Extracellular Vesicles

Scale-up EV production in your laboratory to produce large amounts

High yield production process for Exosomes, Extracellular Vesicles, Microvesicles…

A FiberCell Hollow Fibre Bioreactor produces large amounts of very concentrated EVs 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 without the need for passaging. Yields are so far reported to be 10X to 40X compared to static flask batch processing although the possibility exists for much higher yields. A number of recent studies indicate that the 3D capillarised culture environment of a hollow fibre bioreactor generates a secretome and 3D-EVs more representative of in vivo conditions compared to flask culture.

Request a demo of the procedure for harvesting concentrated EVs (ZOOM) – info@kdbio.com

Advantages:

  • Enables efficient scale-up of extracellular vesicles production
  • Provides 10-40 X more EVs per ml compared to flasks
  • Yields regular EV harvests over several weeks
  • Very high cell density offers more physiological 3D growth conditions
  • Avoids passaging large numbers of cells
  • Makes it easier to switch cells to chemically defined media
  • Fits on the shelf of a standard lab incubator
  • Avoids generating piles of plasticware trash
  • Makes further scale-up very straightforward
Simultaneous stress treatment of a mass of cells in 3D culture

Small, Medium and Large Cartridge Modules available

Catering for pilot scale through to production, FiberCell Systems hollow fibre cartridges are available in volumes of 3.5, 20 and 70ml. Fibre surface areas range from 450cm2 to 1.2m2 for continuous culturing of as many as 5E+10 cells. Two cartridges can be operated per Duet pump. The molecular weight cutoff recommended for exosome production is 20 kD but if a more complete cell secretome is required, to include small proteins, then 5 kD cartridges are available.

Simple routine exosome collection process

A FiberCell Hollow Fibre Bioreactor provides a continuous culture process enabling periodic harvesting of large amounts of EVs in small volumes that are convenient for further processing. No passaging is required. Users of this system avoid batch production in cumbersome monolayer culture set-ups and will significantly reduce the amount of plastic waste generated in the lab. Click on the article below to learn more.

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.

Example: Scaling up EV production from adipose derived MSCs

Flasks were split 1:5 in DMEM/10% FBS until a total of 130 T225 flasks were obtained. DMEM/10% FBS was replaced with DMEM alone and exosomes collected after 2 days.

5X10^8 adipose-derived mesenchymal stem cells were seeded into the hollow fibre bioreactor (C2011 cartridge) and harvests initiated after one week of culture. Medium used in the hollow fibre bioreactors was DMEM/10% FBS in the circulating medium, DMEM without serum in the extra-capillary space.

Total exosomes collected from two HFBR under these conditions equalled that from 3600x T225 flasks in a total volume of 360 mL.

Collection Volume (ml) Total Exosome Protein (mg) Total Exosome Particles (1010)
Hollow Fibre
  Cartridge #1 240 11.82 95.8
  Cartridge #2 120 14.45 326.9
 T-225
 130 flasks 4000 0.9 1.6

Cartridge #1 – 7 weeks, weekly collection
Cartridge #2 – 4 weeks, 6 collections

Total medium consumed:
hollow fibre: 7 L per run
flasks: 24 L

Example: Scaling up production of engineered exosomes from 293T cells

Example of exosome production in HEK293T cells

Scalable, cGMP-compatible purification of extracellular vesicles carrying bioactive human heterodimeric IL-15/lactadherin complexes: Watson,D.C. et al.; J Extracell Vesicles. 2018 Feb 28;7(1) [open acccess] “This method can be used to produce ~40-times more EV per mL medium than conventional cell culture, and in addition these preparations lack animal proteins present in serum-supplemented media. This platform was used in the current study, and allowed the production of several litres of serum-free, EV-rich medium to be generated.”

Starter kit for exosome production

Cat.# Description Qty
P3202 FiberCell® Systems Duet 1
C2011 High Flux PS Cartridge 20 kd (3000 cm2 – 20 ml) 1
A – – – 33mm, 38mm or 45mm Reservoir Cap 1
CDM-HD Serum substitute for high cell density bioreactor (12 week run) 4

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