Biomanufacturing and Curcumin-loading of Human Choroid Plexus Organoid-derived Extracellular Vesicles from a Vertical Wheel Bioreactor to Alleviate Neuro-inflammation

Introduction: : Cerebrospinal fluid (CSF) is secreted by the choroid plexus (ChP). The ChP is a major contributor to the extracellular vesicles (EVs), the membrane-enclosed nanovesicles (50-1000 nm) released by cells through membrane budding, in CSF. Current models don’t provide information regarding the cellular and tissue sources of various secreted proteins. To accomplish this, a human induced pluripotent stem cell (hiPSC)–derived ChP organoid model was developed. EVs are extremely beneficial for drug delivery with a variety of therapeutic agents and biological cargos and are able to cross the blood brain barrier (BBB). A great challenge for the use of stem cells and the secreted EVs in practical applications is the availability of culture systems for large scale production. Bioreactors are a possible solution due to their ability to provide a controlled microenvironment. Vertical Wheel Bioreactors (VWBRs) are showing great promise for both cell expansion and EV production. This is in part due to their unique design features, that result in gentle and uniform mixing and particle suspension with reduced power input and agitation speeds.
This study investigated ChP organoid differentiation from hiPSCs and the EV secretion in a VWBR, with comparison to static cultures. Moreover, this study loaded curcumin into ChP organoid-derived EVs, performed EV lyophilization, and determined the ability of the re-hydrated EVs in alleviating neuro-inflammation. This study presents a novel approach to generate hiPSC-derived ChP organoids and the secreted EVs in VWBRs as well as designing EV-based cell-free therapeutics to treat neural inflammation in neurological disorders.

Materials and

Methods: : ChP organoids were differentiated from Human iPSK3 cell line under Wnt activation and bone morphogenetic protein (BMP) 4 over 24 days in both static and dynamic VWBR conditions. Spent media were collected every 2-3 days for metabolite analysis, including K+, Ca2+, and Na+, glucose, lactate, glutamine, and ammonia. Growth morphology and kinetics during differentiation were monitored. On day 24 the collected media were utilized for EV isolation based on ExtraPEG differential ultracentrifugation and characterization via nanoparticle tracking analysis (NTA), Western Blot, and Transmission Electron Microscopy. mRNA was isolated from cells for Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) analysis of ChP differentiation markers including AQP1, CLIC6, PLYTP etc. and EV biogenesis genes including ALIX, RAB27A, RAB27B etc. EV loading of curcumin was performed using sonication, incubation and freeze-thaw. EVs also underwent a lyophilization and rehydration process and the therapeutic effects was tested in vitro utilizing Amyloid Beta (Aβ) 42 Oligomer stimulated ChP organoids, along with fresh EVs and fresh curcumin-loaded EVs. Aβ42 oligomers (1 μM) were added to the replated ChP organoids for 48h before treating with EVs. Neuroinflammation genes were determined by qRT-PCR.

Results, Conclusions, and Discussions:: The VWBR condition showed larger organoids than the static culture at the end of differentiation. Metabolite analysis shows that K+, Ca2+, and Na+ concentrations were significantly lower in VWBR than the static control (Figure 1A). Glucose consumption and lactate production was significantly higher in the VWBR than the static control. The lactate to glucose ratio for the VWBR was lower over the differentiation period, indicating aerobic metabolism. On day 24, the relative mRNA expression of various ChP markers were measured by RT-qPCR (Figure 1B). It was observed that the VWBR group showed significantly higher expression for 6 of the 10 markers, compared to the static control. The expression of EV biogenesis markers, including both endosomal sorting complexes required for transport (ESCRT)-dependent and -independent biogenesis genes was significantly upregulated for VWBR condition. VWBR condition produced 4-fold higher EVs per mL medium than the static control, consistent with the upregulated gene expression of EV biogenesis. NTA analysis using NanoSight showed that there was no statistical difference in the EV size for the VWBR and static conditions, with expression of exosomal markers and typical exosome morphology. Lyophilization does not negatively affect EV size and stability. For functional testing, lyophilized curcumin-loaded EVs preserve the functional properties of the ChP organoid EVs, alleviating neuro-inflammation induced by exposure to Aβ42 oligomers. Lyophilization process may exert freezing stress on the EVs, leading to mechanical damage due to ice crystal formation, exposure to ice-liquid interfaces, increased iron strength, and pH shifts induced by salt precipitation. The lyophilized EVs downregulated TNFα and IL-6 expression, indicating the effect of reducing pro-inflammatory markers. The anti-inflammatory effect was similar to or better than the fresh EVs. The lyophilized EVs also showed similar effects on the gene expression of matrix remodeling enzymes compared to fresh EVs. This study demonstrates a scalable bioreactor system to promote ChP organoid differentiation and the EV secretion for potential applications in treating ischemic stroke and Alzheimer’s disease. This study also demonstrates the feasibility of drug loading and lyophilization of the ChP organoid EVs to develop cell-free therapeutics for treating neural inflammation in neurological disorders.

Acknowledgements and/or References (Optional):: This work is supported by National Science Foundation (CBET-1917618). Research reported in this publication was also partially supported by the National Institutes of Health (USA) under Award Number R01NS125016.