The lack of effect of glucosamine sulphate on aggrecan mRNA expression and (35)S-sulphate incorporation in bovine primary chondrocytes

Glucosamine and glucosamine sulphate have been promoted as a disease-modifying agent to improve the clinical symptoms of osteoarthritis. The precise mechanism of the action of the suggested positive effect of glucosamine or glucosamine sulphate on cartilage proteoglycans is not known, since the level of glucosamine in plasma remains very low after oral administration of glucosamine sulphate. We examined whether exogenous hexosamines or their sulphated forms would increase steady-state levels of aggrecan and hyaluronan synthase (HAS) or glycosaminoglycan synthesis using Northern blot and (35)S-sulphate incorporation analyses. Total RNA was extracted from bovine primary chondrocytes which were cultured either in 1 mM concentration of glucosamine, galactosamine, mannosamine, glucosamine 3-sulphate, glucosamine 6-sulphate or galactosamine 6-sulphate for 0, 4, 8 and 24 h, or in three different concentrations (control, 100 microM and 1 mM) of glucosamine sulphate salt or glucose for 24 or 72 h. Northern blot assay showed that neither hexosamines nor glucosamine sulphate salt stimulated aggrecan and HAS-2 mRNA expression. Glycosaminoglycan synthesis remained at a control level in the treated cultures, with the exception of mannosamine which inhibited (35)S-sulphate incorporation in low-glucose DMEM treatment. In our culture conditions, hexosamines or their sulphated forms did not increase aggrecan expression or (35)S-sulphate incorporation.     

The lack of effect of glucosamine sulphate on aggrecan mRNA expression and 35S-sulphate incorporation in bovine primary chondrocytes

Glucosamine and glucosamine sulphate have been promoted as a disease-modifying agent to improve the clinical symptoms of osteoarthritis. The precise mechanism of the action of the suggested positive effect of glucosamine or glucosamine sulphate on cartilage proteoglycans is not known, since the level of glucosamine in plasma remains very low after oral administration of glucosamine sulphate. We examined whether exogenous hexosamines or their sulphated forms would increase steady-state levels of aggrecan and hyaluronan synthase (HAS) or glycosaminoglycan synthesis using Northern blot and ³⁵S-sulphate incorporation analyses. Total RNA was extracted from bovine primary chondrocytes which were cultured either in 1 mM concentration of glucosamine, galactosamine, mannosamine, glucosamine 3-sulphate, glucosamine 6-sulphate or galactosamine 6-sulphate for 0, 4, 8 and 24 h, or in three different concentrations (control, 100 μM and 1 mM) of glucosamine sulphate salt or glucose for 24 or 72 h. Northern blot assay showed that neither hexosamines nor glucosamine sulphate salt stimulated aggrecan and HAS-2 mRNA expression. Glycosaminoglycan synthesis remained at a control level in the treated cultures, with the exception of mannosamine which inhibited ³⁵S-sulphate incorporation in low-glucose DMEM treatment. In our culture conditions, hexosamines or their sulphated forms did not increase aggrecan expression or ³⁵S-sulphate incorporation.     

Physiological effects of saline waters on zander

Rapid transfer of zander Stizostedion lucioperca to hypoosmotic brackish water (mean osmolality 230 mOsmol kg–1 , c. 8 psu) significantly increased plasma chloride concentrations after 24 h compared to those transferred to fresh water, although plasma osmolality was not significantly affected. After 6 days, plasma osmolality was slightly elevated but stable plasma glucose and cortisol concentrations and blood haematocrit and haemoglobin suggest a lack of hormonal stress responses and resultant secondary effects. Rapid transfer of zander to a more saline environment, hyperosmotic to plasma (mean osmolality 462 mOsmol kg^‐1, c. 16 psu) induced a greater increase in plasma osmolality and chloride concentrations within 24 h, with a further rise after 6 days exposure, but all fish maintained a state of hypo‐osmoregulation both 24 h and 6 days after transfer. The initial osmotic disturbance (at 24 h) was accompanied by increased plasma glucose, blood haematocrit and haemoglobin and a decreased mean cell haemoglobin concentration (MCHC), suggesting an adrenergic stress response, but these parameters fully recovered within 6 days of exposure to this hyperosmotic environment with MCHC rising to exceed the level in freshwater fish. Zander did not survive rapid transfer to more hyperosmotic conditions (750 or 1001 mOsmol kg^‐1, 26‐35 psu), but they did survive exposure to simulated‘tidal cycles’ of rising and declining salinity, peaking after 6 h at c. 29 or 33 psu. Although osmotic disturbance was apparent after 6 h exposure and other physiological parameters suggested both adrenergic and corticosteroid components of a stress response, rapid recovery was apparent after return to fresh water. The results indicate that the zander, a non‐indigenous species in the U.K., has a high level of osmotic tolerance and a degree of hypo‐osmoregulation in saline environments not found in most stenohaline freshwater teleosts. This osmoregulatory ability could enable invasion of new U.K. river systems by using inshore marine environments of low salinity as saltwater bridges.     

Regulation of matrix synthesis rates by the ionic and osmotic environment of articular chondrocytes

Chondrocytes in cartilage are embedded in a matrix containing a high concentration of proteoglycans and hence of fixed negative charges. Their extracellular ionic environment is thus different from that of most cells, with extracellular Na⁺ being 250–350 mM and extracellular osmolality 350–450 mOsm. When chondrocytes are isolated from the matrix and incubated in standard culture medium (DMEM; osmolality 250–280 mOsm), their extracellular environment changes sharply. We incubated isolated bovine articular chondrocytes and cartilage slices in DMEM whose osmolity was altered over the range 250–450 mOsm by Na⁺ or sucrose addition. ³⁵S-sulphate and ³H-proline incorporation rates were at a maximum when the extracellular osmolality was 350–400 mOsm for both freshly isolated chondrocytes and for chondrocytes in cartilage. The incorporation rate per cell of isolated chondrocytes was only 10% that of chondrocytes in situ both 4 and 24 hours after isolation. For freshly isolated chondrocytes, the rate increased 30–50% in DMEM to which NaCl or sucrose had been added to the increase osmolality. In chondrocytes incubated overnight in DMEM, the rate was greatest in DMEM of normal osmolality and fell from the maximum in proportion to the change in osmolality. The effects of surcrose addition on incorporation rates were similar but not identical to those of Na⁺ addition. Changes in cell volume might be linked to changes in synthesis rates since the cell volume of chondrocytes (measured by Coulter-counter) increased 30–40% when the cells are removed from their in situ environment into DMEM. Synthesis rates can thus be partly regulated by changes in extracellular osmolality, which in cartilage is controlled by proteoglycan concentration. This provides a mechanism by which the chondrocytes can rapidly respond to changes in extracellular matrix composition. © 1993 Wiley-Liss, Inc.     

Proline enhances <Emphasis Type="Italic">Torulopsis glabrata</Emphasis> growth during hyperosmotic stress

This study investigated that the importing of compatible solute proline could enhance the growth of the yeast Torulopsis glabrata under hyperosmotic stress. Osmolarity progressively increased from 860 to 2,603 mOsmol/kg by accumulation of sodium pyruvate in the culture broth, leading to a significant decrease in cell growth. When 1.0 g/L of proline as a compatible solute was added to the culture medium, it was imported and enhanced cell growth by 59.0% at 2,603 mOsmol/kg. By addition of proline during pyruvate production, the concentration, productivity, and yield of pyruvate increased 22.1, 38.4, and 14.3%, respectively. These results suggested that T. glabrata can import proline as an osmoprotectant against high osmotic stress, thus enhance pyruvate productivity. The improvement of yeast growth and viability under hyperosmotic stress by the addition of proline provided an alternative approach to enhance the organic acids production by yeast.     

Competing regulation of matrix biosynthesis by mechanical and IGF-1 signalling in elderly human articular cartilage in vitro

This study investigates the separate and combined effects of IGF-1 and mechanical loads on chondrocytes in elderly human femoral head articular cartilage. Full depth biopsies of articular cartilage were subjected to either no load, static or cyclic (2 s on/2 s off) loading in unconfined compression at a stress of 1 MPa for 48 h with or without IGF-1 (300 ng ml(-1)). Chondrocyte biosynthetic activity was measured using 35S-sulphate and 3H-leucine during the last 24 h of loading. IGF-1 alone increased the rates of isotope incorporation, by 80% for 35S-SO4 and 40% for 3H-leucine, whereas loading alone reduced matrix biosynthesis. Applying load (cyclic or static) in the presence of IGF-1 returned the incorporation rates to their unstimulated levels. This study suggests elderly human articular cartilage is responsive to stimulation by IGF-1 but mechanical factors seem to act sufficiently strongly in the opposite direction to cancel this response.     

The effects of osmotic stress on the viscoelastic and physical properties of articular chondrocytes.

The metabolic activity of chondrocytes in articular cartilage is influenced by alterations in the osmotic environment of the tissue, which occur secondary to mechanical compression. The mechanism by which osmotic stress modulates cell physiology is not fully understood and may involve changes in the physical properties of the membrane or the cytoskeleton. The goal of this study was to determine the effect of the osmotic environment on the mechanical and physical properties of chondrocytes. In isoosmotic medium, chondrocytes exhibited a spherical shape with numerous membrane ruffles. Normalized cell volume was found to be linearly related to the reciprocal of the extracellular osmolality (Boyle van't Hoff relationship) with an osmotically active intracellular water fraction of 61%. In deionized water, chondrocytes swelled monotonically until lysis at a mean apparent membrane area 234 +/- 49% of the initial area. Biomechanically, chondrocytes exhibited viscoelastic solid behavior. The instantaneous and equilibrium elastic moduli and the apparent viscosity of the cell were significantly decreased by hypoosmotic stress, but were unchanged by hyperosmotic stress. Changes in the viscoelastic properties were paralleled by the rapid dissociation and remodeling of cortical actin in response to hypoosmotic stress. These findings indicate that the physicochemical environment has a strong influence on the viscoelastic and physical properties of the chondrocyte, potentially through alterations in the actin cytoskeleton.     

Regulatory volume decrease (RVD) by isolated and in situ bovine articular chondrocytes

Articular chondrocytes in vivo are exposed to a changing osmotic environment under both physiological (static load) and pathological (osteoarthritis) conditions. Such changes to matrix hydration could alter cell volume in situ and influence matrix metabolism. However the ability of chondrocytes to regulate their volume in the face of osmotic perturbations have not been studied in detail. We have investigated the regulatory volume decrease (RVD) capacity of bovine articular chondrocytes within, and isolated from the matrix, before and following acute hypotonic challenge. Cell volumes were determined by visualising fluorescently-labelled chondrocytes using confocal laser scanning microscopy (CLSM) at 21 degrees C. Chondrocytes in situ were grouped into superficial (SZ), mid (MZ), and deep zones (DZ). When exposed to 180mOsm or 250mOsm hypotonic challenge, cells in situ swelled rapidly (within approximately 90 sec). Chondrocytes then exhibited rapid RVD (t(1/2) approximately 8 min), with cells from all zones returning to approximately 3% of their initial volume after 20 min. There was no significant difference in the rates of RVD between chondrocytes in the three zones. Similarly, no difference in the rate of RVD was observed for an osmotic shock from 280 to 250 or 180mOsm. Chondrocytes isolated from the matrix into medium of 380mOsm and then exposed to 280mOsm showed an identical RVD response to that of in situ cells. The RVD response of in situ cells was inhibited by REV 5901. The results suggested that the signalling pathways involved in RVD remained intact after chondrocyte isolation from cartilage and thus it was likely that there was no role for cell-matrix interactions in mediating RVD.     

Osmotic loading of in situ chondrocytes in their native environment

Changes in the osmotic environment cause changes in volume of isolated cells and cells in tissue explants, and the osmotic environment becomes hypotonic in cartilage diseases such as osteoarthritis (OA). However, it is not known how cells respond to a hypotonic osmotic challenge when situated in the fully intact articular cartilage. A confocal laser scanning microscope was used to image chondrocytes of intact rabbit patellae in an isotonic (300 mOsm) and hypotonic (172 mOsm) immersion medium. Cell volumes were calculated before and 5, 15, 60, 120 and 240 minutes after the change in saline concentration. Local tissue strains and swelling of the entire tissue were estimated from the relative movements of cells and displacements of single cells, respectively. Cell volumes increased rapidly (< or = 5 minutes, p<0.05) by approximately 22%, after which they remained constant for an hour (p>0.05). However, two and four hours post the hypotonic challenge, cell volumes were statistically greater (p<0.05) than those at all earlier time points, and swelling of the entire tissue continued throughout the four hour loading period. The results of our study suggest that osmotic loading induced volume changes of in situ chondrocytes in their native environment occur quickly and continue for hours. Understanding the behaviour of cells in their native environment provides novel insigth into the cell mechanics in ostearthritic joints and so may help understand the onset and progression of this disease.     

Hyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cells

Biopharmaceuticals are predominantly produced by Chinese hamster ovary (CHO) cells cultivated in fed‐batch mode. Hyperosmotic culture conditions (≥ 350 mOsmol kg^∑1) resulting from feeding of nutrients may enhance specific product formation rates (q_p). As an improved ATP supply was anticipated to enhance q_p this study focused on the identification of suitable miRNA/mRNA targets to increase ATP levels. Therefor next generation sequencing and a compartment specific metabolomics approach were applied to analyze the response of an antibody (mAB) producing CHO cell line upon osmotic shift (280 → 430 mOsmol kg^‐1). Hyperosmotic culture conditions caused a ～2.6‐fold increase of specific ATP formation rates together with a ～1.7‐fold rise in cytosolic and mitochondrial ATP‐pools, thus showing increased ATP supply. mRNA expression analysis identified several genes encoding glycosylated proteins with strictly tissue related function. In addition, hyperosmotic culture conditions induced an upregulation of miR‐132‐3p, miR‐132‐5p, miR‐182, miR‐183, miR‐194, miR‐215‐3p, miR‐215‐5p which have all been related to cell cycle arrest/proliferation in cancer studies. In relation to a previous independent CHO study miR‐183 may be the most promising target to enhance q_p by stable overexpression. Furthermore, deletion of genes with presumably dispensable function in suspension growing CHO cells may enhance mAB formation by increased ATP levels.     

Reliability of the use of fructose 1-phosphate to detect Hunter cells in fibroblast-cultures of obligate carriers of the Hunter syndrome

Pulse-chase experiments measuring 35S-sulphate incorporation into acid mucopolysaccharides were performed in the presence and absence of fructose 1-phosphate on fibroblasts obtained from one skin-biopsy of 25 obligate Hunter carriers. The presence of fructose 1-phosphate significantly increased the accumulation of 35S-labelled acid mucopolysaccharides in fibroblast cultures of 23 obligate Hunter carriers. In one carrier, the accumulation of labelled acid mucopolysaccharides was significantly increased prior to the addition of fructose 1-phosphate, and in one of the 25 obligate carriers the 35S-sulphate incorporation was normal in the presence as well as in the absence of fructose 1-phosphate. Similar experiments performed on mixtures of Hunter cells and normal cells revealed that 20% Hunter cells should be present to obtain a significantly increased difference in between the incorporation in the presence and in the absence of fructose 1-phosphate. Fructose 1-phosphate had no effect on the accumulation of labelled mucopolysaccharides in fibroblast-cultures of seven women with no family history of mucopolysaccharidosis. The present results show that pulse-chase experiments measuring 35S-sulphate incorporation into fibroblasts, cultured in the presence of fructose 1-phosphate, can identify Hunter carriership, provided that the accumulation is normal prior to the addition of fructose 1-phosphate. Furthermore, 35S-sulphate incorporation in the absence of fructose 1-phosphate, higher than mean +4SD of normal control-fibroblasts indicates carriership.     

Diagnosis of Hunter's syndrome carriers; Radioactive sulphate incorporation into fibroblasts in the presence of fructose 1-phosphate

Summary Mutual correction of co-cultivated fibroblasts from patients with Hunter's and Hurler's syndrome could be inhibited by either fructose 1-phosphate or mannose 6-phosphate. In the presence of fructose 1-phosphate a 50% mixture of fibroblasts from a patient with Hunter's syndrome and a normal homozygous individual showed an increased³⁵S-sulphate incorporation into acid mucopolysaccharides. When fibroblast cultures from one obligate and two possible carriers of Hunter's syndrome were tested for³⁵S-sulphate incorporation, the cultures showed either twice the normal³⁵S-sulphate incorporation into acid mucopolysaccharides in the presence of fructose 1-phosphate or an abnormally high incorporation in the presence as well as in the absence of the sugar phosphate.     

Regulatory volume increase (RVI) by in situ and isolated bovine articular chondrocytes

Metabolism of the matrix by chondrocytes is sensitive to alterations in cell volume that occur, for example, during static loading and osteoarthritis. The ability of chondrocytes to respond to changes in volume could be important, and this study was aimed at testing the hypothesis that chondrocytes can regulate their volume following cell shrinking by regulatory volume increase (RVI). We used single cell fluorescence imaging of in situ bovine articular chondrocytes, cells freshly isolated into 280 or 380 mOsm, or 2-D cultured chondrocytes loaded with calcein or fura-2, to investigate RVI and changes to [Ca2+]i during shrinkage. Following a 42% hyperosmotic challenge, chondrocytes rapidly shrunk, however, only approximately 6% of the in situ or freshly isolated chondrocytes demonstrated RVI. This contrasted with 2D-cultured chondrocytes where approximately 54% of the cells exhibited RVI. The rate of RVI was the same for all preparations. During the 'post-RVD/RVI protocol', approximately 60% of the in situ and freshly isolated chondrocytes demonstrated RVD, but only approximately 5% showed RVI. There was no relationship between [Ca2+]i and RVI either during hyperosmotic challenge, or during RVD suggesting that changes to [Ca2+]i were not required for RVI. Depolymerisation of the actin cytoskeleton by latrunculin, increased RVI by freshly isolated chondrocytes, in a bumetanide-sensitive manner. The results showed that in situ and freshly isolated articular chondrocytes have only limited RVI capacity. However, RVI was stimulated by treating freshly isolated chondrocytes with latrunculin B and following 2D culture of chondrocytes, suggesting that cytoskeletal integrity plays a role in regulating RVI activity which appears to be mediated principally by the Na+ - K+ -2Cl- cotransporter.     

Response of articular chondrocytes to pituitary fibroblst growth factor (FGF)

Rabbit chondrocytes from pooled articular joints have been delineated by their time of attachment of culture flasks after initiation of primary monolayer culture, either attached (48-AT) or floating (48-F) after 48 hours. A general population of chrondrocytes (attached after 72 hours, 72-AT) was also studied. The growth-promoting activity of pituitary fibroblast growth factor (FGF) and its effect on sulfated-proteoglycan synthesis was studied on each chondrocyte population in secondary monolayer culture. ³H-thymidine incorporation during a 1-hour pulse was stimulated by FGF (100 ng/ml) in each chondrocyte population. The response of AT-72 chondrocytes to FGF required an additional fetal bovine serum supplement, while 48-F cells resonded independent of serum. The response of 48-AT chondrocytes to FGF (100 ng/ml) during a 1-hour pulse with ³H-thymidine was increased in low serum (0.5–2.0%) rather than when high serum (8–10%) was present in the culture medium. FGF reduced ³⁵SO₄ incorporation into sulfated-proteoglycans in the 48-AT and 48-F chondrocyte populations, but not in the 72-AT population. The reduction in ³⁵SO₄ incorporation in the 48-AT and 48-F chondrocytes was not characterized by alterations in the hydrodynamic size of the sulfated-proteoglycans as measured by Sepharose CL-2B chromatography nor by changes in the types of sulfated-glycosaminoglycans produced. These results indicated that FGF produced quantitative rather than qualitative alterations in chondrocyte sulfated-proteoglycan synthesis. The latter appears uncoupled from the growth-promoting activity of FGF on chondrocytes.     

The role of BKCa channels on hyperpolarization mediated by hyperosmolarity in human articular chondrocytes

Chondrocytes, the only cell in cartilage, are subjected to hyperosmotic challenges continuously since extracellular osmolarity in articular cartilage increases in response to mechanical loads during joint movement. Hyperosmolarity can affect membrane transport, and it is possible that load modulates matrix synthesis through alterations in intracellular composition. In the present study, the effects of hyperosmotic challenges were evaluated using the whole-cell patch clamp technique, whole cell mode on freshly isolated human and bovine articular chondrocytes. In human chondrocytes, hypertonicity induced the activation of outward Ca(2+)-sensitive K(+) currents, which were inhibited by iberiotoxin and TEA-Cl. The current induced by hypertonic switching (osmolarity from 300 to 400 mOsm/l) caused cell hyperpolarization (from -39 mV to -70 mV) with a reversal potential of -96 ± 7 mV. These results suggest a role for Ca(2+)-activated K(+) channels in human articular chondrocytes, leading to hyperpolarization as a consequence of K(+) efflux through these channels. These channels could have a role in the articular chondrocyte's response to a hyperosmotic challenge and matrix metabolism regulation by load.     

D(+)catechin enhances heparan sulphate content in rat liver

Administration of (D+) catechin (100 mg/kg body wt) to rats resulted in an increase in the amount of total sulphated glycosaminoglycans (GAG) in liver. The increase was more pronounced in the case of heparan sulphate than chondroitin sulphate and dermatan sulphate. The liver slices prepared from catechin-treated rats showed a significant increase in the rate of incorporation of 35S-sulphate into GAG. Similarly there was a concentration-dependent increase in the rate of 35S-sulphate incorporation into GAG by normal liver slices in presence of catechin in vitro. Susceptibility to nitrous acid degradation and chondroitinase ABC digestion showed that more than 80% of the GAG labelled in vivo with 35S-sulphate, was heparan sulphate and about 10% chondroitin sulphate and dermatan sulphate. Gel filtration of the 35S-labelled material isolated from livers of normal and catechin-treated animals over sephacryl S-300 did not show any difference probably excluding the possibility of free GAG chains initiated on catechin or any of its metabolites in vivo. These results indicate that catechin stimulates the synthesis of sulphated GAG, particularly heparan sulphate in liver.     

Cartilage-derived factor (CDF) : II. Somatomedin-like action on cultured chondrocytes

Previously, we showed that fetal bovine cartilage contains a polypeptide that stimulates the incorporation of [³⁵S]sulfate into proteoglycans synthesized by rat and rabbit costal chondrocytes in culture. In this paper, we report that the cartilage-derived factor (CDF) increases not only [³⁵S]sulfate incorporation but also [³H]thymidine incorporation into rabbit chondrocytes in monolayer culture. The dose-response curve of CDF stimulation of DNA synthesis was similar in profile to that of CDF stimulation of proteoglycan synthesis. In addition, CDF markedly enhanced [³H]uridine incorporation into rabbit chondrocytes and significantly enhanced [³H]serine incorporation into total protein. These findings indicate that fetal bovine cartilage contains a factor that shows somatomedin-like activity in monolayer cultures of rabbit chondrocytes.     

Quantitation of autoradiographic grains in different zones of articular cartilage with image analyzer

A novel method is introduced for the estimation of grain numbers in autoradiographic sections of articular cartilage with an image analyzer. It is based on separation of grains from the underlying structures by gray level thresholding and determination of the percentage of total area occupied by grains in a relatively large measuring field. The mean grain size is used as a reference to calculate grain numbers per cell profile and per unit area of tissue in various zones of bovine articular cartilage labelled with 35S-sulphate in tissue culture. The results demonstrate considerable zonal differences as well as site related topographic variation in the rate of 35S-sulphate incorporation. The largest site-related variation in the grain counts was observed in the superficial zone, suggesting a delicate control of proteoglycan synthesis in this zone.     

Enhanced monoclonal antibody production by gradual increase of osmotic pressure

The time length required for the adaptation of AFP-27 hybridoma cells to high osmotic pressure and the effect of a gradual increase of osmotic pressure on monoclonal antibody production were investigated. When the cells were subjected to an increase of osmotic pressure from 300 mOsmol kg-1 to 366 mOsmol kg- 1, the intracellular content of osmoprotective free amino acids reached a maximum level 6 h after the osmotic pressure was increased to 366 mOsmol kg-1. The same time period of 6 h incubation at 366 mOsmol kg-1 was required to obtain a high growth rate of AFP-27 cells at 440 mOsmol kg-1 when the cells were subjected to a two-step increase of osmotic pressure from 300 mOsmol kg-1 to 366 mOsmol kg-1 and then to 440 mOsmol kg-1. The time length for the physiological adaptation of the cells to 366 mOsmol kg-1 was consequently estimated to be 6 h. Osmotic pressure during batch cultivation was gradually increased from 300 mOsmol kg-1 to 400 mOsmol kg-1 with an adaptation time of at least 6 h. The specific growth rates following a gradual increase of osmotic pressure were higher than those at a constant osmotic pressure of 400 mOsmol kg-1, while the specific monoclonal antibody production rate increased with the increase in the mean osmotic pressure. As a result, the cells grown under a gradual increase of osmotic pressure produced higher amounts of monoclonal antibodies than did those grown under constant osmotic pressure.