This technical bulletin describes a protocol for generating extracellular vesicles using MesenCult™-ACF Plus Medium which enables extracellular vesicle (EV)-free culture of mesenchymal stromal cells (also known as mesenchymal stem cells; MSCs). MesenCult™-ACF Plus is animal component- and EV-free and is optimized to derive human MSCs from multiple sources, including bone marrow and adipose tissue. The small EVs generated using this medium are fully functional as indicated by the endothelial tube formation assay. The lack of contaminating EVs in this media leads to reduced experimental variability and results in homogeneous samples when generating MSC-EVs.

MesenCult™-ACF Plus: An EV-Free MSC Culture Medium

To confirm that MesenCult™-ACF Plus is EV-free, the complete medium particle concentration and properties were investigated using several methods. MesenCult™-ACF Plus particle concentration was compared to fetal bovine serum (FBS)- or human platelet lysate (hPL)-containing media (Figure 1). Nanoparticle tracking analysis of different batches from each medium detected at least 94% fewer nanoparticles in MesenCult™-ACF Plus compared to FBS- and hPL-containing media. MesenCult™-ACF Plus was also tested for EV-specific markers using Western blotting. The commonly used EV markers, CD9, CD63, and CD81, were not present in MesenCult™-ACF Plus at detectable levels (Figure 2).

MesenCult™-ACF Plus was also compared to EV-depleted FBS. Nanoparticle tracking analysis indicated that MesenCult™-ACF Plus had a similar particle concentration to EV-depleted FBS-supplemented media (Figure 3A) and led to superior MSC expansion when compared to FBS-containing medium or medium supplemented with EV-depleted FBS (Figure 3B).

MSCs Cultured in MesenCult™-ACF Plus Medium Generate Functional EVs

Since MesenCult™-ACF Plus is animal component- and EV-free, it is an ideal medium for generation of EVs from MSCs. The EV-free nature of MesenCult™-ACF Plus ensures the absence of contaminating EVs. MSC expansion and EV generation is not compromised using this medium. To confirm this, the EVs derived using MesenCult™-ACF Plus were assayed for functionality using a human umbilical vein endothelial cell (HUVEC) tube formation assay (Figure 4). Bioactive molecules, including miRNAs, produced by MSCs and packaged into EVs have been shown to promote angiogenesis in endothelial cultures^1,2, making this assay a suitable method to test MSC-EV functionality. HUVECs treated with EVs isolated from MSCs cultured in MesenCult™-ACF Plus exhibited enhanced angiogenesis with an increased number of branch points. In contrast, HUVECS not treated with MSC-EVs formed fewer branch points and at slower rates (Figure 4). The cargoes carried by MSC-EVs produced in MesenCult™-ACF Plus were analyzed by qPCR. A high abundance of EV-specific markers, let7, miR21, and miR26a, was observed in EVs derived in MesenCult™-ACF Plus (Figure 5).

EV Purification Following Differential Centrifugation

EVs isolated by differential ultracentrifugation can be further purified using size-exclusion columns and sterilized by filtration (Figure 6). These extra purification steps lead to small-EV samples with minimal protein contamination (Figure 7).

NOTE: Perform all steps at 4°C

1. Add the resuspended pellet from the ultracentrifugation step to an EV size-exclusion column.
2. Gradually add PBS (up to 20 mL) to the column and collect 500 μL fractions.
3. Combine 5 fractions from fractions 7 - 12 for a total of ~2.5 mL and discard the others.
4. Pre-wash a 0.2 μm polyethersulfone (PES) membrane syringe filter with 2 mL of PBS and discard flow-through.
5. Pass the 2.5 mL EV solution from Step 6 of the EV Isolation from MSCs protocol through the syringe filter.
6. Wash the filter with 2.5 mL of PBS to collect retained EVs and add to the EV solution from Step 5 of this protocol for a total of 5 mL.
   Note: This step is required if you intend to measure the total EV recovered. Skip this step to achieve a more concentrated EV sample for assays.
7. Analyze the small EVs and use them immediately, or store them at 4°C for short-term storage or -80°C for long-term storage. Avoid repeated freeze-thaw cycles.

HUVEC Tube Formation Assay

The tube formation assay in this technical bulletin was performed using HUVECs that stably express green fluorescent protein (HUVEC-GFP). For details on the maintenance and expansion of MSCs, refer to the PIS for MesenCult™-ACF Plus Medium (Document #10000003462). To culture endothelial cells (i.e. HUVECs) used in the tube formation assay, refer to the PIS for EC-Cult™-XF Culture Kit (Document #DX20709).

1. Culture MSCs in MesenCult™-ACF Plus according to the PIS (Document #10000003462). Once 100% confluent, change the media and allow 3 days for EV production by MSCs.
2. The night before the assay, thaw Corning Matrigel® Matrix on ice in a cold room.
   Note: Keep Matrigel® on ice at all times to prevent polymerization.
3. Isolate MSC-EVs from MesenCult™-ACF Plus after 3 days by differential ultracentrifugation using the EV Isolation Protocol above.
4. Resuspend EVs in PBS to a final concentration of ~2.6 x 10⁹ particles per 50 μL and keep on ice while preparing HUVECs and assay plates.
5. Pipette 30 μL of Matrigel® per well into a 96-well plate kept on ice.
   Note: Use cold pipettes and tips. Pipette slowly and carefully to avoid air bubbles when adding Matrigel® to the wells.
6. Incubate the culture plate for 10 - 20 min at 37°C to allow Matrigel® to polymerize.
7. Add 30 μL of EC-Cult™-XF media to the wells to prevent them from drying out. Then, return the plate to the incubator.
8. Dissociate HUVEC-GFP cells from the flask and add 120 μL of EC-Cult™-XF Medium containing 10,000 cells per well to the Matrigel®-coated 96-well plate.
9. Immediately add 50 μL of MSC-EVs per well to the HUVEC-GFP cells.
10. Image and analyze the plate using an IncuCyte® live-cell imaging instrument running the Angiogenesis Software module or similar software.