Poster Presentation Australasian Extracellular Vesicles Conference 2020

Introduction: Researchers studying extracellular vesicles (EV) often need to grow flasks of cells to gain enough EVs for their experiments. The process is labour intensive; time consuming and can be complicated by bovine serum in the culture medium. Bioreactors that support high-density culture may be an alternative to this problem. Devices that use semipermeable membranes to separate cells and cellular products from bovine contamination are particularly interesting. We tested and compared the performance of two such commercial bioreactors, the FiberCell^TM system and the CELLine^TM Bioreactor, in EV production from Mesenchymal stem cells (MSCs).

Method: CELLine^TM Flask for adherent cells (CELLine^TM 1000AD); FiberCell^TM 2025G and 2025D were used. FiberCell^TM 2025D was further coated with fibronectin to improve adherence. Cells were seeded at 30 million cells/bioreactor, and allowed to grow in DMEM/F12/10% FBS before adapted to serum free DMEM/F12. A chemically defined serum replacement mix CDM-HD was supplemented at this stage. Cell growth was monitored by glucose consumption. EV production was analysed using a Nanosight NS300. EV morphology was confirmed by Transmission Electron Microscopy (TEM). After culture, the bioreactors were disassembled and examined by Scanning Electron Microscopy (SEM).

Results: MSC culture was successfully established in the CELLine^TM Flask whereas no growth was observed in the FiberCell^TM bioreactors. In serum free conditions, EV were harvested from the CELLine^TM at a consistent rate of approximately 0.4-1 x10¹¹ particles/day while EVs harvested from FiberCell^TM cultures were only approximately 1-6 x10⁹ particles/day. TEM indicated normal EV morphology. SEM showed that cells in the FiberCell^TM appeared as clusters with large areas unoccupied. In contrast, cells in the CELLine^TM Flask formed a dense confluent layer that covered the entire growth surface. All cultures were kept for approximately 1.5 months with easy maintenance that generally took less than two hrs/week/bioreactor.

Conclusions: Our results showed CELLine^TM 1000AD is as a good choice for adherent cells under our experimental conditions. The operation is time-efficient and labour friendly. Bioreactors could be a useful tool for high level of EV production in long-term high-density culture with the option of using serum free medium.