TY - JOUR
T1 - Influence of short term storage conditions, concentration methods and excipients on extracellular vesicle recovery and function
AU - van de Wakker, S. I.
AU - van Oudheusden, J.
AU - Mol, E. A.
AU - Roefs, M. T.
AU - Zheng, W.
AU - Görgens, A.
AU - El Andaloussi, S.
AU - Sluijter, J. P.G.
AU - Vader, P.
N1 - Funding Information:
This work is financially supported by the Van Herk Foundation to SvdW and EVICARE (#725229) of the European Research Council (ERC) to JS. Fig. 1A, 2A, 3A, 4A and the graphical abstract were designed using BioRender (BioRender.com). The authors acknowledge Cor Seinen for excellent technical assistance with TEM. AG is an International Society for Advancement of Cytometry (ISAC) Marylou Ingram Scholar 2019-2023. A.G. and S.E.A. are consultants for and have equity interests in Evox Therapeutics Ltd. P.V. serves on the scientific advisory board of Evox Therapeutics Ltd.
Funding Information:
This work is financially supported by the Van Herk Foundation to SvdW and EVICARE (#725229) of the European Research Council (ERC) to JS. Fig. 1 A, 2A, 3A, 4A and the graphical abstract were designed using BioRender (BioRender.com). The authors acknowledge Cor Seinen for excellent technical assistance with TEM. AG is an International Society for Advancement of Cytometry (ISAC) Marylou Ingram Scholar 2019-2023.
Publisher Copyright:
© 2021 The Author(s)
PY - 2022/1
Y1 - 2022/1
N2 - Extracellular vesicles (EVs) are phospholipid bilayer enclosed vesicles which play an important role in intercellular communication. To date, many studies have focused on therapeutic application of EVs. However, to progress EV applications faster towards the clinic, more information about the physical stability and scalable production of EVs is needed. The goal of this study was to evaluate EV recovery and function after varying several conditions in the isolation process or during storage. Physical stability and recovery rates of EVs were evaluated by measuring EV size, particle and protein yields using nanoparticle tracking analysis, microBCA protein quantification assay and transmission electron microscopy. Western blot analyses of specific EV markers were performed to determine EV yields and purity. EV functionality was tested in an endothelial cell wound healing assay. Higher EV recovery rates were found when using HEPES buffered saline (HBS) as buffer compared to phosphate buffered saline (PBS) during EV isolation. When concentrating EVs, 15 ml spinfilters with a 10 kDa membrane cutoff gave the highest EV recovery. Next, EV storage in polypropylene tubes was shown to be superior compared to glass tubes. The use of protective excipients during EV storage, i.e. bovine serum albumin (BSA) and Tween 20, improved EV preservation without influencing their functionality. Finally, it was shown that both 4 °C and −80 °C are suitable for short term storage of EVs. Together, our results indicate that optimizing buffer compositions, concentrating steps, protective excipients and storage properties may collectively increase EV recovery rates significantly while preserving their functional properties, which accelerates translation of EV-based therapeutics towards clinical application.
AB - Extracellular vesicles (EVs) are phospholipid bilayer enclosed vesicles which play an important role in intercellular communication. To date, many studies have focused on therapeutic application of EVs. However, to progress EV applications faster towards the clinic, more information about the physical stability and scalable production of EVs is needed. The goal of this study was to evaluate EV recovery and function after varying several conditions in the isolation process or during storage. Physical stability and recovery rates of EVs were evaluated by measuring EV size, particle and protein yields using nanoparticle tracking analysis, microBCA protein quantification assay and transmission electron microscopy. Western blot analyses of specific EV markers were performed to determine EV yields and purity. EV functionality was tested in an endothelial cell wound healing assay. Higher EV recovery rates were found when using HEPES buffered saline (HBS) as buffer compared to phosphate buffered saline (PBS) during EV isolation. When concentrating EVs, 15 ml spinfilters with a 10 kDa membrane cutoff gave the highest EV recovery. Next, EV storage in polypropylene tubes was shown to be superior compared to glass tubes. The use of protective excipients during EV storage, i.e. bovine serum albumin (BSA) and Tween 20, improved EV preservation without influencing their functionality. Finally, it was shown that both 4 °C and −80 °C are suitable for short term storage of EVs. Together, our results indicate that optimizing buffer compositions, concentrating steps, protective excipients and storage properties may collectively increase EV recovery rates significantly while preserving their functional properties, which accelerates translation of EV-based therapeutics towards clinical application.
KW - Extracellular vesicles
KW - Function
KW - Isolation process
KW - Recovery
KW - Regenerative medicine
UR - http://www.scopus.com/inward/record.url?scp=85120747997&partnerID=8YFLogxK
U2 - 10.1016/j.ejpb.2021.11.012
DO - 10.1016/j.ejpb.2021.11.012
M3 - Article
AN - SCOPUS:85120747997
SN - 0939-6411
VL - 170
SP - 59
EP - 69
JO - European Journal of Pharmaceutics and Biopharmaceutics
JF - European Journal of Pharmaceutics and Biopharmaceutics
ER -