Comparison with T-flasks
The most common method for extracellular vesicle production requires large numbers of T flasks. Cells are expanded and the final collection performed using only basal medium (without serum), as serum contains significant levels of contaminating endogenous exosomes. This batch-mode protocol can entail a final stage of hundreds of flasks and many litres of medium to process. For large scale production of extracellular vesicles the FiberCell Systems Hollow Fibre Bioreactor represents an efficient alternative. The system generates concentrated extracellular vesicles in a space- and time-efficient manner. Practically any type of cell, including mesenchymal stromal cells (MSCs), can be cultured continuously at high density in this system. Yields per millilitre are reported to be 10X to 40X compared to static flask batch processing and 100X is theoretically possible. The total amount of EVs produced will depend on the duration of the production run and the size of cartridge used.
The composition and activity of exosomes reflect the physiologic state of the cells when secreting them and flask culture is far from physiologic or relevant to in vivo conditions. In a FiberCell Systems bioreactor, on the other hand, cells are continuously maintained at high density around porous fibre capillaries by means of which there is a constant flow of nourishing media and the constant removal of waste metabolites. In this way, bioreactor-cultured cells benefit from much more in-vivo like conditions for extracellular vesicles production. Recent publications suggest that exosomes produced in a FiberCell Systems bioreactor have greater potency than those generated using static flask culture. Also, a number of studies with cancer cells indicate that the 3D capillarised culture environment inside a hollow fibre bioreactor generates a secretome that is more representative of in vivo conditions compared to flask culture (5kD MWCO fibres used in this case). In several recent studies cells have been easily weaned off serum under bioreactor conditions and were shown to grow well using a simplified chemically defined media formulation.
With T-flasks it can take several rounds of splitting and culture expansion to attain a final production cell-mass. There can also be technical challenges such as the problem of cells undergoing apoptosis just prior to harvest, contaminating it with difficult-to-remove membrane fragments and protein aggregates.
In conclusion, when compared to the FiberCell Bioreactor, T-flasks are very wasteful in plasticware, more time and space consuming and provide culture conditions with only tenuous relevance to the way cells actually grow in vivo. The FiberCell Systems bioreactor on the other hand provides a way to create a high cell density in vitro model for the purposes of EV production and can be operated completely free of serum for many types of cell. It therefore represents a powerful alternative method to to improve the quality of EVs and enhance laboratory production efficiency.