Comparative investigation of the use of various commercial microcarriers as a substrate for culturing mammalian cells

WOS: 000333527900008PubMed ID: 24357035Microcarriers provide large adhesion area allowing high cell densities in bioreactor systems. This study focused on the investigation of cell adhesion and cell growth characteristics of both anchorage-dependent CHO-K1 and anchorage-independent Ag8 myeloma cell lines cultivated on four different microcarriers (BiosilonA (R), MicrohexA (R), Cytodex 3A (R), Cytoline 2A (R)) by considering the cell kinetics and physiological data. Experiments were performed in both static and agitated cell culture systems by using 24-well tissue culture plates and then 50-ml spinner flasks. In agitated cultures, the highest specific growth rates (0.026 h for CHO-K1 and 0.061 h for Ag8 cell line) were obtained with Cytodex 3A (R) and Cytoline 2A (R) microcarriers for CHO-K1 and Ag8 cell line, respectively. Metabolic characteristics showed some variation among the cultures with the four microcarriers. The most significant being the higher production of lactate with microcarriers with CHO-K1 cells relative to the Ag8 cells. SEM analyses revealed the differences in the morphology of the cells along with microcarriers. On Cytodex 3A (R) and Cytoline 2A (R), CHO-K1 cells attached to the substratum through long, slender filopodia, whereas the cells showed a flat morphology by covering the substratum on the BiosilonA (R) and MicrohexA (R). Ag8 cells maintained their spherical shapes throughout the culture for all types of microcarriers. In an attempt to scale-up, productions were carried out in 50-ml spinner flasks. Cytodex 3A (R) (for CHO-K1 cells) and Cytoline 2A (R) (for Ag8 cells) were evaluated. The results demonstrate that high yield of biomass could be achieved through the immobilization of the cells in each culture system. And cell cultures on microcarriers, especially on Cytodex 3A (R) and Cytoline 2A (R), represented a good potential as microcarriers for larger scale cultures of CHO-K1 and Ag8, respectively. Moreover, owing to the fact that the cell lines and culture media are specific, outcomes will be applicable for other clones derived from the same host cell lines

Comparative investigation of the use of various commercial microcarriers as a substrate for culturing mammalian cells (vol 50, pg 221, 2014)

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Enhanced growth and lipid accumulation by a new Ettlia texensis isolate under optimized photoheterotrophic condition

WOS: 000317024200036PubMed ID: 23353038A green microalgae, named as Ettlia texensis was obtained from local freshwater in Turkey. The effects of autotrophic, photoheterotrophic and heterotrophic cultivations on biomass and lipid production were studied. Searching the preferences of the carbon and nitrogen source revealed that this strain could grow photoheterotrophically well with glucose and yeast extract. In the optimized medium, the highest biomass productivity and total lipid content achieved were 0.97 g/L d and 26% of dry weight basis, respectively. Moreover, the major fatty acid methyl esters were C16:0; C18:1; C18:2 and C18:3. In a scale-up attempt, productions were accomplished in a 3 L stirred tank bioreactor. The final biomass and lipid productivities obtained in bioreactor with 250 rpm agitation rate were 0.92 g/L d and 322 mg/L d, respectively. The biochemical compositions were monitored simultaneously by the FTIR spectroscopy during the production in bioreactor. E. texensis could be potent candidate for commercial production in the bioreactor photoheterotrophically. (C) 2012 Elsevier Ltd. All rights reserved.Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [109M227]; EBILTEM (Ege University Science and Technology Center)Ege University [2010/BIL/013]This research was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) with 109M227 Project Number, and EBILTEM (Ege University Science and Technology Center, 2010/BIL/013 Project Number). The authors wish to thank Prof. Dr. Meltem Conk-Dalay (Department of Bioengineering, Ege University, Izmir, Turkey) for supplying algal culture

Purification of alginate and feasible production of monoclonal antibodies by the alginate-immobilized hybridoma cells

WOS: 000301688200017PubMed ID: 22078279Alginate has an extensive usage in the immobilization of many cell types. Although they have high biocompatibility, commercial alginates contain various degrees of contaminants such as polyphenols, endotoxins and proteins. Thus, these alginates show cytotoxicity against sensitive cell types such as hybridoma cells. In the studies so far, owing to this fact, commercially purchased high-priced ultrapure alginates have been used in the immobilization of hybridoma cells for monoclonal antibody production. However in this study, as a novelty, low-priced commercial alginate was purified, and then the cultivation of alginate-immobilized hybridoma cells was performed for feasible monoclonal antibody production. Low-priced commercial alginate was purified with a profitability ratio of 40%. Then, an optimized immobilization procedure was conducted effectively by using the purified alginate. During more than 25 days of cultivation, serum concentration was kept low, and approximately 2 times greater monoclonal antibody production was achieved, in comparison with its free suspended counterpart. The results showed that the efficiency of monoclonal antibody production via alginate-immobilized hybridoma cultivation can be increased by performing a proved in-house purification method. By shedding light on the efficiency of the in-house purification method, the results also indicated a feasible way of monoclonal antibody production. (C) 2011, The Society for Biotechnology, Japan. All rights reserved

A BioDesign Approach to Obtain High Yields of Biosimilars by Anti-apoptotic Cell Engineering: a Case Study to Increase the Production Yield of Anti-TNF Alpha Producing Recombinant CHO Cells

WOS: 000419607700024PubMed ID: 28685239Recent developments in medical biotechnology have facilitated to enhance the production of monoclonal antibodies (mAbs) and recombinant proteins in mammalian cells. Human mAbs for clinical applications have focused on three areas, particularly cancer, immunological disorders, and infectious diseases. Tumor necrosis factor alpha (TNF-alpha), which has both proinflammatory and immunoregulatory functions, is an important target in biopharmaceutical industry. In this study, a humanized anti-TNF-alpha mAb producing stable CHO cell line which produces a biosimilar of Humira (adalimumab) was used. Adalimumab is a fully human anti-TNF mAb among the top-selling mAb products in recent years as a biosimilar. Products from mammalian cell bioprocesses are a derivative of cell viability and metabolism, which is mainly disrupted by cell death in bioreactors. Thus, different strategies are used to increase the product yield. Suppression of apoptosis, also called anti-apoptotic cell engineering, is the most remarkable strategy to enhance lifetime of cells for a longer production period. In fact, using anti-apoptotic cell engineering as a BioDesign approach was inspired by nature; nature gives prolonged life span to some cells like stem cells, tumor cells, and memory B and T cells, and researchers have been using this strategy for different purposes. In this study, as a biomimicry approach, anti-apoptotic cell engineering was used to increase the anti-TNF-alpha mAb production from the humanized anti-TNF-alpha mAb producing stable CHO cell line by Bcl-xL anti-apoptotic protein. It was shown that transient transfection of CHO cells by the Bcl-xL anti-apoptotic protein expressing plasmid prolonged the cell survival rate and protected cells from apoptosis. The transient expression of Bcl-xL using CHO cells enhanced the anti-TNF-alpha production. The production of anti-TNF-alpha in CHO cells was increased up to 215 mg/L with an increase of 160% after cells were transfected with Bcl-xL expressing plasmid with polyethylenimine (PEI) reagent at the ratio of 1:6 (DNA:PEI). In conclusion, the anti-apoptotic efficacy of the Bcl-xL expressing plasmid in humanized anti-TNF-alpha MAb producing stable CHO cells is compatible with curative effect for high efficiency recombinant protein production. Thus, this model can be used for large-scale production of biosimilars through transient Bcl-xL gene expression as a cost-effective method.TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [2209-A, 1919B011402737]This study has been partly funded by TUBITAK through 2209-A project number 1919B011402737 under the supervision of Sultan GULCE IZ, PhD