Lingling Ye

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.

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

BACKGROUNDnThe 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.nnnMETHODSnIn 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.nnnRESULTSnThe 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.nnnCONCLUSIONSnUsing 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.

Enhancement of plasmid-mediated stable gene expression by woodchuck hepatitis virus post- transcriptional regulatory element (WPRE) in human embryonic kidney (HEK293) cells

Influence of random integration site on the expression of transgene in mammalian cells makes it a major challenge to achieve high productivity of recombinant proteins. Optimization of expression vector is one of the most popular strategies to resolve this problem. Among this, woodchuck hepatitis virusxa0 post-transcriptional regulatory element (WPRE) is a possible enhancer of gene expression in mammalianxa0 cells that promotes efficient export of unspliced (RNA) into the cytoplasm, as has been proved inxa0 transient transfection. In this study, WPRE was evaluated for enhancing stable gene expression levels in two industrial cell lines, human embryonic kidney (HEK293) and CHO-S, using the enhanced green fluorescent protein (EGFP), prourokinase (pro-UK) and protein C (PC) as the reporter gene. Based on the mean fluorescence intensity (MFI), WPRE exerted a clear positive effect on gene expression in HEK293 cells with an increase of EGFP expression level by approximately 2.5- to 3-fold independent of thexa0 promoter used in plasmid vector. In contrast, in Chinese hamster ovary (CHO)-S cells, only a marginal effect on plasmid-mediated EGFP expression by WPRE was observed. The measurable increase of EGFP expression at the protein level was paralleled by an increase of EGFP RNA. Further test of the effect of WPRE on plasmid-mediated gene expression with two therapeutic proteins showed substantial increase of stable pro-UK and PC expression only in HEK293 by about 2.2-fold and 6.1-fold, respectively. The data of PC expression levels obtained from the random HEK293 cell clones transfected with WPRE-containing or lacking vector further demonstrated the enhancement of stable plasmid-mediated gene expression by WPRE in HEK293 cells. These results in stable transfectants show the positive effect of WPRE on transgene expression is cell-type dependent and promoter-independent, and provide valuable information to improve vectors for efficient and stable gene expression in HEK293 cells. Key words : Mammalian cells, plasmid vector, stable gene expression, protein therapeutics, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

Enhancement of Exogenous Gene Expression by the Artificial Transcription Factor in Chinese Hamster Ovary Cells

Abstract 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 VP16 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 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 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.

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.