Wen-song Tan - Academia.edu (original) (raw)
Papers by Wen-song Tan
Chinese Journal of Biotechnology, 2009
As an immunodepressant, anti-CD25 monoclonal antibody has a huge market with wide prospect and ec... more As an immunodepressant, anti-CD25 monoclonal antibody has a huge market with wide prospect and economic value. We developed a low protein serum-free medium for large-scale GS-NS0 myeloma cell culture and anti-CD25 monoclonal antibody production. Further study focused on the characteristics of GS-NSO cell growth, glucose and amino acid metabolism, and antibody production. In the serum-free medium, the maximal viable cell density and antibody concentration reached above 3x10(6) cells/mL and 300 mg/L in batch culture. Compared with the commercial serum-free medium (Excell 620 + 0.2% Primatone), the maximal viable cell density doubled and the maximal antibody concentration increased 46%. Results also showed the specific growth rate decreased when the glucose concentration was lower than 6 mmol/L. And the production of lactate increased when glucose concentration was excessively high (> 30 mmol/L). These results were important to provide technique and theory basis for developing optim...
Additional file 2: Fig. S1. Effects of uridineâ s feed concentration on cell growth and cell viab... more Additional file 2: Fig. S1. Effects of uridineâ s feed concentration on cell growth and cell viability. a Viable cell density; b cell viability. Blank diamond control cultures; Blank circle 0.6 mM uridine fed; Blank triangle 6 mM uridine fed; Blank square 30mM uridine fed. The error bars indicate the standard deviations from three independent experiments.
Additional file 1: Table S1. Reactions in the simplified biochemical network. Table S2. Kinetic r... more Additional file 1: Table S1. Reactions in the simplified biochemical network. Table S2. Kinetic results in different cultures.
Chinese Journal of Biotechnology, 2011
In recent years, there are tremendous economic and social losses across the world because of viru... more In recent years, there are tremendous economic and social losses across the world because of virus-related diseases. It is well known that Madin-Darby canine kidney (MDCK) cells are easily handled, quickly amplified and efficiently infected with influenza virus. Therefore, they are considered as one of the most important cell lines for the production of influenza vaccine. In this work, we first developed a serum-free adherent culture process for MDCK cells with an in-house prepared serum-free medium MDCK-SFM. Next, we derived a cell line named ssf-MDCK, which was amenable for single-cell suspension culture in the serum-free medium. We found that during serum-free batch culture of MDCK cells, the peak viable cell density and maximum specific growth rate were 3.81 x 10(6) cells/mL and 0.056 h(-1), respectively; 3.6- and 1.6-fold increase compared with those in serum-containing adherent batch culture. In addition, we compared growth and metabolic characteristics of MDCK cells in serum-...
Journal of Microbiology and Biotechnology, 2008
Dissolved oxygen (DO) and shear stress have pronounced effects on hyaluronic acid (HA) production... more Dissolved oxygen (DO) and shear stress have pronounced effects on hyaluronic acid (HA) production, yet various views persist about their effect on the molecular weight of HA. Accordingly, this study investigated the effects of DO and shear stress during HA fermentation. The results showed that both cell growth and HA synthesis were suppressed under anaerobic conditions, and the HA molecular mass was only (1.22+/-0.02) x 106 Da. Under aerobic conditions, although the DO level produced no change in the biomass or HA yield, a high DO level favored the HA molecular mass, which reached a maximum value of (2.19+/- 0.05) x 106 Da at 50% DO. Furthermore, a high shear stress delayed the rate of HA synthesis and decreased the HA molecular weight, yet had no clear effect on the HA yield. Therefore, a high DO concentration and mild shear environment would appear to be essential to enhance the HA molecular weight.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 2003
Stirred culture offers a number of advantages over static systems as it maintains a stable, homog... more Stirred culture offers a number of advantages over static systems as it maintains a stable, homogeneous culture environment and is easy to scale-up. This paper focused on the development and application of stirred tank bioreactor to culture hematopoietic cells. Preliminary study of stirred culture of hematopoietic cells was carried out in cord blood mononuclear cells culture in spinner flask. The results showed that the amplification rates of total cell, CFU-GM and BFU-E, with the exception of CFU-Mk, were greater in spinner flask than T-flask. The number of total cells increased 20 fold after 14 days incubation in spinner flask. The amplification rates of CFU-GM, CFU-Mk and BFU-E reached maximum at 10th day, 10th day and 7th day respectively, and the maximal amplification rates were 9.2-fold, 5.5-fold and 2.4-fold respectively, whereas the rate of CD34+ cells in spinner flask was (6.7 +/- 4.0)-fold at day 10. These results indicated that the stirred culture system is better than th...
A Practical Manual for Musculoskeletal Research, 2008
International Journal of Biological Macromolecules, 2021
Epigallocatechin-3-gallate (EGCG) is a kind of flavonoids and has the ability to promote differen... more Epigallocatechin-3-gallate (EGCG) is a kind of flavonoids and has the ability to promote differentiation of mesenchymal stem cells (MSCs) into osteoblasts. However, the EGCG is easily metabolized by cells during cell culture, which reduces its bioavailability. Therefore, in this paper, EGCG-loaded chitosan nanoparticles (ECN) were fabricated and entrapped into chitosan/alginate (CS/Alg) scaffolds to form CS/Alg-ECN scaffolds for improving the bioavailability of EGCG. The human umbilical cord mesenchymal stem cells (HUMSCs) were cultured on CS/Alg-ECN scaffolds to induce osteogenic differentiation. The results indicated that the CS/Alg-ECN scaffolds continuously released EGCG for up to 16 days. Besides, these results suggested that CS/Alg-ECN scaffolds promoted osteoblast differentiation through activating Wnt/β-catenin signaling pathway. Collectively, this study demonstrated that the entrapment ECN into CS/Alg scaffolds was a promising strategy for promoting osteogenesis of MSCs.
Applied Microbiology and Biotechnology, 2020
Tyrosine (Tyr) is crucial to the maintenance of the monoclonal antibody (mAb) titers and quality ... more 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. • 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.
H9N2 subtype avian influenza virus poses a constant threat to the poultry industry and the contro... more H9N2 subtype avian influenza virus poses a constant threat to the poultry industry and the control of the disease leans upon the use of effective vaccines. As an alternative to the conventional chicken embryonated eggs, animal cell culture could overcome the limitations of egg supplies and upgrade the manufacturing of avian influenza vaccines for poultry. Development of serum-free suspension cell culture could allow even higher virus productivity, where a suspension cell line with good growth and production performance is required. In this work, an adherent MDCK cell line was adapted to suspension growth to cell concentration up to 12 × 106 cells/mL in a serum-free medium in batch cultures. Subsequently, the influenza virus propagation in this MDCK cell line was evaluated and was improved with the medium exchange at time of infection as well as optimization of infection conditions in terms of MOI and cell concentration for infection. Furthermore, various feed strategies were tested ...
Biochemical Engineering Journal, 2020
Abstract Bioartificial liver (BAL) comprising a functional bioreactor represents a promising trea... more Abstract Bioartificial liver (BAL) comprising a functional bioreactor represents a promising treatment, bridging patients with acute liver failure (ALF) to liver transplantation. In this research, a packed-bed (PB) bioreactor by utilizing galactosylated poly(ethylene terephthalate) microfibrous carrier (PET-Gal) was fabricated and evaluated in vitro. Human induced hepatocytes (hiHeps) were dynamically seeded into a PET-Gal loaded bioreactor and cultured for 10 days. During perfusion culture, the effects of bioreactor scaffold on hepatocyte seeding, growth and functionality (albumin secretion and urea production) were determined, compared with that of control with non-modified PET carrier. Afterwards, drug metabolism and detoxification ability of hiHeps were measured. Results demonstrated that the seeding rate and proliferation fold within PET-Gal loaded bioreactor were significantly higher than that of control, resulting in a cell density of (8.17 ± 0.52)×107 cells/cm3. In contrast to flattened morphology observed in the control, hiHeps cultured in PET-Gal based bioreactor displayed as three-dimensional (3D) cell aggregates with close cell-matrix and cell-cell interactions and high cell viability, leading to higher albumin secretion and urea production. Besides, there were high levels of phase I drug metabolism and ammonia elimination for PET-Gal cultured cells. Therefore, the PET-Gal based PB bioreactor fabricated herein supports high-density hepatocyte growth and meanwhile preserves hepatic functionality, showing great potential in BAL systems application.
Vaccines, 2020
The miRNA-based strategy has been used to develop live attenuated influenza vaccines. In this stu... more The miRNA-based strategy has been used to develop live attenuated influenza vaccines. In this study, the nucleoprotein (NP) genome segment of the influenza virus was inserted by different perfect miRNA-192-5p target sites, and the virus was rescued by standard reverse genetics method, so as to verify the virulence and protective efficacy of live attenuated vaccine in cells and mice. The results showed there was no significant attenuation in 192t virus with one perfect miRNA-192-5p target site, and 192t-3 virus with three perfect miRNA target sites. However, 192t-6 virus with 6 perfect miRNA target sites and 192t-9 virus with 9 perfect miRNA target sites were both significantly attenuated after infection, and their virulence were similar to that of temperature-sensitive (TS) influenza A virus (IAV) which is a temperature-sensitive live attenuated influenza vaccine. Mice were immunized with different doses of 192t-6, 192t-9, and TS IAV. Four weeks after immunization, the IgG in serum ...
Journal of Industrial Microbiology & Biotechnology, 2019
As the composition of animal cell culture medium becomes more complex, the identification of key ... more 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...
Digestive Diseases and Sciences, 2019
Background and Aims Bioartificial livers (BALs) have attracted much attention as potential suppor... more 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 × 10 5 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 × 10 6 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-β1/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.
Frontiers in Immunology, 2019
Bioresources and Bioprocessing, 2018
Archives of Biochemistry and Biophysics, 2019
The insufficient vascularization is a major challenge in bone tissue engineering, leading to part... more The insufficient vascularization is a major challenge in bone tissue engineering, leading to partial necrosis of the implant. Pre-vascularization is a promising way via in vitro cells co-culture strategies using osteogenic cells and vasculogenic cells, and the cross-talk of cells is essential. In the present study, the effect of rat bone-marrow derived mesenchymal stem cells (BMSCs) on angiogenic capability of human umbilical vein endothelial cells (HUVECs) in growth medium (GM) and osteogenic induction medium (OIM) was investigated. It was demonstrated that cells co-cultured in OIM showed high efficiency in osteogenesis but failed to form capillary-like structure while the results of co-culture in GM were the opposite. By comparing the angiogenic capacity of co-cultures under GM and OIM, chemokine (C-X-C motif) ligand 9 (Cxcl9), secreted by BMSCs in OIM, was identified to be an angiostatic factor to counter-regulate vascular endothelial growth factor (VEGF) and prevent its binding to HUVECs, which abrogated angiogenesis of MSCs-ECs co-culture. Moreover, Cxcl9 was proved to suppress the osteogenic differentiation of BMSCs monoculture. The molecular mechanism of Cxcl9 activation in BMSCs involved mTOR/STAT1 signaling pathway. Therefore, blocking this signaling pathway via rapamycin addition resulted in the inhibition of Cxcl9 and improvement of osteogenic differentiation and angiogenic capacity of co-culture in OIM. These results reveal that Cxcl9 is a negative modulator of angiogenesis and osteogenesis, and its inhibition could promote pre-vascularized of bone tissue engineering.
Applied Microbiology and Biotechnology, 2019
Tyrosine (Tyr), as one of the least soluble amino acids, is essential to monoclonal antibody (mAb... more Tyrosine (Tyr), as one of the least soluble amino acids, is essential to monoclonal antibody (mAb) production in recombinant Chinese hamster ovary (rCHO) cell cultures since its roles on maintaining the specific productivity (qmAb) and avoiding Tyr sequence variants. To understand the effects of Tyr on cell performance and its underlying mechanisms, rCHO cell–producing mAbs were cultivated at various cumulative Tyr addition concentrations (0.6 to 5.5 mM) in fed-batch processes. Low Tyr concentrations gave a much lower peak viable cell density (VCD) during the growth phase and also induced rapid cell death and pH decrease during the production phase, resulting in a low efficient fed-batch process. Autophagy was initiated following the inhibition of mTOR under the Tyr starvation condition. Excessive autophagy subsequently induced autophagic cell death, which was found as the major type of cell death in this study. Additionally, the results obtained here demonstrate that the decrease in culture pH under the Tyr starvation condition was associated with the autophagy and such pH drop might be attributed to the lysosome acidification and cell lysis.
Applied Microbiology and Biotechnology, 2019
Sialylation affects circulating half-life, charge distribution, and other biochemical properties ... more Sialylation affects circulating half-life, charge distribution, and other biochemical properties of therapeutic glycoproteins. Loss of protein sialylation during glycoprotein-producing bioprocesses could lead to a low final protein sialylation level and bring negative effects on subsequent clinical efficacy. In this work, an Fc-fusion protein-producing Chinese hamster ovary cell fed-batch culture process was studied and insights into the loss of protein sialylation during the Fc-fusion protein production phase (days 5 to 13) were presented. The results showed that the decreased total sialic acid content was 13.84 μg/mg during the production phase, which accounted for 24% of the total sialic acid content on day 5. The lost sialic acids were predominantly from α 2-3 sialylation on N- and O-glycans. Through cell-free incubation and kinetics studies, it was found that the decreased sialic acid content caused by extracellular sialic acid degradation and incomplete glycan biosynthesis were 7.79 μg/mg and 6.05 μg/mg, respectively. The two processes had a nearly equal contribution to the loss of final product sialylation. Detailed characterizations revealed that decreases in sialic acid content were due either to extracellular sialic acid degradation via hydrolysis of α 2-3 sialic acids probably by released cytosolic sialidase or to a lack of galactosylated glycan availability for sialylation during late-stage glycosylation. Our work provides a better understanding of losses in protein sialylation during glycoprotein manufacturing.
Journal of Cellular and Molecular Medicine, 2019
Chinese Journal of Biotechnology, 2009
As an immunodepressant, anti-CD25 monoclonal antibody has a huge market with wide prospect and ec... more As an immunodepressant, anti-CD25 monoclonal antibody has a huge market with wide prospect and economic value. We developed a low protein serum-free medium for large-scale GS-NS0 myeloma cell culture and anti-CD25 monoclonal antibody production. Further study focused on the characteristics of GS-NSO cell growth, glucose and amino acid metabolism, and antibody production. In the serum-free medium, the maximal viable cell density and antibody concentration reached above 3x10(6) cells/mL and 300 mg/L in batch culture. Compared with the commercial serum-free medium (Excell 620 + 0.2% Primatone), the maximal viable cell density doubled and the maximal antibody concentration increased 46%. Results also showed the specific growth rate decreased when the glucose concentration was lower than 6 mmol/L. And the production of lactate increased when glucose concentration was excessively high (> 30 mmol/L). These results were important to provide technique and theory basis for developing optim...
Additional file 2: Fig. S1. Effects of uridineâ s feed concentration on cell growth and cell viab... more Additional file 2: Fig. S1. Effects of uridineâ s feed concentration on cell growth and cell viability. a Viable cell density; b cell viability. Blank diamond control cultures; Blank circle 0.6 mM uridine fed; Blank triangle 6 mM uridine fed; Blank square 30mM uridine fed. The error bars indicate the standard deviations from three independent experiments.
Additional file 1: Table S1. Reactions in the simplified biochemical network. Table S2. Kinetic r... more Additional file 1: Table S1. Reactions in the simplified biochemical network. Table S2. Kinetic results in different cultures.
Chinese Journal of Biotechnology, 2011
In recent years, there are tremendous economic and social losses across the world because of viru... more In recent years, there are tremendous economic and social losses across the world because of virus-related diseases. It is well known that Madin-Darby canine kidney (MDCK) cells are easily handled, quickly amplified and efficiently infected with influenza virus. Therefore, they are considered as one of the most important cell lines for the production of influenza vaccine. In this work, we first developed a serum-free adherent culture process for MDCK cells with an in-house prepared serum-free medium MDCK-SFM. Next, we derived a cell line named ssf-MDCK, which was amenable for single-cell suspension culture in the serum-free medium. We found that during serum-free batch culture of MDCK cells, the peak viable cell density and maximum specific growth rate were 3.81 x 10(6) cells/mL and 0.056 h(-1), respectively; 3.6- and 1.6-fold increase compared with those in serum-containing adherent batch culture. In addition, we compared growth and metabolic characteristics of MDCK cells in serum-...
Journal of Microbiology and Biotechnology, 2008
Dissolved oxygen (DO) and shear stress have pronounced effects on hyaluronic acid (HA) production... more Dissolved oxygen (DO) and shear stress have pronounced effects on hyaluronic acid (HA) production, yet various views persist about their effect on the molecular weight of HA. Accordingly, this study investigated the effects of DO and shear stress during HA fermentation. The results showed that both cell growth and HA synthesis were suppressed under anaerobic conditions, and the HA molecular mass was only (1.22+/-0.02) x 106 Da. Under aerobic conditions, although the DO level produced no change in the biomass or HA yield, a high DO level favored the HA molecular mass, which reached a maximum value of (2.19+/- 0.05) x 106 Da at 50% DO. Furthermore, a high shear stress delayed the rate of HA synthesis and decreased the HA molecular weight, yet had no clear effect on the HA yield. Therefore, a high DO concentration and mild shear environment would appear to be essential to enhance the HA molecular weight.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 2003
Stirred culture offers a number of advantages over static systems as it maintains a stable, homog... more Stirred culture offers a number of advantages over static systems as it maintains a stable, homogeneous culture environment and is easy to scale-up. This paper focused on the development and application of stirred tank bioreactor to culture hematopoietic cells. Preliminary study of stirred culture of hematopoietic cells was carried out in cord blood mononuclear cells culture in spinner flask. The results showed that the amplification rates of total cell, CFU-GM and BFU-E, with the exception of CFU-Mk, were greater in spinner flask than T-flask. The number of total cells increased 20 fold after 14 days incubation in spinner flask. The amplification rates of CFU-GM, CFU-Mk and BFU-E reached maximum at 10th day, 10th day and 7th day respectively, and the maximal amplification rates were 9.2-fold, 5.5-fold and 2.4-fold respectively, whereas the rate of CD34+ cells in spinner flask was (6.7 +/- 4.0)-fold at day 10. These results indicated that the stirred culture system is better than th...
A Practical Manual for Musculoskeletal Research, 2008
International Journal of Biological Macromolecules, 2021
Epigallocatechin-3-gallate (EGCG) is a kind of flavonoids and has the ability to promote differen... more Epigallocatechin-3-gallate (EGCG) is a kind of flavonoids and has the ability to promote differentiation of mesenchymal stem cells (MSCs) into osteoblasts. However, the EGCG is easily metabolized by cells during cell culture, which reduces its bioavailability. Therefore, in this paper, EGCG-loaded chitosan nanoparticles (ECN) were fabricated and entrapped into chitosan/alginate (CS/Alg) scaffolds to form CS/Alg-ECN scaffolds for improving the bioavailability of EGCG. The human umbilical cord mesenchymal stem cells (HUMSCs) were cultured on CS/Alg-ECN scaffolds to induce osteogenic differentiation. The results indicated that the CS/Alg-ECN scaffolds continuously released EGCG for up to 16 days. Besides, these results suggested that CS/Alg-ECN scaffolds promoted osteoblast differentiation through activating Wnt/β-catenin signaling pathway. Collectively, this study demonstrated that the entrapment ECN into CS/Alg scaffolds was a promising strategy for promoting osteogenesis of MSCs.
Applied Microbiology and Biotechnology, 2020
Tyrosine (Tyr) is crucial to the maintenance of the monoclonal antibody (mAb) titers and quality ... more 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. • 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.
H9N2 subtype avian influenza virus poses a constant threat to the poultry industry and the contro... more H9N2 subtype avian influenza virus poses a constant threat to the poultry industry and the control of the disease leans upon the use of effective vaccines. As an alternative to the conventional chicken embryonated eggs, animal cell culture could overcome the limitations of egg supplies and upgrade the manufacturing of avian influenza vaccines for poultry. Development of serum-free suspension cell culture could allow even higher virus productivity, where a suspension cell line with good growth and production performance is required. In this work, an adherent MDCK cell line was adapted to suspension growth to cell concentration up to 12 × 106 cells/mL in a serum-free medium in batch cultures. Subsequently, the influenza virus propagation in this MDCK cell line was evaluated and was improved with the medium exchange at time of infection as well as optimization of infection conditions in terms of MOI and cell concentration for infection. Furthermore, various feed strategies were tested ...
Biochemical Engineering Journal, 2020
Abstract Bioartificial liver (BAL) comprising a functional bioreactor represents a promising trea... more Abstract Bioartificial liver (BAL) comprising a functional bioreactor represents a promising treatment, bridging patients with acute liver failure (ALF) to liver transplantation. In this research, a packed-bed (PB) bioreactor by utilizing galactosylated poly(ethylene terephthalate) microfibrous carrier (PET-Gal) was fabricated and evaluated in vitro. Human induced hepatocytes (hiHeps) were dynamically seeded into a PET-Gal loaded bioreactor and cultured for 10 days. During perfusion culture, the effects of bioreactor scaffold on hepatocyte seeding, growth and functionality (albumin secretion and urea production) were determined, compared with that of control with non-modified PET carrier. Afterwards, drug metabolism and detoxification ability of hiHeps were measured. Results demonstrated that the seeding rate and proliferation fold within PET-Gal loaded bioreactor were significantly higher than that of control, resulting in a cell density of (8.17 ± 0.52)×107 cells/cm3. In contrast to flattened morphology observed in the control, hiHeps cultured in PET-Gal based bioreactor displayed as three-dimensional (3D) cell aggregates with close cell-matrix and cell-cell interactions and high cell viability, leading to higher albumin secretion and urea production. Besides, there were high levels of phase I drug metabolism and ammonia elimination for PET-Gal cultured cells. Therefore, the PET-Gal based PB bioreactor fabricated herein supports high-density hepatocyte growth and meanwhile preserves hepatic functionality, showing great potential in BAL systems application.
Vaccines, 2020
The miRNA-based strategy has been used to develop live attenuated influenza vaccines. In this stu... more The miRNA-based strategy has been used to develop live attenuated influenza vaccines. In this study, the nucleoprotein (NP) genome segment of the influenza virus was inserted by different perfect miRNA-192-5p target sites, and the virus was rescued by standard reverse genetics method, so as to verify the virulence and protective efficacy of live attenuated vaccine in cells and mice. The results showed there was no significant attenuation in 192t virus with one perfect miRNA-192-5p target site, and 192t-3 virus with three perfect miRNA target sites. However, 192t-6 virus with 6 perfect miRNA target sites and 192t-9 virus with 9 perfect miRNA target sites were both significantly attenuated after infection, and their virulence were similar to that of temperature-sensitive (TS) influenza A virus (IAV) which is a temperature-sensitive live attenuated influenza vaccine. Mice were immunized with different doses of 192t-6, 192t-9, and TS IAV. Four weeks after immunization, the IgG in serum ...
Journal of Industrial Microbiology & Biotechnology, 2019
As the composition of animal cell culture medium becomes more complex, the identification of key ... more 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...
Digestive Diseases and Sciences, 2019
Background and Aims Bioartificial livers (BALs) have attracted much attention as potential suppor... more 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 × 10 5 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 × 10 6 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-β1/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.
Frontiers in Immunology, 2019
Bioresources and Bioprocessing, 2018
Archives of Biochemistry and Biophysics, 2019
The insufficient vascularization is a major challenge in bone tissue engineering, leading to part... more The insufficient vascularization is a major challenge in bone tissue engineering, leading to partial necrosis of the implant. Pre-vascularization is a promising way via in vitro cells co-culture strategies using osteogenic cells and vasculogenic cells, and the cross-talk of cells is essential. In the present study, the effect of rat bone-marrow derived mesenchymal stem cells (BMSCs) on angiogenic capability of human umbilical vein endothelial cells (HUVECs) in growth medium (GM) and osteogenic induction medium (OIM) was investigated. It was demonstrated that cells co-cultured in OIM showed high efficiency in osteogenesis but failed to form capillary-like structure while the results of co-culture in GM were the opposite. By comparing the angiogenic capacity of co-cultures under GM and OIM, chemokine (C-X-C motif) ligand 9 (Cxcl9), secreted by BMSCs in OIM, was identified to be an angiostatic factor to counter-regulate vascular endothelial growth factor (VEGF) and prevent its binding to HUVECs, which abrogated angiogenesis of MSCs-ECs co-culture. Moreover, Cxcl9 was proved to suppress the osteogenic differentiation of BMSCs monoculture. The molecular mechanism of Cxcl9 activation in BMSCs involved mTOR/STAT1 signaling pathway. Therefore, blocking this signaling pathway via rapamycin addition resulted in the inhibition of Cxcl9 and improvement of osteogenic differentiation and angiogenic capacity of co-culture in OIM. These results reveal that Cxcl9 is a negative modulator of angiogenesis and osteogenesis, and its inhibition could promote pre-vascularized of bone tissue engineering.
Applied Microbiology and Biotechnology, 2019
Tyrosine (Tyr), as one of the least soluble amino acids, is essential to monoclonal antibody (mAb... more Tyrosine (Tyr), as one of the least soluble amino acids, is essential to monoclonal antibody (mAb) production in recombinant Chinese hamster ovary (rCHO) cell cultures since its roles on maintaining the specific productivity (qmAb) and avoiding Tyr sequence variants. To understand the effects of Tyr on cell performance and its underlying mechanisms, rCHO cell–producing mAbs were cultivated at various cumulative Tyr addition concentrations (0.6 to 5.5 mM) in fed-batch processes. Low Tyr concentrations gave a much lower peak viable cell density (VCD) during the growth phase and also induced rapid cell death and pH decrease during the production phase, resulting in a low efficient fed-batch process. Autophagy was initiated following the inhibition of mTOR under the Tyr starvation condition. Excessive autophagy subsequently induced autophagic cell death, which was found as the major type of cell death in this study. Additionally, the results obtained here demonstrate that the decrease in culture pH under the Tyr starvation condition was associated with the autophagy and such pH drop might be attributed to the lysosome acidification and cell lysis.
Applied Microbiology and Biotechnology, 2019
Sialylation affects circulating half-life, charge distribution, and other biochemical properties ... more Sialylation affects circulating half-life, charge distribution, and other biochemical properties of therapeutic glycoproteins. Loss of protein sialylation during glycoprotein-producing bioprocesses could lead to a low final protein sialylation level and bring negative effects on subsequent clinical efficacy. In this work, an Fc-fusion protein-producing Chinese hamster ovary cell fed-batch culture process was studied and insights into the loss of protein sialylation during the Fc-fusion protein production phase (days 5 to 13) were presented. The results showed that the decreased total sialic acid content was 13.84 μg/mg during the production phase, which accounted for 24% of the total sialic acid content on day 5. The lost sialic acids were predominantly from α 2-3 sialylation on N- and O-glycans. Through cell-free incubation and kinetics studies, it was found that the decreased sialic acid content caused by extracellular sialic acid degradation and incomplete glycan biosynthesis were 7.79 μg/mg and 6.05 μg/mg, respectively. The two processes had a nearly equal contribution to the loss of final product sialylation. Detailed characterizations revealed that decreases in sialic acid content were due either to extracellular sialic acid degradation via hydrolysis of α 2-3 sialic acids probably by released cytosolic sialidase or to a lack of galactosylated glycan availability for sialylation during late-stage glycosylation. Our work provides a better understanding of losses in protein sialylation during glycoprotein manufacturing.
Journal of Cellular and Molecular Medicine, 2019