Zhaolie Chen

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.

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.

Impaired formation of homotypic cell-in-cell structures in human tumor cells lacking alpha-catenin expression

Although cell-in-cell structures (CICs) could be detected in a wide range of human tumors, homotypic CICs formed between tumor cells occur at low rate for most of them. We recently reported that tumor cells lacking expression of E- and P-cadherin were incapable of forming homotypic CICs by entosis, and re-expression of E- or P-cadherin was sufficient to induce CICs formation in these tumor cells. In this work, we found that homotypic CICs formation was impaired in some tumor cells expressing high level of E-cadherin due to loss expression of alpha-catenin (α-catenin), a molecular linker between cadherin-mediated adherens junctions and F-actin. Expression of α-catenin in these tumor cells restored cell-cell adhesion and promoted CICs formation in a ROCK kinase-dependent way. Thus, our work identified α-catenin as another molecule in addition to E- and P-cadherin that were targeted to inactivate homotypic CICs formation in human tumor cells.

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.

Ex vivo expansion of human umbilical cord blood hematopoietic progenitor cells in a novel three-dimensional culture system

A novel three-dimensional culture system for the ex vivo expansion of human umbilical cord blood (CB) hematopietic progenitor cells (HPCs) was developed by growing CB mononuclear cells on highly porous CultiSpher G microspheres coated with human bone marrow stromal cells in stirred flasks in the presence of supplemented cytokines. After 12 days, the number of total viable cells, colony-forming units in culture (CFU-C) and CD34+ cells present in the cultures reflected average increases of 7.7, 23.3 and 9.6-fold, respectively, and marked hematopoietic islands were formed on the surface of CultiSpher G.

Effects of Hydrodynamics on Aggregates Formation, Growth and Metabolism of HEK 293 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.

Fluorescence-Activated Cell Sorting Analysis of Heterotypic Cell-in-Cell Structures

Cell-in-cell structures (CICs), characterized by the presence of one or more viable cells inside another one, were recently found important player in development, immune homeostasis and tumorigenesis etc. Incompatible with ever-increasing interests on this unique phenomenon, reliable methods available for high throughput quantification and systemic investigation are lacking. Here, we report a flow cytometry-based method for rapid analysis and sorting of heterotypic CICs formed between lymphocytes and tumor cells. In this method, cells were labeled with fluorescent dyes for fluorescence-activated cell sorting (FACS) by flow cytometry, conditions for reducing cell doublets were optimized such that high purity (>95%) of CICs could be achieved. By taking advantage of this method, we analyzed CICs formation between different cell pairs, and found that factors from both internalized effector cells and engulfing target cells affect heterotypic CICs formation. Thus, flow cytometry-based FACS analysis would serve as a high throughput method to promote systemic researches on CICs.

Cholesterol inhibits entotic cell-in-cell formation and actomyosin contraction

Cell-in-cell structure is prevalent in human cancer, and associated with several specific pathophysiological phenomena. Although cell membrane adhesion molecules were found critical for cell-in-cell formation, the roles of other membrane components, such as lipids, remain to be explored. In this study, we attempted to investigate the effects of cholesterol and phospholipids on the formation of cell-in-cell structures by utilizing liposome as a vector. We found that Lipofectamine-2000, the reagent commonly used for routine transfection, could significantly reduce entotic cell-in-cell formation in a cell-specific manner, which is correlated with suppressed actomyosin contraction as indicated by reduced β-actin expression and myosin light chain phosphorylation. The influence on cell-in-cell formation was likely dictated by specific liposome components as some liposomes affected cell-in-cell formation while some others didnt. Screening on a limited number of lipids, the major components of liposome, identified phosphatidylethanolamine (PE), stearamide (SA), lysophosphatidic acid (LPA) and cholesterol (CHOL) as the inhibitors of cell-in-cell formation. Importantly, cholesterol treatment significantly inhibited myosin light chain phosphorylation, which resembles the effect of Lipofectamine-2000, suggesting cholesterol might be partially responsible for liposomes effects on cell-in-cell formation. Together, our findings supporting a role of membrane lipids and cholesterol in cell-in-cell formation probably via regulating actomyosin contraction.

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).

CDKN2A inhibits formation of homotypic cell-in-cell structures

Cell-in-cell (CIC) structures, characterized by enclosure of one or more cells within another cell, were extensively documented in human cancers. Although elevated CIC formation was found in cancers with CDKN2A inactivation, a causal link between them remains to be established. We reported here that inhibiting CDKN2A expression effectively promoted homotypic CIC formation, whereas ectopic overexpression of p16INK4a or p14ARF, two proteins encoded by CDKN2A gene, significantly suppressed CIC formation in MCF7 cells. The regulation of CIC formation by CDKN2A was tightly correlated with subcellular redistribution of E-cadherin, F-actin rearrangement and reduced phosphorylation of myosin light chain 2 (p-MLC2), consistent with which, CDKN2A expression imparted cells winner/outer identity in competition assay. Moreover, CIC formation negatively correlates with p16INK4a expression in human breast cancers. Thus, our work identifies CDKN2A as the first tumor suppressor whose inactivation promotes homotypic CIC formation in human cancer cells.