Table 4 Isolation methods to obtain EVs from conditioned culture media of AF stem/stromal cells

Exosomes [46]

Aspiration through amniotic sac (primary murine AFSC)

MEM-α

37 °C, 5% CO₂

48

i) 2000g, 20 min

ii) Filtration through 0.22 μm filter

Commercial EV isolation Kit

EVs [47]

Amniocentesis (primary human AFSC)

Sterile phosphate-buffered saline

37 °C, 5% CO₂

24

i) 300g, 5 min

ii) 2000g, 20 min

200,000g, duration not specified

EVs [48]

Amniocentesis (primary human AFSC)

αMEM with 15% FBS, 1% l-glutamine, 1% penicillin/streptomycin, 18% Chang B and 2% Chang C

37 °C, 5% CO₂, 1% O₂ and (hypoxia)

24

i) 3000g, 20 min

ii) 10,000g, 15 min

100,000g, 70 min

Exosomes [49]

Not specified (primary human AFSC)

IDMEM with exosome-free FBS, 100 μg/mL streptomycin/penicillin, 2 mM l-glutamine, 5 ng/mL basic fibroblast growth factor 2

37 °C, 5% CO₂

72

i) 200g, 5 min

ii) 200g, 10 min

iii) 16,500g, 30 min

120,000g, 90 min

EVs [50, 57]

Amniocentesis (primary human AFSC)

FBS free α-MEM with 2 mM l-glutamine, 100 U/ml penicillin/streptomycin

Not specified

96

Concentrating using centrifugal filter unit with 3 K cut-off

Commercial EV isolation Kit

Exosomes [51]

Not specified (cryopreserved murine AFSC)

Serum free MEM-α with 1% penicillin/streptomycin/amphotericin B

Not specified

48

i) 300g, 10 min

ii) 2000g, 10 min

iii) 10,000g, 30 min

100,000g, 70 min

EVs [52]

Aspiration through amniotic sac (primary murine AFSC)

Serum free α-MEM with 20% Chang medium C, 1% penicillin/streptomycin

37 °C, 5% CO₂

24

Commercial EV isolation Kit

Exosomes [56]

Amniocentesis (primary human AFSC)

FBS free α-MEM with 2 mM l-glutamine, 100 units/mL penicillin/streptomycin

37 °C, 5% CO₂

96

Concentration using a 3 KDa cut-off filter

Commercial EV isolation Kit

Exosomes [58]

Amniocentesis (primary human AFSC)

FBS free α-MEM with 2 mM l-glutamine, 100 U/mL penicillin/streptomycin

37 °C, 5% CO₂

96

i) 300g, 10 min

ii) 10,000g, 30 min

100,000g, 1.5 h

Exosomes [59]

Amniocentesis (primary human AFSC)

DMEM: F12 (1:1) with 10 ng/mL bFGF, 10 ng/mL EGF, 10% fetal bovine serum, and 2 mM l-glutamine

37 °C, 5% CO₂

Not specified

i) 500g, 10 min

ii) 16,500g, 20 min

iii) 0.22 μm filtration

118,000g, 70 min

Small EVs [60]

Not specified (primary human AFSC)

Serum free DMEM with 25 mM glucose, 4 mM GlutaMAX, 50 units/mL penicillin/streptomycin

37 °C, 5% CO₂

24 or 48

i) 300g, 10 min

ii) 10,000g, 40 min

iii) Concentration using a 30 kDa filter

Commercial EV isolation Kit

EVs [61, 77]

Amniocentesis (primary human AFSC)

Serum free MSCBM with l-glutamine, gentamicin sulphate/amphotericin B

Not specified

Not specified

i) 300g, 10 min

ii) 2000g, 20 min

iii) 10,000g, 30 min

100,000g, 1 h

EVs [62]

Amniocentesis (primary human AFSC)

DMEM with 10% exosome depleted FBS

Not specified

72

Ultracentrifugation:

10,000g, 45 min

PEG:

Conditioned media mixed 1:1 with PEG 2000, 12 h, 4 °C

Ultracentrifugation:

100,000g, 70 min

PEG:

10,000g, 20 min

EVs [64, 82]

Amniocentesis and caesarean section (primary human AFSC)

Serum free DMEM with 1% l-glutamine and 1% penicillin/streptomycin

37 °C, 5% CO₂, 20% O₂ (normoxia) or 37 °C, 5% CO₂, 1% O₂ and ₍hypoxia)

24

i) 300g, 10 min

ii) 2000g, 20 min

iii) Concentrating using a 3 kDa cut-off filter

iv) 10,000g, 40 min

i) 100,000g, 2 h

ii) Filtration through a 0.22 µm pore filter

EVs [65, 117]

Amniocentesis (primary human AFSC)

α-MEM with 7.5% exosome, depleted FBS

37 °C, 5% CO₂

18

i) 300g, 10 min

ii) 1200g, 10 min

100,000g, 14 h

Nanovesicles [75]

Amniocentesis (primary human AFSC)

Serum free MSCBM medium

Not specified

Not specified

i) 300g, 10 min

ii) 2000g, 20 min

iii) 10,000g, 30 min

100,000g, 1 h

Exosomes [76]

Amniocentesis (cryopreserved human AFSC)

Serum free DMEM with penicillin/streptomycin

37 °C, 5% CO₂

24

2000g, 10 min

High speed, 1 h

EVs [80]

Amniocentesis (cryopreserved human AFSC)

DMEM with 10% EV depleted FBS, 1% glutamine, 1% antibiotics

37 °C, 5% CO₂

Not specified

i) 800g, 30 min

ii) 16,000g, 45 min

i) 100,000g, 2 h

ii) Sucrose gradient centrifugation 100,000g, 16 h

EVs [99, 116]

Aspiration through amniotic sac (primary murine AFSC)

α-MEM with 7.5% exosome-depleted FBS

37 °C, 5% CO₂

18

i) 300g, 10 min

ii) 1200g, 10 min

100,000g, 14 h

Exosomes [102]

Amniocentesis (primary human AFSC)

DMEM with 0.5% exosome-depleted FBS

37 °C, 5% CO₂

48

i) 1000g, 5 min

ii) 3225g, 15 min

iii) Concentrating using a 3 KDa cut-off filter

37,500 rpm, 2 h

Exosomes [104]

Amniocentesis (primary human AFSC)

10% exosome free FBS DMEM

37 °C, 5% CO₂

48

i) 2000g, 10 min

ii) 10,000g, 30 min

100,000g, 70 min

Exosomes [105]

Amniocentesis (primary human AFSC)

Not specified

37 °C, 5% CO₂

Not specified

i) 300g, time not specified

ii) 1000g, 5 min

iii) 10,000g, 10 min

iv) 0.1 μm filtration

100,000g, 1 h

Microvesicles [115]

Not specified (cryopreserved human AFSC)

DMEM with 15–20% FBS, 1% penicillin/streptomycin

Not specified

Not specified

300g, 5 min

100,000g, 2 h

EVs [126]

Amniocentesis (primary human AFSC)

Serum free MEM-α with 1% l-glutamine and 1% penicillin/streptomycin

37 °C, 5% CO₂, 20% O₂ (normoxic) or 37 °C, 5% CO₂, 1% O₂ and ₍hypoxic)

24

i) 3000g, 20 min

ii) 10,000g, 15 min

100,000g, 70 min

EVs [127]

Aspiration through amniotic sac (cryopreserved murine AFSC)

Serum free RPMI-1640

Not specified

Overnight

6000g, 20 min

100,000g, 2 h

EVs [128]

Aspiration through amniotic sac (primary murine AFSC)

Serum free DMEM minimum essential medium with 100 IU/mL penicillin/streptomycin

37 °C, 5% CO₂

48

i) 300g, 5 min

ii) 16,500g, 40 min

iii) Filter through 0.22 μm

120,000g, 70 min

EVs [100]

Not specified (cryopreserved human AFSC)

FBS free α-MEM

37 °C, 5% CO₂

24

i) 180g, 5 min

ii) 1000g, 10 min

iii) 2000g, 10 min

iv) Filter through 0.22 μm

100,000g, 16 h

Exosomes [129]

Caesarean section (primary human AFSC)

10% exosome free FBS supplemented media

37 °C, 5% CO₂

48

i) 3000g, 15 min

ii) 13,000g, 30 min

100,000g, 60 min/commercial EV isolation Kit (unclear)

EVs [103]

Amniocentesis (primary human AFSC)

FBS free α-MEM with 2 mM l-glutamine, 100 U/mL penicillin/streptomycin

37 °C, 5% CO₂

96

i) 300g, 10 min

ii) 10,000g, 30 min

100,000g, 1.5 h

Exosomes [101]

Not specified (cryopreserved human AFSC)

CHANG Amnio (Irvine) culture medium supplemented with 10% exosome-depleted FBS and 1% penicillin/streptomycin

37 °C, 5% CO₂

24–48

i) 750g, 5 min

ii) 1500g, 5 min

iii) 14,000g, 35 min

i) 110,000g, 2 h

ii) 0.22 μm filtration