RESUMO
The global adoption of vaccines to combat disease is hampered by the high cost of vaccine manufacturing. The work described herein follows two previous publications (van der Sanden et al., 2016; Wu et al., 2017) that report a strategy to enhance poliovirus and rotavirus vaccine production through genetic modification of the Vero cell lines used in large-scale vaccine manufacturing. CRISPR/Cas9 gene editing tools were used to knockout Vero target genes previously shown to play a role in polio- and rotavirus production. Subsequently, small-scale models of current industry manufacturing systems were developed and adopted to assess the increases in polio- and rotavirus output by multiple stable knockout cell lines. Unlike previous studies, the Vero knockout cell lines failed to achieve desired target yield increases. These findings suggest that additional research will be required before implementing the genetically engineered Vero cell lines in the manufacturing process for polio- and rotavirus vaccines to be able to supply vaccines at reduced prices.
Assuntos
Técnicas de Cultura Celular por Lotes , Engenharia Genética , Células Vero , Vacinas Virais , Animais , Sistemas CRISPR-Cas , Chlorocebus aethiops , Técnicas de Inativação de Genes , Marcação de Genes , Poliovirus/genética , Poliovirus/imunologia , Vacinas contra Poliovirus/química , Vacinas contra Poliovirus/imunologia , Rotavirus/genética , Rotavirus/imunologia , Vacinas contra Rotavirus/genética , Vacinas contra Rotavirus/imunologiaRESUMO
pH affects many processes on cell metabolism, such as enzyme kinetics. To enhance the understanding of the living cells, it is therefore indispensable to have a method to monitor the pH in living cells. To accomplish this, a dynamic intracellular pH measurement method applying low concentration benzoic acid pulse was developed. The method was thoroughly validated and successfully implemented for measuring fast dynamic intracellular pH of Saccharomyces cerevisiae in response to a glucose pulse perturbation performed in the BioSCOPE set-up. Fast drop in intracellular pH followed by partial alkalinization was observed following the pulse. The low concentration benzoic acid pulse which was implemented in the method avoids the undesirable effects that may be introduced by benzoic acid to cell metabolism.
