. 2019 Dec 5;32(2):113–120. doi: 10.4103/tcmj.tcmj_182_19

Table 1.

Summary of the methods for exosome production in compliance with good manufacturing practice

Cell expansion				Exosome isolation			Exosome validation			Results	Reference

Cell source	Cultivating substrate	Dissociation enzyme	Culture medium	1^st process Removing cells and cell debris	2^nd process Concentration of condition medium	3^rd process Exosome purification	Total protein content	Bio-characterization	Physical characterization
MDDCs	T-175 flasks	Not reported	Serum-free	3/0.8 μm filter	500-kDa MWCO hollow fiber membrane	Sucrose/deuterium UC at 100,000 ×g	ELISA	Tetraspanin proteins, such as CD81, CD63, CD9, and CD82; costimulatory molecule CD86; adhesion proteins, such as CD11b, CD11c, CD58, and CD54	Not reported	Increased quantity (concentration of MHC class II) and protein characterization (using FACS) to standardize exosome vaccine	Journal of immunological methods 2002, 270 (2), 211-26 [18]
BM-MSCs	T225 flask	Not reported	HPL/FBS	0.22 μm filter	UC at 30,000 × g for 20min	UC at 120,000 ×g for 3h	Not reported	CD90, CD14, CD34, CD45, CD73; HLA-II (DR); total RNA; miRNA	NTA	10% HPL-based EV-depleted medium is appropriate for the purification of exclusively human MSC-derived EVs	Cytotherapy, 2017; 19: 458-472 [19]
hCPCs	CellBIND®	TrypLETM Select	Free of nonhuman animal-derived components	Centrifugation (3000 × g) and filtration (0.22 μm)	Amicon Ultra-15 (100 kDa cut-off) or Centricon Plus-70	TFF with a 300-kDa cut-off hollow fiber cartridge	QuantiProTM BCA assay kit	GATA4, TBX5, TBX18, MESP1, TSG101, GRP94, and GAPDH	TEM	High exosome yield, and consistent removal of contaminating proteins (97%)	Front Physiol. 2018; 9: 1169 [20]
HEK293 cell	Hollow-fiber bioreactors (fibercell systems)	Not reported	EV-depleted cell culture medium	Differential centrifugation and filtration (0.22 μm)	TFF device (0.05 μm pore size)	UC at 110,000 ×g for 3 h SEC	Bradford assay	CD63 and calnexin	NTA; immune-TEM; LC-MS	Combination TFF and SEC for large staring volumes	J Extracell Vesicles 2018, 7 (1), 1442088 [22]
BM-MSCs	Hollow-fiber bioreactors (quantum bioreactor)	-	HPL for confluence then HPL-free for collection	Centrifugation (1000 × g) and 0.2-μm filters	Not applied	UC at 110,000 ×g for 3 h	MicroBCA assay	Exosome markers (CD9, CD63, CD81, and CD47); mesenchymal markers (CD29 and CD90); siRNA sequence	NanoSight; TEM	Shelf life, biodistribution, toxicology profile, and efficacy	JCI Insight. 2018 Apr 19; 3 (8) [21]
ADSC; BM-MSCs	Flasks	Trypsin-EDTA	PL	Centrifugation at 3000 × g for 20 min	Not applied	UC: 100,000 ×g for 1 h at 4°C UF: Purified by TFF	Micro BCA-protein assay kit	Cytokine quantification by; Immunogenicity and immunomodulatory properties; Secretome versus. MSC immunomodulatory properties	NTA; phospholipid quantification; FT-IR	UF lead to higher protein, lipid, cytokine, and exosome yield compared with that with UC	Nanomedicine 2019, 14 (6), 753-765 [23]

MDDCs: Monocyte-derived dendritic cells, BM-MSCs: Bone marrow-mesenchymal stem cells, hCPCs: Human cardiac progenitor cells, ADSC: Adipose-Derived Stem Cell, PL: Platelet lysate, HPL: Human platelet lysate, FBS: Foetal bovine serum, EV: Extracellular vesicles, EDTA: Ethylenediaminetetraacetic acid, UC: Ultracentrifugation, TFF: Tangential flow filtration, SWC: Size-exclusion chromatography, UF: Ultrafiltration, NTA: Nanoparticle tracking analyzer, TEM: Transmission electron microscopy, LC-MS: Liquid chromatography-mass spectrometry, SEC: Size-exclusion chromatography, MWCO: Molecular weight cut-off, BCA: Bicinchoninic acid, FACS: Fluorescence-activated cell sorting, MHC: Major histocompatibility complex, HLA-II (DR): Human Leukocyte Antigen II – DR isotype, FT-IR: Fourier-transform infrared spectroscopy