Isolation of Wharton's Jelly Mesenchymal Stem Cells and their Derived Exosomes/Small Extracellular Vesicles

Summary

Umbilical cord tissue is a rich, readily available, non-invasive source of mesenchymal stem cells (MSCs). In this protocol, detailed steps for the isolation and characterization of human Wharton's jelly MSCs (WJ-MSCs) are provided, followed by a simple short protocol for isolating their derived exosomes or small extracellular vesicles.

Abstract

In the field of regenerative medicine, mesenchymal stem cells (MSCs) have sparked particular attention. MSCs from the umbilical cord (UC) tissue, referred to as Wharton's jelly, have been particularly well-established in a variety of applications. In comparison to other MSC sources, Wharton's jelly MSCs (WJ-MSCs) offer a number of benefits and are isolated from UC, which is typically disposed of as medical waste. WJ-MSCs, therefore, do not raise any ethical issues similar to those pertaining to embryonic stem cells. A simple and reproducible explant technique was used to isolate WJ-MSCs from UC tissue. Within five to ten days of isolation, the WJ-MSCs emerge as adherent cells with a fibroblastic shape. After being isolated, the cells reach about 80% confluency in 14-18 days. Following that, in passage 3, the isolated WJ-MSCs was fully characterized by inducing them to differentiate along the adipogenic, osteogenic, and chondrogenic mesenchymal lineages. Additionally, immunophenotyping for negative cluster of differentiation (CD) surface markers, including CD34, CD14, and HLA-DR, as well as distinctive MSC surface markers like CD105, CD90, CD73, and CD44 were tested. Furthermore, precipitating reagent was used in a straightforward approach to isolate exosomes/small extracellular vesicles (sEVs) from the conditioned medium of those WJ-MSCs. After being isolated, exosomes/sEVs were observed under a transmission electron microscope, and particle size analysis was used to calculate their average size. In summary, WJ-MSCs provide a readily available, non-invasive, renewable supply of stem cells that may be used as factories to produce exosomes/sEVs. These WJ-MSCs and their derived exosomes/sEVs can be used in a wide array of therapeutic and downstream research applications.

Introduction

Only a few years back, stem cells were thought to take the lead in medicine^1,2. However, the ethical concern posed by embryonic stem cells (ESCs) was a huge obstacle hindering rapid progress in the field³. This ignited much interest towards exploring alternative renewable sources for stem cells, especially those which can be isolated postnatally from the umbilical cord (UC)⁴. The UC naturally conjoins to the placenta and fetus inside a pregnant mother. The innate role of the UC is to nourish the growing fetus with a continuous and sufficient blood supply through blood vessels and to ensure the protection of these vessels via avoiding any possible torsion, compression, or kinking of the fetus during its movement⁵. Anatomically, the human UC consists of two arteries and a vein that are embedded within a mucoid connective tissue and enclosed within an outer layer of amniotic epithelium⁶. The UC matrix, or the mucoid connective tissue enclosing the three umbilical vessels, is known as "Wharton's jelly" (WJ) and is primarily made of proteoglycans and collagen⁷.

Recently, mesenchymal stem cells (MSCs) have drawn much attention being an effective novel therapeutic modality for a wide range of regenerative medicine applications^{7,8,9,10,11,12}. What is most intriguing about MSCs is their ease of isolation, as well as demonstrated immunomodulatory activities and multipotency^13,14,15. Other superior advantages of MSCs generally and those derived from WJ particularly are that no teratomas manifest upon transplantation, unlike ESCs, and the fact that they maintain their stemness property even after several cell passages^16,17. Thus, it is now believed that MSCs could indeed provide revolutionary therapeutic interventions for various diseases^8,18,19. It's important to point out that in 2006, the International Society for Cell and Gene Therapy (ISCT) defined specific minimal criteria for proper characterization of MSCs²⁰. First, when grown/cultured in standard culture conditions, MSCs are supposed to be plastic-adherent. Second, MSCs must express some characteristic cluster of differentiation (CD) surface markers like CD90, CD105, and CD73, and must not show expression of others like CD14, CD34, and CD45. Third, MSCs must differentiate into osteogenic, adipogenic, and chondrogenic lineages in vitro²⁰. To this day, MSCs have been isolated from multiple sources, including adult tissues such as bone marrow (BM), adipose tissue²¹, as well as fetal/perinatal sources like placenta, UC blood (UCB), and UC matrix^22,23. It's noteworthy here that when comparing the success rate for harvesting MSCs, it has been reported to reach almost 100% from WJ tissue compared to only about 6% from UCB^24,25. Thus, although UCB is indeed a rich source of hematopoietic stem cells, it is also considered a good source of MSCs, however significantly lower than WJ^26,27,28.

WJ-MSCs are exceptional when compared to other types of MSCs because, although they are bona fide (typical) MSCs, possessing similar properties with their adult counterparts, they also possess the expression of various pluripotency/ESCs markers^17,29. Accordingly, WJ-MSCs are believed to be somewhere in between adult and embryonic stem cells¹⁷. Interestingly, WJ-MSCs have been found to express both human MSCs and ESCs markers and are capable of maintaining stemness for several passages¹⁶. Luckily, upon transplantation, they do not form teratomas¹⁷. This can be elucidated by their unique transcriptomic profile as compared to ESCs, as well as other MSCs^29,30. WJ-MSCs present various advantages over adult MSCs, and even over other types of stem cells generally. Their means of isolation is readily available, via the UC that is routinely discarded at birth, and thus is considered medical waste. Therefore, unlike ESCs, WJ-MSCs have no ethical concerns, and unlike BM-MSCs, that is isolated via an invasive technique³¹. Moreover, like other MSC types, WJ-MSCs lack the expression of human leukocyte antigen-D-related (HLA-DR), and particularly have been found to express human leukocyte antigen-G (HLA-G), which provides them with an extra immune-privilege³². This suggests that WJ-MSCs exhibit an immunosuppressive role simulating what usually occurs at the fetus-maternal interface in vivo^33,34,35. Finally, like their analogues isolated from UCB, WJ-MSCs have great potential for banking³⁶. Given all those advantages, WJ-MSCs are thereby advocated to be the new golden standard for MSC-based therapies³⁰.

Interestingly, a great deal of research has revealed that MSCs exhibit their therapeutic effects mainly via paracrine signaling. In many cases, such effects have been reported to occur in response to exosomes/small extracellular vesicles (sEVs) secreted by MSCs³⁷. Exosomes/sEVs are known as bioactive vesicles of nano-size that are acquired from the cell's endomembrane system. They act as shuttles transferring specific cargos of proteins, mRNA, as well as non-coding RNAs like microRNAs and long noncoding RNA (LncRNAs), as a message in a bottle³⁸. As a consequence, they can reprogram the recipient cells and are denoted as "signalosomes" manipulating essential cellular functions³⁹. MSCs-derived exosomes/sEVs have demonstrated their superiority to MSCs therapies in being a possible cell-free alternative. Similar to their parent MSCs, exosomes/sEVs have been found to elicit therapeutic effects and cellular regeneration in several diseases. However, unlike their parent cells, they can cross through biological barriers more easily. Moreover, they are devoid of critical safety concerns; they impart no risk of immune rejection or long-term tumor formation⁴⁰. Furthermore, exosomes/sEVs can be employed for gene therapy applications as a means of a drug delivery system, being naturally equipped to transfer genetic information⁴¹. Thus, exosomes/sEVs have indeed ignited exciting new avenues for novel therapeutic interventions of various diseases^42,43,44.

In this protocol, a thorough stepwise explant-based protocol for the isolation and characterization of WJ-MSCs from UC tissue is presented. This will be followed by a concise protocol for the isolation of exosomes/sEVs from conditioned media of these WJ-MSCs.

Protocol

All the experiments and procedures were carried out in accordance with the approved guidelines and were approved by the Ethical Committees of both the Faculty of Pharmacy, Ain Shams University, Cairo, Egypt (ACUC-FP-ASU RHDIRB2020110301 REC# 220) and the Faculty of Pharmacy, The British University in Egypt, Cairo, Egypt (CL-2309). The UCs were obtained after obtaining signed informed consent from the parent(s).

1. Isolation of WJ-MSCs from human umbilical cord

1. Place about 30-40 mL phosphate-buffered saline (PBS) in a container (sealed collection bag) for collecting the umbilical cord (UC) (about 15 cm long) immediately after delivery. Maintain the collected UC on ice and process it within a maximum of 4-5 h from collection.
   NOTE: The sample cannot be processed after 4-5 h, as this will greatly diminish the MSCs yield. Ensure the PBS and container are sterile.
2. Prepare complete culture media for WJ-MSCs composed of Low Glucose Dulbecco's Modified Eagle Medium (LG-DMEM) complemented with 10% Fetal Bovine Serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, and 0.25 µg/mL amphotericin B.
3. Place 3 mL complete media per well in 6-well plates, then place those 6-well plates having media in a CO₂ incubator set at 37 °C and 5% CO₂ to warm and get prepared.
4. In the biosafety cabinet, prepare eight 10 cm cell culture dishes, each of them filled with 10 mL of sterile PBS.
5. Carefully, using sterilized forceps, transfer the UC from the sealed plastic bag into the first Petri dish containing PBS to wash out the blood (Repeat this step till all blood is washed out from the sample).
6. Spray 70% ethanol on a sterile swab and use it to wipe the UC for 30 s.
7. Transfer the UC to a new Petri dish filled with sterile PBS to remove any residual ethanol.
8. Transfer the UC into another Petri dish priorly filled with sterile PBS and using sterile scissors, cut the UC into smaller pieces (about 3 cm each). Then, move those 3-cm-long pieces into another Petri dish with fresh sterile PBS to avoid blood released during the cutting process.
   NOTE: From this point forward, the UC pieces must be maintained on ice, as well as all the subsequent processing steps. This is really important to avoid the release of gelatinous matter and ensure the success of the isolation procedure of WJ-MSCs from the UC tissue. Processing the collected UC within 4-5 h maximum is crucial for the success of the isolation process. Previously, the UC was processed within longer periods after collection, and unfortunately, the procedure did not show successful results (data not shown).
9. Start processing the UC pieces one by one (on ice) in a Petri dish filled with sterile PBS. Use sterile scissors and forceps to cut away blood vessels and isolate the Wharton's jelly (WJ) tissue. Then place the clear WJ pieces in another Petri dish filled with sterile PBS on ice.
   NOTE: During isolation of WJ tissue from the UC piece, the first incision is done on the clear side of the UC piece, away from the blood vessels, to avoid cutting through the vessel. Afterwards, blood vessels are clamped, cut around, and pulled away.
10. Repeat the previous step on all UC pieces to obtain clear WJ tissue without blood vessels.
    NOTE: Change the PBS Petri dish, if needed, during processing the UC pieces and removal of the blood vessels, if the PBS solution becomes bloody or unclear during WJ tissue isolation.
11. Wash the collected WJ pieces with PBS by transferring them to a fresh 10 cm cell culture dish filled with sterile PBS.
12. Use a sterile, sharp scalpel and forceps to cut the collected WJ tissues into small 10-mm-long pieces.
13. Transfer WJ pieces into the previously prepared 6-well plates in step 1.3. Place 5-6 WJ pieces per well, then incubate the 6-well plates containing WJ tissue explants in a humidified atmosphere with 5% CO₂ at 37 °C in the CO₂ incubator and leave undisturbed for 3 days.
14. On the 4^th day from placing the WJ tissue explants, carefully add 1 mL of media per well, taking care not to disturb the explants.
    NOTE: Avoid touching or scratching the bottom of the plate where the WJ pieces are attached.
15. Repeat the previous step on the 6^th day after placing the WJ tissue explants.
16. On the 8^th day after placing the WJ tissue explants (i.e., almost 1 week after explant placement), start changing half the media per well every other day until the 12^th-14^th day after explant placement. Replace half the culture media from each well by removing about 2.5 mL from every well and adding fresh 2.5 mL of complete warm media (37 °C).
    NOTE: Withdraw the media from the surface of the well and avoid touching or scratching the bottom of the plate where the WJ pieces are attached. The WJ-MSCs usually appear beneath and around the WJ tissue explants within 5-10 days after placing the explants.
17. Observe the 6-well plates routinely under an inverted microscope (10x magnification), and if the released cells are proliferating and their confluency is increasing, reaching 50-60%, proceed to the next step; if not, repeat step 1.16. again. The WJ pieces significantly shrink in size over time.
18. Gently remove the WJ explants attached to the bottom of the wells and remove all the media. Removing WJ explant pieces is usually done on the 12^th-14^th day from initially placing them.
    NOTE: Slightly incline the plates at a 45° angle while removing the media to ensure all media is removed.
19. Wash each well with 2 mL of PBS, gently swirl the plate, and then discard PBS. Repeat this washing step twice.
    NOTE: Gently add PBS to the wall of the well and avoid touching the bottom of the wells to ensure no disturbance of the cells at all.
20. Add 2 mL of warm complete media per well gently (media with exact composition as that described in step 1.2) and incubate the 6-well plates under humidified conditions with 5% CO₂ at 37 °C_.
21. Change the media completely every other day (2 mL of complete media per well) until the cells reach about 80% confluency.
22. When cells reach about 80% confluency, passage the cells using trypsin-EDTA to T25 cell culture flasks (the total cells obtained from one 6-well plate can be passaged to one T25 culture flask, use 8-9 mL of complete media per T25 flask). These WJ-MSCs are denoted as P1 cells.
    NOTE: Do not leave cells for too long with Trypsin-EDTA to avoid toxic effects; usually, 3 min is sufficient for cells' detachment. Different suppliers can provide Trypsin-EDTA with variable potency. Accordingly, this needs to be considered when specifying the incubation time.
23. Change the media every other day. Once the cells have reached about 80% confluency, passage the cells using Trypsin-EDTA to T75 cell culture flasks (the total cells obtained from one T25 culture flask can be passaged to one T75 culture flask; use 15-18 mL of complete media per T75 flask); these WJ-MSCs are denoted as P2 cells.
24. Change the media every other day. Once the cells have reached about 80% confluency, detach the cells using Trypsin-EDTA, then seed WJ-MSCs at a density of about 0.75 x 10⁶-1 x 10⁶ cells per T75 flask. These WJ-MSCs are denoted as P3 cells.
    NOTE:Use cells between P3-P5 for future experiments described in this protocol.
25. Cryopreserve the isolated WJ-MSCs in liquid nitrogen using cryopreservation media composed of 80% FBS, 10% complete media (as described in step 1.2), and 10% dimethyl sulfoxide (DMSO).
    NOTE: A schematic presentation of the entire steps for WJ-MSCs isolation is provided in Figure 1.

2. Characterization of isolated WJ-MSCs by immunophenotyping using flow cytometric analysis for various CD surface markers

1. Detach the cells using Trypsin-EDTA at passage 4. Wash the cells with PBS twice and count their number using a hemocytometer.
   NOTE: Minimal incubation time (for 3 min) during trypsinization is critical to detach cells successfully without exerting any damage. Prolonged incubation with Trypsin-EDTA might result in damaging cell surface markers and negatively affecting the immunophenotyping assay results.
2. Incubate about 100,000 cells (in dark conditions) for 20 min at 4 °C with human monoclonal antibodies labeled with either phycoerythrin (PE) or fluorescein isothiocyanate (FITC) as follows: Anti-CD90 PE, Anti-CD73 PE, Anti-CD105 FITC, Anti-CD44 PE, Anti-CD34 PE, Anti-CD14 PE, and Anti-HLA-DR FITC, and use unstained cells as controls
3. Rinse the cells with PBS and resuspend them afterwards in 500 µL of FACS buffer; analyze using flow cytometry⁴⁵.

3. Assessment of the differentiation potential of isolated WJ-MSCs towards various mesenchymal lineages

1. Adipogenic differentiation
   1. Resuspend the cells in complete proliferation media (as described in step 1.2). Afterwards, culture the cells at a density of 3.7 x 10⁴ cells/well (0.5 mL medium/well) in a 24-well tissue culture plate. Then, incubate under humidified conditions of 5% CO₂ at 37 °C_. Next_,Next, every other day, change the media till the cells reach almost 100% confluency; this usually takes about 3-4 days.
   2. Remove the proliferation media and replace it with adipogenic differentiation media to induce adipogenesis. Complement the LG-DMEM media with 100 µg/mL streptomycin, 100 U/mL penicillin, 0.25 µg/mL amphotericin B, and the adipogenic supplement provided in the mesenchymal stem cell functional identification kit. Afterwards, change the media gently (0.5 mL/well) every 3 days and avoid the disruption of the lipid vacuoles.
   3. After about 21 days, using microscopic examination, stain the lipid vacuoles with Oil Red to assess the adipogenic differentiation.
   4. Prepare a fresh stock solution of Oil Red; dissolve 30 mg of Oil Red stain in 10 mL of isopropanol and rest for at least 30 min at room temperature. Next, prepare a working solution by mixing 2 parts double-distilled water (ddH₂O) with 3 parts stock solution, mixing them well, and letting it rest for 10 min at room temperature. Afterwards, filter using a 0.22 or 0.45-µm filter.
      NOTE: For best staining results, freshly prepare the Oil Red stain.
   5. For successful adipogenic differentiation documentation, wash the cells twice with PBS and fix them with neutral-buffered 10% formalin (0.75 mL/well) for 15 min. Afterwards, wash the cells twice with PBS using 1 mL/well (before discarding the PBS after each wash, incubate the cells for about 5 min).
      NOTE: Cells maintained in proliferation media (described in step 1.2) are used as control, uninduced cells. Add neutral-buffered formalin slowly to the walls, not directly on the cells, to avoid cellular damage. After the final wash with PBS, complete aspiration of PBS is crucial prior to adding the Oil Red stain working solution to avoid inefficient staining.
   6. After the final wash, stain the cells (1 mL/well) with Oil Red working solution and incubate them for 60 min at 37 °C. Next, withdraw and discard the stain solution and gently rinse the cells twice with PBS. Then, under the microscope, observe the stained lipid droplets.
2. Osteogenic differentiation
   1. Resuspend the cells in complete proliferation media (as described in step 1.2). Afterwards, in a 24-well tissue culture plate, seed 7.4 x10³ cells/well (add 0.5 mL medium/well), and incubate them for 3 days under humid conditions at 5% CO₂ at 37 °C to reach almost 70% confluency.
   2. At about 70% confluency, replace proliferation media with differentiation-induction media incorporating the osteogenic supplement - provided in the mesenchymal stem cell functional identification kit - in LG-DMEM media complemented with 10% FBS, together with 100 U/mL Penicillin, 100 µg/mL Streptomycin, 2 mM L-glutamine, and 0.25 µg/mL Amphotericin B.
   3. Replace the differentiation media once every 3 days, and continue the osteogenic induction for about 21 days.
   4. Prepare a working solution of Alizarin Red S stain; bring 9 mL of ddH₂O and dissolve 200 mg of Alizarin Red S stain in it, and then use ammonium hydroxide and HCl to adjust pH to 4.1-4.3. Next, complete the volume to 10 mL with ddH₂O. Afterwards, filter the solution using a filter (0.22 or 0.45 µm) to remove any precipitates.
   5. At the end of the induction period, rinse the cells twice with PBS, and then fix them using neutral buffered 10% formalin (0.75 mL/well) and let them rest for 15 min. Afterwards, rinse the cells twice using PBS (1 mL/well), and ensure to incubate the cells for 5 min with each rinse.
      NOTE: Cells maintained in proliferation media (described in step 1.2) are used as control, uninduced cells.
   6. Add the 2% Alizarin-Red S working solution prepared in step 3.2.4 to those fixed cells from the previous step, and incubate in the dark for about 45-60 min at room temperature.
   7. Subsequently, discard the stain solution, and rinse the cells with ddH₂O twice and then with PBS once, then assess successful osteogenic differentiation under the microscope by observing the stained calcium-rich extracellular matrix.
3. Chondrogenic differentiation
   1. Resuspend the cells in complete proliferation media (as described in step 1.2). Afterwards, in a 24-well tissue culture plate, seed 7.4 x 10³ cells/well (add 0.5 mL of medium/well) and incubate these under humid conditions at 5% CO₂ at 37 °C for 3 days to reach almost 90% confluency.
   2. At about 90% confluency, start inducing the chondrogenic differentiation using serum-free DMEM/F12 supplemented with insulin-transferrin selenium (ITS), and chondrogenic supplement - provided with the mesenchymal stem cell functional identification kit - together with 0.25 µg/mL Amphotericin B, 100 U/mL Penicillin, 2 mM L-glutamine, and 100 µg/mL Streptomycin⁴⁶.
      NOTE: The complete chondrogenic differentiation induction media is kept in the dark at 2-8 °C.
   3. Continue chondrogenic differentiation induction for about 21 days; meanwhile, change the media every 3 days.
   4. Prepare Alcian Blue stain working solution as follows: First, prepare 3% glacial acetic acid in ddH₂O (add 3 mL glacial acetic acid and fill up to 100 mL with ddH₂O). Then, prepare an Alcian Blue stain working solution by adding 20 mg to 20 mL of 3% glacial acid solution, keeping it standing for about 30 min, and subsequently filtering using a 0.22 µm or 0.45 µm filter to remove any precipitates.
   5. At the end of the induction period, rinse the cells twice with PBS, then fix them using 10% neutral buffered formalin (0.75 mL/well) and let them rest for 15 min. After that, wash the cells twice using PBS (1 mL/well). Ensure to incubate the cells for 5 min each time.
      ​NOTE: Cells maintained in proliferation media (as described in step 1.2) are used as control, uninduced cells.
   6. Incubate the fixed cells in the 0.1% Alcian blue prepared working solution for about 60 min at 37 °C. Rinse the cells with ddH₂O twice and with PBS once to remove the residual stain. Then, using microscopic examination, observe the stained sulfated proteoglycans.

4. Isolation of exosomes/sEVs from WJ-MSCs conditioned media

1. Resuspend WJ-MSCs in complete proliferation media (as described in step 1.2) and seed 6-well tissue culture plates with these WJ-MSCs at a density of 150,000 cells/well (2 mL media/well) and incubate them at 37 °C under humid conditions at 5% CO₂.
2. Prepare serum-free (SF) exosomes collection media as follows: Low glucose Dulbecco's Modified Eagle Medium (LG-DMEM) supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B.
   NOTE: The exosomes collection media has the same composition as the complete proliferation media (described in step 1.2) but without FBS.
3. Follow the 6-well plates (prepared in step 4.1) and replace the media every other day using the proliferation media (as described in step 1.2). Observe the cells under a microscope until they reach about 75-80% confluency.
4. When 70% confluency is reached, wash the cells twice with PBS (1 mL/well), then add serum-free (SF) exosomes collection media (1.5 mL/well) (described in step 4.2), and incubate for 48 h at 37 °C under humid conditions at 5% CO₂.
5. After 48 h, collect the WJ-MSCs conditioned media from the 6-well tissue culture plates placed in the previous step in 15 mL centrifugation collection tubes. Then, carry out the following sequential centrifugation and filtration steps to clear the collected conditioned media⁴⁷.
   ​NOTE: Using trypan blue exclusion dye followed by counting of cells using the hemocytometer, assess the viability percentage of the cells initially used to derive the conditioned media collected; the viable cells should ideally be more than 90%. Afterwards, store the collected cell pellet in -80 °C in case it is needed for any future investigations.
   1. Centrifuge the collected conditioned media at 300 x g for 10 min at 4 °C to get rid of any suspended cells. Then, collect the supernatant and perform the next centrifugation step for this collected supernatant (conditioned media).
   2. Centrifuge the conditioned media obtained from step 4.5.1 at 3000 x g for 15 min at 4 °C to get rid of necrotic cells as well as cell debris.
      NOTE: The centrifugation steps are preferably done at 4 °C, but can also be done at room temperature with similar results.
   3. Filter the supernatant obtained from the previous step by passing it through a 0.22 µm filter into a new collection 50 mL polypropylene tube to get rid of any residual cell debris and large EVs.
      ​NOTE: The filtered conditioned media can be immediately used for subsequent steps to isolate exosomes/sEVs, stored in the fridge at 4 °C and used for no longer than 3 days, or stored at -80 °C for long-term storage until isolating exosomes/sEVs is required. The cleared conditioned media stored at -80 °C is preferred to be processed within a maximum duration of 1-3 months; the sooner the better.
6. Place 7.5 mL of cleared conditioned media (obtained from step 4.5) in a 15 mL centrifuge tube, and add 1.5 mL of reagent. Add the reagent to the media in a 1:5 ratio (i.e., 1 mL of Exoquick-TC precipitating reagent for every 5 mL of media). Afterwards, invert the tube up and down, and flick it several times to ensure proper mixing of the precipitating reagent with the media.
   NOTE: The precipitating reagent is a heavy, viscous reagent. Thereby, the volume required should be withdrawn slowly and carefully to ensure the intended volume is transferred completely.
7. Store the tube having the media with the precipitating reagent 4 °C in an upright standing position overnight for about 16 h (at least 12 h).
8. Next day, centrifuge the media/precipitating reagent mixture at 1500 x g for 30 min at 4 °C. Following centrifugation, the exosomes/sEVs pellet appears as a very small white or beige pellet that has settled at the bottom of the tube.
   NOTE: This centrifugation step can also be done at room temperature with similar results. In case the exosome/sEVs pellet is not visible at the end of this centrifugation step, additional centrifugation time at 3000 x g for an extra 20-30 min at 4 °C can be added. Sometimes the exosomes/EVs pellet remains invisible even after the extended centrifugation time, which means that there is a small amount of exosomes/sEVs. In this case, proceed with the protocol by leaving the bottom 5-10 µL of the tube.
9. Aspirate the supernatant, and centrifuge down residual media/precipitating reagent mixture at 1500 x g for 5 min at 4 °C. Then, very carefully, aspirate the remaining traces of fluid without disturbing the precipitated exosomes/sEVs pellet.
10. Suspend the exosomes/sEVs pellet in 100-300 µL of sterile PBS according to the pellet size and visibility. Afterwards, these exosomes/sEVs can either be immediately utilized or stored in an ultra-freezer at -80 °C for subsequent experiments.
    NOTE: In case of starting with more than one centrifuge tube for the same media (described in step 4.9), and the exosomes/sEVs pellets (obtained in step 4.10) are invisible, the exosomes/sEVs pellets collected from all of them can be pooled using about 100-300 μL of sterile PBS to suspend all of them pooled together.
11. Quantify the collected exosomes/sEVs relative to their protein content using a commercially available bicinchoninic acid (BCA) protein quantification kit, as stated by the manufacturer.
    ​NOTE: A schematic presentation of all the isolation steps of WJ-MSCs-derived exosomes/sEVs is provided in Figure 2.

5. Imaging of isolated exosomes/sEVs using transmission electron microscopy (TEM)

1. Suspend freshly isolated exosomes/sEVs in 100-300 µL of sterile PBS according to the pellet size and visibility (as described previously in step 4.11).
   ​NOTE: It is much preferred to use freshly isolated rather than frozen exosomes/sEVs for TEM imaging
2. Pipette 20 µL of exosomes/sEVs suspension onto a carbon-coated grid and allow to dry for 10 min.
3. Fix the placed exosomes/sEVs using glutaraldehyde solution (2% in distilled water) for about 15 min.
4. Afterward, stain the fixed exosomes/sEVs with 2% uranyl acetate and leave for about 3 min in the dark, then examine them under a transmission electron microscope (TEM) (20000 magnification) using the default TEM settings.

6. Determination of the average size of isolated exosomes/sEVs using a particle size analyzer

1. Dilute freshly isolated exosomes/sEVs with sterile PBS pH 7.4 at a ratio of 1:250.
2. Measure the average particle size distribution of isolated exosomes/sEVs using a particle size analyzer at 25 °C.

Results

Isolation and characterization of WJ-MSCs

As demonstrated in Figure 3, small fibroblast-like cells initially appeared beneath and surrounding the WJ explant tissues within 5-10 days after seeding those explants (Figure 3A). These released WJ-MSCs continue to proliferate and usually reach about 80% confluency within 14-18 days post-isolation. It is notew...

Discussion

In this protocol, we were able to present detailed steps for isolating WJ-MSCs from human UC tissue and also to isolate their derived exosomes/sEVs. UC is an easily attainable untapped non-invasive source to obtain MSCs, and no special equipment for collection or processing is required ^{11,15,30,32,48}. Various protocols have been utilized to isolate WJ-MSCs fro...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alcian Blue 8GX	Sigma-Aldrich, USA	A3157
Alizarin Red S	Sigma-Aldrich, USA	A5533
Ammonium hydroxide	Fisher Scientific, Germany	1336-21-6
Antibody for human CD-105, FITC	R&D systems Inc., MN, USA	FAB10971F
Antibody for human CD-14, PE	Beckman Coulter, USA	IM0650U
Antibody for human CD-34, PE	Beckman Coulter, USA	IM1420
Antibody for human CD-44, PE	Beckman Coulter, USA	A32537
Antibody for human CD-73, PE	R&D systems Inc., MN, USA	FAB5795P
Antibody for human CD-90, PE	R&D systems Inc., MN, USA	FAB2067P
Antibody for human HLA-DR, FITC	Beckman Coulter, USA	IM1638U
Bicinchoninic acid (BCA) protein quantification kit	Thermofisher	23227
Biosafety cabinet, Laminar flow, MSC Advantage	Thermo Scientific, USA
CO2 Incubator, Direct Forma	Thermo Scientific, USA
Cooling Centrifuge	Sigma, Germany
Cooling Centrifuge	Eppendorf, Germany
CytoFLEX Flow Cytometer	Beckman Coulter, FL, USA
Dimethyl sulfoxide (DMSO)	Fisher Scientific, Germany	BP231-100
DMEM - Low Glucose 1 g/L	Lonza, Switzerland	12-707F
DMEM/F12	Lonza, Switzerland	12-719F
DNAse/RNAse free water	Gibco Thermo Fisher, USA	10977-035
Ethanol absolute, Molecular biology grade	Sigma-Aldrich, Germany	24103
ExoQuick-TC	System Biosciences (SBI), USA	EXOTC50A-1	Precipitating agent
Fetal Bovine Serum (FBS)	Gibco Thermo Fisher, Brazil	10270-106
Formaldehyde 37%	Fisher Scientific	BP531-500
Glacial Acetic acid	Sigma Aldrich, Germany	A38S-500
Glutaraldehyde solution	Sigma	G7776
Hydrochloric acid (HCl)	Fisher Scientific, Germany	A144-212
Inverted microscope	Olympus, Japan
L-Glutamine	Gibco Thermo Fisher, USA	25030-024
Mesenchymal Stem Cell Functional identification kit	R&D systems Inc., MN, USA	SC006
Microplate reader	Chromate, Awareness Technologies, USA
Oil Red Stain	Sigma-Aldrich, USA	O0625
Penicillin-Streptomycin-Amphotericin	Gibco Thermo Fisher, USA	15240062
Phosphate buffered saline, 1x, without Ca/Mg	Lonza, Switzerland	BE17-516F
Shaking water-bath	Daihan Wisebath, Korea
Syringe filter, 0.2 micron	Corning, USA	431224
Transmission electron microscope	JEM-1200EX II, Jeol, Germany
Trypan blue	Gibco Thermo Fisher, USA	15250061
Trypsin-Versene-EDTA, 1x	Lonza, Switzerland	CC-5012
Uranyl acetate solution (2%)	Electron Microsopy Sciences, USA	22400-2
UV-Visible spectrophotometer	Shimadzu, Japan
Vortex	SciLogex
Zeta-sizer Nano ZN	Malvern Panalytical Ltd, United Kingdom		Particle size analyzer