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Abstract

In this work a recombinant BHK21 clone producing a fusion protein with potential application in tumour target therapy was adapted to five different serum-free media (SFM) and to a protein-free medium (PFM). Only the PFM did not require a gradual adaptation to cell growth in the absence of serum. All tested SFM required a gradual adaptation (up to 35 days). For the majority of the SFM tested, cell specific productivity was not affected by the decrease in serum concentration during adaptation; however, cell growth was significantly affected by the serum decrease. Both cell growth and productivity were increased when PFM SMIF6 was used instead of the control medium. Long term measurements (approximately 100 days) of cell specific productivity for PFM and the two best SFM showed that productivity was maintained. This indicates the media capability to be used in long term production processes.

Semliki Forest Virus replicon particles production in serum-free medium BHK-21 cell cultures and their use to express different proteins

“…Growth kinetic parameters including cell viability, density, and maximum specific growth rate were evaluated in several passages, with similar results observed for the BHK/SFM and BHK/FBS cells. The values obtained were in agreement with results reported previously for BHK-21 cells cultured in DMEM supplemented with FBS (Cruz et al 1998;Fernández-Núñez et al 2015), and were higher than BHK/SFM and BHK/FBS cells were infected in parallel with VRP-RVGP-SFM or VRP-RVGP-DMEM, using a MOI of 50 for a and 100 for b, c. The experiments was performed in 6-well plates, the cells were collected 3; 6; 12 and 24 h post-infection for a and 24 h post-infection for b, c. In a mRNA-RVGP expression was evaluated by qPCR. The results are average of duplicate.…”

“…Major morphological alterations were observed in the cells, due to the new culture conditions, including the formation of cell aggregates, loss of adhesion capacity (semiadherent growth), and rounded morphology (data not shown). These features are common in adaptation processes and have been reported for BHK cells (Cruz et al 1998;Hesse and Wagner 2000;Kallel et al 2002).…”

“…This, was achieved using BHK-21 cells that are widely used for the propagation of viruses and protein expression by infection and transfection (Barteling 1976;Cruz et al 1998;Kallel et al 2002;Frazatti-Gallina et al 2004;Hernandez and Brown 2010). The ability of BHK-21 cells to grow in serum-free media has been reported previously (Barteling 1976;Cruz et al 1998;Kallel et al 2002;Frazatti-Gallina et al 2004). Despite accumulated literature on serum-free formulations, one must test different available SFM in order to determine which is the most suitable for a specific application/cell line (Sinacore et al 2000).…”

“…Most commercial serum-free culture media are unable to provide the necessary conditions for cell growth after an internal adaptation (Link et al 2004). Hence, the sequential adaptation technique was selected, which has been used in several previous studies (Cruz et al 1998;Costa et al 2013;Huang et al 2015;do Amaral et al 2015). Major morphological alterations were observed in the cells, due to the new culture conditions, including the formation of cell aggregates, loss of adhesion capacity (semiadherent growth), and rounded morphology (data not shown).…”

Fetal bovine serum: geographic origin and regulatory relevance of viral contamination

“…On the other hand, it has the disadvantage of high costs and the need for testing and the elimination of adventitious viruses, as do other media of animal origin. Serum-free media have been developed to avoid the use of animals for cell culture media, and have met with great success, especially in the production of some proteins for medical uses (Cruz et al 1998). However, pharmaceutical companies, diagnostic labs and researchers still depend heavily on FBS for most of their cell culture needs.…”