TY - JOUR
T1 - A simple benchtop filtration method to isolate small extracellular vesicles from human mesenchymal stem cells
AU - Koh, Benson
AU - Tan, Kian Leong
AU - Chan, Hong Hao
AU - Looi, Qi Hao Daniel
AU - Lim, Moon Nian
AU - How, Chee Wun
AU - Law, Jia Xian
AU - Foo, Jhi Biau
N1 - Funding Information:
The publication of this video was supported by My CytoHealth Sdn. Bhd.
Funding Information:
The publication of this video was supported by My CytoHealth
Publisher Copyright:
© 2022 JoVE.
PY - 2022/6
Y1 - 2022/6
N2 - The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs' size was 30-200 nm, with a particle concentration of 7.75 × 1010 particles/mL and a protein concentration of 80 µg/ mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).
AB - The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs' size was 30-200 nm, with a particle concentration of 7.75 × 1010 particles/mL and a protein concentration of 80 µg/ mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).
UR - http://www.scopus.com/inward/record.url?scp=85133319225&partnerID=8YFLogxK
U2 - 10.3791/64106
DO - 10.3791/64106
M3 - Article
C2 - 35816005
AN - SCOPUS:85133319225
SN - 1940-087X
VL - 2022
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
IS - 184
M1 - e64106
ER -