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Abstract

Human Embryonic Kidney 293 (HEK293) cells were adapted into a serum-free suspension medium through steps of gradual serum weaning for the production of adenoviral (AdV) gene therapy vectors. The presence of sodium heparin in the medium formulation reduced cell clumping dramatically in suspension culture. The adapted cells were ready to grow either in serum-containing medium as an attached culture or in serum-free medium in suspension culture. A scalable production process was developed in shake flasks and was then evaluated in stirred tank bioreactors. This process includes a growth phase in batch-mode followed by a production phase involving medium perfusion and supplementation. Fortification with calcium chloride post viral inoculation resulted in an increase in virus production by at least one fold. Addition of stimulating agents such as sodium butyrate, N-acetyl-L-cysteine (NAC), dimethyl sulfoxide(DMSO), or ethyl alcohol post infection was shown to further improve virus production in a dose-dependent manner. The serum-free suspension process described here should be suitable for the manufacturing of other E1-deleted AdV vectors and could potentially be used for the production of recombinant proteins by HEK293 cells.

FGF, Insulin, and SMAD Signaling Cooperate for Avian Primordial Germ Cell Self-Renewal

“…We initially attempted to propagate chicken PGCs without feeder cells or conditioned medium in KO-DMEM basal medium containing the growth factors FGF2, Activin A, and IGF-1 and supplemented with B-27 serum-free supplement, a stem cell supplement containing insulin. The proteoglycan heparin, also was added to increase FGF signaling and reduce cell-cell adhesion ( Furue et al., 2008 , Tsao et al., 2001 ). Embryonic blood (1 μl) from a single chicken embryo was placed in a single well and cultured for 3 weeks, and the PGCs present at the end of this culture period were counted.…”

Advances and Drawbacks of the Adaptation to Serum-Free Culture of CHO-K1 Cells for Monoclonal Antibody Production

“…The transition to serum-free conditions is not straightforward. However, once cells have been adapted, researchers can benefit from improved control over culture conditions, improved reproducibility between cultures, consistency of media that avoids the need to screen batches, potential ease in scale-up operation, simplicity in downstream processing, reduced risk of virus contamination, and reduced costs [3,5,8,25,27,29].…”

Comparison of Vero and MDCK cell lines transfected with human siat7e gene for conversion to suspension culture

“…The industry has overcome this limitation by using microcarriers such as Cytodex 1 and 3, but microcarriers are expensive and the process burdensome compared to suspension culture [11]. Successful attempts at changing anchorage-dependent cells to anchorage independent cells have been previously reported [11,[18][19][20][21][22].…”