Human iPS cell culture
1. Add 1ml ice cold Matrigel solution (diluted with DMEM/F12; dilution fold as suggested by the manufacturer) into one well of a 6-well cell culture plate and incubate at 37℃ for 20 min.
2. Remove cell culture medium, wash twice with 1ml PBS, add 1ml Accutase, and incubate at 37℃ for 5min.
3. Gently pipette mix and transfer into a conical tube with 4 ml DMEM/F12. Centrifuge at 300rcf for 4 min.
4. Remove supernatant and gently resuspend cell pellet using 2ml mTeSR1 + 10μM Y-27362. Load 20 μl cell mix into the cell counting chamber, determine cell density using the automatic cell counter, and calculate the volume needed for 500,000 cells.
5. Remove all medium in the Matrigel-coated plate and add 1.5ml mTeSR1 + 10μM Y-27362. Add 500,000 cells into the well, shake the plate well to mix evenly, and put it into the 37℃ incubator with 5.5% CO2 and humidity.
6. Next day, change medium with 2 ml mTeSR1.
7. Next day, change medium with 2.5 ml mTeSR1.
8. Next day, change medium with 3.5 ml mTeSR1.
9. Next day, start again from Step 1.
Somitoid protocol
(Day -1)
1. Remove the medium of iPS cell culture (~80% confluency), wash twice with PBS, add 1ml Accutase, and incubate at 37℃ for 5min. More confluent culture tends to result in increased organoid death in this protocol.
2. Gently pipette mix and transfer into a conical tube with 4 ml DMEM/F12. Centrifuge at 300rcf for 4 min.
3. Remove supernatant and gently resuspend cell pellet using 2ml mTeSR1 + 10μM Y-27362. Load 20 μl cell mix into the cell counting chamber, determine cell density using the automatic cell counter.
4. Calculate and dilute cell mix using mTeSR1 medium + 10 μM Y-27362, so that 100 μl diluted cell mix contain 3,000 cells. Dispense 100 μl into each well of the 96-well U-bottom non-treated plates (Greiner bio-one 650185 or Falcon 351177). Do not use the cell-repellent plate as used in the segmentoid protocol.
5. Centrifuge the plate at 300 rcf for 2 min and put it into the incubator without disturbing the pellet.
(Day 0)
1. Prepare DICL medium: DMEM/F12 supplemented with 1% insulin-transferrin-selenium (ITS; Gibco 41400045), 3 μM CHIR99021 (Tocris 4423) and 0.5 μM LDN193189 (Stemgent 04-0074).
2. Remove all medium from each well under a stereo dissection microscope in the cell culture hood. The aggregate often appears as a flat disk attached at the bottom, which makes it easy to remove as much medium as possible. Sometimes several small aggregates are formed but they will fuse in subsequent culture.
3. Add 150 μl DICL medium. Gently pipette the aggregate under a stereo dissection microscope to make sure it is unattached from the well. Put the plate back into the incubator.
(Day 1)
1. The aggregate should appear as a sphere resting in suspension on the bottom of the well. Remove 130 μl medium from each well and add in 150 μl fresh DICL medium. This step is optional as we did not observe any difference when no medium change is performed on Day 1.
(Day 2)
1. Add 1ml 0.1% gelatin solution (Stemcell Technologies 07903) or other coating solution to a well of 6-well TC plate. Incubate at 37℃ for 1h.
2. Prepare somite-inducing medium (SIM): DMEM/F12 supplemented with 1% ITS, 1.5 μM CHIR99021, 0.5 μM LDN193189, and 5% Fetal Bovine Serum (FBS; Sigma-Aldrich EmbryoMax ES-009-B). Note that the concentration of CHIR99021 is lowered by half from what is used in DICL medium.
3. Remove all gelatin solution from the well and add in 2 ml SIM medium.
4. Transfer the organoids one by one from the U-bottom wells into the flat TC plate well, using the 200 μl pipette. Under the stereo microscope, position the organoid at the very tip so very small release with little carry-over medium is sufficient for the transfer. Up to 20 organoids can be put into one well.
5. Shake the plate to mix well the organoids and incubate without further medium change.
Segmentoid protocol
(Day -1)
1. Add 1ml ice cold Matrigel solution (diluted with DMEM/F12; dilution fold as suggested by the manufacturer) into one well of a 6-well cell culture plate and incubate at 37℃ for 20 min.
2. Remove the medium of iPS cell culture (~100% confluency), wash twice with PBS, add 1ml Accutase, and incubate at 37℃ for 5min.
3. Gently pipette mix and transfer into a conical tube with 4 ml DMEM/F12. Centrifuge at 300rcf for 4 min.
4. Remove supernatant and gently resuspend cell pellet using 2ml mTeSR1 + 10μM Y-27362. Load 20 μl cell mix into the cell counting chamber, determine cell density using the automatic cell counter, and calculate the volume needed for 200,000 cells.
5. Remove all medium in the Matrigel-coated plate and add 1.5ml mTeSR1 with 10μM Y-27362. Add 200,000 cells into the well, shake the plate well to mix evenly, and put it into the 37℃ incubator with 5.5% CO2 and humidity. The cell number per well is the most important parameter throughout this protocol.
(Day 0)
1. Prepare DICL medium: DMEM/F12 supplemented with 1% insulin-transferrin-selenium (ITS; Gibco 41400045), 3 μM CHIR99021 (Tocris 4423) and 0.5 μM LDN193189 (Stemgent 04-0074).
2. Remove all medium from each well and add 2 ml DICL medium.
(Day 1)
1. Prepare N2B27 medium: 1:1 mix of DMEM/F12 and Neurobasal medium (Gibco 21103049), supplemented with 1% N2 Supplement-B (Stemcell Technologies 07156), 2% B-27 Supplement (Gibco 17504044), and 0.1% 2-Mercaptoethanol (Gibco 21985023).
2. Remove all DICL medium and wash once with PBS. Add 1ml Accutase and incubate at 37℃ for 5min.
3. Gently pipette mix and transfer into a conical tube with 4 ml DMEM/F12. Centrifuge at 300rcf for 4 min.
4. Remove supernatant and gently resuspend cell pellet using 300 μl N2B27 + 10μM Y-27362. Load 20 μl cell mix into the cell counting chamber, determine cell density using the automatic cell counter.
5. Add 150 μl N2B27 + 10μM Y-27362 into each well of the 96-well cell-repellent U-bottom plate (Greiner bio-one, 650970). Note that the plate is different from what is used in the somitoid protocol.
6. Calculate volume and add 6,000-8,000 cells into each well.
7. Centrifuge the plate at 300 rcf for 2 min and put it into the incubator without disturbing the pellet.
(Day 2)
1. Thaw Matrigel (Corning CB-40234A) on ice and prepare ice-cold 10% Matrigel solution using N2B27 media.
2. A single spheroid should be formed in each well. Remove medium under a dissection microscope and add 30 μl ice-cold 10% Matrigel. Gently tap the plate to facilitate each organoid to sink to the bottom.
3. Incubate the plate at 37°C for one hour.
4. Carefully add 150 μl N2B27 medium to each well. Lean the tip against the inside wall to avoid breaking the solidified Matrigel. Incubation and no further medium change is needed.