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In 2008, Hahn et al. presented a method for cultivating a 3D organ culture of the cochlea. Although this method is well established, it is currently only applied to early postnatal animals. Given the known differences in regeneration and repair abilities between early postnatal and adult mammalian cochleae, our goal was to further develop and optimize this method to extend it beyond early postnatal animals to include adult mammalian cochleae. After rapidly dissecting the cochlea, it is opened and placed in a neurotrophin-containing culture medium. The culture is then maintained at 32 °C in a rotating bioreactor for 24 h. The combination of mild hypothermia (32 °C), quick cochlea dissection, and the addition of 10 ng/mL of Brain-derived neurotrophic factor (Bdnf) and 5 ng/mL of Neurotrophin 3 (Ntf3) to the culture medium ensures the complete cell survival of all cochlear cell types in 10-day-old mice. The modifications to the established method include the incorporation of neurotrophins (Bdnf and Ntf3) into the culture medium and cultivation under mild hypothermic conditions (32 °C). By introducing neurotrophins and cultivating at 32 °C, a 3D organ culture of the cochlea can also be established with 10-day-old mice. This in vitro model preserves all cochlear cell types under conditions similar to those found in vivo. Full article

CD19 is an essential protein in personalized CD19-targeting chimeric antigen receptor (CAR)-T cell-based cancer immunotherapies and CAR-T cell functionality evaluation. However, the recombinant expression of this “difficult to-express” (DTE) protein is challenging, and therefore, commercial access to the protein is limited. We have previously described the successful stable expression of our soluble CD19-AD2 fusion protein of the CD19 extracellular part fused with human serum albumin domain 2 (AD2) in CHO-K1 cells. The function, stability, and secretion rate of DTE proteins can be improved by culture conditions, such as reduced temperature and a shorter residence time. Moreover, glycosylation, as one of the most important post-translational modifications, represents a critical quality attribute potentially affecting CAR-T cell effector function and thus impacting therapy’s success. In this study, we increased the production rate of CD19-AD2 by 3.5-fold through applying hypothermic culture conditions. We efficiently improved the purification of our his-tagged CD19-AD2 fusion protein via a Ni-NTA-based affinity column using a stepwise increase in the imidazole concentration. The binding affinity to commercially available anti-CD19 antibodies was evaluated via Bio-Layer Interferometry (BLI). Furthermore, we revealed glycosylation patterns via Electrospray Ionization Mass Spectrometry (ESI–MS), and five highly sialylated and multi-antennary N-glycosylation sites were identified. In summary, we optimized the CD19-AD2 production and purification process and were the first to characterize five highly complex N-glycosylation sites. Full article