TITLE: Differentiation of Mesenchymal Stromal Cells to Endothelial-like cells in Spheroidal Culture
AUTHORS: Simeng Li, Isabel Arias Quiros, Guenther Eissner

[DESCRIPTION]
In this protocol, an easy and cost-friendly method to form mesenchymal stromal cell (MSC) spheroids was specified. The MSC spheroids would form in 1-3 days and are suitable for endothelial differentiation. Accutase treatment can be used to disassociate the spheroids into single cells, for further analysis.
Multiple growth factors were used to differentiate MSC Spheroids into endothelial-like spheroids, which express endothelial markers such as von willebrand factors (vWF) and CD31 detected by immunofluorescence. 

[STEPS]
SECTION: Mesenchymal stromal cell spheroids formation
7.  Split and resuspend MSC in fully-supplemented MesenCult-ACF plus medium. 
SECTION: Mesenchymal stromal cell spheroids formation
8. Count cells with Trypan blue and dilute cell to 3x10e5/ml or 12x10e5/ml. Cells need to be over 95% viable in order to form spheroids.
SECTION: Mesenchymal stromal cell spheroids formation
9. Seed 50uL of cell suspension to each well of a 96-well round-bottom plate. There will be 15,000 cells per well.
SECTION: Mesenchymal stromal cell spheroids formation
10. Keep MSC in incubator for 1-3 days until one spheroid is formed in each well. Depending on the size of the spheroids, they can be observed either with eyes or underneath a microscope. Culture condition is 37 degree, 5% CO2 and 80% humidity.
SECTION: Endothelial differentiation of MSC spheroids
11. Make endothelial differentiation medium by adding 10ng/ml (final concentration) VEGF, EGF, bFGF and 2% (v/v) human serum (HS) into fully supplemented MesenCult ACF-plus medium.
SECTION: Immunofluorescence to check endothelial markers of differentiated spheroids
15. Remove medium in each well carefully. Before fixation and permeabilization, wash spheroids three times with PBS, 5 min each.
SECTION: Immunofluorescence to check endothelial markers of differentiated spheroids
16. The same 96-well round-bottom plate was used for fixation. Fixation was done with 4% PFA for 1 hour at room temperature (RT). Fixation time varies depending on the size of spheroids.
SECTION: Immunofluorescence to check endothelial markers of differentiated spheroids
17. Wash spheroids with 100uL PBS per well three times, 5 min each.
SECTION: Immunofluorescence to check endothelial markers of differentiated spheroids
19. Dilute primary antibodies 1:100–1:200  (or according to manufacturor's instruction) in the blocking buffer. Add 50ul of primary antibody to each well and incubated overnight at 2-8 °C. Primary antibodies produced from different species can be used together.
SECTION: Immunofluorescence to check endothelial markers of differentiated spheroids
20. Remove solution from each well and wash Spheroids with PBST (0.05% Tween‱ 20 detergent in PBS) for 10 min at RT, three times.
SECTION: Mesenchymal stromal cells (MSC) culture
1. Coat culture flasks with Animal Component-Free Cell Attachment Substrate prior to cell seeding. Dilute Animal Component-Free Cell Attachment Substrate 1:15 in D-PBS (without Ca++ and Mg++) and coat the culturewares 2 hours at room temperature (15-25 °C), using the sufficient volume to cover the culture surface. Alternatively, coat flasks overnight at 2-8 °C with flask lid sealed by parafilm.
SECTION: Immunofluorescence to check endothelial markers of differentiated spheroids
21. Add corresponding secondary antibodies at 1:250 in blocking buffer for 2 h at RT. 
SECTION: Immunofluorescence to check endothelial markers of differentiated spheroids
22. Remove solution from each well and wash spheroids with TBST (0.1% Tween‱ 20 detergent in Tris-buffered saline), 20 min each time, 3 times.
SECTION: Immunofluorescence to check endothelial markers of differentiated spheroids
18. Block spheroids using blocking buffer (PBS with 3% FBS, 1% BSA, 0.5% Triton X-100 and 0.5% Tween) for 2 hours at RT on a shaker.
SECTION: Immunofluorescence to check endothelial markers of differentiated spheroids
23. Stain Spheroids with 300nM DAPI for 20 min at 37 degree. Then wash the spheroids once with 50 uL PBST for 5 min.
SECTION: Immunofluorescence to check endothelial markers of differentiated spheroids
24. Use a 1 ml pipette to take the spheroid up together with the solution from each well. Transfer the spheroid to a well of ibidi µ-Slide 15 Well 3D. 
SECTION: Immunofluorescence to check endothelial markers of differentiated spheroids
25. Remove PBST from each well of ibidi µ-Slide, add a drop of ibidi mounting medium to mount the spheroids. Control the amount of mounting medium added, so that the spheroids will be located at the bottom of each well.
SECTION: Immunofluorescence to check endothelial markers of differentiated spheroids
26. Image the spheroids with a Olympus FV1000 confocal microscope (Olympus Lifescience), or other suitable confocal microscope.
SECTION: Endothelial differentiation of MSC spheroids
12. Carefully remove the media from each well, avoid the spheroid stucking at the end of the tips.
SECTION: Endothelial differentiation of MSC spheroids
13. Add 50ul of differentiation medium into each well. Keep some spheroids as untreated control by adding normal culture medium.
SECTION: Endothelial differentiation of MSC spheroids
14. Differentiation duration can be between 5-11 days. It can be determined by how advanced the differentiated cells need to be. 
SECTION: Mesenchymal stromal cells (MSC) culture
2. Bring flasks with attachment substrate to room temperature if flasks were coated at 2-8 °C. Remove attachment substrate completely by tilting the flask and gathering the substrate to the edge of the flask.
SECTION: Mesenchymal stromal cells (MSC) culture
3. Wash flasks once with D-PBS. Then the flasks are ready to use.
SECTION: Mesenchymal stromal cells (MSC) culture
5. Trypsinze MSCs with controlled treatment time. Normally 30s-1min is sufficient to detach MSC from flasks. Use Trypsin neutralizing solution to neutralize trypsin since there is no serum in the cell culture medium.
SECTION: Mesenchymal stromal cells (MSC) culture
6. Count cells with Trypan Blue exclusion and seed MSC in fully-supplemented MesenCult-ACF Plus Medium.  
SECTION: Mesenchymal stromal cells (MSC) culture
4. Complete 500 mL MesenCult-ACF Plus Medium by adding 1 mL of MesenCult-ACF Plus 500X Supplement and 2 mM of L-glutamine.
SECTION: Disassociation of MSC from Spheroids for flow cytometry analysis
27. Harvest 10 spheroids from each condition into one 1.5 mL Eppendorf tubes. Centrifuge at 300g, 4 min. Then remove and discard the supernatant.
SECTION: Disassociation of MSC from Spheroids for flow cytometry analysis
28. Add 400 uL accutase solution (Sigma/ Cat: 6964) to each tube and incubate the tube on a Thermomixer comfort (Eppendorf, Hamburg, Germany) at 37 °C, 1400 rpm for 10min. Use a 200uL pipette to pipette up and down for ten times to further disassociate the spheroids. This is considered as one disassociation cycle.
SECTION: Disassociation of MSC from Spheroids for flow cytometry analysis
29. Repeat the disassociation cycle 1-3 times more until no visible cell clumps remaining in the tube.
SECTION: Disassociation of MSC from Spheroids for flow cytometry analysis
30. Wash cells once with 1 mL PBS. Centrifuge at 300g for 5 min and then cells can be resuspend in flow cytometry buffer for antibody labelling.