1. Objective
Seeding of cells against extracellular matrix (ECM) gel in an OrganoPlate® 2-lane for tubule formation.
2. Background
Tubular structures, such as endothelial or epithelial barrier tissues, are established in the OrganoPlate® by growing cells against an ECM gel. Morphology and function of the tubule can be assessed by microscopy, a barrier integrity assay, or other functional assays.
This protocol describes the culture of a tubule against ECM in the top lane of an OrganoPlate® 2-lane (see figure 1 and 2). In case both apical and basal access to the tubule are required for your experiment, the OrganoPlate® 3-lane should be used (see protocol OrganoPlate® 3-lane tubule seeding).
3. Materials
• OrganoPlate® 2-lane (MIMETAS, 9605-400-B)
• Collagen-I 5 mg/mL (AMSbio Cultrex® 3D collagen I rat tail, 5 mg/mL, #3447-020-01)
• 1 M HEPES (Life Technologies 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)
• Medium (12 mL per OrganoPlate® plate)
• Cells: seeding density is dependent on the cell type
• Repeating pipette for gel loading and cell seeding, we recommend:
o The Eppendorf® Multipette® M4 with the Eppendorf® Biopur® 0.1 mL tip (VWR #613-2067) for dispensation of 2 μL, or
o The Sartorius eLINE® electronic pipette (Sartorius, #735021) for accurate dispensation of volumes ranging from 0.2 to 10 μL. Use with corresponding Sartorius tips or with Eppendorf® ep Dualfilter tips (Eppendorf, 022491211 / 0030077512)
• Multichannel pipette (1200 μL and 300 μL) with tips
• Crushed ice
4. Tubule seeding in the OrganoPlate®
A collagen-I ECM gel is loaded in the gel inlet of the OrganoPlate® and fills the gel channel. After polymerization of the gel, a cell suspension is seeded in the medium inlet and fills the medium channel. After cell attachment, medium perfusion is started to aid the formation of a tubule (see figure 3).
Load ECM gel in the OrganoPlate®
Note: avoid touching the bottom glass plate of the OrganoPlate®
1. Take the OrganoPlate® from the packaging
2. Add 50 μL of HBSS to all observation windows (columns 3, 7, 11, 15, 19, 23) using a multichannel repeating pipette
3. Prepare the required amount of ECM gel (e.g. 2 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, quickly spin the tube down (~5 seconds)
vi. Use gel immediately after preparation (within 10 minutes)
4. Dispense the gel into the gel inlet (columns 1, 5, 9, 13, 17, 21) 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 μL of gel per gel inlet
d. In case of incomplete gel filling, increase the loading volume (i.e. to 2.3 μL)
e. In case the gel overflows from the gel channel into the adjacent medium channel, reduce the loading volume (i.e. to 1.7 μL)
f. For examples of correct gel filling in the OrganoPlate® 2-lane, see figure 5 below.
Both the Eppendorf® Multipette® M4 and the Sartorius eLINE electronic pipette can successfully be used to load gel in the OrganoPlate®. Table 1 shows an overview of each pipette’s advantages and disadvantages for gel loading.
5. Place the OrganoPlate® in a humidified incubator (i.e. 37°C, 5% CO2) for 15 minutes to allow polymerization of the collagen-I gel
6. Add 30 μL of HBSS to the gel inlet (columns 1, 5, 9, 13, 17, 21) to prevent the gel from drying out
7. Place the OrganoPlate® 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 cell types show optimal results when seeded one day after gel loading.
Seed cells against the ECM gel
Note: avoid touching the bottom glass plate of the OrganoPlate®
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® and pellet them
a. The optimal cell density for seeding against ECM in the OrganoPlate® is cell type dependent (generally between 5,000 and 20,000 cells/μL)
b. For example:
i. Number of chips to seed: 96
ii. Volume of cell solution to seed per chip: ~2 μL
iii. Seeding density: 10,000 cells/μL
iv. You need: 96 x 2 x 10,000 = 1.92*106 cells
v. Prepare 25% extra: pellet 2.4*106 cells
4. Resuspend pellet in [2.4*106 / 10,000 =] 240 μL medium to obtain a 10,000 cells/μL cell suspension
5. Remove HBSS from the gel inlets
6. Seed 2 μL of cell suspension in the medium inlet (columns 2, 6, 10, 14, 18, 22) 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, seed 2 μL of medium without cells in the medium inlet of these chips (instead of the cell suspension)
7. Add 50 μL of medium to the medium inlet (columns 2, 6, 10, 14, 18, 22)
8. Place the OrganoPlate® on its side in the MIMETAS plate stand (see figure 6) in the incubator to allow cells to attach
a. The time cells need to attach is cell type dependent and generally varies between 2-6 hours.
9. After cells have attached, add 50 μL of medium to the medium outlet (columns 4, 8, 12, 16, 20, 24)
a. Ensure that the medium has filled the channel completely
b. Ensure that no air bubbles are trapped on medium inlet and outlet. If bubbles are trapped, remove the bubbles gently with a pipette tip
10. Place the plate on the OrganoFlow® in a humidified incubator to start cell culture (see figure 7)
a. An inclination of 7° and an interval of 8 minutes is optimal for most cultures.
11. 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
12. An example of a tubule culture against ECM gel in the OrganoPlate® 2-lane is shown in figure 7.
5. Troubleshooting
Cell invasion
In case of undesired cell invasion into the gel, the use of MMP inhibitors is recommended (e.g. addition of 10 μM of MMP-I inhibitor GM6001 (Abcam, ab120845) to the culture medium).