Exosomes

P9031651Hollow fiber bioreactors are the ideal method for the production of exosomes from stem cells and other cell types such as 293 cells. Exosomes are retained to high concentration in the extra capillary space as they will not cross the fiber. If needed, serum can be used in the circulating medium and the endogenous exosomes in serum will not cross the fiber so no serum starvation is used. Cell debris is significantly reduced for simplified purification. Stem cells do not differentiate over time as no cell splitting is required, and exosomes can be harvested over long periods of time with no change in activity.

Comparison of Exosomes Produced in T-225 flasks vs. FiberCell Systems C2011 20 mL 20 kd MWCO PS 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. 5X108 adipose derived mesenchymal stem cells were seeded into the hollow fiber bioreactor and harvests initiated after one week of culture. Medium used in the hollow fiber bioreactors was DMEM/10% FBS in the circulating medium, DMEM without serum in the ECS. Contaminating exosomes in serum cannot cross the fiber allowing it’s use in culture. Total exosomes collected from two HFBR under these conditions equaled that from 3600 T225 flasks in a total volume of 360 mL.

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

Total medium consumed:  HFB: 7 L per run.   Flasks:  24 L

 

Incorporation of exosomes into human endothelial cells. In order for exosomes to transfer their cargo (nucleic acid, protein), they must somehow be incorporated into the recipient cell. Lipophilic dye transferred from exosomes and incorporated into cultured cells has been used to support the function of exosomes to deliver cargo into cells . We incubated Vybrant DiI cell labeling solution (Life Technologies) with approximately 5 x 10E8 exosomes for 20min at 37oC, followed by untracentrifugation at 100,000 xg for 2 hr to remove unincorporated dye. The labeled exosome pellet was resupended in PBS and added to cultured endothelial cells. Following incubation at 37oC for the experimental, and 4oC for the negative control, cells were removed from the plate by trypsinization and analyzed by flow cytometry . As shown by the shift in the population in the right panel, the cells incorporated DiI from the labeled exosomes, supporting the notion that these exosome preparations from the bioreactor are capable of delivering their cargo (i.e.: nucleic acid, protein) into the cell.

Incorporation of exosomes into human endothelial cells. In order for exosomes to transfer their cargo (nucleic acid, protein), they must somehow be incorporated into the recipient cell. Lipophilic dye transferred from exosomes and incorporated into cultured cells has been used to support the function of exosomes to deliver cargo into cells . We incubated Vybrant DiI cell labeling solution (Life Technologies) with approximately 5 x 10E8 exosomes for 20min at 37oC, followed by untracentrifugation at 100,000 xg for 2 hr to remove unincorporated dye. The labeled exosome pellet was resupended in PBS and added to cultured endothelial cells. Following incubation at 37oC for the experimental, and 4oC for the negative control, cells were removed from the plate by trypsinization and analyzed by flow cytometry . As shown by the shift in the population in the right panel, the cells incorporated DiI from the labeled exosomes, supporting the notion that these exosome preparations from the bioreactor are capable of delivering their cargo (i.e.: nucleic acid, protein) into the cell.

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