Reducing Culture Temperature Research Articles

Systematic study of microzooplankton in mass culture of the green microalga Scenedesmus acuminatus and quantitative assessment of its impact on biomass productivity throughout a year

The green microalgae Scenedesmus spp. can grow rapidly and produce significant amounts of protein or lipid. However, frequent microzooplankton contamination leading to reduced biomass productivity has hindered the microalgae commercialization. Here, a comprehensive investigation into harmful microzooplankton species in mass cultures of a commercially promising species Scenedesmus acuminatus were conducted throughout the year. Twenty-five microzooplankton species were identified, with the amoeba Vannella sp. and the ciliate Vorticella convallaria being the most harmful to algal cells. The results indicated that it was the harmful grazers, rather than the overall microzooplankton diversity, led to culture deterioration and reduced biomass yield. Increasing the concentration of algal inoculants or reducing culture temperature during hot summer days were found to be effective in mitigating the impact of these harmful grazers. The findings will contribute to the best management protocol for monitoring and controlling the harmful microzooplankton in mass cultures of S. acuminatus.

Reduced culture temperature attenuates oxidative stress and inflammatory response facilitating expansion and differentiation of adipose-derived stem cells

BackgroundAdipose-derived stem cell (ASC) expansion under atmospheric oxygen levels (21%) was previously shown to cause increased reactive oxygen species (ROS) accumulation and genetic instability compared to cells cultured under physiological oxygen levels (2–8%). However, since culture under physiological oxygen levels is costly and complicated, a simpler method to reduce ROS accumulation is desirable. The current study aimed to determine whether lower culture temperature can reduce ROS production in ASCs without impairing their culture expansion.MethodsProliferation, differentiation, ROS accumulation, and gene expression were compared between ASC cultures at 35 °C and 37 °C. ASCs isolated either from rat fat depots or from human lipoaspirates were examined in the study.ResultsRat visceral ASCs (vASCs) cultured at 35 °C demonstrated reduced ROS production and apoptosis and enhanced expansion and adipogenic differentiation compared to vASCs cultured at 37 °C. Similarly, the culture of human ASCs (hASCs) at 35 °C led to reduced ROS accumulation and apoptosis, with no effect on the proliferation rate, compared to hASCs cultured at 37 °C. Comparison of gene expression profiles of 35 °C versus 37 °C vASCs uncovered the development of a pro-inflammatory phenotype in 37 °C vASCs in correlation with culture temperature and ROS overproduction. This correlation was reaffirmed in both hASCs and subcutaneous rat ASCs.ConclusionsThis is the first evidence of the effect of culture temperature on ASC growth and differentiation properties. Reduced temperatures may result in superior ASC cultures with enhanced expansion capacities in vitro and effectiveness in vivo.

Characterization of human skin equivalents developed at body's core and surface temperatures.

Human skin equivalents (HSEs) are in vitro developed three‐dimensional models resembling native human skin (NHS) to a high extent. However, the epidermal lipid biosynthesis, barrier lipid composition, and organization are altered, leading to an elevated diffusion rate of therapeutic molecules. The altered lipid barrier formation in HSEs may be induced by standardized culture conditions, including a culture temperature of 37°C, which is dissimilar to skin surface temperature. Therefore, we aim to determine the influence of culture temperature during the generation of full thickness models (FTMs) on epidermal morphogenesis and lipid barrier formation. For this purpose, FTMs were developed at conventional core temperature (37°C) or lower temperatures (35°C and 33°C) and evaluated over a time period of 4 weeks. The stratum corneum (SC) lipid composition was analysed using advanced liquid chromatography coupled to mass spectrometry analysis. Our results show that SC layers accumulated at a similar rate irrespective of culture temperature. At reduced culture temperature, an increased epidermal thickness, a disorganization of the lower epidermal cell layers, a delayed early differentiation, and an enlargement of granular cells were detected. Interestingly, melanogenesis was reduced at lower temperature. The ceramide subclass profile, chain length distribution, and level of unsaturated ceramides were similar in FTMs generated at 37°C and 35°C but changed when generated at 33°C, reducing the resemblance to NHS. Herein, we report that culture temperature affects epidermal morphogenesis substantially and to a lesser extent the lipid barrier formation, highlighting the importance of optimized external parameters during reconstruction of skin.

Alterations in lipidome and metabolome profiles of Nannochloropsis salina in response to reduced culture temperature during sinusoidal temperature and light

Metabolic response to four suboptimal cultivation temperatures was explored in Nannochloropsis salina, with an emphasis on fatty acid and lipid metabolism. Cultures were cultivated in controlled environmental photobioreactors. Physiological performance, characterized by growth rate and photosynthetic output, were reduced at all three cold stress temperatures (5 °C, 10 °C, 15 °C). However, biomass productivity and overall growth for cultures at 20 °C was like that of those grown at the optimal temperature (25 °C). Fatty acid productivity was elevated at all suboptimal temperatures, and both total fatty acid and eicosapentaenoic acid content were statically elevated at 20 °C. Lipid accumulation was observed exclusively in response to cold stress; however, lipid turnover and enrichment in polyunsaturated fatty acids occurred in all temperature variants. In addition, extensive lipid remodeling occurred in both polar and neutral lipid pools, specifically in monogalactosyl diacylglycerol and triacylglycerol pools. While 20 °C is optimal for eicosapentaenoic acid and total fatty acid productivity, cold stress is necessary to induce both productivity and polyunsaturated fatty acid enrichment in triacylglycerol pools.

Mild hypothermia upregulates myc and xbp1s expression and improves anti-TNFα production in CHO cells.

Chinese hamster ovary (CHO) cells are the most frequently used host for commercial production of therapeutic proteins. However, their low protein productivity in culture is the main hurdle to overcome. Mild hypothermia has been established as an effective strategy to enhance protein specific productivity, although the causes of such improvement still remain unclear. The self-regulation of global transcriptional regulatory factors, such as Myc and XBP1s, seems to be involved in increased the recombinant protein production at low temperature. This study evaluated the impact of low temperature in CHO cell cultures on myc and xbp1s expression and their effects on culture performance and cell metabolism. Two anti-TNFα producing CHO cell lines were selected considering two distinct phenotypes: i.e. maximum cell growth, (CN1) and maximum specific anti-TNFα production (CN2), and cultured at 37, 33 and 31°C in a batch system. Low temperature led to an increase in the cell viability, the expression of the recombinant anti-TNFα and the production of anti-TNFα both in CN1 and CN2. The higher production of anti-TNFα in CN2 was mainly associated with the large expression of anti-TNFα. Under mild hypothermia myc and xbp1s expression levels were directly correlated to the maximal viable cell density and the specific anti-TNFα productivity, respectively. Moreover, cells showed a simultaneous metabolic shift from production to consumption of lactate and from consumption to production of glutamine, which were exacerbated by reducing culture temperature and coincided with the increased anti-TNFα production. Our current results provide new insights of the regulation of myc and xbp1s in CHO cells at low temperature, and suggest that the presence and magnitude of the metabolic shift might be a relevant metabolic marker of productive cell line.

Model-based investigation of intracellular processes determining antibody Fc-glycosylation under mild hypothermia.

ABSTRACTDespite the positive effects of mild hypothermic conditions on monoclonal antibody (mAb) productivity (q mAb) during mammalian cell culture, the impact of reduced culture temperature on mAb Fc‐glycosylation and the mechanism behind changes in the glycan composition are not fully established. The lack of knowledge about the regulation of dynamic intracellular processes under mild hypothermia restricts bioprocess optimization. To address this issue, a mathematical model that quantitatively describes Chinese hamster ovary (CHO) cell behavior and metabolism, mAb synthesis and mAb N‐linked glycosylation profile before and after the induction of mild hypothermia is constructed. Results from this study show that the model is capable of representing experimental results well in all of the aspects mentioned above, including the N‐linked glycosylation profile of mAb produced under mild hypothermia. Most importantly, comparison between model simulation results for different culture temperatures suggests the reduced rates of nucleotide sugar donor production and galactosyltransferase (GalT) expression to be critical contributing factors that determine the variation in Fc‐glycan profiles between physiological and mild hypothermic conditions in stable CHO transfectants. This is then confirmed using experimental measurements of GalT expression levels, thereby closing the loop between the experimental and the computational system. The identification of bottlenecks within CHO cell metabolism under mild hypothermic conditions will aid bioprocess optimization, for example, by tailoring feeding strategies to improve NSD production, or manipulating the expression of specific glycosyltransferases through cell line engineering. Biotechnol. Bioeng. 2017;114: 1570–1582. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc.

Human Laminin Isotype Coating for Creating Islet Cell Sheets.

Our experimental approach toward the development of new islet-based treatment for diabetes mellitus has been the creation of a monolayered islet cell construct (islet cell sheet), followed by its transplantation into a subcutaneous pocket. Previous studies describe rat laminin-5 (chain composition: α3, β3, γ2) as a suitable extracellular matrix (ECM) for surfaces comprised of a coated temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm). To progress toward the clinical application of this approach, the present study attempted to identify an optimal human ECM as a coating material on PIPAAm surfaces, which allowed islet cells to attach on the surfaces and subsequently to be harvested as a monolithic cell sheet. Dispersed rat islet cells were seeded onto PIPAAm dishes coated with various human laminin isotypes: human laminin (HL)-211, HL-332, HL-411, HL-511, and HL-placenta. Plating efficiency at day 1, the confluency at day 3, and glucose-stimulated insulin secretion test at day 3 were performed. The highest value of plating efficiency was found in the HL-332-PIPAAm group (83.1 ± 0.7%). The HL-332-PIPAAm group also showed the highest cellular confluency (98.6 ± 0.5%). Islet cells cultured on the HL-332-PIPAAm surfaces showed a positive response in the glucose-stimulated insulin secretion test. By reducing culture temperature from 37°C to 20°C in the HL-332-PIPAAm group, cells were able to be harvested as a monolithic islet sheet. The present study showed that HL-332 was an optimal human-derived ECM on a PIPAAm coating for preparing islet cell sheets.

An ex vivo model of the human airways to study the effects of long-term exposure to Space conditions

An innovative 3D model of the human bronchial mucosa, a result of a long- term collaboration with Prof. Davies from the University of Southampton, UK, will be used to study, on board the International Space Station (ISS), the chang- es occurring in lungs exposed to the adverse environmental conditions found in Space. Our research group has developed a tissue engineered human airway model, providing a viable alternative to the use of animal models. The bronchial outgrowth offers numerous advantages compared to traditional approaches that use human tissue, including: the morphological analogy with the bronchial mucosa; the possibility to study long-term exposure (several months) to environmental factors (cigarette smoke, respiratory viruses, etc.) being able to analyze eventual changes in the long term; the possibility to add cellular and humoral components of the immune system in order to evaluate their effects. Recently, our application to the ILSRA 2014 solicitation by NASA/ESA, in which we propose to study the effects of prolonged stay on board the ISS on the development and function of human airway cells in our model, was successful and our project was selected. This project will involve several international partners, including, in addition to the University of Palermo and Southampton, the Euro-Mediterranean Institute of Science and Technology (IEMEST, Palermo), the University of Leiden (NL), the University of Galveston (Texas, USA), the Wyle Laboratories (Texas, USA) and NASA itself. The results obtained will be particularly useful in view of exploratory missions of our solar system planned in the near future. To verify the resilience of the model to the extreme environmental conditions on board the vectors that transport the samples to the ISS, and therefore collect essential data prior to the flight experiments, differentiated outgrowths obtained from healthy patients were exposed for up to 1 week to the following stress conditions: withdrawal of growth medium, reduced culture temperature (10 and 20°C), absence of CO2, intense rolling and shaking motions. TER values were measured 1 week prior to 1 week after the exposures to evaluate the effects of these stimuli. The data obtained show that even though the stimuli determined a slight degree of tissue alteration (reduction of TER) during exposure, the outgrowths promptly recovered their origi- nal structure and function.

Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures.

The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures.

Reduced Culture Temperature Differentially Affects Expression and Biophysical Properties of Monoclonal Antibody Variants.

Reduced culture temperature is an increasingly popular practice to improve recombinant protein yields in CHO cells. Recent studies have attributed the enhancement of protein titers at sub-physiological temperatures to increased mRNA levels as well as extended stationary phase. We observed that reducing the culture temperature arrested cell growth, prolonged viability, and increased cell size. However, the reduced culture temperature had a differential effect on protein and mRNA expression of closely related antibody mutants from stable cell lines. The highly expressing mutant (Ala) exhibited similar or decreased specific productivity and decreased volumetric productivity over the culture lifetime at 32 °C compared to 37 °C. In contrast, the specific and volumetric productivity of the poorly expressing mutant (Gly) was enhanced at the lower culture temperature. The difference in specific productivity was reflected in the amounts of heavy- and light-chain mRNA. Analysis of the secondary and tertiary configurations of the purified antibodies by circular dichroism revealed fundamental structural differences imposed by the Ala to Gly mutation as well as reduced culture temperature. We propose that the effect of reduced culture temperature on expression is protein-dependent; protein folding fidelity and assembly is improved at lower temperatures, enhancing the expression of proteins that have a propensity to misfold.

Glycyrrhetinic Acid-Based Thermoresponsive Hydrogel As a Synthetic Extracellular Matrix for Hepatocyte Culture and Recovery

Glycyrrhetinic acid-based thermoresponsive hydrogels P(NIPAAm-co-GAA) were prepared and used to promote initial cell adhesion, growth, and recovery by lowering temperature for further tissue engineering or bioartificial liver applications. The compositions, morphologies and properties of hydrogels were characterized by element analysis, scanning electron microscopy (SEM), and water contact angle measurement (WCA), respectively. P(NIPAAm-co-GAA) hydrogels exhibited a shift between hydrophilicity and hydrophobicity by controlling temperature. Adhesion, growth, detachment, and physiological functions of human hepatocyte (HL-7702) cultured on P(NIPAAm-co-GAA) hydrogels were examined. The introduction of glycyrrhetinic acid (GA) with specific affinity to hepatocyte promoted the adhesion and proliferation of HL-7702 cells effectively. Importantly, HL-7702 cells cultured on P(NIPAAm-co-GAA) hydrogels maintained high levels of urea and albumin synthesis. HL-7702 cells grown on P(NIPAAm-co-GAA) hydrogels can also be recovered spontaneously by only reducing culture temperature without any damage. Furthermore, the behaviors of HL-7702 and human hepatoma cells (HepG2) on P(NIPAAm-co-GAA) hydrogels were evaluated comparatively. The result revealed that P(NIPAAm-co-GAA) hydrogels facilitated HL-7702 but inhibited the growth of HepG2. Glycyrrhetinic acid-based thermoresponsive hydrogel was shown to be a promising matrix for cultivation of functional hepatocytes.

Creation of Human Islet Sheets for Generating a New Islet-Based Therapy

Introduction: Our experimental approach toward the development of novel treatments for diabetes mellitus has allowed us to create a monolayered contiguous sheet (islet cell sheet), which can be transplanted and engrafted subcutaneously (Shimizu et al, Biomaterials, 2009). For fabricating an islet cell sheet, dispersed islet cells were seeded onto a temperature responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), grafted culture dish coated with rat laminin 5 (RL5). The transplant of islet cell sheets allows hyper glycaemia in diabetes mellitus mouse to return to normal glycaemia (Saito et al, Transplantation, 2011). For establishing functional human islet cell sheets for clinical application, the present study explored various human extracellular matrixes (ECMs) as coating materials on PIPAAm dishes and investigates the status of human islet cells cultured on the dishes. Methods: Human islet, classified as unsuitable for islet transplantation therapy in University of Alberta, was dispersed by trypsin-EDTA digestion. Dispersed islet cells were seeded onto PIPAAm dishes coated with various ECMs; human laminin-511 (HL-511), human laminin-411 (HL-411), human laminin-211 (HL-211), human laminin-332 (HL-332), human laminin-placenta (HL-pl) and human collagenIV (Col IV), at a density of 5 × 106 cells/cm2. The dishes were incubated in an incubator with 5% CO2 at 37°C. Dish coated with RL5 was used as the positive control, and non-coated dish was also used as the negative control. Plating efficiency at day1 (n =3), the confluency of dish at day3 (n =3), and glucose stimulated insulin secretion test at day 3 (n =3) were determined. Culture islet cells reached to confluence were harvested from PIPAAm dishes by decreasing temperature from 37 to 20°C for 30 min. Results: Plating efficiency on HL-511 at day 1 was found to be 65.4 ± 4.9%; HL-411,26.8 ± 14.6%; HL-211, 9.3 ± 7.3%; HL-332, 90.3 ± 5.3%; HL-pl, 12.9 ± 4.5%; Col IV, 18.9 ± 4.5 %. The efficiencies on the negative (non-coated) and positive (RL5) controls were found to be 19.5 ± 0.6 % and 80.3 ± 8.1%, respectively. Confluency of the cells was also determined at day 3 (Figure). By reducing culture temperature on HL-322 groups, cells were able to be harvested as a monolithic islet cell sheet. Amount of insulin secretion at a glucose concentration 20mmol/L in culture media was more than that at a glucose concentration of 3.3mmol/L.Figure: [Cell confluency at day 3]Conclusion: The data indicated that a surface coated with HL-322 was found to clearly stimulate islet cells to adhere and expand on the surfaces, leading to the formation of a confluent monolayer structure.

Shear stress-dependent cell detachment from temperature-responsive cell culture surfaces in a microfluidic device

A new approach to quantitatively estimate the interaction between cells and material has been proposed by using a microfluidic system, which was made of poly(dimethylsiloxane) (PDMS) chip bonding on a temperature-responsive cell culture surface consisted of poly(N-isopropylacrylamide) (PIPAAm) grafted tissue culture polystyrene (TCPS) (PIPAAm-TCPS) having five parallel test channels for cell culture. This construction allows concurrently generating five different shear forces to apply to cells in individual microchannels having various resistance of each channel and simultaneously gives an identical cell incubation condition to all test channels. NIH/3T3 mouse fibroblast cells (MFCs) and bovine aortic endothelial cells (BAECs) were well adhered and spread on all channels of PIPAAm-TCPS at 37 °C. In our previous study, reducing culture temperature below the lower critical solution temperature (LCST) of PIPAAm (32 °C), cells detach themselves from hydrated PIPAAm grafted surfaces spontaneously. In this study, cell detachment process from hydrated PIPAAm-TCPS was promoted by shear forces applied to cells in microchannels. Shear stress-dependent cell detachment process from PIPAAm-TCPS was evaluated at various shear stresses. Either MFCs or BAECs in the microchannel with the strongest shear stress were found to be detached from the substrate more quickly than those in other microchannels. A cell transformation rate constant Ct and an intrinsic cell detachment rate constant k0 were obtained through studying the effect of shear stress on cell detachment with a peeling model. The proposed device and quantitative analysis could be used to assess the possible interaction between cells and PIPAAm layer with a potential application to design a cell sheet culture surface for tissue engineering.

Dual Responsive Nanostructured Surfaces for Biomedical Applications

In this paper, we describe the construction and characteristics of thermoresponsive, thin nanostructured films prepared by layer-by-layer sequential assembly of chitosan-graft-NIPAAm and alginate. FTIR and (1)H NMR spectra have confirmed the introduction of NIPAAm moieties onto the chitosan backbone. The LCST of the synthesized copolymer was found to be around 31-33 °C. The formation of the polyelectrolyte multilayers containing the copolymer and alginate was followed in situ by quartz crystal microbalance with dissipation monitoring technique and ex situ by UV-vis measurements. Our results revealed the linear increase of the multilayer film growth and the influence of the presence of salt. Moreover, AFM analysis has confirmed that PNIPAAm is able to reconform upon temperature swaps even when combined with other layers in a polyelectrolyte multilayer, demonstrating that the nanoassemblies are thermoresponsive. Preliminary results showed that, upon reducing culture temperature below PNIPAAm LCST, a gradual detachment of cell sheets from these PNIPAAm-based coatings has occurred.

Immobilization of galactose ligands on thermoresponsive culture surface and its influence on cell adhesion/detachment

A novel bio-functionalized thermoresponsive surface was prepared by UV-induced copolymerization of N-isopropylacrylamide (NIPAAm) and acrylic acid (AAc) and grafting onto tissue culture polystyrene (TCPS) dishes, followed by immobilization of galactose ligands. The results indicated that the roughness of surfaces was increased after copolymerization with NIPAAm and immobilization of galactose ligands. The amount of monomers grafted on TCPS was increased with increasing photografting time. The temperature sensitivity of surface was improved with increasing amount of NIPAAm grafted on TCPS, and surface hydrophilicity was enhanced by the introduction of carboxyl groups and galactose ligands, which accelerated cell detachment. Adhesion, proliferation, detachment and transshipment of human hepatoma cell line (HepG2) on the modified surfaces were examined. The immobilization of galactose ligands facilitated the cell adhesion and proliferation. HepG2 cells growing on the modified surfaces could be recovered spontaneously by only reducing culture temperature. The activity of cells obtained by temperature induction was higher than that obtained by enzymatic digestion.

Rescue of a Trafficking Defective Human Pacemaker Channel Via a Novel Mechanism: Roles of Src, Fyn, Yes Tyrosine Kinases

Therapeutic strategies such as using channel blockers and reducing culture temperature have been used to rescue some long-QT associated voltage-gated potassium Kv trafficking defective mutant channels. A hyperpolarization-activated cyclic nucleotide-gated HCN4 pacemaker channel mutant (D553N) has been recently found in a patient associated with cardiac arrhythmias including long-QT. D553N showed the defective trafficking to the cell surface, leading to little ionic current expression (loss-of-function). We show in this report that enhanced tyrosine phosphorylation mediated by Src, Fyn, and Yes kinases was able to restore the surface expression of D553N for normal current expression. Src or Yes, but not Fyn, significantly increased the current density and surface expression of D553N. Fyn accelerated the activation kinetics of the rescued D553N. Co-expression of D553N with Yes exhibited the slowest activation kinetics of D553N. Src, Fyn, and Yes significantly enhanced the tyrosine phosphorylation of D553N. A combination of Src, Fyn, and Yes rescued the current expression and the gating of D553N comparable to those of wild-type HCN4. In conclusion, we demonstrate a novel mechanism using three endogenous Src kinases to rescue a trafficking defective HCN4 mutant channel (D553N) by enhancing the tyrosine phosphorylation of the mutant channel protein.

Rescue of a Trafficking Defective Human Pacemaker Channel via a Novel Mechanism: ROLES OF Src, Fyn, AND Yes TYROSINE KINASES

Therapeutic strategies such as using channel blockers and reducing culture temperature have been used to rescue some long QT-associated voltage-gated potassium Kv trafficking defective mutant channels. A hyperpolarization-activated cyclic nucleotide-gated HCN4 pacemaker channel mutant (D553N) has been recently found in a patient associated with cardiac arrhythmias including long QT. D553N showed the defective trafficking to the cell surface, leading to little ionic current expression (loss-of-function). We show in this report that enhanced tyrosine phosphorylation mediated by Src, Fyn, and Yes kinases was able to restore the surface expression of D553N for normal current expression. Src or Yes, but not Fyn, significantly increased the current density and surface expression of D553N. Fyn accelerated the activation kinetics of the rescued D553N. Co-expression of D553N with Yes exhibited the slowest activation kinetics of D553N. Src, Fyn, and Yes significantly enhanced the tyrosine phosphorylation of D553N. A combination of Src, Fyn, and Yes rescued the current expression and the gating of D553N comparable with those of wild-type HCN4. In conclusion, we demonstrate a novel mechanism using three endogenous Src kinases to rescue a trafficking defective HCN4 mutant channel (D553N) by enhancing the tyrosine phosphorylation of the mutant channel protein.

Enhanced interferon‐β production by CHO cells through elevated osmolality and reduced culture temperature

For efficient production of native interferon-beta (IFN-beta) in recombinant CHO cell culture, the IFN-beta molecular aggregation that occurs during culture needs to be minimized. To do so, we investigated the effect of hyperosmolality and hypothermia on IFN-beta production and molecular aggregation in rCHO cell culture. Both hyperosmolality (470 mOsm/kg) and hypothermia (32 degrees C) increased specific native INF-beta productivity q(IFN-beta). Furthermore, they decreased the IFN-beta molecular aggregation, although severe IFN-beta molecular aggregation could not be avoided in the later phase of culture. To overcome growth suppression at hyperosmolality and hypothermia, cells were cultivated in a biphasic mode. Cells were first cultivated at 310 mOsm/kg and 37 degrees C for 2 days to rapidly obtain a reasonably high cell concentration. The temperature and osmolality were then shifted to 32 degrees C and 470 mOsm/kg, respectively, to achieve high q(IFN-beta) and reduced IFN-beta molecular aggregation. Due to the enhanced q(IFN-beta) and delayed molecular aggregation, the highest native IFN-beta concentration achieved on day 6 was 18.03 +/- 0.61 mg/L, which was 5.30-fold higher than that in a control batch culture (310 mOsm/kg and 37 degrees C). Taken together, a combination of hyperosmolality and hypothermia in a biphasic culture is a useful strategy for improved native IFN-beta production from rCHO cells.

Temperature-dependent changes of cell shape during heterophyllous leaf formation in Ludwigia arcuata (Onagraceae)

Although elongation of epidermal cells in submerged leaves is thought to be a common feature of heterophyllous aquatic plants, such elongation has not been observed in Ludwigia arcuata Walt. (Onagraceae). In this study we found that reduced culture temperature induced the elongation of epidermal cells of submerged leaves in L. arcuata. Since such submerged leaves also showed a reduction in the number of epidermal cells aligned across the leaf transverse axis, these data indicate that heterophyllous leaf formation in L. arcuata is partially temperature sensitive, i.e., the elongation of epidermal cells was temperature sensitive while the reduction in the number of epidermal cells did not show such temperature sensitivity. To clarify the mechanisms that cause such temperature sensitivity, we examined the effects of ethylene, which induced the formation of submerged-type leaves on aerial shoots at the relatively high culture-temperature of 28 degrees C. At 23 degrees C, ethylene induced both cell elongation and reduction in the number of epidermal cells across the leaf transverse axis, while cell elongation was not observed at 28 degrees C. Moreover, both submergence and ethylene treatment induced a change in the arrangement of cortical microtubules (MTs) in epidermal cells of developing leaves at 23 degrees C. Such changes in the arrangement of MTs was not induced at 28 degrees C. Factors involved in the temperature-sensitive response to ethylene would be critical for temperature-sensitive heterophyllous leaf formation in L. arcuata.

Enhanced production of recombinant rabies virus glycoprotein (rRVGP) by Drosophila melanogaster S2 cells through control of culture conditions

Culture conditions that affect product quality are important to the successful operation and optimization of recombinant protein production. The objective of this study was to optimize culture conditions for growth of recombinant Drosophila melanogaster S2 cells (S2AcRVGP) in order to enhance the production of rRVGP. The addition of DMSO and glycerol to the medium and growth at a reduced temperature (22 degrees C) were the culture condition variations selected to be tested. Experimental cultures were first performed in serum-free Sf900 II medium in 250 ml Schott flasks. The most promising conditions identified in these experiments were also tested on a higher scale in a 3l bioreactor. In the Schott flasks experiments, all the changes in culture conditions resulted in an increase of rRVGP production. The protein concentration was 3.6-fold higher with addition of 1% DMSO and 1% glycerol and 9.3-fold higher when the cells were cultured at 22 degrees C instead of the standard 28 degrees C. The maximum concentration of rRVGP reached was 591 mug l(-1). In bioreactor experiments, with control of pH at 6.20 and DO at 50%, the reduced culture temperature (22 degrees C) was the strategy that promoted the highest glycoprotein production, 928 mug l(-1).