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Benchtop Isolation and Characterisation of Small Extracellular Vesicles from Human Mesenchymal Stem Cells

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Abstract

The objective of this study is to develop a simple protocol to isolate and characterise small extracellular vesicles (sEVs) from human umbilical cord-derived MSCs (hUC-MSCs). hUC-MSCs were characterised through analysis of morphology, immunophenotyping and multidifferentiation ability. SEVs were successfully isolated by ultrafiltration from the conditioned medium of hUC-MSCs. The sEVs’ size distribution, intensity within a specific surface marker population were measured with zetasizer or nanoparticle tracking analysis. The expression of surface and internal markers of sEVs was also assessed by western blotting. Morphology of hUC-MSCs displayed as spindle-shaped, fibroblast-like adherent cells. Phenotypic analysis by flow cytometry revealed that hUC-MSCs expressed MSC surface marker, including CD90, CD73, CD105, CD44 and exhibited the capacity for osteogenic, adipogenic and chondrogenic differentiation. Populations of sEVs with CD9, CD63 and CD81 positive were detected with size distribution in the diameter of 63.2 to 162.5 nm. Typical sEVs biomarkers such as CD9, CD63, CD81, HSP70 and TSG101 were also detected with western blotting. Our study showed that sEVs from hUC-MSCs conditioned medium were successfully isolated and characterised. Downstream application of hUC-MSCs-sEVs will be further explored.

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Acknowledgements

This work was funded by Ming Medical Sdn. Bhd. Industrial Grant (MING/2019/SOP/001). We would like to appreciate the technical support provided by product specialist from Particle Metrix during NTA data acquisition with ZetaView.

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Author notes

  1. WuiChuen Chia is the Co-first author

Authors and Affiliations

  1. School of Biosciences, Faculty of Health & Medical Sciences, Taylor’s University, No. 1, Jalan Taylor’s, 47500, Subang Jaya, Malaysia

    Kian Leong Tan & Yin Sim Tor

  2. Ming Medical Services Sdn Bhd, D3-7 (1st Floor), Block D3, Dana 1 Commercial Centre, Jalan PJU 1A/46, 47301, Petaling Jaya, Selangor, Malaysia

    Wui Chuen Chia & Qi Hao Daniel Looi

  3. School of Pharmacy, Monash University Malaysia, 47500, Bandar Sunway, Selangor, Malaysia

    Chee Wun How

  4. Department of Chemical and Environmental Engineering, Faculty of Engineering, University of Nottingham Malaysia Campus, Jalan Broga, Semenyih, Selangor Darul Ehsan, Malaysia

    Pau Loke Show

  5. My Cytohealth Sdn Bhd, 18-2, Jalan Radin Bagus 1, Bandar Seri Petaling, 57000, Kuala Lumpur, Malaysia

    Qi Hao Daniel Looi

  6. Welltech Utopia Sdn Bhd, 6th floor, Vida Bukit Ceylon 1D, Jalan Ceylon, Bukit Ceylon, 50200, Kuala Lumpur, Malaysia

    Qi Hao Daniel Looi

  7. School of Pharmacy, Faculty of Health & Medical Sciences, Taylor’s University, No. 1, Jalan Taylor’s, 47500, Subang Jaya, Malaysia

    Jhi Biau Foo

  8. Centre for Drug Discovery and Molecular Pharmacology (CDDMP), Faculty of Health & Medical Sciences, Taylor’s University, 1, Jalan Taylors, 47500, Subang Jaya, Selangor, Malaysia

    Yin Sim Tor & Jhi Biau Foo

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  1. Kian Leong Tan
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Contributions

JBF, QHDL and YST contributed to the design of the study and interpretation of data. KLT and WCC performed most of the data collection. CWH and PLS assisted in the data collection from Zeta Sizer and NTA. This manuscript is compiled by KLT, JBF and critically revised by YST, QHDL and CWH.

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Correspondence to Qi Hao Daniel Looi or Jhi Biau Foo.

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Tan, K.L., Chia, W.C., How, C.W. et al. Benchtop Isolation and Characterisation of Small Extracellular Vesicles from Human Mesenchymal Stem Cells. Mol Biotechnol 63, 780–791 (2021). https://doi.org/10.1007/s12033-021-00339-2

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