1. Objective
Loading extracellular matrix (ECM) gel and seeding of endothelial cells in the OrganoPlate® Graft for preparation of a vascular network.
2. Background
The OrganoPlate® Graft is a 3D cell culture platform that can be used to vascularize tissues such as organoids, spheroids, and explants. This protocol describes the formation of a vascular network in the OrganoPlate® Graft (see figure 1 and 2). Endothelial cells are seeded in the perfusion channels against an ECM gel. A gradient of angiogenic factors is added to the graft chamber and induces the formation of angiogenic sprouts, resulting in a vascular network. Morphology and function of the vessels can be assessed by microscopy, a perfusion assay, or other functional assays.
This protocol describes the formation of a vascular network using primary human umbilical vein endothelial cells (HUVECs) from Lonza. Different cell types may require optimization of the culture medium and angiogenic cocktail composition.
3. Materials
• OrganoPlate® Graft (MIMETAS, 6401-400-B)
• Collagen-I 5 mg/mL (AMSbio Cultrex® 3D collagen I rat tail, 5 mg/mL, #3447-020-01)
• 1 M HEPES (ThermoFisher 15630-122, pH 7.2-7.5)
• 37 g/L NaHCO3 (Sigma S5761-500G, dissolve in sterile MilliQ water, adjust pH to 9.5 using NaOH)
• Human umbilical vein endothelial cells (HUVECs, Lonza, C2519AS)
• EGM-2 medium (Lonza, CC-3162)
• Components for angiogenic cocktail (see table 1)
• Repeating pipette for gel loading and cell seeding. We recommend the eLINE® electronic pipette (Sartorius, #735021 (previously #730021)) and corresponding pipette tips or the Eppendorf® ep Dualfilter tips (Eppendorf, 022491211 / 0030077512)
• Multichannel pipette (1200 μL and 300 μl)
• Multichannel tips
• Crushed ice
4. Procedure
An ECM gel is loaded in the gel inlet of the OrganoPlate® Graft and fills the graft chamber. After gel polymerization, endothelial cells are seeded in the perfusion channels and perfusion is started to aid the formation of endothelial vessels. A gradient of pro-angiogenic factors is then added to the graft chamber to induce formation of a vascular network (see figure 3).
Load ECM gel in the OrganoPlate® Graft
Note: avoid touching the bottom glass plate of the OrganoPlate® Graft
1. Take the OrganoPlate® Graft from the packaging
2. Prepare the required amount of ECM gel (e.g. 2.5 μL gel per chip + 40% extra)
a. Collagen-I 4 mg/mL preparation
i. Place an Eppendorf tube on ice
ii. The collagen-I 4 mg/mL gel is prepared by mixing 1 M HEPES, 37 g/L NaHCO3, and 5 mg/mL collagen-I in a 1:1:8 ratio. For example, to prepare 100 μL of gel:
▪ Place an Eppendorf tube on ice
▪ Mix 10 μL of 1 M HEPES with 10 μL of 37 g/L NaHCO3
▪ Add 80 μL of collagen-I 5 mg/mL to the HEPES/NaHCO3 mixture
iii. Prepare at least 100 μL of total gel volume to ensure proper mixing of all components
iv. Mix well by pipetting the mixture up and down >20 times, while keeping it on ice
v. If bubbles are formed, briefly spin the tube down (~5 seconds)
vi. Use gel immediately after preparation (within 10 minutes)
3. Dispense the gel into the gel inlet (columns 2, 5, 8, 11, 14, 17, 20, 23; rows A, C, E, G, I, K, M, O) using the Sartorius eLINE® electronic pipette
a. Gently place your pipette tip on top of the hole in the bottom of the well and dispense the gel. Contact between the pipette tip and the hole is essential for gel loading. Correct positioning of the gel on top of hole allows capillary forces to pull the gel into the microfluidic gel channel (see figure 4)
b. The optimal loading volume depends on several factors, such as the viscosity of the gel and the temperature in the lab
c. Start by loading 2.5 μL gel per gel inlet for the OrganoPlate® Graft (6401-400-B)
d. In case of incomplete gel filling, increase the loading volume (i.e. to 3 μL)
e. In case the gel overflows from the gel chamber into the adjacent perfusion channels, reduce the loading volume (i.e. to 2 μL)
f. For examples of correct gel filling in the OrganoPlate® Graft, see figure 5
4. Place the OrganoPlate® in a humidified incubator (i.e. 37°C, 5% CO2) for 15 minutes to allow polymerization of the collagen-I gel
5. Add 50 μL of HBSS to the gel inlet (columns 2, 5, 8, 11, 14, 17, 20, 23; rows A, C, E, G, I, K, M, O) to prevent the gel from drying out
a. For examples of dried out gel, see section Trouble Shooting, figure 8.
6. Place the OrganoPlate® Graft back in the incubator and proceed to cell seeding
a. You can choose to proceed to cell seeding immediately or to wait until the next day. While cells generally form tubules with either option, some cells show optimal results when seeded one day after gel loading
Endothelial cell seeding
1. Harvest cells according to their dissociation protocol
2. Count the number of live cells in the cell suspension
3. Calculate the required number of cells for seeding in the OrganoPlate® Graft and pellet them
a. The optimal cell density for seeding against ECM in the OrganoPlate® is cell type dependent (10,000 cells/μL for Lonza HUVECs)
b. For example:
i. Number of chips to seed: 64
ii. Volume of cell solution to seed per perfusion channel: 2 μL → per chip: 4 μL
iii. Seeding density: 10,000 cells/μL
iv. You need: 64 x 4 x 10,000 = 2.56*106 cells
v. Prepare up to 25% extra: pellet 3.2*106 cells
4. Resuspend pellet in [3.2*106 / 10,000 =] 320 μL medium to obtain a 10,000 cells/μL cell suspension
5. Seed 2 μL of cell suspension in the perfusion medium inlet (columns 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24; rows A, C, E, G, I, K, M, O, see plate layout on pg.8) using the same pipetting procedure as previously used for gel loading (see figure 4)
a. Regularly resuspend the cell suspension during seeding to ensure homogenous cell density
b. In case you want to include cell-free controls, introduce 2 μL of medium without cells in the top medium inlet of these chips (instead of the cell suspension)
6. Add 50 μL of medium to the perfusion medium inlet (columns 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24; rows A, C, E, G, I, K, M, O)
7. Place the OrganoPlate® Graft static in the incubator to allow cells to attach
a. The time cells need to attach is cell type dependent and generally varies between 0.5-3 hours
8. After cells have attached, add 50 μL of medium to the perfusion medium outlet (columns 1, 3, 4, 6, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24; rows B, D, F, H, J, K, L, N, P).
9. Add 50 μl of media to the Graft chamber (columns 2, 5, 8, 11, 14, 17, 20, 23; rows B, D, F, H, J, K, L, N, P)
10. Remove HBSS from the gel inlet (columns 2, 5, 8, 11, 14, 17, 20, 23; rows A, C, E, G, I, K, M, O)
11. Place the plate on the MIMETAS Rocker in a humidified incubator to start cell culture (see figure 6)
a. An inclination of 14° and an interval of 8 minutes is optimal for most vessels
12. Refresh medium every 2-3 days by aspirating and replacing the medium from medium inlets and outlets (50 μL in each) using a repeating multichannel pipette
a. Note: to avoid disruption of the culture, insert the aspiration tip in the corner or side of each well. Avoid placing the aspiration tip directly on the holes connected to the channels/chambers.
13. An example of a tubule culture against ECM gel in the OrganoPlate® Graft is shown in figure 7.
Induction of angiogenic sprouts
Angiogenic sprouting can be induced after endothelial vessels have formed in the perfusion channels. The time it takes for vessels to form is dependent on the cell source. For Lonza HUVEC, this takes approximately 3 days
1. Prepare the stock solutions of the six angiogenic compounds as described in column 4 of table 1.
2. Prepare the final angiogenic cocktail (you need 50 μL per chip) by diluting the six angiogenic factors in EGM-2 medium according to the final concentration listed in the last column of table 1.
a. Prepare 50 μL per chip plus 20% extra volume (for complete OrganoPlate® Graft use ~4 mL)
b. If stored at 4°C, the cocktail can be used for up to a week after preparation
c. When using other cell types than Lonza HUVECs, optimization of medium and angiogenic cocktail composition may be required
3. Aspirate media from all wells of the OrganoPlate® Graft
4. Add 50 μL of angiogenic cocktail in all Graft chambers (columns 2, 5, 8, 11, 14, 17, 20, 23; rows B, D, F, H, J, K, L, N, P)
5. Add 50 μL of medium without angiogenic factors in all perfusion inlets and outlets
6. Place the plate on the MIMETAS Rocker in a humidified incubator to continue cell culture (see figure 6)
a. An inclination of 14° and an interval of 8 minutes works well for vascular network formation
The angiogenic potential of endothelial vessels grown in OrganoPlate® Graft can vary between endothelial cell sources and media compositions. Usually angiogenic sprouts can reach the glass Graft chamber opening within 3-7 days after sprouting induction. Observe cultures daily to monitor sprouting. An example of a vascular network in the OrganoPlate® Graft is shown in figure 8.
5. Troubleshooting
ECM drying
In some cases, the ECM gel can dry out during the gel loading and polymerization process (see figure 9). This generally happens when the loading process takes longer than expected and the gel in the chips that were loaded first has been incubated much longer than the gel in the chips that were loaded last. When gel loading goes smoothly, this problem doesn’t occur. However, if loading takes longer than expected (> 10 min), check regularly under the microscope to see if the gel starts to dry out and if you observe that it is, quickly add HBSS to the gel inlet of those chips to prevent further drying.
Sprout regression
In some cases, the sprouts regress or do not develop further after tissue has been added to the vascular network. Possible causes may include:
1. the media used for culturing the tissue has a composition that does not support the endothelial cells and/or tissue survival.
2. the tissue does not release the angiogenic growth factors necessary to support vascular network development and/or stabilization.
It is important to understand if the regression is due to the media composition or the tissue itself, therefore we recommend inclusion of a media only control (chips cultured with tissue specific media but without tissue) to the experiments.
1. If the media causes sprout regression, additional media optimization is required to find the best media configuration for the co-culture.
2. If the tissue causes sprout regression:
a. First, evaluate if the tissue requires presence of extra ECM gel to support the functionality. If so, perform additional ECM embedding of the tissue after placement in the graft chamber.
b. If no additional ECM embedding is needed, or regression of sprouts is still observed, supplement the media (for the graft chamber) with relevant pro-angiogenic growth factors. Additional growth factors can be added for the entire duration of the co-culture. The growth factors used for the angiogenic cocktail can be used as a starting point.