Pilot-Scale Production of Lyophilized Inactivated Rabies Vaccine Candidate in Vero Cells under Fully Animal Component-Free Conditions Using Microcarrier Technology and Laboratory Animal Trials

The upstream process was carried out in an animal component-free medium on Cytodex 1 microcarriers. Recombinant trypsin is a non-animal derived protease used as an alternative to animal-derived trypsin. To inactivate recombinant trypsin, a soybean trypsin inhibitor (STI) should be added to the medium. A protocol was first tested in T-flasks and then passaged to 500 mL and 3 L spinner flasks. Cell detachment was completed in 10 - 12 min, and 0.4 g/L STI was added to a 3L spinner, and cells were transferred into a 30 L stirred tank bioreactor. On day 5, the cell density had reached its maximum (around 1.8 × 106 cells/mL). At an MOI of 0.3 with serum-free medium conditions, cell infection yielded a maximal rabies virus titer of 1.82 × 107 FFU/mL at 5 days. All cell culture conditions and virus growth kinetics in serum-free media were investigated. In conclusion, Vero cells were grown on Cytodex 1 with serum-free media and a high amount of rabies virus was obtained. A mouse challenge was used to determine the immune response to an inactivated rabies virus vaccine candidate. Also, we evaluated inactive rabies vaccine candidate safety, and immunogenicity in mice, sheep, horses, and cattle. We found that no horses, sheep, or cattle who were given vaccine IM at 3.2 IU/dose exhibited any clinical sign of disease and all developed high VNA titers (up to 10.03 IU/mL) by 3 - 4 WPI. After the accelerated stability studies, the lyophilized inactivated rabies vaccine candidate showed enough antigenic potency (2.6 IU/mL) in the mouse challenge test. Also, 18-month long-term stability studies showed enough immune response (1.93 IU/mL) on day 14. The activity of the vaccine candidate showed a good immune response and safety criteria that meet WHO requirements. This is the first pilot-scale mammalian cell-based viral rabies vaccine production study in Türkiye that used microcarriers.

Ketamine effect on intracellular and mitochondrial calcium mobilization

The suppressive effects of ketamine on intracellular calcium has been reported in a variety of cells although the mechanisms involved are not well understood.The aim of this work was to evaluate the ketamine effect on the mitochondrial Ca^(2+)accumulation and the cellular Ca^(2+)mobilization using FLUO4-AM and flow cytometry.The results showed that mitochondria from ketamine injected animals presented a lower ability to retain calcium at concentrations higher than 20μM,as compared with controls(saline injected animals).In ad-dition,ketamine showed a significant decreased KCl-induced intracellular calcium concentration.KCl increased calcium influx through cellular depolarization.According to the data presented herein,ketamine presents a clear inhibitory effect on cytosolic Ca2+transport mechanisms,independently from their action on the calcium channel associated NMDA receptor.