DMEM (ThermoFisher), 10% Fetal Bovine Serum (FBS, ThermoFisher), 1% Nonessential amino acids (ThermoFisher), 1mM GlutaMAX (ThermoFisher), 1% Penicillin-streptomycin (ThermoFisher), 55μM 2-mercaptoethanol (ThermoFisher) and 1mM sodium pyruvate (ThermoFisher).
Add 10ml of LSGS (ThermoFisher) to 500ml medium 106 basal (ThermoFisher); supplement with 1% Pen-strep (ThermoFisher).
In vitro culture 1 (IVC1) medium7,8
Advanced DMEM/F-12 (ThermoFisher), 1% ITS-X supplement (ThermoFisher), 2mM L-Glutamine (ThermoFisher), 0.5% Penicillin-streptomycin (ThermoFisher), 20% Fetal Bovine Serum (FBS, ThermoFisher), 25 μM N-acetyl-L-cysteine (Sigma), 8 nM β-estradiol (Sigma) and 200 ng/ml progesterone (Sigma).
Human iBlastoid medium
2:1:1 mixture of IVC1 medium, iBlastoid basal medium 1 [50:50 mixture of DMEM/F-12 (ThermoFisher) and Neurobasal medium (ThermoFisher), supplemented with 2mM L-Glutamine (ThermoFisher), 0.1mM 2-mercaptoethanol (ThermoFisher), 0.5% N2 supplement (ThermoFisher), 1% B27 supplement (ThermoFisher), 1% Penicillin-streptomycin (ThermoFisher)] and iBlastoid basal medium 2 [DMEM/F-12, GlutaMAX (ThermoFisher) supplemented with 0.3% BSA (Sigma), 0.2% FBS (ThermoFisher), 1% ITS-X supplement (ThermoFisher), 0.1mM 2-mercaptoethanol (ThermoFisher), 0.5% Penicillin-streptomycin (ThermoFisher), 1.5 μg/ml L-ascorbic acid (Sigma)] supplemented with 2 μM CHIR99021 (Miltenyi Biotec), 0.5 μM A83-01 (Sigma), 1 μM SB431542 (Selleckchem), 0.8 mM Valproic acid (VPA, Sigma), 50 ng/ml EGF (Peprotech) and 10ng/ml BMP4 (Miltenyi Biotec).
All culture media were filtered with a 0.22μm filter (Corning) before use.
Preparing human dermal fibroblasts for reprogramming
1. Primary human adult dermal fibroblasts (HDFa) from different donors can be obtained from Gibco (Catalogue number, C-013-5C) (lot#1029000 for 38F, lot#1528526 for 55F and lot#1569390 for 32F were used in this study).
2. For recovery of the commercially purchased cryopreserved stock vials, cells are to be thawed quickly at 37ºC and then pelleted at 200xg for 5 minutes.
3. Resuspend the cell pellet in 10 ml of 106 medium (Gibco) supplemented with LSGS (Gibco) and plated into a T75 flask (one cryovial to one T75).
4. Culture the cells in a 37ºC, 5% O2 and 5% CO2 incubator for expansion. Media are changed every other day and cultures split at a 1:3-1:5 ratio using Trypsin/EDTA Solution (TE) (ThermoFisher) upon confluency.
5. For cryo-preservation, freeze cells in 90% FBS (Gibco) with 10% DMSO (Santa cruz); we suggest generating a large number of early passage stock for future experiments.
Human iBlastoids generation
1. For somatic cell reprogramming with the Cytotune 2.0 kit, experiments were performed according to the manufacturer’s instructions (Invitrogen).
2. To prepare the primary HDFa for reprogramming, seed the cells at a density of ~5-10x104 cells per well of a 12-well plate in fibroblast medium.
3. After 36 hours, transduce the attached primary HDFa with Sendai viruses in fibroblast medium at the multiplicity of infection (MOI) as follows, KOS MOI=5, c-MYC MOI=5, KLF4 MOI=6 in 500 μl of fibroblast medium per well. The cells are cultured in a 37ºC, 5% O2 and 5% CO2 incubator. The day of Sendai virus transduction is counted as day 0.
4. After 24 hours of transduction (day 1), perform a media replacement with 1 ml/well of fresh and warmed fibroblast medium to remove the viruses.
5. Perform a media replacement every other day from day 1 of transduction. Starting from day 8 onwards when the reprogramming cells are becoming highly proliferative, daily media replacement with 2 ml/well of fibroblast medium is required.
6. On day 21 of reprogramming, the cells would be ready for the generation of iBlastoids.
7. Before dissociating the reprogrammed cells, prepare the AggreWell 400 24-well plate (Stem cell technologies) according to the manufacturer’s instructions. Briefly, add 500 μL of Anti-Adherence Rinsing Solution (Stem cell technologies) into each well of the AggreWell 400 24-well plate to be used and centrifuge the plate at 1300xg for 5 minutes. Ensure that no air bubbles are present after centrifugation and subsequently aspirate the rinsing solution from the wells. Perform an additional rinsing step with warmed basal medium, then add 1mL of Human iBlastoid medium supplemented with Y-27632 into each well. Place the plate in the incubator to be used later.
8. For the dissociation of day 21 reprogrammed cells,
a. First aspirate the spent medium and perform a washing step with DPBS.
b. Aspirate the DPBS and add 500 μl of TrypLE Express Enzyme into each well.
c. Place the plate back into the incubator for 5 minutes.
d. After 5 minutes, gently pipette the contents in each well to ensure that they have been well dissociated.
e. Add 500 μL of fibroblast medium to stop the enzymatic reaction.
f. Collect the cellular contents into a conical tube and centrifuge at 500xg for 3 minutes. If undissociated cellular patches are visible, we recommend filtering the cell suspension using a cell strainer size 40 μm (Corning) before centrifugation.
g. Aspirate the supernatant and resuspend the cell pellet in appropriate volume of Human iBlastoid medium supplemented with Y-27632 for cell counting.
9. Perform a cell count on the collected cells and dilute accordingly to a cell concentration of 1.2x105 cells/ml with Human iBlastoid medium supplemented with Y-27632.
10. Take the prepared AggreWell plate out from the incubator and transfer 1 mL of well mixed cell suspension into each well of the plate. Ensure that the cells seeded into the Aggrewell are evenly distributed by pipetting the cell suspension in the well for several times carefully as per manufacturer’s instructions.
11. Centrifuge the plate at 100xg for 3 minutes to capture the cells at the bottom of microwells.
12. Observe the plate under the microscope to confirm that cells have been evenly distributed among the microwells.
13. Carefully transfer the plate back to the 37ºC, 5% O2 and 5% CO2 incubator.
14. 24 hours later, carefully perform a media replacement with warmed Human iBlastoid medium without Y-27632.
15. On day 6 of iBlastoid formation in the AggreWell, the iBlastoids would be clearly visible and could be collected into a 15 mL conical tube. We recommend using wide bore p1000 pipette tips for the collection of iBlastoids to prevent any distortion. Alternatively, cut the end of a standard p1000 pipette tips for a wider bore to collect the iBlastoids.
16. Let the iBlastoids in the conical tube to sediment for 5-10 minutes and carefully aspirate the majority of the supernatant to remove most of the cell debris.
17. Transfer the sedimented iBlastoids into a 60 mm TC-treated Cell Culture Dish (Corning) filled with 3 mL of Human iBlastoid medium.
18. Using a dissection microscope, carefully pick the cavitated iBlastoids for subsequent analysis.