TITLE: Generation of 3-dimensional spheroids of human umbilical vein endothelial cells and human amnion-derived mesenchymal stem cells in human platelet lysate-based gels
AUTHORS: Eva Rossmanith, Sabrina Summer

[DESCRIPTION]
Generation of 3-dimensional spheroids containing human umbilical vein endothelial cells and human amnion-derived mesenchymal stem cells. The protocol uses an optimized cell ratio to obtain stable spheroids that can be transferred to human platelet lysate-based gels. Within the gels the spheroids form sprouts and interconnect with each other. After grwoth, the spheroids can be fixed in the gel and stained for confocal microscopy.

[BEFORE_START]
Isolate the human umbilical vein endothelial cells and human amnion-derived mesenchymal stem cells and culture them in their corresponding medium until 70-80% confluency (passage 0). https://doi.org/10.3389/fbioe.2019.00338

[GUIDELINES]
Critical steps:
1) Use primary cells at an early passage
2) do not handle the hanging drop culture too roughly, also carefully open/close the incubator door
3) take your time while transferring the spheroids to the gel

[STEPS]
SECTION: Harvesting of the cells
1. 
 Aspirate the medium and wash the cells with 1x PBS


0.24. 
SECTION: Harvesting of the cells
2. Add 300 µl accutase to a T-25 flask and incubate at 37 °C, 5% CO2 for 3-5 minutes
SECTION: Harvesting of the cells
3. Resuspend the cells in 10 ml PBS and transfer the cell suspension to a 15-ml falcon tube
SECTION: Harvesting of the cells
4. Centrifuge at 500g for 5 minutes at 20 °C
SECTION: Harvesting of the cells
5. Aspirate the supernatant and dissolve the pellet in 10 ml M-199 medium (serum-free)
SECTION: Harvesting of the cells
6. Count the cells using a Luna Cell Counter

SECTION: Hanging drop
7. Prepare cell suspension for hanging drops

 5000 cells in 25 µl drop

 For 40 spheroids:
 Kultur	                                        HUVEC	 hAMSC	  Medium
 10% HUVECs + 90% hAMSCs	2*104	1.8*105	  1000 µl

For 10 ml medium:
 Reagent	Stock solution	End concentration	Volume for 10 ml
 M-199	        8 ml
 FBS	       100%	                20%	                                  2 ml
 ECGS	       10 mg/ml	        10 µg/ml	                10 µl
 Heparin        5000 I.E./ml	        15 I.E./ml	                30 µl

SECTION: Hanging drop
8. Pipette 25 µl drops on the inner side of the lid of a pertridish (hydrophobic)
SECTION: Hanging drop
9. Carefully flip the lid
SECTION: Hanging drop
10. Put the lid on the bottom of the petridish filled with 20 ml PBS
SECTION: Hanging drop
11. Incubate for 48 hours at 37 °C, 5% CO2

  
  

SECTION: Culturing of the spheroids in HPL-gels
12. Prepare gel mix (500 µl/µ-dish)

 Gel mix for 2 gels
 Reagent	                Volume for 1000 µl
 HPL	                        200 µl
 Aprotinin                 	  58 µl
 M-199	                        741 µl
 ECGS	                             1 µl

Aprotinin is added to prevent fibrinolysis. 
SECTION: Culturing of the spheroids in HPL-gels
13. Mix 500 µl Gel mix with 100 µl thrombin
SECTION: Culturing of the spheroids in HPL-gels
14. Carefully pipette the gel mix on the window of the µ-dish (avoid bubbles)
SECTION: Culturing of the spheroids in HPL-gels
15. Polymerize the gels at 37 °C for at least 20 minutes
SECTION: Culturing of the spheroids in HPL-gels
16. Pick a spheroid with a 200 µl pipette tip (avoid transferring too much medium) and place the spheroid on the gel (positioning according to the picture)

  


SECTION: Culturing of the spheroids in HPL-gels
17. Repeat step 16 until 5 spheroids are transferred to the gel
SECTION: Culturing of the spheroids in HPL-gels
18. Put the µ-dish in a bigger pertridish with PBS (to prevent dehydration of the gels) and incubate at 37 °C, 5% CO2 overnight
SECTION: Culturing of the spheroids in HPL-gels
19. After 24 hours sprouts are visible
SECTION: Culturing of the spheroids in HPL-gels
20. Add medium to the gel (500 µl/gel), pipette carefully at the border of the dish

 For 2 ml medium:
 Reagent	Stock solution	End concentration	Volume for 2 ml
 M-199	                                                                                1.6 ml
 HPL	       100%	                20%                          	400 µl
 ECGS	       10 mg/ml	        10 µg/ml	                     2 µl

SECTION: Culturing of the spheroids in HPL-gels
21. Incubate at 37 °C, 5% CO2