RESUMO
Antigenically divergent H7N9 viruses pose a potential threat to public health, with the poor immunogenicity of candidate H7N9 vaccines demonstrated in clinical trials underscoring the urgent need for more-effective H7N9 vaccines. In the present study, mice were immunized with various doses of a suspended-MDCK-cell-derived inactivated H7N9 vaccine, which was based on a low-pathogenic H7N9 virus, to assess cross-reactive immunity and cross-protection against antigenically divergent H7N9 viruses. We found that the CRX-527 adjuvant, a synthetic TLR4 agonist, significantly enhanced the humoral immune responses of the suspended-MDCK-cell-derived H7N9 vaccine, with significant antigen-sparing and immune-enhancing effects, including robust virus-specific IgG, hemagglutination-inhibiting (HI), neuraminidase-inhibiting (NI), and virus-neutralizing (VN) antibody responses, which are crucial for protection against influenza virus infection. Moreover, the CRX-527-adjuvanted H7N9 vaccine also elicited cross-protective immunity and cross-protection against a highly pathogenic H7N9 virus with a single vaccination. Notably, NI and VN antibodies might play an important role in cross-protection against lethal influenza virus infections. This study showed that a synthetic TLR4 agonist adjuvant has a potent immunopotentiating effect, which might be considered worth further development as a means of increasing vaccine effectiveness.
Assuntos
Anticorpos Antivirais , Imunidade Humoral , Subtipo H7N9 do Vírus da Influenza A , Vacinas contra Influenza , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae , Receptor 4 Toll-Like , Vacinas de Produtos Inativados , Animais , Subtipo H7N9 do Vírus da Influenza A/imunologia , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/imunologia , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/administração & dosagem , Camundongos , Anticorpos Antivirais/imunologia , Cães , Células Madin Darby de Rim Canino , Vacinas de Produtos Inativados/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/imunologia , Feminino , Anticorpos Neutralizantes/imunologia , Proteção Cruzada/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Adjuvantes de Vacinas , Imunoglobulina G/imunologia , Imunoglobulina G/sangueRESUMO
Cryopreservation is a crucial step in the supply process of off-the-shelf chimeric antigen receptor engineered natural killer (CAR-NK) cell products. Concerns have been raised over the clinical application of dimethyl sulfoxide (Me2SO) due to the potential for adverse reactions following infusion and limited cell-specific cytotoxic effects if misapplied. In this study, we developed a Me2SO-free cryopreservation medium specifically tailored for CAR-NK cells to address this limitation. The cryopreservation medium was formulated using human serum albumin (HSA) and glycerol as the base components. Following initial screening of seven clinically-compatible solutions, four with cryoprotective properties were identified. These were combined and optimized into a single formulation: IF-M. The viability, phenotype, and function of CAR-NK cells were evaluated after short-term and long-term cryopreservation to assess the effectiveness of IF-M, with Me2SO serving as the control group. The viability and recovery of CAR-NK cells in the IF-M group were significantly higher than those in the Me2SO group within 90 days of cryopreservation. Moreover, after 1 year of cryopreservation the cytotoxic capacity of CAR-NK cells cryopreserved with IF-M was comparable to that of fresh CAR-NK cells and significantly superior to that of CAR-NK cells cryopreserved in Me2SO. The CD107a expression intensity of CAR-NK cells in IF-M group was significantly higher than that of Me2SO group. No statistical differences were observed in other indicators under different cryopreservation times. These results underscore the robustness of IF-M as a suitable replacement for traditional Me2SO-based cryopreservation medium for the long-term cryopreservation and clinical application of off-the-shelf CAR-NK cells.
Assuntos
Criopreservação , Receptores de Antígenos Quiméricos , Humanos , Criopreservação/métodos , Crioprotetores/farmacologia , Crioprotetores/metabolismo , Receptores de Antígenos Quiméricos/genética , Dimetil Sulfóxido/farmacologia , Dimetil Sulfóxido/metabolismo , Células Matadoras Naturais , Sobrevivência CelularRESUMO
PURPOSE: The accumulation of carbon dioxide during large-scale culture of animal cells brings adverse effects, appropriate aeration strategies alleviate CO2 accumulation while improper reactor operation may lead to the presence of low CO2 partial pressure (pCO2) condition as occurs in many industrial cases. Thus, this study aims to reveal the in-depth influence of low pCO2 on Chinese Hamster Ovary (CHO) cells for providing a reference for design space determination of CO2 control with regard to the Quality by Design (QbD) guidelines. METHODS AND RESULTS: The headspace air over purging caused the ultra-low pCO2 (ULC) where the monoclonal antibody production as well as the aerobic metabolic activity were reduced. Intracellular metabolomics analysis indicated a less efficient aerobic glucose metabolic state under ULC conditions. Based on the increase of intracellular pH and lactate dehydrogenase activity, the shortage of intracellular pyruvate could be the cause of the deficient aerobic metabolism, which could be partially mitigated by pyruvate addition under ULC conditions. Finally, a semi-empirical mathematical model was used to better understand, predict and control the occurrence of extreme pCO2 conditions during the cultures of CHO cells. CONCLUSION: Low pCO2 steers CHO cells into a defective metabolic state. A predictive relation among pCO2, lactate, and pH control was applied to get new insights into CHO cell culture for better and more robust metabolic behavior and process performance and the determination of QbD design space for CO2 control.
Assuntos
Dióxido de Carbono , Ácido Láctico , Cricetinae , Animais , Cricetulus , Células CHO , Dióxido de Carbono/metabolismo , Pressão Parcial , Ácido Láctico/metabolismo , Ácido PirúvicoRESUMO
High metal ion concentrations and low pH cause severely inhibit the activity of an acidophilic microbial consortium (AMC) in bioleaching. This work investigated the effects of exogenous spermine on biofilm formation and the bioleaching efficiency of LiCoO2 by AMC in 9K medium. After the addition of 1 mM spermine, the activities of glutathione peroxidase and catalase increased, while the amount of H2O2, intracellular reactive oxygen species (ROS) and malondialdehyde in AMC decreased. These results indicated that the ability of AMC biofilm to resist oxidative stress introduced by 3.5 g/L Li+ and 30.1 g/L Co2+ was improved by spermine. The activity of glutamate decarboxylase was promoted to restore the intracellular pH buffering ability of AMC. Electrochemical measurements showed that the oxidation rate of pyrite was increased by exogenous spermine. As a result, high bioleaching efficiencies of 97.1% for Li+ and 96.1% for Co2+ from a 5.0% (w v-1) lithium cobalt oxide powder slurry were achieved. This work demonstrated that Tafel polarization can be used to monitor the AMC biofilm's ability of uptaking electrons from pyrite during bioleaching. The corrosion current density increased with 1 mM spermine, indicating enhanced electron uptake by the biofilm from pyrite.
Assuntos
Peróxido de Hidrogênio , Consórcios Microbianos , Ácidos , Lítio , Estresse Oxidativo , EsperminaRESUMO
BACKGROUND: Mesenchymal stem cells have been widely used in the treatment of diabetes mellitus. However, hyperglycemia associated with DM promotes cell apoptosis and affects osteogenic differentiation of MSCs in varying degrees, leading to osteoporosis in DM patients. Therefore, in this paper, the effect of high glucose on apoptosis and osteogenesis of MSCs was investigated and underlying mechanism was further determined. METHODS AND RESULTS: Intracellular ROS levels were determined using probe DCFH-DA. MMP was detected using JC-1 staining. Cell apoptosis was detected using Annexin V-FITC/PI and Flow Cytometer. The expression of genes and protein was detected by qRT-PCR and Western blot respectively. The results showed high glucose induced MSC apoptosis but promoted its osteogenesis. Western blot analysis revealed that high glucose downregulated AKT-Sirt1-TWIST pathway. Activation of Sirt1 via SRT1720 increased TWIST expression, alleviated MSC apoptosis and promoted osteogenesis of MSCs. TWIST knockdown studies demonstrated that inhibition of TWIST intensified high glucose-induced apoptosis but promoted osteogenesis differentiation of MSCs. TWIST is likely to be a new regulator for cross talk between Sirt1 and its downstream targets. CONCLUSION: Our data demonstrates that high glucose induces MSC apoptosis and enhances osteogenesis differentiation via downregulation of AKT-Sirt1-TWIST.
Assuntos
Osteogênese , Sirtuína 1 , Apoptose , Diferenciação Celular , Regulação para Baixo , Glucose/farmacologia , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismoRESUMO
Hematopoietic stem/progenitor cells (HSPCs) ex vivo expansion is critical in facilitating their widespread clinical application. NF-κB pathway is implicated in the energy homeostasis and metabolic adaptation. To explore the effect of NF-κB pathway on the ex vivo HSPC expansion and metabolism, the 50 nM-1 µM inhibitor of NF-κB pathway TPCA-1 was used to expand cord blood derived CD34+ cells in serum-free culture. The expansion folds, function, mitochondrial profile and metabolism of HSPCs were determined. After 10 days of culture with 100 nM TPCA-1, the expansion of total cellsï¼ CD34+CD38- cellsï¼ and CD34+CD38-CD45RA-CD90+CD49f+ cells were significantly increased compared to the cytokine priming alone. Notably, TPCA-1 treatment generated ~ 2-fold greater percentage of CD34+EPCR+ and CD34+CD38-CD45RA-CD90+CD49f+ cells compared to cytokine only conditions. Moreover, TPCA-1 expanded CD34+ cells displayed enhanced serial colonies forming potential and secondary expansion capability. NF-κB inhibition increased the expression of self-renewal related genes, while downregulated the expression of mitochondrial biogenesis regulator (Pgc1α) and mitochondrial chaperones and proteases (ClpP, Hsp10, Hsp60). Mitochondrial mass and membrane potential were markedly decreased with TPCA-1 treatment, leading to the reduced mitochondrial reactive oxygen species (ROS) level in HSPCs. NF-κB inhibition displayed augmented glycolysis rate with compromising mitochondrial metabolism. This study demonstrated that NF-κB pathway inhibition improved glycolysis and limited ROS production that promoted the ex vivo expansion and maintenance of functional HSPCs.
Assuntos
Amidas/farmacologia , Metabolismo Energético/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Tiofenos/farmacologia , Antígenos CD34/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Respiração Celular/efeitos dos fármacos , Respiração Celular/genética , Células Cultivadas , Metabolismo Energético/genética , Glicólise/efeitos dos fármacos , Glicólise/genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Humanos , Proteínas I-kappa B/fisiologia , Imunofenotipagem , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NF-kappa B/metabolismo , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Subunit vaccines with high purity and safety are gradually becoming a main trend in vaccinology. However, adjuvants such as interferon-gamma (IFN-γ) are required to enhance immune responses of subunit vaccines due to their poor immunogenicity. The conjugation of antigen with adjuvant can induce more potent immune responses compared to the mixture of antigen and adjuvant. At the same time, the selection of linker, indispensable in the construction of the stable and bioactive fusion proteins, is complicated and time-consuming. The development of immunoinformatics and structural vaccinology approaches provides a means to address the abovementioned problem. Therefore, in this study, a E2-IFN-γ fusion protein with an optimal linker (E2-R2-PIFN) was designed by bioinformatics approaches to improve the immunogenicity of the classical swine fever virus (CSFV) E2 subunit vaccine. Moreover, the E2-R2-PIFN fusion protein was expressed in HEK293T cells and the biological effects of IFN-γ in E2-R2-PIFN were confirmed in vitro via Western blotting. Here, an alternative method is utilized to simplify the design and validation of the antigen-adjuvant fusion protein, providing a potential subunit vaccine candidate against CSFV. KEY POINTS: ⢠An effective and simple workflow of antigen-adjuvant fusion protein design and validation was established by immunoinformatics and structural vaccinology. ⢠A novel E2-IFN-γ fusion protein with an optimal linker was designed as a potential CSFV vaccine. ⢠The bioactivity of the newly designed fusion protein was preliminarily validated through in vitro experiments.
Assuntos
Vírus da Febre Suína Clássica , Peste Suína Clássica , Vacinas Virais , Adjuvantes Imunológicos , Animais , Anticorpos Antivirais , Peste Suína Clássica/prevenção & controle , Vírus da Febre Suína Clássica/genética , Células HEK293 , Humanos , Interferon gama , Suínos , Vacinas de Subunidades Antigênicas/genética , Vacinologia , Proteínas do Envelope Viral/genética , Vacinas Virais/genéticaRESUMO
The absence of blood vessels in tissue engineered bone often leads to necrosis of internal cells after implantation, ultimately affecting the process of bone repair. Herein, mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) were cocultured to induce osteogenesis and angiogenesis. Based on the findings, the number of HUVECs in the coculture system increased in the growth medium group, but decreased in the osteogenic induction medium (OIM) group. Considering that the paracrine effects of MSCs had changed, we tested the genes expression of osteogenically differentiated MSCs. The expression of osteogenic genes in MSCs increased during osteogenesis. Further, the expression levels of pigment epithelial-derived factor (PEDF) gene and protein, an antivascular factor, were also increased. To verify whether MSCs promote HUVECs apoptosis via PEDF, PEDF was silenced via siRNA. The conditioned medium of differentiated MSCs with PEDF silencing significantly improved the proliferation and apoptosis of HUVECs. Based on further experiments, PEDF mediated the apoptosis and proliferation of HUVECs through p53, BAX/BCL-2, FAS, and c-Caspase-3. However, when PEDF was silenced with siRNA, the osteogenic potential of MSCs was affected. The results of this study provide a theoretical basis for the construction of prevascularized bone tissues in vitro.
Assuntos
Células-Tronco Mesenquimais , Humanos , Células Endoteliais da Veia Umbilical Humana , RNA Interferente Pequeno/metabolismo , Células-Tronco Mesenquimais/metabolismo , Diferenciação Celular , Técnicas de Cocultura , Apoptose , Células Cultivadas , Neovascularização FisiológicaRESUMO
Haematopoietic stem/progenitor cell (HSPC) integrates intracellular signal network from growth factors (GFs) and utilizes its proliferation feature to generate high yields of transplantable cells upon ex vivo culture. However, the molecular basis for HSPC activation and proliferation is not completely understood. The goal of this study was to investigate proliferation regulator in the downstream of GFs and develop HSPC expansion strategy. Microarray and Ingenuity Pathway Analysis were performed to evaluate differentially expressed genes in cytokine-induced CD34+ cells after ex vivo culture. We identified that MEK1 was a potential HSPC proliferation regulator, which represented indispensable roles and MEK1 silence attenuated the proliferation of HSPC. Notably, 500 nM MEK1 agonist, PAF C-16, increased the numbers of phenotypic HSPC and induced cell cycling of HSPC. The PAF C-16 expanded HSPC demonstrated comparative clonal formation ability and secondary expansion capacity compared to the vehicle control. Our results provide insights into regulating the balance between proliferation and commitment of HSPC by targeting the HSPC proliferation-controlling network. This study demonstrates that MEK1 critically regulates HSPC proliferation and cell production in the ex vivo condition for transplantation.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Antígenos CD34 , Proliferação de Células , Células CultivadasRESUMO
The development of an effective cryopreservation method to achieve off-the-shelf and bioactive tissue-engineered constructs (TECs) is important to meet the requirements for clinical applications. The trehalose, a non-permeable cryoprotectant (CPA), has difficulty in penetrating the plasma membranes of mammalian cells and has only been used in combination with other cell penetrating CPA (such as DMSO) to cryopreserve mammalian cells. However, the inherent cytotoxicity of DMSO results in increasing risks with respect to cryopreserved cells. Therefore, in this study, permeable trehalose glycopolymers were synthesised for cryopreservation of TECs. The trehalose glycopolymers exhibited good ice inhibiting activities and biocompatibilities. Furthermore, the viability and function of TECs after cryopreservation with 5.0 wt% S2 were similar to those of the non-cryopreserved TECs. We developed an effective preservation strategy for the off-the-shelf availability of TECs.
Assuntos
Criopreservação , Trealose , Animais , Sobrevivência Celular , Criopreservação/métodos , Crioprotetores/metabolismo , Crioprotetores/farmacologia , Dimetil Sulfóxido , Mamíferos/metabolismo , Trealose/metabolismo , Trealose/farmacologiaRESUMO
Influenza is a global public health issue leading to widespread morbidity and mortality with devastating economic loss annually. Madin-Darby Canine Kidney (MDCK) cell line has been a major cell line for influenza vaccine applications. Though many details of the host metabolic responses upon influenza A virus (IAV) infection have been documented, little is known about the metabolic reprogramming features of a hyper-productive host for IAV vaccine production. In this study, a MDCK cell clone H1 was shown to have a particular high productivity of 30 × 103 virions/cell. The glucose and amino acid metabolism of H1 were evaluated, indicating that the high producer had a particular metabolic reprogramming phenotype compared to its parental cell line (P): elevated glucose uptake, superior tricarboxylic acid cycle flux, moderate amino acid consumption, and better regulation of reactive oxygen species. Combined with the stronger mitochondrial function and mild antiviral and inflammatory responses characterized previously, our results indicated that the high producer had a sufficient intracellular energy supply, and balanced substrate distribution for IAV and host protein synthesis as well as the intracellular redox status. Understanding of these metabolic alterations paves the way for the rational cell line development and reasonable process optimization for high-yield influenza vaccine production.
Assuntos
Vírus da Influenza A , Vacinas contra Influenza , Influenza Humana , Cães , Animais , Humanos , Células Madin Darby de Rim Canino , Oxirredução , AminoácidosRESUMO
Seasonal influenza epidemics occur both in northern and southern hemispheres every year. Despite the differences in influenza virus surface antigens and virulence of seasonal subtypes, manufacturers are well-adapted to respond to this periodical vaccine demand. Due to decades of influenza virus research, the development of new influenza vaccines is relatively straight forward. In similarity with the ongoing coronavirus disease 2019 pandemic, vaccine manufacturing is a major bottleneck for a rapid supply of the billions of doses required worldwide. In particular, egg-based vaccine production would be difficult to schedule and shortages of other egg-based vaccines with high demands also have to be anticipated. Cell culture-based production systems enable the manufacturing of large amounts of vaccines within a short time frame and expand significantly our options to respond to pandemics and emerging viral diseases. In this study, we present an integrated process for the production of inactivated influenza A virus vaccines based on a Madin-Darby Canine Kidney (MDCK) suspension cell line cultivated in a chemically defined medium. Very high titers of 3.6 log10 (HAU/100 µl) were achieved using fast-growing MDCK cells at concentrations up to 9.5 × 106 cells/ml infected with influenza A/PR/8/34 H1N1 virus in 1 L stirred tank bioreactors. A combination of membrane-based steric-exclusion chromatography followed by pseudo-affinity chromatography with a sulfated cellulose membrane adsorber enabled full recovery for the virus capture step and up to 80% recovery for the virus polishing step. Purified virus particles showed a homogenous size distribution with a mean diameter of 80 nm. Based on a monovalent dose of 15 µg hemagglutinin (single-radial immunodiffusion assay), the level of total protein and host cell DNA was 58 µg and 10 ng, respectively. Furthermore, all process steps can be fully scaled up to industrial quantities for commercial manufacturing of either seasonal or pandemic influenza virus vaccines. Fast production of up to 300 vaccine doses per liter within 4-5 days makes this process competitive not only to other cell-based processes but to egg-based processes as well.
Assuntos
COVID-19 , Técnicas de Cultura de Células , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vacinas contra Influenza/metabolismo , SARS-CoV-2/crescimento & desenvolvimento , Animais , Cães , Células Madin Darby de Rim CaninoRESUMO
Natural killer-92 cells (NK-92 cells) need to be efficiently expanded by serum-free culture in vitro to meet clinical requirements. Fatty acids mainly provide substrates for energy production, which is of crucial importance to meet the energy demands of highly proliferating cells. This study optimized the medium (EM) for NK-92 cells by designing an experiment to expand cells efficiently. EM, an in-house designed chemically defined serum-free medium, was used as the basal medium. Fatty acids as additive ingredients were screened and optimized by the experimental design method. Three additives, arachidonic acid, myristic acid and palmitoleic acid, were screened; therefore, the optimized medium was named EM-FA. The total cell expansion of NK-92 cells in EM-FA was 72.61±11.95-fold on day 8, which was significantly higher than the 28.55±8.67-fold expansion in EM. To explore the mechanism by which fatty acids promote NK-92 cell expansion, the cell growth kinetics and metabolic characteristics in EM-FA were analyzed. The results showed that NK-92 cells in EM-FA were rapidly expanded while maintaining their cell phenotype and cytotoxicity and enhancing the oxygen consumption rate and mitochondrial function. Fatty acids promoted ATP production to elevate the energy flux for better cell expansion. This study developed an expansion strategy of NK-92 cells in vitro to facilitate their clinical application. KEY POINTS: ⢠Arachidonic acid, myristic acid and palmitoleic acid in serum-free medium were optimized by experimental design to enable the rapid expansion of NK-92 cells in vitro. ⢠Fatty acids upregulated oxidative phosphorylation levels and improved the energy metabolism of NK-92 cells.
Assuntos
Ácidos Graxos , Células Matadoras Naturais , Proliferação de Células , Meios de Cultura , Metabolismo EnergéticoRESUMO
Similar to the recent COVID-19 pandemic, influenza A virus poses a constant threat to the global community. For the treatment of flu disease, both antivirals and vaccines are available with vaccines the most effective and safest approach. In order to overcome limitations in egg-based vaccine manufacturing, cell culture-based processes have been established. While this production method avoids egg-associated risks in face of pandemics, process intensification using animal suspension cells in high cell density perfusion cultures should allow to further increase manufacturing capacities worldwide. In this work, we demonstrate the development of a perfusion process using Madin-Darby canine kidney (MDCK) suspension cells for influenza A (H1N1) virus production from scale-down shake flask cultivations to laboratory scale stirred tank bioreactors. Shake flask cultivations using semi-perfusion mode enabled high-yield virus harvests (4.25 log10(HAU/100 µL)) from MDCK cells grown up to 41 × 106 cells/mL. Scale-up to bioreactors with an alternating tangential flow (ATF) perfusion system required optimization of pH control and implementation of a temperature shift during the infection phase. Use of a capacitance probe for on-line perfusion control allowed to minimize medium consumption. This contributed to a better process control and a more economical performance while maintaining a maximum virus titer of 4.37 log10(HAU/100 µL) and an infectious virus titer of 1.83 × 1010 virions/mL. Overall, this study clearly demonstrates recent advances in cell culture-based perfusion processes for next-generation high-yield influenza vaccine manufacturing for pandemic preparedness. KEY POINTS: ⢠First MDCK suspension cell-based perfusion process for IAV produciton was established. ⢠"Cell density effect" was overcome and process was intensified by reduction of medium use and automated process control. ⢠The process achieved cell density over 40 × 106 cells/mL and virus yield over 4.37 log10(HAU/100 µL).
Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , Cultura de Vírus/métodos , Replicação Viral/fisiologia , Animais , Reatores Biológicos , Cães , Células Madin Darby de Rim CaninoRESUMO
For successful engineering of pre-vascularized bone tissue in vitro, understanding the interactions between vasculogenic cells and bone-forming cells is a prerequisite. Mounting evidence indicates that microRNAs can serve as intercellular signals that allow cell-cell communication. Here, the role of the transfer of the microRNA miR-200b between vasculogenic and osteogenic cells was explored in a co-culture system. Rat bone-marrow derived mesenchymal stem cells (BMSCs) formed functional gap junctions composed of connexin 43 (Cx43, also known as GJA1) with human umbilical vein endothelial cells (HUVECs), through which miR-200b could transfer from BMSCs to HUVECs to regulate osteogenesis and angiogenesis. As a negative regulator, the decrease in miR-200b level in BMSCs derepressed the expression of VEGF-A, leading to increased osteogenic differentiation. Once inside HUVECs, miR-200b reduced the angiogenic potential of HUVECs through downregulation of ZEB2, ETS1, KDR and GATA2 Additionally, TGF-ß was found to trigger the transfer of miR-200b to HUVECs. Upon adding the TGF-ß inhibitor SB431542 or TGF-ß-neutralizing antibody, the formation of capillary-like structures in co-culture could be partially rescued. These findings may be fundamental to the development of a cell-based bone regeneration strategy.
Assuntos
Junções Comunicantes/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Neovascularização Fisiológica , Osteogênese , Animais , Células Cultivadas , Técnicas de Cocultura , Conexina 43/genética , Conexina 43/metabolismo , Junções Comunicantes/genética , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , Ratos , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
During bioleaching of Cobalt from waste lithium-ion batteries, the biooxidation activity of acidophilic bacteria is inhibited by a high concentration of Co ion in the liquid phase. However, the mechanism for Co2+ toxicity to acidophilic bacteria has not been fully elucidated. In this study, the effects of Co2+ concentration on the biooxidation activity for Fe2+, intracellular reactive oxygen species (ROS) level and antioxidant defense systems in a mixed-culture of acidophilic bacteria (MCAB) were investigated. The results showed that the biooxidation activity of the MCAB was inhibited by Co2+. Furthermore, it was indicated that the intracellular ROS contents of the MCAB under conditions of 0.4 M and 0.6 M Co2+ were 2.60 and 3.34 times higher than that under the condition of 0 M Co2+. The increase in intracellular malondialdehyde content indicated that the oxidative damage was induced by Co2+. Moreover, the antioxidant systems in MCAB were affected by Co2+. It was observed that the Co2+ exposure increased the catalase and glutathione peroxidase activities while reducing the superoxide dismutase activity and the intracellular glutathione (GSH) content. It was found that an exogenous GSH supplementation eliminated excess intracellular ROS and improved the biooxidation activity of the MCAB.
Assuntos
Estresse Oxidativo , Antioxidantes , Bactérias , Catalase , Glutationa , Superóxido DismutaseRESUMO
BACKGROUND AND AIMS: Bioartificial livers (BALs) have attracted much attention as potential supportive therapies for liver diseases. A serum-free microcarrier culture strategy for the in vitro high-density expansion of human-induced hepatocyte-like cells (hiHeps) suitable for BALs was studied in this article. METHODS: hiHeps were transdifferentiated from human fibroblasts by the lentiviral overexpression of FOXA3, HNF1A, and HNF4A. Cells were cultured on microcarriers, their proliferation was evaluated by cell count and CCK-8 assays, and their function was evaluated by detecting liver function parameters in the supernatant, including urea secretion, albumin synthesis, and lactate dehydrogenase levels. The expressions of hepatocyte function-associated genes of hiHeps were measured by qRT-PCR in 2D and 3D conditions. The expression of related proteins during fibronectin promotes cell adhesion, and proliferation on microcarrier was detected by western blotting. RESULTS: During microcarrier culture, the optimal culture conditions during the adherence period were the use of half-volume high-density inoculation, Cytodex 3 at a concentration of 3 mg/mL, a cell seeding density of 2.0 × 105 cells/mL, and a stirring speed of 45 rpm. The final cell density in self-developed, chemically defined serum-free medium (SFM) reached 2.53 × 106 cells/mL, and the maximum increase in expansion was 12.61-fold. In addition, we found that fibronectin (FN) can promote hiHep attachment and proliferation on Cytodex 3 microcarriers and that this pro-proliferative effect was mediated by the integrin-ß1/FAK/ERK/CyclinD1 signaling pathway. Finally, the growth and function of hiHeps on Cytodex 3 in SFM were close to those of hiHeps on Cytodex 3 in hepatocyte maintenance medium (HMM), and cells maintained their morphology and function after harvest on microcarriers. CONCLUSIONS: Serum-free microcarrier culture has important implications for the expansion of a sufficient number of hiHeps prior to the clinical application of BALs.
Assuntos
Técnicas de Cultura de Células/métodos , Proliferação de Células , Transdiferenciação Celular , Hepatócitos/citologia , Fígado Artificial , Albuminas/biossíntese , Adesão Celular , Técnicas de Reprogramação Celular/métodos , Meios de Cultura Livres de Soro , Ciclina D1/metabolismo , Dextranos , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 3-gama Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/genética , Hepatócitos/metabolismo , Hepatócitos/fisiologia , Humanos , Integrina beta1/metabolismo , L-Lactato Desidrogenase/metabolismo , Sistema de Sinalização das MAP Quinases , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ureia/metabolismoRESUMO
Tyrosine (Tyr) is crucial to the maintenance of the monoclonal antibody (mAb) titers and quality attributes in fed-batch cultures of recombinant Chinese hamster ovary (rCHO) cells. However, the relation between tyrosine and these aspects is not yet fully defined. In order to further elucidate such a relation, two groups of fed-batch experiments with high tyrosine (H-T) or low tyrosine (L-T) additions producing an IgG1 monoclonal antibody against CD20 were implemented to investigate the intracellular and extracellular effects of tyrosine on the culture performance. It was found that the scarcity of tyrosine led to the distinctive reduction in both viable cell density and antibody specific production rate, hence the sharply reduced titer, possibly related to the impaired translation efficiency caused by the substrate limitation of tyrosine. In addition, alterations to the critical quality attributes were detected in the L-T group, compared to those in the H-T condition. Notable decrease in the contents of intact antibody was found under the L-T condition because of the elevated reductive level in the supernatant. Moreover, the aggregate content in the L-T condition was also reduced, probably resulting from the accumulation of extracellular cystine. In particular, the lysine variant content noticeably increased with tyrosine limitation owing to the downregulation of two carboxypeptidases, i.e., CpB and CpH. Overall, understanding the role of tyrosine in these aspects is fundamental to the increase of product titers and control of critical quality attributes in the monoclonal antibody production of rCHO cell fed-batch cultures. KEY POINTS: ⢠Tyrosine is essential in the maintenance of product titers and the control of product qualities in high cell density cultivations in rCHO cell. ⢠This study revealed the bottleneck of decreased qmAbupon the deficiency of tyrosine. ⢠The impact of tyrosine on the critical product qualities and the underlying mechanisms were also thoroughly assessed.
Assuntos
Anticorpos Monoclonais/biossíntese , Meios de Cultura/química , Tirosina/farmacologia , Animais , Antígenos CD20/imunologia , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Células CHO , Cricetulus , Imunoglobulina G/biossínteseRESUMO
As the composition of animal cell culture medium becomes more complex, the identification of key variables is important for simplifying and guiding the subsequent medium optimization. However, the traditional experimental design methods are impractical and limited in their ability to explore such large feature spaces. Therefore, in this work, we developed a NRGK (nonparametric regression with Gaussian kernel) method, which aimed to identify the critical components that affect product titres during the development of cell culture media. With this nonparametric model, we successfully identified the important components that were neglected by the conventional PLS (partial least squares regression) method. The superiority of the NRGK method was further verified by ANOVA (analysis of variance). Additionally, it was proven that the selection accuracy was increased with the NRGK method because of its ability to model both the nonlinear and linear relationships between the medium components and titres. The application of this NRGK method provides new perspectives for the more precise identification of the critical components that further enable the optimization of media in a shorter timeframe.
Assuntos
Algoritmos , Animais , Células CHO , Cricetulus , Meios de Cultura , Análise dos Mínimos Quadrados , Projetos de PesquisaRESUMO
Transdifferentiated hepatocytes are potential seeding cells for bioartificial liver (BAL) treatment, and it is important to obtain a sufficient number of functional hepatocytes in serum-free medium (SFM). Although insulin plays an essential role in promoting cell proliferation and metabolism, the functions of insulin in transdifferentiated cells remain poorly understood. Here, we found that 1.0 mg/L insulin significantly increased human-induced hepatocyte-like cells (hiHeps) proliferation and viability in SFM. The pro-proliferative effect of insulin on these cells occurred via augmented cyclin D1 expression that was mediated by activation of the Akt1/mTOR/p70S6K and Akt1/P53 pathways. Further studies revealed that insulin also enhanced the specific liver function of hiHeps in SFM. Additionally, Western blotting and siHNF1A transfection analysis showed that insulin increased the protein expression of Albumin (ALB) and UDP-glucuronosyltransferase1A1 ï¼UGT1A1 ï¼ in hiHeps via HNF1A. Finally, hiHep proliferation and the expression of specific genes were maintained during long-term passaging in SFM supplemented with 1.0 mg/L insulin. Collectively, our findings show that insulin promotes transdifferentiated hiHep proliferation and specific functional expression. These findings have important implications for the expansion of functional hiHeps prior to clinical applications of BALs.