Culture of ESCs
1. Coat 12-well plates with 1 ml of 0.2% gelatin solution per well and place at room temperature (RT) for over 30 minutes.
2. Remove 0.2% gelatin solution by aspirating.
3. Seed mitomycin treated mouse embryonic fibroblast (MEF) feeder cells (100,000 cells per well, 12-well plate) in 1 ml of MEF medium and incubate at 37℃, 5% CO2 incubator for 1 day.
4. Wash MEF plates with 1 ml of PBS (-) and remove PBS (-).
5. Seed murine ESCs (100,000 cells per well, 12-well plate) on MEF plates in 1 ml of KSR based ESC medium (ES medium) supplemented with CHIR99021 (final 3mM), PD0325901(final 1mM), and LIF (1000 Unit/ml) and incubate at 37℃, 5% CO2 incubator.
6. Change ES medium every day.
7. After 2 days, aspirate culture medium and wash the cells with 1 ml of PBS (-).
8. Add 0.4 ml of 0.25% Trypsin-EDTA solution and incubate at 37℃ for 3 minutes.
9. Add 1 ml of 10% FBS-DMEM medium and pipette cells with 1000 ml tip.
10. Count the number of cells by using hemacytometer.
11. Pool the cells into a 15 ml tube and centrifuge at 1,500 rpm for 5 minutes.
12. Aspirate medium and resuspend the cells with ES medium supplemented with CHIR99021 (final 3mM), PD0325901(final 1mM), and LIF (1000 Unit/ml).
13. Seed murine ESCs (100,000 cells per well, 12-well plate) on MEF plates and incubate at 37℃, 5% CO2 incubator.
1. Prepare 6-well culture plates coated with 0.2% gelatin solution, before trypsinization of ESCs.
2. Aspirate culture medium from 2 days cultured ESCs and wash the cells with 1 ml of PBS (-).
3. Add 0.2 ml of 0.25% Trypsin-EDTA solution and incubate at 37℃ for 2-3 minutes.
4. Add 1 ml of 10% FBS-DMEM medium and pipette cells with 1000 ml tip.
5. Pool the cells and transfer the cells into 6-well plates coated with 0.2% gelatin and incubated at 37°C for 45 minutes to remove feeder cells.
6. Collect floating ESCs gently without contamination of feeder cells and count the number of cells by using hemacytometer.
7. Centrifuge at 1,000 rpm for 5 minutes.
8. Aspirate medium and resuspend the cells with warm FBS-supplemented ES medium without LIF (FBS ES medium).
9. Seed 1,000-5,000 ESCs in 200 ml of FBS ES medium onto each well of a U-bottomed 96-well plate by using 8-channel pipettor.
10. Incubate at 37°C, 5% CO2 incubator for 4 days without changing medium. The cells form aggregate spheres on the next day of seeding.
Generation of heart organoids
Plate coating with LN/ET gel (85.7 μg/cm2)
1. Before coating plates, thaw 2 mg/ml of LN/ET complex on ice.
2. Add 30 ml of LN/ET complex into a well of 8-well chamber slides.
3. Incubate 8-well chamber slides at RT for 2 hours.
In vitro culture of EBs for the generation of heart organoids
6. Warm the heart organoid medium and FGF4.
7. Mix the heart organoid medium with FGF4 (30-60 ng/ml).
8. Add 200 ml of the medium in each well of LN/ET gel coated 8-well chamber slides.
9. Carefully transfer an EB (showing sphere morphology) cultured for 4 days by using 200 ml tips, into a well of LN/ET gel coated 8-well chamber slides with the medium.
10. Maintain cultures in 5% CO2 incubator at 37℃.
11. Replace medium at day3, 5, 7, 9, 11,13,14, and 15.
12. From day9, use FGF4-heart organoid medium additionally supplemented BMP4 (50 ng/ml), BIO (2.5 μM), and LIF (1,000 units/ml). In case of long term culture of heart organoids, such as over 15 days culture, carefully transfer heart organoids into iMatrix-411-coated (10.7 μg/cm2) chamber slides in 200 μl of heart organoid medium.
13. Observe the morphogenesis of heart organoids under microscope. These heart organoids usually show the initial beating movement by 3 days, and continue to grow with morphogenetic changes.
14. Usually collect heart organoids over 9 days of culture after chamber formation. When transfer heart organoids for collecting or performing further analysis, use wide bore tip and/or fine dissection forceps under a stereomicroscope.