1. Objective
Seeding of a co-culture of Caco-2/HT29-MTX-E12 cells against extracellular matrix (ECM) gel in an OrganoPlate® 3-lane.
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 culturing of a tubule of Caco-2 and HT29-MTX-E12 cells against ECM in the top lane of an OrganoPlate® 3-lane (see figure 1 and 2). The resulting tubular culture allows for access to apical and basal side of the barrier tissue.
3. Materials
• OrganoPlate® 3-lane (MIMETAS, 4004-400-B)
• OrganoFlow® S or L (MIMETAS, MI-OFPR-S or MI-OFPR-L)
• Human colon adenocarcinoma cell line Caco-2 (Sigma-Aldrich, 86010202)
• Human colon cell line HT29-MTX-E12 (Sigma-Aldrich, 12040401)
• Caco-2 medium components: EMEM (ATCC, 30-2003) + 10% FBS HI (ThermoFisher, 16140-071) + 1% Non-Essential Amino Acid (ThermoFisher, 11140050) + 1% Pen/Strep (Sigma, P4333)
• 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)
• Medium: 10 mL per OrganoPlate®
• 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)
• HBSS (Sigma H6648)
• Multichannel pipette (1200 μL and 300 μl) and multichannel tips
• Crushed ice
4. Tubule seeding in the OrganoPlate®
A collagen-I ECM gel is loading in the gel inlet of the OrganoPlate® and fills the gel channel. After polymerization of the gel, a cell suspension is seeded in the top medium inlet and fills the top medium channel. After cell attachment, medium perfusion is started to aid the formation of a tubule (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 wells in columns 2, 5, 8, 11, 14, 17, 20, and 23 using a multichannel repeating pipette. These columns contain the chips’ observation windows
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, 4, 7, 10, 13, 16, 19, 22; rows B, E, H, K, N) using the Eppendorf® Multipette® M4 or 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 allow 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 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)
5. For examples of correct gel filling in the OrganoPlate® 3-lane, see figure 5.
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.
6. Place the OrganoPlate® in a humidified incubator (i.e. 37°C, 5% CO2) for 15 minutes to allow polymerization of the collagen-I gel
7. Add 30 μL of HBSS to the gel inlet (columns 1, 4, 7, 10, 13, 16, 19, 22; rows B, E, H, K, N) to prevent the gel from drying out
a. For examples of dried out gel, see section 5. Trouble Shooting, figure 8.
8. 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, Caco-2 cells show optimal results when seeded one day after gel loading.
Seed Caco-2/HT29-MTX-E12 cells (ratio of 6:1) against the ECM gel
1. Harvest the Caco-2 and HT29-MTX-E12 cells according to their dissociation protocol
2. Before pelleting the cells by centrifugation, 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. For example:
a. Number of chips to seed: 40
b. Volume of cell solution to seed per chip: ~2 μL
c. Seeding density: 10,000 cells/μL
d. You need: 40 x 2 x 10,000 = 8.0*105 cells (80 μL)
e. Prepare 25% extra: 1.0*106 cells (100 μL)
f. Live cell count Caco-2 = 2.75*106 cells/mL
g. Live cell count HT-29-MTX = 7.05*106 cells/mL
h. Centrifuge them according to protocol
i. Prepare 1x concentration for both cell lines = 10.000 cells/μL
i. Caco-2 pellet from 1 mL solution (see f) + 275 μL media = 10.000 cells/μL
ii. HT-29-MTX pellet from 1 mL solution (see g) + 705 μL media = 10.000 cells/μL
j. Premix the Caco-2 and HT-29-MTX in 6:1 (v/v) (85.7 μL + 14.3 μL= 100 μL total) in a new Eppendorf tube
4. Remove HBSS from the gel inlets
5. Seed 2 μL of cell suspension in the top medium inlet (columns 1, 4, 7, 10, 13, 16, 19, 22; rows A, D, G, J, M) using the same pipetting procedure as previously used for gel loading (see figure 3)
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 top medium inlet of these chips (instead of the cell suspension)
6. Add 50 μL of medium to the top medium inlet (columns 1, 4, 7, 10, 13, 16, 19, 22; rows A, D, G, J, M)
7. Place the OrganoPlate® on its side in the MIMETAS plate stand for 3-4 hours in the incubator to allow the cells in the top channel to settle onto the ECM gel and attach (see figure 6).
8. After cells have attached, add 50 μL of medium to the top medium outlet (columns 3, 6, 9, 12, 15, 18, 21, 24; rows A, D, G, J, M).
9. Add 50 μL of medium to the bottom medium inlet (columns 1, 4, 7, 10, 13, 16, 19, 22; rows C, F, I, L, O)
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. Add 50 μL of medium to the bottom medium outlet (columns 3, 6, 9, 12, 15, 18, 21, 24; rows C, F, I, L, O)
11. 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
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
13. An example of a tubule culture against ECM in the OrganoPlate® 3-lane 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 (figure 9). This generally happens when the gel 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 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.
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).