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Extracellular Vesicles

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Extracellular Vesicles

HFBRs are the method of choice for EV production at clinical scale

HOLLOW FIBER BIOREACTORS (HFBR) OFFER SIGNIFICANT ADVANTAGES OVER FLASK CULTURE

Extracellular Vesicles have very high specific activity, but progress in understanding and applying them has been hampered by their low levels of secretion making it difficult to produce large quantities for experimentation and research. The most common method for producing exosomes uses 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. The composition and activity of exosomes reflect the physiologic state of the cells when secreting them and this method is far from physiologic or relevant to in-vivo conditions. This method is wasteful, time-consuming, inefficient, and not amenable to scale-up for clinical applications.

HFBRs are the most in-vivo like way to culture large numbers of cells and harvest what they secrete. They are ideal for the collection and concentration of large numbers of exosomes under physiologic cell culture conditions. Large numbers of cells can be supported in 3-D, at high densities. The molecular weight cut-off of the fiber allows nutrients and waste products to pass through the fibers but concentrate the larger secreted products and exosomes in the small volume of the ECS (extra-capillary space).

If serum is required it can be used in the circulating medium only, while the ECS containing the cells and secreted extracellular vesicles can be maintained serum free. The HFBR can facilitate the use of a protein free medium, such as CDM-HD. The cells are also bound to a porous support so that splitting is not required, cells are free to grow post-confluent and can form spheroids. The collection of secreted extracellular vesicles can be maintained for several weeks or months of continuous production.

High speed EV pellet from 275ml of harvested supernatant from the C2011 cartridge (so about 2 weeks of harvests). Yield is about 20mg. With FiberCell hollow fiber bioreactors you can see what you are working with.

This is an example of the amount of plastic waste used to generate 1×109 cells.

Plastic trash generated by the culture of 1X109 cells

The C2011 cartridge can support large numbers of MSC and other cells, replacing 2-10 CellStacks.

Hollow fiber bioreactors (HFBR) offer significant advantages over flask culture

HFBRs are the most in-vivo like way to culture large numbers of cells and harvest what they secrete. They are ideal for the collection and concentration of large numbers of extracellular vesicles under physiologic cell culture conditions. Large numbers of cells can be supported in 3-D, at high densities. The molecular weight cut-off of the fiber allows nutrients and waste products to pass through the fibers but concentrate the larger secreted products and extracellular vesicles in the small volume of the ECS (extra-capillary space).

If serum is required it can be used in the circulating medium only, while the ECS containing the cells and secreted extracellular vesicles can be maintained serum free. The HFBR can facilitate the use of a protein free medium, such as CDM-HD. The cells are also bound to a porous support so that splitting is not required, cells are free to grow post-confluent and can form spheroids. The collection of secreted extracellular vesicles can be maintained for several weeks or months of continuous production.

There is an ever-growing body of work in the published literature demonstrating the utility of hollow fiber bioreactors for extracellular vesicle production. A reference list can be found here.

Hollow fiber bioreactors are the method of choice for the production of extracellular vesicles to clinical scale

Gram quantities of exosomes can be produced in HFBRs.

  • Extracellular vesicles are concentrated by a factor of 10–100X.
  • No interference from endogenous extracellular vesicles present in serum.
  • Reduction in apoptosis reduces cell debris.
  • No serum starvation, more physiologic cell culture conditions.
  • Continuous production over several months.
  • No passaging of cells required.
  • Closed, single use system.
  • Manipulations such as heat shock can easily be applied uniformly to large numbers of cells.
  • Continuous biomanufacturing process.
  • Clinical scale under cGMP possible.
  • Production at the gram scale possible using current technology.

Scalability of 3D hollow fiber reactor

To find out more about the scalability of 3D hollow fiber reactor, click on each image to to read the research results.

Serum Starvation vs. 10% CDM-HD

Standard EV production can entail the use of T-flasks and the use of basal medium without serum for EV collection. In the examples below basal medium has been supplemented with 10% CDM HD for the collection phase. EV output is significantly enhanced by CDM HD.

Harvests were taken from confluent T175 flasks with roughly 2.5×106 total cells in each flask. Serum starved flasks were grown up in 10% FBS+DMEM and serum starved for 48 hours prior to collection. CDM-HD media flasks were grown up in 10% FBS+DMEM and switched to 10% CDM-HD+DMEM for 48 hours prior to collection. Each flask contained 35 mL of media. Particle concentrations are as follows (particles/mL): MSC-Serum Starved (4.6×108), MSC-CDM-HD (3.7×109), Prostate-Serum Starved (3.0×109, Prostate-CDM-HD (2.9×1010).

Comparison of flask culture to a C2011 HFBR for the production of extracellular vesicles from mesenchymal stem cells
Collection Volume (mL) Total Exosome Protein (mg) Total Exosome Particles (1010)
HFBR
Cartridge #1 (7 weeks, collection every week) 240 11.82 95.78
Cartridge #2 (4 weeks , 6 collections) 120 14.45 326.9
Flasks
130 T225 4000 0.9 1.6
Suggested Ordering For Extracellular Vesicles:
Cartridges
Catalog No. Size Surface Area Fiber Type Packing Density ECS Vol MWCO 50% Max. Cell#
C2025D Small 450 cm2 High flux PS 50% 3.2 mL 20 kD 108 more info
C2011 Medium 4000 cm2 high flux PS 50% 15 mL 20 kD 109 more info
C2018 Large 1.2 m2 high flux PS 50% 70 mL 20 kD 5 x 1010 more info
Reservoir Caps
A1005 33 mm Reservoir Cap more info
A1006 38 mm Reservoir Cap more info
A1008 45 mm Reservoir Cap more info

Click here for our US list pricing for 2023. We offer tiered discounted pricing for quantity purchases of our products, 10-25 units, 25+ units, and 50+ units. Please enquire for details. We recommend the most appropriate products for your applications so please feel free to email or phone us to discuss your application and provide you with a quote at info@fibercellsystems.com, 301-471-1269, 240-440-2662. We can accept credit cards and purchase orders from accredited institutions.

Case Studies

1. Comparison of flask culture to a C2011 HFBR for the production of extracellular vesicles from mesenchymal stem cells

5 X 108 human adipose derived adult MSC were cultured in a FiberCell Systems HFBR for 8 weeks using DMEM/10% FBS in the circulating medium only, with basal DMEM in the ECS. At the end of 8 weeks the cartridge was cut open and cells recovered for phenotypic analysis. This demonstrated no change in cell phenotype over the period of culture. See results here.

2. Production of IL-15 RC decorated EVs from HEK 293 cells using CDM-HD.

CD63 and Alix were greatly enriched from the bioreactor compared to flask culture. EV/protein ratio was 10-fold higher in harvests from the bioreactor suggesting a significant reduction in contaminating cell membrane fragments. Purified HEK 293 cells retained their IL-15 biological activity. See results here.

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For detailed information on producing exosomes using HFBRs in your laboratory, contact us or request for a quote.