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. Add PBS gradually (up to 20 mL) to the column and collect 500 μL fractions.
3. Combine 5 fractions from the range of 7 - 12 fraction numbers for a total of ~2.5 mL and discard 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 previous protocol (EV Isolation From MSCs) through the syringe filter.
6. Wash filter with 2.5 mL of PBS to collect retained EVs and add to EV solution from step 5 for a total of 5 mL.
   Note: Skip this step to achieve a more concentrated EV sample for assays. If you intend to measure the total EV recovered, this step is required.
7. Analyze small-EVs and use them immediately, or store them at 4°C for short-term and-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 maintenance and expansion of MSCs, refer to the product PIS for MesenCult™-ACF Plus Medium (Document #10000003462). For culture of endothelial cells (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 media and allow 3 days for EV production by MSCs.
2. Thaw Corning Matrigel® Matrix the night before the assay on ice in the 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 approximately 2.6 x 109 particles/50 μl and keep on ice while preparing HUVECs and assay plates.
5. Pipet 30 μl of Matrigel® into the 96-well plate kept on ice.
   Note: Use cold pipettes and tips. To avoid air bubbles when adding Matrigel® to the wells, pipette slowly and carefully.
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, and then return the plate to the incubator.
8. Dissociate HUVEC-GFP cells from flask and add 120 μl of EC-Cult™-XF media containing 10,000 cells/well in the Matrigel®-coated 96-well plate.
9. Immediately add 50 μl of MSC-EVs to the HUVEC-GFP cells.
10. Image and analyze plate using the live cell imaging IncuCyte® instrument running the Angiogenesis Software module.