Scale-up production of extracellular vesicles

Up to 1E14 particles in each daily 20ml harvest

MSCs, cancer cell lines, amniotic fluid stem cells, HEK293, suspension cell lines… A FiberCell Hollow Fibre Bioreactor enables any cell culture laboratory to generate large amounts of very concentrated Extracellular Vesicles in a space- and time-efficient manner.

High yield, high purity process

EV yields (per ml CM) are reported to be 10X to 40X higher compared to static flask batch processing. A daily 20ml harvest of CM from a C2011 cartridge might contain between 1E13 and 1E14 particles when growing an immortalised cell type.

Gentle, continuous 3D culture in a hollow fibre bioreactor are reported to result in EV proteome / miRNA profiles consistent with a more in-vivo like cell microenvironment.

Recent emailers on the topic of EV production

Advantages:

  • Enables efficient scale-up of extracellular vesicles production
  • Provides 10-40 X more EVs per ml compared to flasks
  • Regular high yield and high purity EV harvests over several weeks
  • MSCs can be maintained under serum-free conditions (see protocol)
  • Cell lines can be maintained in protein-free chemically defined media
  • Very high cell density offers more physiological 3D growth conditions

Small, Medium and Large Cartridges

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 to permit continuous culturing of as many as 5E+10 cells in a standard incubator. 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.

FiberCell System in incubator producing concentrated monoclonal antibody with hybridoma.

FiberCell DUET Pump and C2011 Cartridge Module

Simple routine collection process

A FiberCell Hollow Fibre Bioreactor provides a continuous culture process enabling periodic harvesting of large amounts of Extracellular Vesicles 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.

Production of Exosomes in Hollow Fiber Bioreactor - John Cadwell

Production of Exosomes in a

Hollow Fiber Bioreactor – PDF

Hollow-fiber bioreactor production of extracellular vesicles from human bone marrow mesenchymal stromal cells yields nanovesicles that mirrors the immuno-modulatory antigenic signature of the producer cell: Gobin, J. et al. Stem Cell Research and Therapy(2021) 12:127 [open access]… “The hollow-fiber bioreactor from FiberCell Systems allows for seeding large amounts of adherent cells based on its hollow-fiber technology which consequently increases the cell seeding surface area (medium-sized cartridge offers 4000 cm2 of surface area). In turn, this configuration allows for a scale-up of continuous EV production sampled over time from the EV-rich cell-conditioned medium produced by millions of cells, thus allowing anticipated clinical doses to be achieved.”

Engineered extracellular vesicle decoy receptor-mediated modulation of the IL6 trans-signalling pathway in muscle Mariana Conceicao et al. [open access]… “For in vivo experiments, FiberCell hollow fiber bioreactors with 20 kDa cutoff hydrophilic fibers (KDBIO, Berstett, France) were used for large-scale production of MSC-derived EVs, according to manufacturer’s instructions.”

Hypoxia-induced tumor exosomes promote M2-like macrophage polarization of infiltrating myeloid cells and microRNA-mediated metabolic shift: Park, JE et al.; Oncogene (2019) [abstract].. “B16-F0, A375, A431, orA549 cells were inoculated into the extra-capillary space of each cartridge and allowed to attach to the fiber surface over 24 h. Cancer cells were maintained in DMEM supplemented with 5% Chemically Defined Medium for High Density cell culture (CDM-HD; Fibercell). CDM-HD, a chemically defined protein-free serum replacement, was used to avoid contamination of FBS-derived exosomes”. Author describes use of hollow fibre culture system and workflow of exosome isolation.

Efficient production and enhanced tumor delivery of engineered extracellular vesicles: Watson DC et al.; Biomaterials 105, 2016: 195-205 [open access]“…these data suggest that the use of hollow-fiber bioreactor, serum-free media [CDM-HD] and an optimized purification protocol represent a superior method to achieve high yield of purified EV (yields were >3 mg/week from a single lab-scale [20ml, Medium Size] culture cartridge)..”

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 #124011.8295.8
Cartridge #212014.45326.9
T-225
130 flasks40000.91.6

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

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

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.”

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