Biomedical subjects

Chunnuan Chen

Publications and source records attributed to Chunnuan Chen.

4 recordsLinked to original sources

PubMed2005

Allele quantification using molecular inversion probes (MIP).

Detection of genomic copy number changes has been an important research area, especially in cancer. Several high-throughput technologies have been developed to detect these changes. Features that are important for the utility of technologies assessing copy number changes include the ability to interrogate regions of interest at the desired density as well as the ability to differentiate the two homologs. In addition, assessing formaldehyde fixed and paraffin embedded (FFPE) samples allows the utilization of the vast majority of cancer samples. To address these points we demonstrate the use of molecular inversion probe (MIP) technology to the study of copy number. MIP is a high-throughput genotyping technology capable of interrogating >20 000 single nucleotide polymorphisms in the same tube. We have shown the ability of MIP at this multiplex level to provide copy number measurements while obtaining the allele information. In addition we have demonstrated a proof of principle for copy number analysis in FFPE samples.

Alleles↗

PubMed2004

A fibrous-bed bioreactor for continuous production of monoclonal antibody by hybridoma.

A fibrous-bed bioreactor (FBB) has been developed to culture hybridoma cells for long-term continuous production of monoclonal antibody (MAb). A non-woven polyester fibrous matrix was used to immobilize the cells to reach a high viable cell density of 3 x 10(8) cells cm(-3) packed bed, which gave a high volumetric MAb productivity of 1 g L(-1) day(-1) under continuous feed conditions with the medium containing 10% serum. Reducing the medium serum content to 1% increased MAb production to 6.5 g L(-1) day(-1) in a repeated batch FBB culture. MAb production was higher at higher dissolved oxygen (DO) levels in the range between 10% and 70% of air saturation, although DO did not significantly affect glucose metabolism and lactate production. The medium LDH (lactate dehydrogenase) level increased dramatically when the DO level was decreased from 30% to 10%, suggesting that a critical DO level of approximately 30% is necessary for maintaining the FBB culture for long-term operation. Compared with suspension cultures in T-flasks and spinner flasks, the FBB culture had a lower lactate yield from glucose (0.80 vs. 0.91 g g(-1), produced MAb at a higher concentration (up to 442 mg L(-1) vs. 83.5 mg L(-1), and was stable for continuous long-term operation (more than 1 month). The superior FBB performance was attributed to the highly porous fibrous matrix that enabled the efficient mass transfer, cell immobilization, and continued growth and regeneration that are critical to maintaining a high density of viable and productive cell populations. The cells immobilized in the fibrous matrix had high viability (>85%) even though many of them were in growth arrest (G1/G0 phase) as indicated by their smaller cell size (<10 microm). Scanning electron microscopic studies of the cell-matrix showed that the high density of cells formed large clumps within the interstitial spaces of the fibrous matrix. Their close contact and interaction with each other might have contributed to their ability to survive well under adverse conditions such as low DO and low serum content in the medium. It was also found that the cells present inside the fibrous matrix had a higher viability and lower apoptosis than those present in the liquid suspension, indicating that the fibrous matrix had selectively retained healthy, nonapoptotic cells and dislodged apoptotic and dead cells; this also might have contributed to the stability of the long-term culture. This work demonstrated that the FBB originally developed for microbial fermentation also gave excellent results in achieving high cell density, productivity, and product concentrations, and should have a good potential for industrial animal cell culture applications.

Antibodies, Monoclonal↗

PubMed2002

A fibrous-bed bioreactor for continuous production of developmental endothelial locus-1 by osteosarcoma cells.

Genetically engineered human osteosarcoma cells containing developmental endothelial locus-1 (del-1) gene were studied for production of Del-1, a protein that has the properties of an extracellular matrix protein and can regulate vascular morphogenesis and remodeling. Del-1 has been studied as a potential anti-angiogenesis drug targeting solid tumors. In this study, osteosarcoma cells were cultured in a fibrous-bed bioreactor (FBB) to continuously produce Del-1. The FBB was constructed by packing a polyester fibrous matrix into a 1.5-l spinner flask. The effects of media composition, including the serum content in the medium, and dilution rate on cell growth, metabolism, and Del-1 production were studied. A gradual reduction of serum content from 10% (v/v) to 0.5% (v/v) caused no loss in Del-1 production. However, the production of Del-1 decreased significantly in a serum-free medium, suggesting some nutrients present in the serum were important to culture viability and Del-1 production. The continuous FBB culture was stable for long-term production of Del-1, with a higher Del-1 titer than that normally obtained in T-flask cultures and overall productivity similar to the total production from 300 25-cm(2) T-flasks. Reducing geneticin in the medium from 250 microg ml(-1) to zero at later culturing stages had no significant effect on Del-1 production. The FBB was operated for a period of more than 4 months without any notable degeneration, and reached a final cell density of 3 x 10(8) cells ml(-1) of packing volume with >90% cell viability. The good reactor performance can be attributed to the three-dimensional environment provided by the fibrous matrix that allows for efficient mass transfer and cell immobilization and growth. Scanning electron microscopic and confocal scanning laser microscopic studies of the cell-matrix showed that cells formed large aggregates in the fibrous matrix and cell density was relatively uniform in the matrix.

Anti-Bacterial Agents↗

PubMedDate not supplied

Effects of three-dimensional culturing on osteosarcoma cells grown in a fibrous matrix: analyses of cell morphology, cell cycle, and apoptosis.

Osteosarcoma cells were cultured in stirred tank bioreactors with either a fibrous matrix or nonporous microcarriers to study the environmental effects on cell growth, morphology, cell cycle, and apoptosis. Cell cycle and apoptosis were analyzed using flow cytometry and visualized using confocal laser scanning microscopy and fluorescence microscopy. The three-dimensional (3-D) fibrous culture had better cell growth and higher metabolic rates than the two-dimensional (2-D) microcarrier culture because cells in the fibrous matrix were protected from shear stress and had lower apoptosis and cell death even under suboptimal conditions (e.g., nutrient depletion). The polyester fibrous matrix used in this study also exhibited the capability of selectively retaining viable and nonapoptotic cells and disposing apoptotic and nonviable cells. Consequently, very few apoptotic cells were found in the fibrous matrix even in the long-term (1 month) T-flask culture. In the continuous culture with packed fibrous matrixes for cell support, most cells were arrested in the G1/G0 phase after 4 days. Decreasing the dissolved oxygen level from 60 to 10% air saturation did not significantly change cell cycle and apoptosis, which remained low at approximately 15%. These results could explain why the fibrous bed bioreactor had good long-term stability and was advantageous for production of non-growth-associated proteins by animal cell cultures.

Apoptosis↗