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.
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Protocol
The overview of the protocol steps is shown in Figure 1. The details of the reagents and equipment used in the study are listed in the Table of Materials.
1. Media preparation
- Prepare a culture medium for ADSC without exosomes using 450 mL of basal culture medium, 50 mL of fetal bovine serum without exosomes, and 5 mL of triple antibiotics.
2. Resuscitating and culturing ADSCs
NOTE: Resuscitate ADSCs.
- Turn on the UV radiation of the ultra-clean table beforehand and disinfect it for 30 min.
- Preheat the medium in a 37 °C water bath beforehand.
- Retrieve 2 tubes of frozen ADSCs (P3) from liquid nitrogen and agitate them in a 37 °C water bath with constant temperature until fully thawed.
NOTE: The opening of the freezer tube should not be in contact with water to prevent contamination. - Disinfect the cryopreservation tube using 75% alcohol and then move it to the ultra-clean table. Aspirate the cell suspension using a pipette and place it in a 15 mL centrifuge tube.
- Place the centrifuge tube into a low-speed centrifuge machine and centrifuge at 250 x g for 5 min at 4 °C.
- Prepare four 10 cm culture dishes and put 9 mL of medium into each dish. Mark the name, cell type, generation, and experimental date, and then preheat them in a 37 °C thermostatic cell incubator.
- Disinfect the centrifuge tube and move it to the ultra-clean table. Remove the supernatant with a pipette.
- Add 4 mL of medium and gently agitate the solution (1 x 105 cells/mL) until it is evenly dispersed.
NOTE: Take care not to eliminate the cell pellet when extracting the supernatant. - Add 1 mL of cell suspension to each dish. Shake the dishes with a cross method.
NOTE: Position the plate on a level surface and agitate it in a cross pattern (moving it up, down, left, and right) for 5-6 repetitions. Avoid circular motions, as they may cause cell accumulation in the center. - Observe the cells at 40x magnification with the optical microscope, and then place the dishes into the incubator.
NOTE: The primary purpose of observing the cells is to determine if they are evenly distributed, as uneven distribution will affect subsequent growth. - Prepare exchange medium for the cells.
- Turn on the UV radiation of the ultra-clean table beforehand and disinfect it for 30 min.
- Preheat the medium in a 37 °C water bath beforehand.
- Observe the cell state under the optical microscope at 40x magnification. The confluence level is approximately 50%.
NOTE: The confluence level is calculated as the ratio of the area occupied by the cells to the surface area of the petri dish after the cells adhere to the wall and fully stretch. - Transfer the culture dish to the ultra-clean table (Biosafety level 2) after disinfection and remove the medium.
- Rinse each culture dish twice with 2 mL of PBS solution gently.
- Add 10 mL of medium to each dish. Transfer the dishes to the incubator.
3. Collecting the ADSCs supernatant and extracting the EVs
- Turn on the UV radiation of the ultra-clean table beforehand and disinfect it for 30 min.
- Observe the cell state under a microscope. The confluence level is approximately 80%.
- Transfer the dish to the ultra-clean table after disinfection. Carefully collect the supernatant with a pipette and place it in a 50 mL centrifuge tube.
NOTE: Cells can be either frozen or discarded. If sterility is not required, the following steps can be performed on the operating table. At this point, the experiment can be paused and then restarted later. If the experiment is halted, store the supernatant in a 4 °C refrigerator for not more than one week. Long-term storage is not recommended as it may affect the activity of EVs. The optimal approach is to extract EVs immediately. - Centrifuge (300 x g, 10 min, at room temperature) to remove suspended cells. Collect the supernatant with a pipette.
NOTE: Do not pour. Leave a little supernatant to prevent the pellet from being sucked up. - Centrifuge to remove cellular debris (2000 x g, 10 min, 4 °C), then continue to collect the supernatant with a pipette.
NOTE: The key points are the same as in step 3.4. - Filter the supernatant with a 0.22 um membrane to remove large particles and cell debris.
NOTE: Filter the supernatant with 2 mL of sterile PBS to moisten the filter membrane prior to filtration. - Centrifuge in the ultracentrifuge machine (10,000 x g, 30 min, 4 °C).
- Next, centrifuge at 1,00,000 x g for 70 min at 4 °C to remove the pellet and collect the supernatant.
NOTE: The obtained EVs were purified by centrifugation multiple times to increase their purity. The pellet may not be visible to the naked eye. Therefore, it is crucial to operate the pipette gently and to leave a little supernatant at the bottom to avoid pellet collection. - Remove the supernatant with a pipette. Resuspend the pellet with PBS and centrifuge (1,00,000 x g, 70 min, 4 °C).
NOTE: The pellet may not be visible to the naked eye. Therefore, when aspirating the supernatant with a pipette, it is critical to gently and thoroughly remove the supernatant to maximize the purity of EVs. - Remove the supernatant and obtain the EVs pellet. Resuspend it with 1 mL of PBS, transfer it into a 1 mL centrifuge tube, and store it in a 4 °C refrigerator for subsequent detection.
NOTE: The pellet may not be visible to the naked eye. Consequently, when aspirating the supernatant with a pipette, it is crucial to gently and thoroughly remove the supernatant to maximize EVs purity. If the samples are not needed for an extended period, store them in a -80 °C refrigerator.
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Representative Results
Firstly, ADSCs were characterized and identified, including their morphology10 and surface antibodies. Based on Figure 2A, it is apparent that ADSCs are arranged in a spindle shape and form a vortex after dense growth. The cultured cells were differentiated into adipogenic, osteogenic, and chondrogenic cells and stained with Oil Red O, Alizarin Red, and Alcian Blue11. The induced differentiation experiment and flow cytometry validated that they are indeed ADSCs with the potential for multi-directional differentiation. However, 22% of cells are CD45 positive in the results, which won't affect the conclusion as sufficient positive and negative antibodies support it (Figure 2A,B).
Next, the identification of the extracted EVs is essential. It mainly includes three aspects: morphology, particle size, and marker proteins. The TEM results clearly reveal the cup-shaped EV structure with a bilayer membrane12 (Figure 3A). NTA indicates that EVs have a particle size distribution of around 50-150 nm, with a concentration of approximately 1.6 x 1010 particles/mL (Figure 3B). Western Blot analysis also shows that the marker proteins of EVs, including CD9, CD63, and TSG10113, are expressed smoothly (Figure 3C).
Figure 1: Overview of the protocol steps. Please click here to view a larger version of this figure.
Figure 2: Characterization of ADSCs. (A) The morphology of ADSCs is fusiform. ADSCs were cultured in the medium for adipogenic, osteogenic, and chondrogenic differentiation, respectively. Adipogenic differentiation was identified by Oil Red O staining. Osteogenic and chondrogenic differentiation was confirmed by Alizarin Red staining and Alcian Blue staining, respectively. Scale bar: 100 µm. (B) Flow cytometry analysis of ADSCs for MSC surface markers (CD90, CD105, CD29, and CD73) and hematopoietic cell-specific markers (CD31, CD34, CD45, and HLA-DR). The gating strategy: circle the blank group first. Based on that, circle CD90, CD105, CD29, CD73, CD31, CD34, CD45, and HLA-DR successively according to the labeled antibodies of each group. This figure is adapted from Han, Y. D. et al.13. Please click here to view a larger version of this figure.
Figure 3: Characterization of EVs. (A) The TEM results clearly reveal the cup-shaped EV structure of the bilayer membrane. Scale bar: 200 nm. (B) NTA indicates that EVs have a particle size distribution of around 50-150 nm, with a concentration of approximately 1.6 x 1010 particles/ mL. (C) Detection of TSG101, CD9, and CD63 expressions in exosomes by Western blot. This figure is adapted from Han, Y. D. et al.13. Please click here to view a larger version of this figure.
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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 90% to ensure sufficient secretion of as many EVs as possible. To maintain the maximum purity of EVs, the researchers must be cautious during the first three centrifugations and ensure no liquid residue is left to prevent impurities and pellets from aspirating. During the last two centrifugations, ensure that the supernatant is completely removed while protecting the pellet.
Ultracentrifugation is cost-effective and requires no further treatment. This technique has been widely used in EV studies and has demonstrated satisfactory efficiency in obtaining EVs, as confirmed by characterization experiments. Nonetheless, there are some inevitable drawbacks. First, the EVs recovery rate is low. Second, EVs tend to aggregate sometimes. Additionally, non-cellular components can be isolated, and some EVs may rupture during the process, leading to debris accumulation14,15,16. To address these defects, most research focuses on purifying EVs12,13. However, this protocol covers the entire process from cell culture to exosome purification because we believe that the entire process impacts the status and purity of the final extracted EVs. It is crucial to adhere to the aforementioned guidelines during operation. Additionally, collecting more supernatant and implementing 3D cell culture may be beneficial, as numerous studies have demonstrated that 3D culture is more effective in stimulating the secretion of EVs compared to 2D culture17,18.
This protocol outlines the process of extracting EVs from ADSCs using ultracentrifugation. It provides practical guidance and enables experimenters to avoid details during the operation as much as possible. In summary, the protocol assists experimenters in improving the quality of samples and serves as a reminder that we should not only focus on the details of each step but also consider the experiment as a whole.
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Disclosures
The authors have no financial interest to declare.
Acknowledgments
This work was partially supported by the National Natural Science Foundation of China (82202473).
Materials
| Name | Company | Catalog Number | Comments |
| 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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