Comparison of Production and Activity of Amniotic Fluid Stem Cell Extracellular Vesicles from 3D Hollow Fiber Bioreactor and 2D culture.
Laura Perin et al.
;Introduction: EV clinical translation is limited by scale-up of EVs production. Hollow fiber bioreactors (HFBR) support culture of large numbers of cells at high densities. Culture conditions may affect EV composition and potency. Here we compare production, potency, identity and therapeutic potential of EVs collected from cells grown in culture dish (2D) versus HFBR (3D).
Methods: Human clonal AFSC were derived from patient-consented amniotic fluids. 1x10e6 hAFSC were seeded in 2D (145cm2), and 1.6X10e7 hAFSC on a small 20kd MWCO HFBR (FiberCell-C2025D, 450cm2) with fibronectin coating; both cultured in Chang medium with 20% of ES-FBS, starved for 24hr and then EVs collected. The effect of harvest frequency was tested (8hrs, 24hr, 72hrs,1wk). 2D-EVs and 3D-EVs were compared by Nanosight, potency assay (by WB), identity (by Exoview analysis) and therapeutic effect (in vivo in an animal model of kidney disease, Alport Syndrome).
Results: 2D production was ~5.5X10e9EV/ml/24hrs while 3D was ~2.8X10e10EV/ml (first four 24hrs) and ~4.4X10e10EV/ml (two days of hourly harvests). Very little difference in EV concentration and very similar size distribution (~130nm) were observed during harvest intervals; possibly indicating either significant EV re-uptake or inhibition of EV secretion dependent upon free EV in the supernatant. 3D-EVs trapped VEGF (an in vitro established potency assay) as efficiently as 2D-EVs, and expressed CD9, CD81, CD63, CD80, CD86 and VEGFR1 as 2D-EVs.
Summary/Conclusion: 3D-EVs had comparable properties and bio-activity to 2D-EVs, but the HFBR produced 10x more EVs. HFBR cell culture conditions for hAFSC still need optimization, however an available 1.2m2 cartridge provides a 50X scale up potential. The HFBR, a cGMP closed system, can produce sufficient numbers of EV to support pre-clinical and clinical applications with at least similar properties to EVs produced by conventional 2D methods.
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