Large-scale production of engineered Extracellular Vesicles (EVs) in a serum-free continuous culture bioreactor
Bruce, Kelsey
Bruce, Kelsey
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Publication Date
2024-02-26
Type
Thesis
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Abstract
Extracellular vesicles (EVs) are nanoparticles produced by cells that facilitate intercellular communication through cargo transfer. When isolated from a biocompatible cell source, EVs can deliver therapeutic interventions directly to tumor sites. The study of EVs is limited by suitable approaches for scale up of production in reproducible, serum free conditions. This work addresses the need for large scale, dynamic serum free production of EVs.Triple negative breast cancer cells (MDA-MB-231) were transduced with lentivirus containing the sodium iodide symporter (NIS). PCR was used at multiple timepoints to confirm stable mRNA expression, with protein expression confirmed via immunocytochemistry. 1 x 109 cells were inoculated into a hollow fiber bioreactor and glucose consumption monitored daily. When stable 3D culture was established, the transition to serum free conditions was made. Glucose levels were used as an indicator of cell health, media requirements, and harvesting frequency. Cells were extracted at 5 time points, and 16 harvests of EV rich cell conditioned media (CCM) were performed. EVs were isolated from CCM via size exclusion chromatography (SEC) and analysed via nanoparticle tracking analysis and microBCA assay. Expression of NIS mRNA was confirmed in transduced cells (log102.6 fold increase), with protein localization to the cell membrane observed. Cells in the bioreactor consumed 367 mg of glucose within 24 hours, which decreased to an average of 240 mg/24 h over the next 6 days. By day 12, cells were established in the bioreactor and tolerated the transition to serum free culture well, indicated by increasing glucose consumption. Cells sampled from the 3D bioreactor retained original morphology and proliferative ability in 2D. Over the total 46 day culture period, cells retained elevated expression of NIS. EVs isolated via SEC were <200 nm in size and early SEC fractions contained 1.76 x 109 - 8.90 x 1010 EVs. Protein in these fractions did not exceed 16 μg/mL while later fractions contained up to 593 μg/mL. The total yield over the culture period was 4.30 x 1012 EVs. Both the yield and purity of EVs isolated via SEC from cells cultured in 3D dynamic serum free conditions were significantly improved compared to those isolated via ultracentrifugation from cells in 2D. This scalable approach to reproducible, serum free EV production will support advancement of this exciting field towards clinical translation.
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NUI Galway