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Setting a Successful Sorting for Extracellular Vesicle Isolation
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Secretion pattern of canine amniotic stem cells derived extracellular vesicles
Rafael Garcia Karam1, Lina Castelo Branco Motta1, Matheus Ferreira de Almeida2
1Departamento de Cirurgia, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, SP, Brasil.
Animal Reproduction
|November 25, 2022
Summary
Canine amniotic membrane stem cells (MSCs) produce more extracellular vesicles (EVs) in early passages (P0). This suggests P0 MSCs are optimal for preclinical studies, though further research on EV content is needed.
Area of Science:
- Stem Cell Biology
- Extracellular Vesicles
- Regenerative Medicine
Background:
- Extracellular vesicles (EVs) from stem cells (SCs) show regenerative potential for chronic diseases.
- EVs offer lower tumorigenicity risk and ease of isolation/storage compared to SCs.
- Canine amniotic membrane-derived stem cells are a potential source for therapeutic EVs.
Purpose of the Study:
- To compare the morphological characteristics of EVs derived from canine amniotic membrane stem cells across different in vitro culture passages (P0-P2).
- To determine the optimal passage of canine MSC-derived EVs for potential therapeutic applications.
Main Methods:
- Isolation and characterization of canine amniotic membrane stem cells (MSCs) via differentiation and flow cytometry (CD90+, CD105+, CD34-, CD45-).
- Generation of conditioned media from MSCs at different passages (P0-P2) with normalized cell counts.
- Separation and characterization of EVs using nanoparticle tracking analysis (NTA), western blot (Alix+, Cytochrome C-), and transmission electron microscopy (TEM).
Main Results:
- EV concentration was significantly higher in passage 0 (P0) compared to passages 1 (P1) and 2 (P2).
- No significant differences in EV size were observed across passages (P0: 132nm, P1: 130nm, P2: 120nm).
- TEM confirmed the characteristic cup-shape morphology of the isolated EVs.
Conclusions:
- Passage 0 canine MSCs yield a higher concentration of EVs compared to later passages.
- This suggests P0 MSC-derived EVs may be more suitable for preclinical therapeutic evaluations.
- Further studies are required to elucidate EV content and cellular responses to EV treatment.
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