Construction of vascularized cardiac tissue from genetically modified mouse embryonic stem cells.

BACKGROUND: The aim of myocardial tissue engineering is to repair or regenerate damaged myocardium with engineered cardiac tissue constructed by a combination of cells and scaffolds in vitro. However, this strategy has been hampered by the lack of cardiomyocytes and the significant cell death after transplantation in vivo. METHODS: In this study we explored the feasibility of in vitro construction of vascularized cardiac muscle using genetically modified mouse embryonic stem cells (ESCs) transfected by pMHC-neo/SV40-hygro. A stirred bioreactor was used to facilitate the formation of a large number of ESC-derived cardiomyocytes, which were then mixed with human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblasts (MEFs) in a liquid collagen scaffold to construct highly vascularized cardiac tissue in vitro. RESULTS: The resulting tissue constructs were transplanted into dorsal subcutaneous sites of nude mice. Tumor formation was not detected in all samples and vascularized cardiac tissue could survive after transplantation. Vascularization of the implanted cardiac muscle was significantly enhanced by the addition of HUVECs and MEFs, which resulted in a thicker myocardium. The combination of genetically modified ESCs and stirred bioreactor cultivation not only benefited the large-scale production of pure ESC-derived cardiomyocytes, but also effectively controlled the potential risk of undifferentiated ESCs. CONCLUSIONS: Using liquid collagen as scaffold, the enriched cardiomyocytes derived from genetically modified ESCs mixed with HUVECs and MEFs in 3-dimensional culture resulted in highly vascularized cardiac tissues.

[Analysis of the transcriptional profiling of cell cycle regulatory networks of recombinant Chinese hamster ovary cells in batch and fed-batch cultures].

In the light of Chinese hamster ovary (CHO) cell line 11G-S expressing human recombinant pro-urokinase, the differences of gene expression levels of the cells in different growth phases in both batch and fed-batch cultures were revealed by using gene chip technology. Then, based on the known cell cycle regulatory networks, the transcriptional profiling of the cell cycle regulatory networks of the cells in batch and fed-batch cultures was analyzed by using Genmapp software. Among the approximate 19 191 target genes in gene chip, the number of down-regulated genes was more than those of up-regulated genes of the cells in both batch and fed-batch cultures. The number of down-regulated genes of the cells in the recession phase in fed-batch culture was much more than that of the cells in batch culture. Comparative transcriptional analysis of the key cell cycle regulatory genes of the cells in both culture modes indicated that the cell proliferation and cell viability of the cells in both batch and fed-batch cultures were mainly regulated through down-regulating Cdk6, Cdk2, Cdc2a, Ccne1, Ccne2 genes of CDKs, Cyclin and CKI family and up-regulating Smad4 gene.

[Evaluation of the critical process parameters for the cultivation of recombinant Chinese hamster ovary cells in serum-free fed-batch mode].

Taking a suspension adapted recombinant CHO cell line, 11G-S expressing human Pro-urokinase (Pro-UK) as the object of study, the impacts of different feeding nutrients, the start time of feeding and cell inoculation density on the growth and Pro-UK production of 11G-S cells in serum-free fed-batch culture were evaluated in 100 mL shacking flasks. The results indicated that amino acids, serum-free supplements and inorganic salts played important role in cell growth, cell viability and protein expression. And the effects of cells fed-batch culture was much better with the initial cell inoculation density at 3 x 10(5)-4 x 10(5) cells/mL and the start time of feeding set at 72 h, a maximum viable cells density of 7.8 x 10(6) cells/mL with a peak Pro-UK activity at 8570 IU/mL was achieved during 12 d fed-batch culture. Further, the mu of the 11G-S cells at the middle phase of the fed-batch culture, and both the q(glu) and q(gln) of the 11G-S cells at the middle and later phases of the fed-batch culture was higher than that of the 11G-S cells at the same phase of the batch culture, respectively.

Different strategies for preparation of non-tagged rV270 protein and its efficacy against Yersinia pestis challenge.

OBJECTIVE: LcrV is an important component for the development of a subunit vaccine against plague. To reduce immunosuppressive activity of LcrV, a recombinant LcrV variant lacking amino acids 271 to 326 (rV270) was prepared by different methods in this study. METHODS: A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a, or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a. After Co(2+) affinity chromatography, a purification strategy was developed by cleavage of His tag on column, following Sephacryl S-200HR column filtration chromatography. RESULTS: Removal of His tag by thrombin, enterokinase and factor Xa displayed a yield of 99.5%, 32.4% and 15.3%, respectively. Following Sephacryl S-200HR column filtration chromatography, above 97% purity of rV270 protein was obtained. Purified rV270 that was adsorbed to 25% (v/v) Al(OH)3 adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to rV270 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 106 CFU of Y. pestis virulent strain 141. CONCLUSION: The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa, but they exhibited extremely low cleavage activity to the corresponding recognition site. Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy. The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.

[Serum-free medium for suspension culture of recombinant Chinese hamster ovary (11G-S) cells].

With suspension adapted recombinant Chinese hamster ovary (CHO) cell lines 11G-S expressing human pro-urokinase (pro-UK) as the object of study, a serum-free medium for the cultivation of recombinant CHO cells in suspension was formulated by using Plackett-Burman design and response surface methodology. The two-level Plackett-Burman design was used to evaluate the effect of 10 medium supplements on the growth of the 11G-S cells in suspension culture. Among the 10 medium supplements, insulin, transferrin, and putrescine were identified as the most significant factors (P < 0.05). The response surface methodology with three factors and three levels was used to determine the optimal levels of these factors. And a serum-free medium, SFM-CHO-S for recombinant CHO cells suspension culture was formulated. The maximum cell density of 11G-S cells in SFM-CHO-S in suspension batch culture reached 4.12 x 10(6) cells/mL with a maximum pro-UK activity at 5614 IU/mL, which was superior to the commercial serum-free medium for recombinant CHO cells.

[Metabolic characteristics and kinetic model of recombinant CHO cells in serum-free suspension batch culture].

By using the cell density, cell viability, Pro-UK activity, specific consumption rate of glucose (q(glc)), specific production rate of lactate (q(lac)), yield of lactate to glucose (Y(lac/glc)) and as the evaluation indexes, the growth and metabolism characteristics of pro-urokinase (Pro-UK) expressing CHO cells in serum-free suspension batch culture were examined and compared to those in serum-containing suspension batch culture. We observed hardly differences in growth and metabolism characteristics between the CHO cell populations grown in serum-free suspension batch culture and serum-containing suspension batch culture. The optimal mathematical model parameters for the CHO cells grown in suspension batch culture were obtained by non-linear programming of data representing the growth, substrate consumption and product formation of the CHO cells during logarithmic growth phase using MATLAB software, and the kinetic model of the cell growth and metabolism in serum-free culture were established.

Comparison of mouse, guinea pig and rabbit models for evaluation of plague subunit vaccine F1+rV270.

In this study, a new subunit vaccine that comprised native F1 and recombinant rV270 was evaluated for protective efficacy using mouse, guinea pig and rabbit models in comparison with the live attenuated vaccine EV76. Complete protection against challenging with 10(6) colony-forming units (CFU) of virulent Yersinia pestis strain 141 was observed for mice immunized with the subunit vaccines and EV76 vaccine. In contrast, the subunit vaccine recipes VII (F1-20 microg+rV270-10 microg) and IX (F1-40 microg+rV270-20 microg) and EV76 vaccine provided 86%, 79% and 93% protection against the same level of challenge in guinea pigs and 100%, 83% and 100% protection in rabbits, respectively. The immunized mice with the vaccines had significantly higher IgG titres than the guinea pigs and rabbits, and the immunized guinea pigs developed significantly higher IgG titres than the rabbits, but the anti-F1 response in guinea pigs was more variable than in the mice and rabbits, indicating that guinea pig is not an ideal model for evaluating protective efficacy of plague subunit vaccine, instead the rabbits could be used as an alternative model. All the immunized animals with EV76 developed a negligible IgG titre to rV270 antigen. Furthermore, analysis of IgG subclasses in the immunized animals showed a strong response for IgG1, whereas those receiving EV76 immunization demonstrated predominant production of IgG1 and IgG2a isotypes. The subunit vaccine and EV76 vaccine are able to provide protection for animals against Y. pestis challenge, but the subunit vaccines have obvious advantages over EV76 in terms of safety of use.

A high-yield and scaleable adenovirus vector production process based on high density perfusion culture of HEK 293 cells as suspended aggregates.

Cells of the human embryonic kidney cell line (HEK 293) were grown as suspended aggregates in stirred vessels and infected with a recombinant adenovirus vector (Ad-TH-GFP). Regular spherical aggregates with the mean diameter less than 300 microm and a viable cell density greater than 5 x 10(6) cells x ml(-1) were readily achieved after 9 day culture in spinner flasks. The HEK 293 cells growing as suspended aggregates could be efficiently infected by Ad-TH-GFP at an MOI of 10 with a prolonging infection time up to 144 hour post-infection (hpi). The time profile of Ad-TH-GFP production was strongly corresponding to the infection process with a virus concentration peak occurred consistently at 144 h after infection. And the infected aggregates essentially maintained spherical in shape, the portion of dissociated cells from the infected aggregates was less than 5% at 144 hpi. Perfusion culture of HEK 293 cells grown as suspended aggregates in a 7.5 L stirred tank bioreactor and infected with Ad-TH-GFP at a density higher than 1 x 10(7) cells x ml(-1) resulted in a similar Ad-TH-GFP production kinetics, but a much higher virus yield approximately at 5.7 x 10(11) GTU ml(-1) at 144 hpi to that of the infected spinner flask cultures. These results demonstrate the feasibility for using suspended cell aggregates as an immobilization system to facilitate perfusion in stirred tank bioreactors, and improve volumetric productivities by eliminating the cell density effect.

[Evaluation of immunization protection efficacy of plague subunit vaccine].

OBJECTIVE: To evaluate the protective efficacy of plague subunit vaccine, BALB/c mice, guinea pigs and rabbits were used in this study. METHODS: Groups of mice (10 per group), guinea pigs (14 per group) and rabbits (6 per group) were immunized with F1 + rV270 vaccine, EV76 vaccine and alum adjuvant by intramuscular route, respectively. Serum antibody titres of mice, guinea pigs and rabbits were determined by ELISA and the immunized animals were challenged with 10(6) CFU of Y. pestis strain 141 at the 8th week after the primary immunization. RESULTS: The immunized mice, guinea pigs or rabbits with subunit vaccine developed anti-F1 IgG titre of 41 587.3 +/- 2.1, 11 543.7 +/- 2.1 or 522.4 +/- 22.4 and elicited statistical anti-F1 IgG titre difference among them (F = 17.58, P < 0.01). The immunized mice, guinea pigs or rabbits with subunit vaccine had anti-rV270 IgG titre of 15 748.7 +/- 1.6, 12.6 +/- 1.4 or 1648.0 +/- 5.0 and induced statistical anti-rV270 IgG titre difference among them (F value was 16.34, P < 0.01). There was significant anti-F1 IgG titre difference among mice, guinea pigs and rabbits immunized with EV76 vaccine that developed anti-F1 IgG titre of 913.4 +/- 4.5, 937.0 +/- 2.0 or 342.0 +/- 12.0 (F = 23.67, P < 0.01), whereas the immunized mice, guinea pigs and rabbits with EV76 vaccine developed anti-rV270 IgG titre of 12.0 +/- 1.0, 447.0 +/- 10.0, 40.0 +/- 11.0 and there was no anti-rV270 IgG titre difference between them (F = 2.20, P = 0.1314). The immunized mice with subunit vaccine developed significantly higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 30.57 and 19.04, respectively, P < 0.01), and there were no anti-F1 IgG titre differences between the immunized guinea pigs and rabbits (q = 0.04, P = 0.8485). The immunized mice with subunit vaccine developed significantly higher anti-rV270 IgG titres than immunized guinea pigs and rabbits (q value was 27.10 and 19.49, respectively, P < 0.01), and there were no anti-rV270 IgG titre differences between the immunized guinea pigs and rabbits with the subunit vaccine (q = 0.25, P = 0.6187). The immunized mice with EV76 elicited higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 40.67 and 29.10, respectively, P < 0.01), whereas there was no difference of F1 IgG titer between immunized guinea pigs and rabbits (q = 0.06, P = 0.8098). The immunized mice, guinea pigs and rabbits with subunit vaccine provided 100% (10/10), 86% (12/14) and 100% (5/5) protection against 10(6) CFU Y. pestis of challenge, respectively. The immunized mice, guinea pigs and rabbits with EV76 vaccine gave 100% (6/6), 93% (13/14) and 100% (6/6) protection against 10(6) CFU Y. pestis of challenge respectively. CONCLUSION: BALB/c mice is the best small animal model for valuation of protective efficacy of plague subunit vaccine. The guinea pigs showed a high individual variation for this purpose. The rabbits can be used as an alternative model for evaluating plague subunit vaccine.

A new purification strategy for fraction 1 capsular antigen and its efficacy against Yersinia pestis virulent strain challenge.

F1 antigen is an attractive candidate for the development of a subunit vaccine against plague. In previous study, the extraction of this antigen from Yersinia pestis is characterized by using organic solvents. In this work, a new purification strategy that produced high-purity F1 antigen from Y. pestis EV76 was developed by the substitution of physical disruption for organic solvent one, followed by a combination of ammonium sulfate fractionation and Sephacryl S-200HR column filtration chromatography. As revealed in this study, this purification procedure is simple and effective, and avoids potential adverse effect on the antigen by organic solvents. Highly purified F1 that adsorbed to 25% (v/v) Al(OH)3 adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to F1 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 10(4) CFU of Y. pestis virulent strain 141.

[Enhancement of exogenous gene expression by artificial transcription factor in CHO cells].

Using the amino acids 1-147 of the yeast transcriptional activator GAL4 as the DNA-binding domain and four tandem repeats of the 12-aa peptide (DALDDFDLDMLG) of the herpesvirus as the activation domain, an artificial transcription factor, GVP4,was constructed via the linkage of the nuclear localization signal sequence of SV40. And then, GVP4 was cloned into expression vector pcDNA3 . 1/Hygro ( + ) . Various amounts of targeting sites of artificial transcription factor were linked to the upstream of promoter CMV in exogenous gene expression vector pcDNA3.1 ( + ) that separately harbored EGFP cDNA and t-PA cDNA.The CHO cells were then co-transfected with GVP4 expression vector and EGFP or t-PA expression vector. The effect of GVP4 on exogenous gene expression was evaluated by measuring the fluorescence intensity of EGFP in CHO cells and the concentration of t-PA in the supernatant. GVP4 showed positive effect on the enhancement of exogenous gene expression in CHO cells integrated with targeting sites of artificial transcription factor. And, CHO cells integrated with 10 targeting sites of GVP4 was more favorable to foreign gene expression, which resulted in 2-3-fold increase in both EGFP and t-PA expressions. These results indicated that artificial transcription factor is potent in the enhancement of exogenous gene expression in mammalian cells.

Cultivation of recombinant Chinese hamster ovary cells grown as suspended aggregates in stirred vessels.

Recombinant Chinese hamster ovary (rCHO) cells capable of producing a prourokinase mutant (mPro-uk) grown as suspended aggregates in stirred vessels were described and characterized. The addition of chitosan to a mixture of DMEM and Ham's F12 (D-MEM/F-12) medium promoted cell aggregation and spheroid formation efficiently. Multicellular aggregates formed immediately after the rCHO cells were inoculated into the chitosan-added medium, and the mean diameter of the cell aggregates reflecting the aggregate size increased with culture time, shifting from 65 to 163 mum after 2 and 9 d of culture in spinner flasks. No significant difference in the metabolism performance of the rCHO cells was observed between suspended aggregates and anchored monolayers. However, the cells cultured as suspended aggregates showed a marked decrease in growth rate as evaluated from specific growth rate (mu). Replacing D-MEM/F-12 medium with CD 293 medium caused compact spherical cell aggregates to dissociate into small irregular aggregates and single cells without apparent effects on cell performance in subcultures. The perfusion culture of the rCHO cells grown as suspended aggregates in a 2-l stirred tank bioreactor for 15 d resulted in a maximum viable cell density of 5.6 x 10(6) cells ml(-1) and an mPro-uk concentration of about 2.6 x 10(3) IU ml(-1), and cell viability was remained at roughly 90% during the entire run.

[In vitro cytolysis of B-lymphoma cells mediated by an anti-CD3/anti-CD20 bispecific single-chain antibody].

After having successfully constructed and expressed the gene of the anti-CD3/anti-CD20 bispecific single-chain antibody (bscCD3 x CD20), here we analyzed its in vitro bioactivity of mediating the lysis of Ramous human B-lymphoma cells in the presence of T-enriched human peripheral blood lymphocytes (PBL). Obvious opoptosis characters were observed by Annexin V/PI(AV/PI) stained and scanning electron microscope. As evaluated by non-radioactive cytotoxity assay, the bscCD3 x CD20 showed potent bioactivity of mediating human B-lymphoma cells lysis in the presence of T-enriched human PBL. The potency of cytotoxicity depended on the ratios of effect cells to target cells (E:T) used. Further, the antibody showed a dose and time-dependent effect on mediating Ramous cells lysis. The specific lysis reached about 87.3% at an antibody concentration of 5microg/mL and E:T used at 10:1. Clear changes in apoptogenes expression profiles were detected by apoptosis gene array after Ramous cells were treated with the antibody and PBL. Among the upregulated apoptogenes, ATM and P53 showed an increase of 187 times and 15 times respectively, which suggested that ATM-p53 pathway may be the main apoptosis way of Ramous cells induced by T cells in the presence of the bscCD3 x CD20.

[Effects of hydrodynamic on aggregates formation, growth and metabolism of HEK293 cells in suspension culture].

By using the size distribution of cell aggregates, viable cell density, cell viability, specific consumption rate of glucose (q(glc)), specific production rate of lactate (q(lac)) and lactate transform rate (Y(lac/glc)) as the evaluation indexes, the effects of hydrodynamic on aggregates formation, growth and metabolism of HEK293 cells in suspension culture were examined in 250mL spinner-flasks by setting the agitation rates at 25, 50, 75 and 100r/min, respectively. It was found that agitation plays an important role in HEK293 cell aggregates formation and cell aggregates size distribution. After 7d cultivation in spinner-flasks operated at 50r/min and 75r/min, the average diameter of HEK293 cell aggregates was 201 microm and 175 microm, respectively, with the fraction of aggregates larger than 225 microm less than 10%. The cell viability was kept above 90% with the metabolic indexes, including q(glc), q(lac) and Y(lac/glc) kept constant. These results demonstrated that hydrodynamic derived from the proper agitation play a decisive role in controlling the formation and size distribution of HEK293 cell aggregates, and provided sufficient mass transfer to support the normal growth and metabolism of HEK293 cells in suspended aggregates.

[Expression, identification and bioactivity characterization of an anti-CD3/anti-CD20 bispecific single-chain antibody].

The synthetic gene with 1640bp encoding for the anti-CD3/anti-CD20 bispecific single-chain antibody was designed and obtained by SOE (splicing by overlap extension) PCR. The cDNA was cloned into Flp-In expression vector pcDNA5/FRT and transfeced into Flp-In CHO cells to generate a stable expression cell line with a capacity for expressing anti-CD3/anti-CD20 bispecific single-chain antibody at 300 microg/L. The protein, which had a molecular weight of about 70 kD,was purified by Ni-NTA affinity chromatography and identified by SDS-PAGE and Western-blot analysis. Immunofluorescence assay and cellular rosetting showed that it can react specifically on Jurkat (CD3+) and Ramous (CD20+) cells. The lysis of human PBL against CD20-positive lymphoma Ramous cells in the presence of the anti-CD3/anti-CD20 bispecific single-chain antibody can observed by microscope. All these results would lighten the further study of its biological functions in vitro and in vivo.

Temperature shift as a process optimization step for the production of pro-urokinase by a recombinant Chinese hamster ovary cell line in high-density perfusion culture.

Based on the effects of temperature shift on the cell cycle, apoptosis and metabolism of a recombinant Chinese hamster ovary (rCHO) cell line (CL-11G) producing pro-urokinase (pro-UK) in batch cultures, the potential of temperature shift as a tool in the optimization of the perfusion culture of CL-11G cells for the production of pro-UK was examined. The proportion of CL-11G cells in the G0/G1 phase in static cultures increased from 56.4% to 82.8% following a temperature shift from 37 degrees C to 31 degrees C. Conversely, the proportion of CL-11G cells in the S phase decreased from 34.8% to 11.6%. The specific growth rate of CL-11G cells reflected the effect of temperature on the cell cycle and decreased from 0.024 h(-1) at 37 degrees C to 0.006 h(-1) at 31 degrees C. Continuous exposure to the non-permissive temperature of 31 degrees C led to a marginal increase in apoptosis. The specific pro-UK productivity of CL-11G cells increased by 74% at 34 degrees C compared with controls at 37 degrees C in batch cultures. CL-11G cells immobilized with Cytopore 1 in a 5-l bioreactor initiated at 37 degrees C and temperature shifted to 34 degrees C exhibited an average 17% increase in viable cell density and an average 47% increase in pro-UK production. These results demonstrated that temperature shift offers the prospect of enhancing the productivity of pro-UK in high-density perfusion culture.