Protocol 1 : Differentiation of human iPSC-derived VF mucosa .
Human iPS-GFP IMR-90-4 reporter cells were maintained in an undifferentiated state in mTesr1 media on plates coated with Matrigel and they were routinely passaged with Versene in a ratio of 1:6.
Day 1 - 3 : Definitive Endoderm Differentiation
Day 1
When human iPS cell colonies reach 80% confluence, DE induction is performed.
1) Mix freshly RPMI medium/Glutamax with Activin A (100ng/ml) and Wnt 3a (25ng/ml).
2) Aspirate to remove mTesr1 medium, wash cells twice with RPMI medium/Glutamax to remove the rest of mTesr1 medium and add freshly prepared medium (step 1) to the plate. Return the plates to the 370C 5% CO2 tissue culture incubator overnight.
Day 2
3) Mix freshly RPMI medium/Glutamax with Activin A (100ng/ml) and 0.2% FBS.
4) Aspirate to remove the old medium and add the freshly prepared medium (step 3) to the plate. Return the plates to the 370C 5% CO2 tissue culture incubator overnight.
Day 3
5) Mix freshly RPMI medium/Glutamax with Activin A (100ng/ml) and 0.2% FBS.
6) Aspirate to remove the old medium and add the freshly prepared medium (step 5) to the plate. Return the plates to the 370C 5% CO2 tissue culture incubator overnight.
Note: For better cell survival, we recommend adding rock inhibitor Y-27632 (10uM) to the day 1 medium (step 1). We used a modified protocol for the DE derivation as published elsewhere (1, 2)
Days 4 – 8: Anterior Foregut Endoderm differentiation
Day 4
7) Mix DMEM/F12/Glutamax medium 500ml with 2% B12 (10ml), 1% N2 (5ml) and 1% P/S (1ml) to form DMEM/F12 basal medium. Keep it in the fridge for 8 weeks.
8) Make ascorbic acid 1000x stock solution (50 mg/ml). Prepare a 50 mg/ml ascorbic acid solution by dissolving 500 mg of l-ascorbic acid powder in 10 ml of water, and then filter it with a 0.22-µM filter. Make 100-µl aliquots and store them at- 20 °C for up to 6 months. Store thawed aliquots at 4 °C and use them within 24 h.
9) Mix freshly DMEM/F12 basal medium with ascorbic acid (0.05mg/ml), 1-Thioglycerol (0.4mM), Noggin (200ng/ml) and SB-431542 (10uM).
10) Aspirate to remove the old medium and add the freshly prepared medium (step 9) to the plate. Return the plates to the 370C 5% CO2 tissue culture incubator overnight.
Days 5 - 8
11) Continue culturing AFE cells for an additional 3 days, by replacing the old medium with freshly prepared medium (step 9) every day.
Note: We used a modified protocol for AFE derivation as previously published (1, 3).
Day 8 - VBP differentiation
12) Mix freshly DMEM/F12 basal medium with ascorbic acid (0.05mg/ml), 1-Thioglycerol (0.4mM), FGF2 (250ng/ml), FGF7 (100ng/ml) and FGF10 (100ng/ml)
13) Aspirate the old medium and replace it with freshly prepared medium (step 12). Return the plates to the 370C 5% CO2 tissue culture incubator for 48hours.
14) Meanwhile prepare collagen-fibroblast constructs and conditional FAD medium (See Sections 2 and 3).
Day 10 – Re-plating VBP on collagen-fibroblast constructs
15) Mix freshly DMEM basal medium with ascorbic acid (0.05mg/ml), 1-Thioglycerol (0.4mM), FGF2 (250ng/ml), FGF7 (100ng/ml) and FGF10 (100ng/ml)
16) Collect and transfer the old medium from the culture plate into a 15ml conical tube. Filter sterile the medium. (Do a few wells at a time to avoid drying of the wells). Briefly digest cells with warm (0.025%, wt/vol) trypsin for 3 - 5 min.
17) Aspirate the trypsin and add the collected medium (step 16) back to the wells. Mechanically detach the cells with 1,000-µl barrier tips or a glass pipette and transfer cells to a new 15 ml conical tube (usually 2 wells at a time). Wash the wells to collect remaining cells with freshly prepared medium (step 15). Optional : we recommend adding 0.2% FBS to the cell mixture to inhibit the trypsin activity.
18) Pipette the medium up and down and allow cells to settle for a few minutes or centrifuge cells briefly. Aspirate gently to remove the supernatant. Resuspend the cells in 100ul freshly prepared medium (step 15). Re-plate the cells in 2x 50ul volume on the top of collagen-fibroblast constructs. Return the plates to the 370C 5% CO2 tissue culture incubator for 2 hours.
19) After 2 hours, cells should be attached to the collagen matrix. Flood the cells with new freshly prepared medium (step 15). For one insert/well add 1ml of media into the upper chamber (insert) and 2 ml into the lower chamber (well). Return the plates to the 370C 5% CO2 tissue culture incubator for 48h.
Note: If the colonies of VBP in a plate at day 10 grow slowly and do not cover the whole plate and/or pile up, remove the old medium and add the freshly prepared medium to the cells (step 15). Re-plate cells on the top of collagen-fibroblast constructs on day 11.
Day 12 (or day 13, when the cells are re-seeded on day 11)
20) Mix conditional FAD medium with FGF2 (250ng/ml), FGF7 (100ng/ml) and FGF10 (100ng/ml). Instructions how to prepare FAD and conditional FAD media are listed in the Section 3.
21) Aspire old medium from the upper and lower chambers and add freshly prepared conditional FAD medium (step 20) to the plate. Return the plates to the 370C 5% CO2 tissue culture incubator for 48h.
Days 14 – 18: Air/Liquid interface with conditional FAD and FGFs
Day 14
22) Mix conditional FAD medium with FGF2 (250ng/ml), FGF7 (100ng/ml) and FGF10 (100ng/ml)
23) Aspire old medium from the upper and lower chambers and add freshly prepared medium (step 22) to the lower chamber only (1.5ml per insert/well). Return the plates to the 370C 5% CO2 tissue culture incubator for 48h.
Day 16
24) Mix conditional FAD medium with FGF2 (250ng/ml), FGF7 (100ng/ml) and FGF10 (100ng/ml)
25) Aspire old medium and add freshly prepared medium (step 24) to the lower chamber (1.5ml per insert/well). Return the plates to the 370C 5% CO2 tissue culture incubator for 48h.
Day 18 - 32 (or Day 19, if the cells are re-seeded on day 11): Air/Liquid interface in regular FAD medium.
Day 18
26) Aspire old medium and add FAD medium to the lower chamber only (1.5ml per an insert/well). Return the plates to the 370C 5% CO 2 tissue culture incubator.
27) Culture the plates for additional 14 days, changing the FAD medium three times a week in the basolateral chamber only. The cultivation at the A/Li interface can be extended up to three weeks.
Day 32 (or Day 33)
Set the wells aside for RNA extraction and IF staining or flow cytometry cell sorting.
Protocol 2: Preparation of collagen – fibroblast constructs
Prepare collagen-fibroblast constructs 1 or 2 days before VBP are re-plated on day 10). VF fibroblasts are routinely cultivated in culture medium composed of DMEM high glucose supplemented with 10%FBS, 1% MEM NEAA 100x and 1% P/S. The medium is changed every other day and cells are passaged with trypsin.
Day 8 / 9 (If the cells are re-plated on day 10)
1) On ice, combine high-concentration rat tail collagen (4mg/ml; 80% final volume) and 10xDMEM (10% final volume) and adjust pH with 1N NaOH to 7.2 - 7.4.
2) Resupend VF primary fibroblasts 2T1 cells, passage P5 - P6, in ice-cold FBS (10% final volume; 500 000 cells/ml final volume) and add to a collagen mixture.
3) Plate a mixture of collagen gel and VF fibroblasts on a cell culture insert, 2ml per a 6-well culture insert
4) Let it solidified for one hour in a tissue incubator at 5% CO2, 370C degrees.
5) After one hour, gently detach collagen with a pauster pipette and flood constructs with DMEM basal medium (Section 1, step 7). Return constructs into an incubator and left them at least 24 hours to allow for gel contraction.
Day 10 - Re-plating VBP on collagen-fibroblast constructs on day 10
6) In 1 or 2 days, mildly trypsinize VBP and plate them on collagen constructs at high density in 100ul DMEM basal medium supplemented with high concentration of FGF2 (250ng/ml), FGF10 (100ng/ml) and FGF7 (100ng/ml) as described in Section 1.
Note: To make 4mg/ml rat tail collagen follow the manufacture's instructions and calculations. Briefly, to make 10ml of gel, mix 1.25ml 10xPBS, 4.17ml of rat tail collagen and 6.12ml of sterile HyPure water. Keep cold and adjust pH with 1N NaOH to pH=7.2 - 7.4. The calculations vary according to the Lot# of the rat tail collagen. The protocol for the gel preparation has been previously described by others (4, 5, and 6).
Protocol 3: FAD medium preparation.
FAD medium is freshly prepared every week, as previously described (4, 5, and 6)
1) Mix DMEM medium high glucose and Ham's F12 in ratio 1:3, with 2.5 ml FBS, 0.4 mg/ml hydrocortisone, 8.4ng /ml cholera toxin, 5 mg/ml insuline, 24mg/ml adenine, 10ng/ml epidermal growth factor, 1% penicillin-streptomycin.
2) In submerged cultures, apply 1ml of FAD on the transwells with collagen constructs and 2 ml of FAD into the basolateral chamber. FAD is changed every other day.
3) To create the A/Li, FAD medium was placed in the basolateral chamber only and changed three times a week.
4) Conditional FAD medium was formed by cultivation of FAD with human primary VFF 21T cells for 24 hours in 370C in 5% CO2-humidified atmosphere. After 24 hours the medium was collected, sterile-filtered and stored at -200 C. The ratio of 30:70 (30% for conditional and 70% for fresh FAD medium) was used in the experiment.