adenine (3-MA), developed for a tissue-plasminogen activator (t-PA) expressing DHFR based CHO cell line, was successfully adapted to a monoclonal antibody (MAb) expressing CHOK1-SV based CHO cell line. By optimizing the timing and dose of 3-MA treatment, the cell-specific productivity was increased 4-fold, resulting in 2-fold increased total MAb production. The positive effect of the 3-MA treatment appeared to be reduced when the amino acid feed concentration was increased 5-fold. Further investigation revealed that by slowly increasing osmolality up to ∼450 mOsm/kg, both the cell-specific productivity and the total MAb almost doubled. This effect was replicated with a DUXB-based CHO cell line expressing a human–llama chimeric antibody. The positive effect of gradually increasing osmolality was then combined with the positive effects of the 3-MA treatment, however their combined effect were not additive. Thus, either increased osmolality or 3-MA treatment were equally effective in increasing MAb-CHO cell fed-batch production on the cell lines tested. Analysis of protein glycosylation showed that both of these fed-batch modifications did not substantially influence the overall glycan profiles of the MAb product.     

Considerations for osmolality measurement under elevated pCO(2): comparison of vapor pressure and freezing point osmometry

Osmolality increases with pCO(2) in bioreactors with pH control, and it has been shown that osmolality compensation by decreasing the basal NaCl concentration partially mitigates the adverse effects of elevated pCO(2) on animal cell growth, protein production, and glycosylation. Thus, measurement of osmolality is important for a complete characterization of the culture environment under elevated pCO(2). However, osmolality measurement may be compromised by CO(2) evolution. Freezing point depression and vapor pressure depression osmometry were directly compared for the measurement of osmolality in samples at elevated pCO(2) (up to 250 mmHg) and at a variety of pH values (6.7-7.5). More extensive degassing may be expected with the vapor pressure osmometer due to the smaller sample volume and larger surface area employed. However, both types of osmometer yielded similar results for all pCO(2) and pH values studied. Moreover, the measured values agreed with osmolality values calculated using a semi-empirical model. Further analysis showed that, while sample degassing may result in a large decrease in pCO(2), there is little associated decrease in osmolality. The great majority of total CO(2) in solution is present as bicarbonate (HCO(3)(-)). Although a small amount of HCO(3)(-) is converted to CO(2) to compensate for CO(2) evolution, further depletion of HCO(3)(-) is inhibited by the associated increase in medium pH and by the need for HCO(3)(-) to maintain charge neutrality in solution. This explanation is consistent with the observed similarity in osmolality values for the two types of osmometer. It was also observed that osmolality did not change in samples that were frozen at -20 degrees C for up to 1 year.     

Responses of GS-NS0 Myeloma cells to osmolality: Cell growth, intracellular mass metabolism, energy metabolism, and antibody production

The influence of osmolality on growth, metabolism, and antibody production of mammalian cells has been widely reported in the past. However, more information about the responses of GS-NS0 Myeloma cells to osmolality, especially regarding the intracellular mass and energy metabolism, has not been available in detail. Fed-batch cultures started at different osmolalities in the range of 280∼370 mOsm/kg were designed to investigate the effects. As the osmolality and cell status changed during the process, cell performance was evaluated in the comparable periods with similar growth rates, nutrition concentrations, and relatively consistent environments. Metabolic flux analysis indicated most of extra consumed glucose at higher osmolalities flowed into lactate formation pathway. The proportion of glucose flux flowed into glycolysis pathway remained approximately 90% and the need of glucose for biomass synthesis was constantly. Also, more than 88% of the glutamine was used in biomass synthesis and the absolute flux remained constant. The specific consumption rate of glutamine declined significantly when cells were cultured in hypo-osmolality (276 mOsm/kg) and a portion of glutamine was synthesized from glutamate. Furthermore, cells were in the state of high energy production at osmolality of 276 mOsm/kg. More glucose flowed into TCA circle with the high efficiency of energy production to meet the demand. Thus, the IVC, the specific antibody production rate, and maximal antibody concentration in fed-batch culture started at 280 mOsm/kg decreased by 35, 36, and 48% compared to those in the culture started at 330 mOsm/kg.     

Effect of osmotic pressure on the production of retroviral vectors: Enhancement in vector stability

The use of Moloney murine leukaemia virus (MoMLV) derived retroviral vectors in gene therapy requires the production of high titer preparations. However, obtaining high titers of infective MoMLV retroviral vectors is difficult due to the vector inherent instability. In this work the effect of the cell culture medium osmotic pressure upon the virus stability was studied. The osmolality of standard medium was raised from 335 up to 500 mOsm/kg using either ionic (sodium chloride) or non-ionic osmotic agents (sorbitol and fructose). It was observed that, independently of the osmotic agent used, the infectious vector inactivation rate was inversely correlated with the osmolality used in the production media; therefore, the use of high medium osmolalities enhanced vector stability. For production purposes a balance must be struck between cell yield, cell productivity and retroviral stability. From the conditions tested herein sorbitol addition, ensuring osmolalities between 410 and 450 mOsm/kg, yields the best production conditions; NaCl hampered the viral infectious production while fructose originates lower cell yields. Lipid extractions were performed for cholesterol and phospholipid analyses showing that more stable viral vectors had a 10% reduction in the cholesterol content. A similar reduction in cholesterol was observed in the producer cells. A detailed analysis of the major phospholipids composition, type and fatty acid content, by mass spectrometry did not show significant changes, confirming the decrease in the cholesterol to phospholipids ratio in the viral membrane as the major reason for the increased vector stability.     

A pilot study on post-thawing quality of Iberian red deer spermatozoa (epididymal and electroejaculated) depending on glycerol concentration and extender osmolality

The optimization of cryopreservation extenders is a fundamental issue for adequately performing germplasm banking on wild species. We have tested two glycerol concentrations (4 and 8%), and three extender osmolalities (320, 380 and 430 mOsm/kg; before adding cryoprotectants), for cryopreservation of epididymal and ejaculated sperm samples from Iberian red deer. All the extenders were based on Tes-Tris and fructose (for osmolality adjustment), and complemented with 20% egg yolk. Epididymal and ejaculated sperm samples were obtained from the cauda epididymis (post-mortem) and using electroejaculation, respectively. Samples were diluted 1:1 with each extender and equilibrated for 2 h at 5 degrees C. Then, they were diluted down to 100x10(6) sperm/mL and frozen at -20 degrees C/min. Post-thawed samples were assessed for motility (CASA), HOS test, proportion of swollen (osmotically challenged) cells in the untreated sample, viability and acrosomal status. For epididymal samples, 8% glycerol rendered a slightly higher proportion of intact acrosomes on viable spermatozoa than 4%; regarding extender osmolality, 380 and 430 mOsm/kg rendered higher motility results, and the 430 mOsm/kg yielded the lowest proportion of swollen spermatozoa. For ejaculated samples, 4% glycerol yielded more viable spermatozoa than 8%; for extender osmolality, 320 mOsm/kg rendered the highest percentages of progressively motile and viable spermatozoa, although 380 mOsm/kg extender was not significantly different. These results show that sample source influences extender suitability, and that extenders should be isoosmotic or rather slightly hyperosmotic. Future studies should test multiple glycerol concentrations and extender osmolalities in order to adjust them to these kinds of sample.     

Effects of nitrogen source, calcium concentration and osmotic stress on protoplasts and protoplast-derived cell cultures of Pinus Pinaster cotyledons

The experiments described emphasize the effects of several factors crucial to the maintenance of cell divisions leading to increased cell numbers in suspension and colony formation from cotyledon protoplasts of Pinus Pinaster Ait. Osmotic potential of the incubation and culture media are critical. Reducing the osmolality from 680 mOsm kg H₂O⁻¹ during protoplast isolation to 610 mOsm kg H₂O⁻¹ during washing and culture was essential to achieve a high frequency of cell division. Survival of the cells beyond 3 weeks of culture occurs only if the calcium concentration is decreased from 5.6 m M to 1.5 m M . Glutamine as sole source of nitrogen shortens the lag phase of response of the protoplasts and increases their plating efficiency. After 6 weeks of culture, a combination of low osmolality (225 mOsm kg H₂O⁻¹) and high level of glutamine (40 m M ) is a prerequisite for obtaining actively growing cell suspensions.     

The role of osmotic resistance on equine spermatozoal function

Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. The goal of this experiment was to determine the osmotic tolerance levels of equine sperm by analyzing motility, viability, mitochondrial membrane potential (MMP), and mean cell volume (MCV). Spermatozoa were incubated at 22 degrees C for 10 min in isosmolal TALP (300 mOsm/kg), or a range of anisosmolal TALP solutions (75-900 mOsm/kg), for initial analysis, and then returned to isosmolal conditions for 10 min for further analysis. Total sperm motility was lower (P < 0.05) in anisosmolal conditions compared to sperm motility in control medium. When cells were returned to isosmolal conditions, only sperm previously incubated in 450 mOsm/kg TALP were able to recover to control levels of motility. Sperm viability and MMP were lower (P < 0.05) when exposed to hypotonic solutions in comparison to control solutions. Sperm suspensions that were returned to isosmolal conditions from 75, 150, and 900 mOsm/kg had lower (P < 0.05) percentages of viable sperm than control suspensions (300 mOsm/kg). MMP was lower (P < 0.05) in cells previously incubated in 75 and 900 mOsm/kg when returned to isosmolal, as compared to control cells. MCV differed (P < 0.05) from control cell volume in all anisosmolal solutions. Cells in all treatments were able to recover initial volume when returned to isosmolal medium. Although most spermatozoa are able to recover initial volume after osmotic stress, irreversible damage to cell membranes may render some sperm incapable of fertilizing an oocyte following cryopreservation.     

A novel osmolality-shift fermentation strategy for improving acarbose production and concurrently reducing byproduct component C formation by Actinoplanes sp. A56

Component C (Acarviosy-1,4-Glc-1,1-Glc) was a highly structural acarbose analog, which could be largely formed during acarbose fermentation process, resulting in acarbose purification being highly difficult. By choosing osmolality level as the key fermentation parameter of acarbose-producing Actinoplanes sp. A56, this paper successfully established an effective and simplified osmolality-shift strategy to improve acarbose production and concurrently reduce component C formation. Firstly, the effects of various osmolality levels on acarbose fermentation were firstly investigated in a 50-l fermenter. It was found that 400–500 mOsm/kg of osmolality was favorable for acarbose biosynthesis, but would exert a negative influence on the metabolic activity of Actinoplanes sp. A56, resulting in an obviously negative increase of acarbose and a sharp formation of component C during the later stages of fermentation (144–168 h). Based on this fact, an osmolality-shift fermentation strategy (0–48 h: 250–300 mOsm/kg; 49–120 h: 450–500 mOsm/kg; 121–168 h: 250–300 mOsm/kg) was further carried out. Compared with the osmolality-stat (450–500 mOsm/kg) fermentation process, the final accumulation amount of component C was decreased from 498.2 ± 27.1 to 307.2 ± 9.5 mg/l, and the maximum acarbose yield was increased from 3,431.9 ± 107.7 to 4,132.8 ± 111.4 mg/l.