Osmolarity Of Medium Research Articles

Physiological osmolarities do not enhance long-term tissue synthesis in chondrocyte-laden degradable poly(ethylene glycol) hydrogels.

Encapsulating chondrocytes in synthetic and degradable hydrogels for cartilage tissue engineering enables tuning of scaffold degradation, but provides no biological cues. Culture medium that recapitulates the physiological osmolarity of the interstitial fluid in cartilage can enhance matrix synthesis in the short term, but long-term benefits remain to be determined. This study investigates the long-term effect of culture medium osmolarity on tissue synthesis using chondrocytes isolated from three skeletally mature bovine donors encapsulated in degradable poly(ethylene glycol) hydrogels. The cell-laden hydrogels were cultured up to 4 weeks in standard chondrocyte-specific medium (330 mOsm) or medium adjusted by sucrose or salts (NaCl and KCl) to reach a physiological osmolarity (400 mOsm). Neocartilaginous matrix synthesis and matrix catabolism were evaluated by quantitative and immunofluorescence methods. Hydrogel degradation kinetics of acellular constructs were not affected by medium osmolarity or osmolyte. Matrix composition was predominantly aggrecan and collagen type II for all conditions. One day after encapsulation, total collagen accumulated in the constructs was increased by 80-90% in 400 mOsm medium, regardless of osmolyte. However, this effect did not persist, and at 4 weeks, total collagen synthesized and released to the medium was more than three times higher in 330 mOsm medium. Medium osmolarity had minimal effects on sulfated glycosaminoglycan content and did not affect catabolic activity. These findings suggest that culture medium at physiological osmolarities may not be beneficial for long-term chondrocyte culture in degradable hydrogels, but that initially culturing chondrocytes at a higher osmolarity may enhance early tissue deposition.

How medium osmolarity influences dielectrophoretically assisted on-chip electrofusion

Cells submitted to an electric field gradient experience dielectrophoresis. Such a force is useful for pairing cells prior to electrofusion. The latter event is induced by the application of electric field pulses leading to membrane fusion while cells are in physical contact. Nevertheless, the efficiency of dielectrophoretic pairing and electrofusion of cells are highly dependent on medium properties (osmolarity and conductivity). In this paper, we examine the effect of medium osmolarity on volume, viability and electrical properties of cells. Then we characterize in real time the impact of electropermeabilization of cells on their dielectrophoretic response. To do so, a microfluidic device, inducing particular field topologies is used. These real time observations are correlated to numerical analysis of the Clausius–Mossotti factor. Taking into account the identified changes, an electrofusion protocol adequate to the optimal medium (100mOsm, 0.03S/m) is defined. Up to 75% simultaneous binuclear rapid electrofusions were achieved and monitored with average membrane fusion duration lower than 12s.

The self-assembly of a cyclic lipopeptides mixture secreted by a B. megaterium strain and its implications on activity against a sensitive Bacillus species.

Cyclic lipopeptides are produced by a soil Bacillus megaterium strain and several other Bacillus species. In this work, they are detected both in the Bacillus intact cells and the cells culture medium by MALDI-TOF mass spectrometry. The cyclic lipopeptides self-assemble in water media producing negatively charged and large aggregates (300–800 nm of mean hydrodynamic radius) as evaluated by dynamic light scattering and zeta-potential analysis. The aggregate size depends on pH and ionic strength. However, it is not affected by changes in the osmolarity of the outer medium suggesting the absence of an internal aqueous compartment despite the occurrence of low molecular weight phospholipids in their composition as determined from inorganic phosphorus analysis. The activity against a sensitive Bacillus cereus strain was evaluated from inhibition halos and B. cereus lysis. Essential features determining the antibiotic activity on susceptible Bacillus cereus cells are the preserved cyclic moiety conferring cyclic lipopeptides resistance to proteases and the medium pH. The aggregates are inactive per se at the pH of the culture medium which is around 6 or below. The knock out of the sensitive cells only takes place when the aggregates are disassembled due to a high negative charge at pH above 6.

In Vitro Tissue Regeneration by Human Degenerated Nucleus Pulposus Cells in Hyperosmotic Culture Medium

Introduction Healthy intervertebral disc (IVD) cells normally reside in a high osmolality environment of 450 to 550 mOsm/kg and extracellular matrix production by MSC cells has been shown to increase with increasing osmolality. However, it is not known how human degenerated disc cells are affected by osmolality. The aim of the current study was to determine the effects of osmolality on tissue regeneration by human degenerated NP cells. Materials and Methods The osmolality of standard chondrogenic culture medium (DMEM, 2% ascorbic acid-2 phosphate, 2% human serum albumin, 2% insulin-transferring selenium-X, 1% penicillin/streptomycin, 10 ng/mL transforming growth factor β2 [TGF-β2]) was adjusted with NaCl and sucrose from 340 to 400, 450, and 500 mOsm/kg. IVD cells from four human donors (Thompson grade III) were cultured for 28 days in high density (1*10^6 cells/cm2) on collagen II coated filters. LDH analysis was performed to analyze toxicity. Samples were stained with Safranin-O for glycosaminoglycans (GAGs) and with immunohistochemistry for collagen II. Gene expression was evaluated by qPCR and a collagen low-density array. Matrix content as reflected by glycosaminoglycan (GAG) production was measured with a dimethylmethylene blue (DMMB) assay and DNA content with a Picogreen assay. Statistical analysis included univariate analysis of variance with randomized block design and post hoc Dunnet t-test. Results No cytotoxic effects were found in any of the conditions tested. Gene expression showed an osmolality-dependent increase compared with standard medium in collagen II and aggrecan only when NaCl was used (see Fig. 1A, B for p-values). However, at the protein level, no effect of increased osmolality using NaCl was found. This could not be explained by MMP gene expression levels, as these seemed lower at high osmolality. Tissues cultured in the presence of the osmolyte sucrose appeared to contain less proteoglycans, but this difference was not significant. Conclusion In conclusion, in the current culture system, the increased expression of matrix genes with osmolality appears to be osmolyte dependent rather than osmolality dependent. Moreover, the absence of an effect at the protein level suggests adjusting osmolality with NaCl to enhance matrix production by IVD cells is not required. Disclosure of Interest None declared

Increased Osmolarity and Cell Clustering Preserve Canine Notochordal Cell Phenotype in Culture

Introduction Degeneration of the intervertebral disc (IVD) is associated with a loss of notochordal cells (NCs) from the nucleus pulposus (NP) and their replacement by chondrocyte-like cells. NCs have a stimulatory effect upon other cells, such as native IVD chondrocyte-like cells and mesenchymal stem cells, and therefore they are an interesting focus for regenerative tissue engineering strategies. However, optimal conditions, such as osmolarity and other characteristics of the culture media, for long-term culture of NCs are not known. The purpose of this study was to investigate the effects of different culture media and osmolarity on the physiology of NCs in vitro. Materials and Methods NC clusters isolated from canine IVDs were suspended in alginate beads and cultured at 37°C under normoxic conditions for 28 days. Three different culture conditions were investigated; (1) DMEM/F12 (300 mOsm/L), (2) α-MEM (300 mOsm/L), and (3) α-MEM adjusted to 400 mOsm/L to mimic a hyperosmolar environment. NC morphology, expression of genes related to NC markers, matrix production and remodeling, and DNA- and GAG analyses were performed on 1, 7, 14, and 28 days in culture. Data analysis were performed with the aid of a mixed linear model which incorporated fixed effects (i.e., “time in culture” 1, 7, 14, 21, and 28 days, and NP tissue), “culture condition” (DMEM/F12, α-MEM, and α-MEM 400 mOsm/L), and the interaction between these factors; in the random part of the mixed models, the factor “dog” (dogs 1-6) was incorporated to take the correlation within each dog into account. Benjamini-Hochberg correction was applied to correct for multiple comparisons. Results Large, vesicle-containing cells organized in clusters, characterized as NCs, remained present during 28 days for all culture conditions. However, the proportion of the NC clusters decreased over time, whereas the proportion of spindle-shaped cells increased. When comparing the different culture conditions, on day 28 in culture, the cell population cultured in α-MEM 400 mOsm/L appeared to contain more vacuolated cells (NCs) compared with the DMEM/F12 and α-MEM and hence fewer spindle and polygonal cells (Fig.). Gene expression profiling at 7, 14, and 28 days in culture compared with day 1 indicated an initial loss of NC phenotype followed by recovery of brachyury and aggrecan expression after 28 days of culture supporting a potential recovery of NC phenotype. NCs cultured in α-MEM adjusted to 400 mOsm/L showed the highest expression of brachyury, cytokeratin 18, and aggrecan, the highest GAG production, and the lowest collagen 1α1 gene expression. Conclusion In conclusion, NCs cultured in alginate in native cell clusters under normoxic conditions, partially retained their characteristic morphology and recovered their phenotype in long-term culture. In comparison to using DMEM/F12 (300 mOsm/L) culture medium, the use of α-MEM (300 mOsm/L) culture medium for culturing NCs has a beneficial effect on preserving the NC phenotype in culture, which is further facilitated by increasing the culture medium osmolarity to physiological levels (400 mOsm/L). This may be explained by the difference in composition of DMEM/F12 and α-MEM culture media: α-MEM contains a lower concentration of D-glucose (dextrose) than DMEM/F12 (5.5 vs. 17.5 mM, respectively). Since IVD cells are subject to low glucose concentrations in vivo, the relatively low glucose content in α-MEM may facilitate NCs to retain their phenotype. Moreover, hyperglycemia has been positively correlated with IVD degeneration in diabetic rats and recently it has been shown that a high glucose environment stimulates apoptosis and inhibits proliferation of NCs in vitro. Furthermore, preservation of the NC clusters may facilitate the NC physiology and the preserved extracellular matrix may participate in the presentation of growth factors to the cultured NCs. These findings provide additional information concerning the maintenance of NCs in vitro that may set the stage for further mechanistic inquiry into the biology of NCs and their potential role in designing new regenerative strategies. Acknowledgments This work was supported by AO Spine International, through an AOSpine Reserach Network grant (# 104982). Disclosure of Interest None declared

Abstract 37: Secretion of von Willebrand Factor by Endothelial Cells Links Salt to Hypercoagulability and Thrombosis

Hypercoagulability increases the risk of thrombi that cause cardiovascular events. Dehydration and hypernatremia are often accompanied by thrombosis, but the mechanisms are not clear. Von Willebrand Factor is secreted by endothelium, affecting aggregation of platelets and promoting activation of the coagulation cascade and formation of thrombi. Here we show that in culture of primary Human Umbilical Vein Endothelia Cells, elevating medium osmolality to 320-380 mosmol/kg by adding NaCl reversibly increases both vWF mRNA and vWF secretion. The high NaCl increases expression of tonicity regulated transcription factor NFAT5 and its binding to promoter of vWF gene, suggesting that vWF upregulation is caused by hypertonic signaling. To elevate NaCl in vivo, we modeled mild dehydration, subjecting mice to water restriction (WR) for 9 days by feeding them with gel food containing 30% of water. Such WR elevates blood sodium from 145.1±0.5 to 150.2±1.3 mmol/l and activates hypertonic signaling as evidenced from increased expression of NFAT5 in tissues. WR increased vWF mRNA in liver and lung and raised vWF protein in blood. Immunostaining of liver revealed increased production of vWF protein by endothelium and increased number of microthrombi inside capillaries. WR also increased blood level of D-dimer, a fibrinogen degradation product indicative of ongoing coagulation and thrombolysis. We conclude that elevation of extracellular sodium within the physiological range raises expression and secretion of von Willebrand Factor sufficiently to increase coagulability of blood and risk of thrombosis. The results suggest that hydration and salt intake are modifiable factors that affect coagulability and thrombosis through high salt-dependent secretion of vWF from endothelial cells.

Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensitivity to osmotic and freezing-induced injury, but respond favorably to dimethyl sulfoxide

We assessed the influences of medium osmolality, cryoprotectant and cooling and warming rate on maned wolf (Chrysocyon brachyurus) spermatozoa. Ejaculates were exposed to Ham’s F10 medium (isotonic control) or to this medium plus NaCl (350–1000mOsm), sucrose (369 and 479mOsm), 1M glycerol (1086mOsm) or dimethyl sulfoxide (Me2SO, 1151mOsm) for 10min. Each sample then was diluted back into Ham’s medium and assessed for sperm motility and plasma membrane integrity. Although glycerol and Me2SO had no influence (P>0.05), NaCl and sucrose solutions affected sperm motility (P<0.05), but not membrane integrity. Motility of sperm exposed to <600mOsm NaCl or sucrose was less (P<0.05) than fresh ejaculate, but comparable (P>0.05) to the control. As osmolality of the NaCl solution increased, motility decreased to <5%. In a separate study, ejaculates were diluted in Test Yolk Buffer containing 1M glycerol or Me2SO and cooled from 5°C to −120°C at −57.8°C, −124.2°C or −67.0°C/min, frozen in LN2, thawed in a water bath for 30s at 37°C or 10s at 50°C, and then assessed for motility, plasma- and acrosomal membrane integrity. Cryopreservation markedly (P<0.05) reduced sperm motility by 70% compared to fresh samples. Higher (P<0.05) post-thaw motility (20.0±1.9% versus 13.5±2.1%) and membrane integrity (51.2±1.7% versus 41.5±2.2%) were observed in samples cryopreserved in Me2SO than in glycerol. Cooling rates influenced survival of sperm cryopreserved in glycerol with −57.8°C/min being advantageous (P<0.05). The findings demonstrate that although maned wolf spermatozoa are similar to domestic dog sperm in their sensitivity to osmotic-induced motility damage, the plasma membranes tolerate dehydration, and the cells respond favorably to Me2SO as a cryoprotectant.

Endothelin, Ang II, vasopressin and osmolality regulate hyaluronan turnover by renomedullary interstitial cells suggesting an involvement of this matrix component in fluid handling (860.9)

The content of the matrix component hyaluronan (HA) change in the medullary interstitium in relation to hydration status in vivo: elevated during hydration, reduced during dehydration suggesting a role in fluid handling by affecting interstitial fluid flux. We examined the effect of osmolality and fluid regulating hormones on HA‐turnover by RMIC in culture. Reducing media osmolality to simulate hydration, increased supernatant HA 4‐fold. Cell HA synthase (HAS) 2 and Hyaluronidase (Hyal) 2 mRNA expressions were unaltered while Hyal 1 mRNA was reduced by 35%. Supernatant Hyal activity was reduced by 44%. An inhibitor of Hyal activity (Asc‐P) elevated HA to similar high levels as low osmolality. AngII reduced HA by 58% and combined treatment with vasopressin reduced HA even further (69%). HAS and Hyal mRNA were stable while Hyal activity increased by 38% by AngII and 67% by the combination. Low dose endothelin (ET, 10‐10M) increased HA 3‐fold which was inhibited by ET‐B receptor blocker BQ788. On the contrary, high dose ET (10‐6M) reduced HA by 47%. The ET‐A receptor antagonist BQ123 not only reversed the reducing effect of high dose ET on HA but elevated it to the same level as low dose ET suggesting importance of ET‐B receptors. The study demonstrates a regulatory influence of osmolality and fluid regulating hormones on HA turnover, supporting the concept of an involvement of medullary interstitial HA in renal fluid handling by affecting interstitial permeability.Grant Funding Source: Supported by the Swedish Research Council‐Medicine

Increased Osmolarity and Cell Clustering Preserve Canine Notochordal Cell Phenotype in Culture

Degeneration of the intervertebral disc (IVD) is associated with a loss of notochordal cells (NCs) from the nucleus pulposus (NP) and their replacement by chondrocyte-like cells. NCs are known to maintain extracellular matrix quality and stimulate the chondrocyte-like NP cells, making NCs attractive for designing new tissue engineering approaches for IVD regeneration. However, optimal conditions, such as osmolarity and other characteristics of the culture media, for long-term culture of NCs are not known. The purpose of this study was to investigate the effects of different culture media and osmolarity on the physiology of NCs in vitro. NC clusters isolated from canine IVDs were suspended in alginate beads and cultured at 37°C under normoxic conditions for 28 days. Three different culture conditions were investigated; (1) Dulbecco's modified Eagle's medium (DMEM)/F12 (300 mOsm/L), (2) α-MEM (300 mOsm/L), and (3) α-MEM adjusted to 400 mOsm/L to mimic a hyperosmolar environment. NC morphology, expression of genes related to NC markers, matrix production and remodeling, and DNA- and glycosaminoglycan (GAG) analyses were performed on 1, 7, 14, and 28 days in culture. Large, vesicle-containing cells organized in clusters, characterized as NCs, remained present during 28 days for all culture conditions. However, the proportion of the NC clusters decreased over time, whereas the proportion of spindle-shaped cells increased. Gene expression profiling at 7, 14, and 28 days in culture compared to day 1 indicated a initial loss of NC phenotype followed by some recovery of brachyury and aggrecan gene expression after 28 days of culture supporting a potential recovery of NC phenotype. NCs cultured in α-MEM adjusted to 400 mOsm/L showed the highest gene expression of brachyury, cytokeratin 18, and aggrecan, the highest GAG production, and the lowest collagen 1α1 gene expression. In conclusion, NCs cultured in alginate in native cell clusters, partially retained their characteristic morphology and recovered their phenotype in long-term culture. The type of culture medium and medium osmolarity appear to be important factors for culturing NC clusters. These findings provide additional information concerning the maintenance of NCs in vitro that may aid further mechanistic inquiry into the biology of NCs.

Effects of polyvinylpyrrolidone both as a binder and pore-former on the release of sparingly water-soluble topiramate from ethylcellulose coated pellets

Delivering sparingly water-soluble drugs from ethylcellulose (EC) coated pellets with a controlled-release pattern remains challenging. In the present study, hydrophilic polyvinylpyrrolidone (PVP) was used both as a binder and a pore-former in EC coated pellets to deliver sparingly water-soluble topiramate, and the key factors that influenced drug release were identified. When the binder PVP content in drug layers below 20% w/w was decreased, the physical state of topiramate changed from amorphous to crystalline, making much difference to drug solubility and dissolution rates while modifying the drug release profile from first-order to zero-order. In addition, without PVP in drug layering solution, drug layered particles were less sticky during layering process, thus leading to a shorter process and higher loading efficiency. Furthermore, PVP level as a pore-former in EC coating layers mainly governed drug release from the coated pellets with the sensitivity ranging from 23% to 29%. PVP leaching rate and water permeability from EC/PVP film increased with the PVP level, which was perfectly correlated with drug release rate. Additionally, drug release from this formulation was independent of pH of release media or of the paddle mixing speed, but inversely proportional to the osmolality of release media above the physiological range.

Effect of Nerve Growth Factor on the In Vitro Induction of Apoptosis of Human Conjunctival Epithelial Cells by Hyperosmolar Stress

To evaluate the effects of nerve growth factor (NGF), which is activated during inflammatory episodes of ocular diseases, on the apoptotic response in cultured human primary conjunctival epithelial cells (pHCECs). Levels of NGF transcripts and NGF protein in pHCEC grown in medium with normal osmolarity (307 mOsm/L) or hyperosmolar medium (350, 400, and 450 mOsm/L) were determined using RT-PCR or ELISA, respectively. To assess apoptosis, pHCEC were cultured in normal or 400 mOsm/L hyperosmolar medium with neutralizing anti-NGF antibody or recombinant human NGF for 6 hours before analysis by flow cytometry. Levels of Bcl-xL, Bax, phosphorylated JNK, and cleaved caspase-3 were detected using Western blotting. Levels of the inflammatory cytokine IL-6 was analyzed using ELISA. Hyperosmolar conditions increased NGF levels in cultured pHCECs. Hyperosmolarity and exposure to neutralizing anti-NGF antibody significantly increased the number of apoptotic cells. Addition of recombinant human NGF to 400 mOsm/L medium decreased the number of apoptotic cells, reduced the expression of phosphorylated JNK, Bax, and cleaved caspase-3 and increased the expression of Bcl-xL. Levels of IL-6 were increased by hyperosmotic stress but decreased by exposure to recombinant human NGF. Hyperosmolarity induces apoptosis of pHCECs by activating JNK signaling. Increased levels of NGF under hyperosmolar conditions may contribute, at least in part, to the reduced apoptosis of pHCECs and may be beneficial in recovering conjunctival damage due to chronic hyperosmolar stress.

TolC promotes ExPEC biofilm formation and curli production in response to medium osmolarity.

While a high osmolarity medium activates Cpx signaling and causes CpxR to repress csgD expression, and efflux protein TolC protein plays an important role in biofilm formation in Escherichia coli, whether TolC also responds to an osmolarity change to regulate biofilm formation in extraintestinal pathogenic E. coli (ExPEC) remains unknown. In this study, we constructed ΔtolC mutant and complement ExPEC strains to investigate the role of TolC in the retention of biofilm formation and curli production capability under different osmotic conditions. The ΔtolC mutant showed significantly decreased biofilm formation and lost the ability to produce curli fimbriae compared to its parent ExPEC strain PPECC42 when cultured in M9 medium or 1/2 M9 medium of increased osmolarity with NaCl or sucrose at 28°C. However, biofilm formation and curli production levels were restored to wild-type levels in the ΔtolC mutant in 1/2 M9 medium. We propose for the first time that TolC protein is able to form biofilm even under high osmotic stress. Our findings reveal an interplay between the role of TolC in ExPEC biofilm formation and the osmolarity of the surrounding environment, thus providing guidance for the development of a treatment for ExPEC biofilm formation.

Is contrast medium osmolality a causal factor for contrast-induced nephropathy?

The exact pathophysiology of contrast-induced nephropathy (CIN) is not fully clarified, yet the osmotic characteristics of contrast media (CM) have been a significant focus in many investigations of CIN. Osmotic effects of CM specific to the kidney include transient decreases in blood flow, filtration fraction, and glomerular filtration rate. Potentially significant secondary effects include an osmotically induced diuresis with a concomitant dehydrating effect. Clinical experiences that have compared the occurrence of CIN between the various classes of CM based on osmolality have suggested a much less than anticipated advantage, if any, with a lower osmolality. Recent animal experiments actually suggest that induction of a mild osmotic diuresis in association with iso-osmolar agents tends to offset potentially deleterious renal effects of high viscosity-mediated intratubular CM stagnation.

Chemical chaperone suppresses the antibody aggregation in CHO cell culture

Background Aggregation of therapeutic antibodies could be generated at different steps of the manufacturing process, posing the problem for quality control of produced antibodies. It has been well known that secreted antibodies from recombinant mammalian cells into culture medium can aggregate due to the physicochemical stresses such as media pH and osmolality, cultivation temperature [1,2]. The antibody aggregation during the cell culture process is difficult to suppress because the cell culture conditions for antibody production are generally optimized for cell culture and growth and not for suppressing the aggregate formation. Here we show the novel strategy to suppress the antibody aggregation; application of chemical chaperone to the cell culture process. It is well established that an addition of some cosolutes serves as chemical chaperone to suppress the protein aggregation. Trehalose, nonreducing sugar formed from two glucose units with a-1,1 linkage, is known as an effective chemical chaperone. In this study, we investigated the anti-aggregation effect of trehalose in the culture process of recombinant Chinese hamster ovary cell (CHO) line producing Ex3-humanized IgG-like bispecific single-chained diabody with Fc (Ex3scDb-Fc). Ex3-scDb-Fc shows the remarkable anti-tumor activity based on anti-EGFR and anti-CD3 bispecificity [3]. However, our in-house results showed that Ex3scDb-Fc shows aggregation tendency, demonstrating the necessity of developing a bioprocess for suppressing the aggregation of the bispecific diabody.

Quantifying SOCE fluorescence measurements in mammalian muscle fibres. The effects of ryanodine and osmotic shocks

We have quantified Ca(2+) entry through store operated calcium channels in mice muscle fibres, measuring the rates of change of myoplasmic [Ca(2+)], d[Ca(2+)](myo)/dt, and of Ca(2+) removal, d[Ca(2+)](Removal)/dt, turning store operated calcium entry (SOCE) ON, and OFF, by switching on or off external Ca(2+). In depleted fibres, poisoned with 10 μM cyclopiazonic acid SOCE influx was about 3 μM/s. Ryanodine (50 μM) caused a robust, nifedipine (50 μM) independent, increase in SOCE activation to 8.6 μM/s. Decreasing medium osmolarity from 300 to 220 mOsm/L, decreased SOCE to 0.9 μM/s, while increasing osmolarity from 220 to 400 mOsm/L potentiated SOCE to 43.6 μM/s. Ryanodine inhibited the effects of hypotonicity. Experiments using 2-aminoethoxydiphenyl borate, nifedipine, or Mn(2+) quenching, strongly suggest that the increased [Ca(2+)](myo) by ryanodine or hypertonic shock is mediated by potentiated SOCE activation. The Ca(2+) response decay, quantified by d[Ca(2+)](Removal)/dt, indicates a robust residual Ca(2+) removal mechanism in sarco-endoplasmic reticulum calcium ATPase poisoned fibres. SOCE high sensitivity to osmotic shocks, or to ryanodine receptor (RyR) binding, suggests its high dependency on the structural relationship between its molecular constituents, Orai1 and stromal interaction molecule and the sarcoplasmic reticulum and plasma membranes, in the triadic junctional region, where RyRs, are conspicuously present. This study demonstrates that SOCE machinery is highly sensitive to structural changes caused by binding of an agonist to its receptor or by imposed osmotical volume changes.

Amphipathic polymer-mediated uptake of trehalose for dimethyl sulfoxide-free human cell cryopreservation

For stem cell therapy to become a routine reality, one of the major challenges to overcome is their storage and transportation. Currently this is achieved by cryopreserving cells utilising the cryoprotectant dimethyl sulfoxide (Me2SO). Me2SO is toxic to cells, leads to loss of cell functionality, and can produce severe side effects in patients. Potentially, cells could be frozen using the cryoprotectant trehalose if it could be delivered into the cells at a sufficient concentration. The novel amphipathic membrane permeabilising agent PP-50 has previously been shown to enhance trehalose uptake by erythrocytes, resulting in increased cryosurvival. Here, this work was extended to the nucleated human cell line SAOS-2. Using the optimum PP-50 concentration and media osmolarity, cell viability post-thaw was 60±2%. In addition, the number of metabolically active cells 24h post-thaw, normalised to that before freezing, was found to be between 103±4% and 91±5%. This was found to be comparable to cells frozen using Me2SO. Although reduced (by 22±2%, p=0.09), the doubling time was found not to be statistically different to the non-frozen control. This was in contrast to cells frozen using Me2SO, where the doubling time was significantly reduced (by 41±4%, p=0.004). PP-50 mediated trehalose delivery into cells could represent an alternative cryopreservation protocol, suitable for research and therapeutic applications.

Accumulated Polymer Degradation Products as Effector Molecules in Cytotoxicity of Polymeric Nanoparticles

Polymeric nanoparticles (PNPs) are a promising platform for drug, gene, and vaccine delivery. Although generally regarded as safe, the toxicity of PNPs is not well documented. The present study investigated in vitro toxicity of poly-ε-caprolactone, poly(DL-lactic acid), poly(lactide-cocaprolactone), and poly(lactide-co-glycide) NPs and possible mechanism of toxicity. The concentration-dependent effect of PNPs on cell viability was determined in a macrophage (RAW 264.7), hepatocyte (Hep G2), lung epithelial (A549), kidney epithelial (A498), and neuronal (Neuro 2A) cell lines. PNPs show toxicity at high concentrations in all cell lines. PNPs were efficiently internalized by RAW 264.7 cells and stimulated reactive oxygen species and tumor necrosis factor-alpha production. However, reactive nitrogen species and interleukin-6 production as well as lysosomal and mitochondrial stability remained unaffected. The intracellular degradation of PNPs was determined by monitoring changes in osmolality of culture medium and a novel fluorescence recovery after quenching assay. Cell death showed a good correlation with osmolality of culture medium suggesting the role of increased osmolality in cell death.

Induction medium osmolality improves microspore embryogenesis in wheat and triticale

The objective of this study was to determine the effect of induction medium osmolality on embryogenesis and green plant production in wheat and triticale. Isolated microspores of wheat and triticale were subjected to a range of osmolality (300–500 mOsm kg−1) using mannitol. In both species, the maximum number of embryo-like structures (ELS) and green plants were obtained at 350 mOsm kg−1 when the induction medium was supplemented with 9.1 g L−1 of mannitol. A sharp decline in microspore response was observed at higher osmolality. These results demonstrate the effect of osmolality on induction of ELS and production of green plants indicating that the process of microspore embryogenesis can be improved in wheat and triticale by increasing osmolality of the induction medium to 350 mOsm kg−1.

ABCC1 Is Related to the Protection of the Distal Nephron against Hyperosmolality and High Sodium Environment: Possible Implications for Cancer Chemotherapy

AimsGlutathione (GSH) plays an important role in protecting cells against oxidative damage. ABCC1 protein transports GSH. Although this protein is largely studied in cancer, due to multidrug resistance phenotype, its role in the tubular cells of the kidney is unknown. The goal of this study was to find out whether ABCC1 has a role in protecting cells from the distal nephron against the stress caused by high medullar osmolality.Main MethodsMA104 cells were treated with high concentrations of sodium chloride, urea, or both to raise the osmolality of the culture medium. Cell viability was accessed by MTT and trypan blue assays. ABCC1 expression and extrusion of carboxi-fluorescein (CF), a fluorescent ABCC1 substrate, were measured by flow cytometry.Key FindingsIncubation of MA104 cells in a high sodium concentration medium resulted in changes in cell granularity and altered expression and activity of ABCC1. Urea did not alter ABCC1 expression or activity, but reversed the observed NaCl effects. High sodium concentrations also had a negative effect on cell viability and urea also protected cells against this effect.SignificanceOur findings demonstrate that ABCC1 plays a significant role in the protection of kidney epithelial cells against the stress caused by high sodium environment present in renal medulla.

Osmotic Stress Changes the Expression and Subcellular Localization of the Batten Disease Protein CLN3

Juvenile CLN3 disease (formerly known as juvenile neuronal ceroid lipofuscinosis) is a fatal childhood neurodegenerative disorder caused by mutations in the CLN3 gene. CLN3 encodes a putative lysosomal transmembrane protein with unknown function. Previous cell culture studies using CLN3-overexpressing vectors and/or anti-CLN3 antibodies with questionable specificity have also localized CLN3 in cellular structures other than lysosomes. Osmoregulation of the mouse Cln3 mRNA level in kidney cells was recently reported. To clarify the subcellular localization of the CLN3 protein and to investigate if human CLN3 expression and localization is affected by osmotic changes we generated a stably transfected BHK (baby hamster kidney) cell line that expresses a moderate level of myc-tagged human CLN3 under the control of the human ubiquitin C promoter. Hyperosmolarity (800 mOsm), achieved by either NaCl/urea or sucrose, dramatically increased the mRNA and protein levels of CLN3 as determined by quantitative real-time PCR and Western blotting. Under isotonic conditions (300 mOsm), human CLN3 was found in a punctate vesicular pattern surrounding the nucleus with prominent Golgi and lysosomal localizations. CLN3-positive early endosomes, late endosomes and cholesterol/sphingolipid-enriched plasma membrane microdomain caveolae were also observed. Increasing the osmolarity of the culture medium to 800 mOsm extended CLN3 distribution away from the perinuclear region and enhanced the lysosomal localization of CLN3. Our results reveal that CLN3 has multiple subcellular localizations within the cell, which, together with its expression, prominently change following osmotic stress. These data suggest that CLN3 is involved in the response and adaptation to cellular stress.