Generation of 3-dimensional spheroids of human umbilical vein endothelial cells and human amnion-derived mesenchymal stem cells in human platelet lysate-based gels
Eva Rossmanith, Sabrina Summer
Abstract
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
Steps
Harvesting of the cells
Aspirate the medium and wash the cells with 1x PBS
Add 300 µl accutase to a T-25 flask and incubate at 37 °C, 5% CO2 for 3-5 minutes
Resuspend the cells in 10 ml PBS and transfer the cell suspension to a 15-ml falcon tube
Centrifuge at 500 g for 5 minutes at 20 °C
Aspirate the supernatant and dissolve the pellet in 10 ml M-199 medium (serum-free)
Count the cells using a Luna Cell Counter
Hanging drop
Prepare cell suspension for hanging drops
5000 cells in 25 µl drop
For 40 spheroids: Kultur HUVEC hAMSC Medium um
10% HUVECs + 90% hAMSCs 104 1.8* 4104 1.85105 1000 µl
For 10 ml medium: Reagent Stock solution End concentration Volume for 10 ml l
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
Pipette 25 µl drops on the inner side of the lid of a pertridish (hydrophobic)
Carefully flip the lid
Put the lid on the bottom of the petridish filled with 20 ml PBS
Incubate for 48 hours at 37 °C, 5% CO2
Culturing of the spheroids in HPL-gels
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.
Mix 500 µl Gel mix with 100 µl thrombin
Carefully pipette the gel mix on the window of the µ-dish (avoid bubbles)
Polymerize the gels at 37 °C for at least 20 minutes
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)
Repeat step 16 until 5 spheroids are transferred to the gel
Put the µ-dish in a bigger pertridish with PBS (to prevent dehydration of the gels) and incubate at 37 °C, 5% CO2overnight
After 24 hours sprouts are visible
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
Incubate at 37 °C, 5% CO2
