Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells
Summary
This protocol describes all procedures, from culturing human adipose-derived mesenchymal stem cells (ADSCs) and collecting supernatant to extracting extracellular vesicles (EVs) using ultracentrifugation.
Abstract
Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research suggests that their regenerative function may be attributed to cell-cell contact and cell paracrine effects. The paracrine effect is an important way for cells to interact and transfer information over short distances, in which extracellular vesicles (EVs) play a functional role as carriers. There is significant potential for ADSC EVs in regenerative medicine. Multiple studies have reported on the effectiveness of these methods. Various methods for extracting and isolating EVs are currently described based on principles such as centrifugation, precipitation, molecular size, affinity, and microfluidics. Ultracentrifugation is regarded as the gold standard for isolating EVs. Nevertheless, a meticulous protocol to highlight precautions during ultracentrifugation is still absent. This study presents the methodology and crucial steps involved in ADSC culture, supernatant collection, and EV ultracentrifugation. However, even though ultracentrifugation is cost-effective and requires no further treatment, there are still some inevitable drawbacks, such as a low recovery rate and EV aggregation.
Introduction
Most ADSCs are derived from adipose tissue and have been demonstrated to promote the regeneration and reconstruction of various tissues and organs, including the myocardium, bone, and skin1. Recent research suggests that the regenerative function of ADSCs may be due to intercellular contact and the paracrine effects of the cells2. The paracrine effect is an important means for cells to interact and transfer information over short distances, and this function is achieved through extracellular vesicles (EVs).
EVs are double-layer membrane structures produced by cells, with a diameter ranging from 40 nm to 160 nm (with an average of about 100 nm). They affect different cellular functions, such as cytokine production, cell proliferation, apoptosis, and metabolism3,4. Numerous studies have been conducted on the functions of ADSC EVs, including promoting bone regeneration, oral mucosal tissue regeneration, adipose tissue survival after tissue transplantation, and skin wound repair5,6,7,8. The enormous potential of ADSC EVs in regenerative medicine is evident. Various methods exist for extracting and separating EVs from the supernatant, such as techniques based on centrifugation, precipitation, molecular size, affinity, and microfluidics. The ultracentrifugation method is widely considered the gold standard for isolating EVs9. The fundamental principle of ultracentrifugation for EV separation is based on the fact that particles in the sample have varying sedimentation coefficients, resulting in their sedimentation and aggregation in distinct separation layers. Nevertheless, a detailed protocol emphasizing precautions during ultracentrifugation has not yet been established.
Therefore, this study objectively outlines the ADSC culture, supernatant collection, and EVs ultracentrifugation procedures and key points with a logical flow of information and clear, formal language. This provides a valuable reference for future experiments.
Protocol
Representative Results
Discussion
During the formal experimental process, several points are crucial for achieving the best experimental results. Based on our previous experience, it is recommended to opt for passage 3-6 ADSCs, as they ensure the best possible cell state. Before P3, red blood cells, endothelial cells, and other miscellaneous cells may not have been screened out. After P6, the cells may gradually age, which can affect the state of secreted EVs. Secondly, the supernatant must be collected when the cell confluence degree is between 80% and …
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was partially supported by the National Natural Science Foundation of China (82202473).
Materials
| Acrodisc Needle Filter of Supor Membrane | Acros Organics | 4652 | 0.22 μm |
| Basal Medium For Cell Culture | OriCell | BHDM-03011 | |
| Fetal Bovine Serum Without EXO + Culture Supplement (For Human Adipose-derived Mesenchymal Stem Cells) | OriCell | HUXMD-05002 | |
| Inverted Microscope | OLYMPUS | Lx70-S8F2 | |
| Low Speed Centrifuge | Anhui USTC Zonkia Scientific Instruments Co.,Ltd. | SC-3612 | |
| Normocin | InvivoGen | ant-nr-05 | |
| Optima Max-XP Tabletop Ultracentrifuge | Beckman Coulter | 393315 | |
| Penicillin-Streptomycin-Gentamicin Solution (100x) | Solarbio | P1410 |
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