BIOPROCESS DESIGN AND OPTIMIZATION FOR EXTRACELLULAR VESCILES DERIVED FROM MESENCHYMAL STEM CELLS

• Published in: Cytotherapy (Oxford, England) England), 2024-06, Vol.26 (6), p.S87-S87
• Main Authors: Dehghani, M., Black, E., Zhao, Z., Haridas, N., Saha, S., Splan, D., Szczypka, M.S.
• Format: Article
• Language: English
• Subjects: Bioprocessing; Extracellular Vesicles; Mesenchymal Stem Cells
• ISSN: 1465-3249; 1477-2566
• DOI: 10.1016/j.jcyt.2024.03.164

Extracellular Vesicles (EVs) derived from mesenchymal stem cells (hMSCs) have been investigated in several clinical trials for therapeutic applications. Optimization of upstream and downstream process for EV production is a critical step to support clinical trials. The USP consisted of expansion, growth, and collection phases for the total of 10 days in single use bioreactor using microcarriers. MSCs were seeded at 3000 cells/cm2 with microcarrier density of 10 cm2/mL in MSC NutriStem® media. At day 4, 50% media exchange was done and at day 7, the EV collection phase was started by switching to EV depleted media. The DSP started with a clarification step using 5 and 0.6 filters. Tangential flow filtration was then performed using 750 KDa hollow fibers to achieve 5X concentration and diafiltration. The impurities were further removed in an anion exchange chromatography step. Second TFF step was performed to exchange the buffer and concentrate the EVs and was followed by sterile filtration. The MSC harvest was collected at day 10 with the final cell density of 1-5 cells/mL and particle concentration of 1-5 particles/mL measured by scattering mode of NTA. More than 93% cell viability was measured throughout the upstream processing. Multiple analytical techniques were used to measure recovery and purity before and after each unit operations in DSP. We optimized each step using orthogonal single particle analysis techniques such as NTA (scattering and fluorescence) and flow cytometry as recommended by MISEV guidelines. We further analyzed each sample by Analytical HPLC, a semi-quantitative technique to characterize EV samples using 3 different detectors; multi angle light scattering, UV and fluorescence. The purity was assessed by monitoring the protein and DNA concentration throughout the process. Western blot showed the presence of tetraspanin markers and cargo protein. The functionality of the purified MSC-EV was then confirmed by measuring the wound recovery in a dose dependent manner. We have developed a bioprocess for MSC derived EVs which provides a scalable and end-to-end platform for EV production. We have optimized each step using multiple orthogonal analytical techniques to achieve the highest yield and purity.