Derivation of three dimensional human induced pluripotent stem cell derived vocal fold mucosa for clinical and pharmacological applications

Abstract

Healthy vocal fold mucosa is composed of two major cell types, non-keratinized stratified squamous epithelium and vocal fold fibroblasts. Although dysfunction of the epithelium may play a significant pathogenic role in vocal fold diseases, studies at the genetic and molecular level using primary epithelial cells or models of human vocal fold mucosa have been significantly limited by the availability of relevant tissue types, poor growth, and heterogeneity of primary vocal fold epithelial cells. Here, we describe in vitro developmental differentiation of human induced pluripotent stem cells into vocal fold basal epithelial progenitors that were reseeded on collagen-fibroblast constructs to induce stratification and generate a three-dimensional model of human VF mucosa. The engineered VF mucosa represents physiologically relevant and clinically useful model that can be used as a tool for disease modeling and testing of therapeutic approaches for the treatment of laryngeal and VF inflammation.

1. Introduction

The vocal folds (VF) are housed in the larynx, the crossroad between the digestive and respiratory tracts. They perform multiple functions related to breathing, protection and phonation. Due to their strategic position they guard the entrance into the trachea and lower airways. Edematous laryngeal structures caused by mucosal inflammation, injuries and/or pathological mucosal remodeling can result in acute life-threatening airway obstruction and respiratory distress. Acute or chronic VF diseases can lead to voice problems and loss of phonation (1,2).

Healthy VF mucosa is composed of two major cell types, non-keratinized stratified squamous epithelium and fibroblasts that reside in the underlying lamina propria. Although dysfunction of the epithelium may play a significant pathogenic role in VF diseases (3), studies at the genetic and molecular level using primary VF epithelial cells or models of human VF mucosa have been significantly limited by the availability of relevant tissue types derived from a biopsy or cadaveric sources, poor growth, and heterogeneity of primary VF epithelial cells. A recently developed three-dimensional (3D) models of human VF mucosa using human induced pluripotent stem cell-derived (hiPSC-derived) VF epithelium and primary VF fibroblasts embedded in collagen matrix provide a powerful platform for dissecting molecular and cellular mechanisms of VF diseases and pathologies (4).

Here we describe in vitro developmental differentiation of hiPS cells into VF basal epithelial progenitors that were reseeded on collagen-fibroblast constructs to induce stratification and to generate a 3D model of human VF mucosa. The novel 3D system of human VF mucosa was used to model keratotic changes in the VF epithelium in response to cigarette smoke extract and induce VF epithelial injury and inflammation in response to electronic cigarette vaping extracts.

The 3D in vitro system of VF mucosa represents physiologically relevant and clinically useful model that can be used as a tool for disease modeling and testing of therapeutic approaches for the treatment of laryngeal and VF inflammation.

2. Material

2.1. hiPS Cell Culture

1. IMR-90-4 cell line obtained from WiCell (Madison, Wi, USA). Research involving human induced pluripotent stem cells was conducted in accordance with relevant ethical guidelines and applicable institutional and funding agency regulations.

2. Culture medium for IMR-90-4 cell line expansion: mTeSR 1 (WiCell, Madison, WI).

3. Growth factor reduced Matrigel (Corning Discovery Labware, BD Biosciences,, San Jose, CA, cat #: 354230).

4. 6-well tissue culture plates (Corning BD Falcon, Tewksbury, MA; cat #: 353046).

5. Sterile glass disposable pipets (5ml and 10ml) (Fisher Scientific, Hampton, NH; cat #: 13-678-27E and 13-678-27F).

6. Disposable Pasteur pipets (Fisher Scientific, Hampton, NH; cat #: 13-678-20C) for medium aspiration.

7. Versene (1x) (Gibco, Fisher Scientific, Hampton, NH, cat #: 15040-066).

2.2. Differentiation of hiPS cells into Definitive endoderm (Days 1-3)

1. 6-well tissue culture plates with hiPS cell colonies.

2. Disposable Pasteur pipets (Fisher Scientific, Hampton, NH; cat #: 13-678-20C) for medium aspiration.

3. Sterile glass disposable pipets (5ml and 10ml) (Fisher Scientific, Hampton, NH; cat #: 13-678-27E and 13-678-27F).

4. Sterile DPBS without Ca ²⁺ and Mg ²⁺ (Lonza, Morristown, NJ; cat #: 17-512F).

5. Culture medium for Definitive Endoderm (DE) differentiation:

   • RPMI medium supplemented with Glutamax (Gibco, Life Technologies, cat #: 61870-036).
   • 100ng/ml Activin A (Peprotech, Rocky Hill, NJ, cat #: 120-14).
   • 25ng/ml Wnt3a (R&D Systems, Minneapolis, MN; cat #: 5036-WN).
   • 10 μM dihydrochloride (rho-associated kinase (ROCK) inhibitor Y-27632 (R&D Minneapolis, MN; cat #: 1254/10).
   • Fetal bovine serum (FBS) (Ppeak Serum, Wellington, CO; cat #: PSFB3).

2.3. Differentiation of Definitive Endoderm into Anterior Foregut Endoderm (Days 4-7)

1. 6-well tissue culture plates with DE cell cultures (Day 4).

2. Disposable Pasteur pipets (Fisher Scientific, Hampton, NH; cat #: 13-678-20C) for medium aspiration.

3. Sterile glass disposable pipets (5ml and 10ml) (Fisher Scientific, Hampton, NH; cat #: 13-678-27E and 13-678-27F).

4. 25mm GD/X sterile disposable filters; pore size 0.2 μm (GE Healthcare Life Sciences, Whatman, Chicago, IL; cat #: 6901-2502).

5. HyClone HyPure cell culture grade water (Hyclone, GE Healthcare Life Sciences, Chicago, IL: cat#: SH 3052903).

6. Culture medium for Anterior Foregut Endoderm (AFE) differentiation

   • DMEM/F-12 with Glutamax (Gibco, Life Technologies; cat #: 10565-018).
   • 2% B27 supplement (Gibco, Life Technologies; cat #: 17504044).
   • 1% N2 supplement (Gibco, Life Technologies; cat #: 17502048).
   • 1% Penicillin-streptomycin P/S (Gibco, Invitrogen, Carlsbad, CA; cat #: 15-140-122).

Supplements:

• 50μg/ml l-Ascorbic acid (Millipore Sigma, St. Louis, MO; cat #: A82902).

• 0.4mM 1-Thioglycerol (MTG; Millipore Sigma, St. Louis, MO; cat #: M6165).

• 200ng/ml NOGGIN (R&D Systems, Minneapolis, MN; cat #: 8057-NG).

• 10μM transforming growth factor beta pathway inhibitor SB-431542 (R&D Systems, Minneapolis, MN; cat #: 1614/10).

2.4. Differentiation of Anterior Foregut Endoderm into Vocal Fold Basal progenitors (Days 8-12)

1. 6-well culture tissue plates with AFE cell cultures (Day 8)

2. Disposable Pasteur pipets (Fisher Scientific, Hampton, NH; cat #: 13-678-20C) for medium aspiration.

3. Sterile glass disposable pipets (5ml and 10ml) (Fisher Scientific, Hampton, NH; cat #: 13-678-27E and 13-678-27F).

4. Culture medium for differentiation of Vocal Fold Basal Progenitors (VBP)- DMEM basal medium (Subheading 2.3) + supplements

Supplements:

• 50μg/ml l-Ascorbic acid (Millipore Sigma, St. Louis, MO; cat #: A82902).

• 0.4mM 1-Thioglycerol (MTG; Millipore Sigma, St. Louis, MO; cat #: M6165).

• 250ng/ml FGF2 (R&D Systems, Minneapolis, MN; cat #: 233-FB).

• 100ng/ml FGF10 (R&D Systems, Minneapolis, MN; cat #: 345-FG).

• 100ng/ml FGF7/ keratinocyte growth factor (KGF; R&D Systems, Minneapolis, MN; cat #: 251-KG).

2.5. Dissociation of Vocal Basal Progenitors (VBP) and reseeding of the cells on collagen-fibroblast constructs (Day 10)

1. 6-well tissue culture plates with VBP cell cultures (Day 10).

2. 6-well plate tissue culture inserts with collagen-fibroblast constructs prepared at Day 9.

3. 25mm GD/X sterile disposable filters; pore size 0.2μm (GE Healthcare Life Sciences, Whatman, Chicago, IL; cat #: 6901-2502).

4. BD 5 ml syringes (Becton Dickinson, East Rutherford, NJ: cat #: 309646).

5. Sterile glass disposable pipets (5ml and 10ml) (Fisher Scientific, Hampton, NH; cat #: 13-678-27E and 13-678-27F).

6. Disposable Pasteur pipets (Fisher Scientific, Hampton, NH; cat #: 13-678-20C) for medium aspiration.

7. 15ml conical tubes for the cell collection.

8. Hank’s Balanced Salt Solution 1x (HBSS) (Gibco, Fisher Scientific, Hampton, NH; cat #: 14175-079) for washing of cells and trypsin dilution.

9. Trypsin - TrypLE Express 1x (Gibco, Fisher Scientific, Hampton, NH cat #: 12604-013) for cell dissociation. Dilute TrypLE Express with HBSS in the ratio 1:5.

10. Culture medium for VBP reseeding - DMEM basal medium (Subheading 2.3) + supplements.

Supplements:

• 50μg/ml l-Ascorbic acid (Millipore Sigma, St. Louis, MO; cat #: A82902).

• 0.4mM 1-Thioglycerol (MTG; Millipore Sigma, St. Louis, MO; cat #: M6165).

• 250ng/ml FGF2 (R&D Systems, Minneapolis, MN; cat#: 233-FB).

• 100ng/ml FGF10 (R&D Systems, Minneapolis, MN; cat#: 345-FG).

• 100ng/ml FGF7/ keratinocyte growth factor (KGF; R&D Systems, Minneapolis, MN; cat#: 251-KG).

2.6. Differentiation of Vocal Fold Basal Progenitors into VF stratified epithelium (Days 12 – 32)

1. Collagen-fibroblast culture inserts seeded with VBP (Day 12)

2. Disposable Pasteur pipets (Fisher Scientific, Hampton, NH; cat #: 13-678-20C) for medium aspiration.

3. Sterile glass disposable pipets (5ml and 10ml) (Fisher Scientific, Hampton, NH; cat #: 13-678-27E and 13-678-27F).

4. Culture medium for VBP stratification – Flavonoid Adenine Dinucleotide medium (FAD) (Days 12-32):

   • DMEM medium high glucose (Millipore Sigma, St. Louis, MO; cat #: D6429-500ml).
   • F12 Ham 1x supplement (Gibco, Fisher Scientific, Hampton, NH; cat #: 11765-054).
   • 2.5 ml FBS (2.5% FBS) (Peak Serum, Wellington, CO; cat #: PSFB3).
   • 0.4 μg/ml hydrocortisone (Millipore Sigma, St. Louis, MO; cat #: H0888).
   • 8.4ng /ml cholera toxin (Millipore Sigma, St. Louis, MO; cat #: C9903).
   • 5 μg/ml of insulin (Millipore Sigma, St. Louis, MO; cat #: 3068855).
   • 24μg/ml adenine (Millipore Sigma, St. Louis, MO; cat #: A8626).
   • 10ng/ml epidermal growth factor (EGF; R&D Systems, cat #: 2028-EG-200).
   • 1% penicillin-streptomycin P/S (Gibco, Fisher Scientific, Hampton, NH; ca t#: 15-140-122).
   • All supplements must be sterile. Keep FAD medium in the fridge for one week. Prepare fresh FAD medium every week.

Supplements (Days 12-18 or 20):

• 250ng/ml FGF2 (R&D Systems, Minneapolis, MN; cat#: 233-FB).

• 100ng/ml FGF10 (R&D Systems, Minneapolis, MN; cat#: 345-FG).

• 100ng/ml FGF7/ keratinocyte growth factor (KGF; R&D Systems, Minneapolis, MN; cat#: 251-KG).

No supplements at days 18 or 20-32

2.7. VF fibroblast cell cultures for collagen-fibroblast constructs and FAD conditional medium preparation

1. Primary VF fibroblasts, passage P5-6.

2. 10 cm culture dishes (Corning BD Falcon, Tewksbury, MA; cat #: 353003).

3. Sterile glass disposable pipets (5ml and 10ml) (Fisher Scientific, Hampton, NH; cat #: 13-678-27E and 13-678-27F).

4. Disposable Pasteur pipets (Fisher Scientific, Hampton, NH; cat #: 13-678-20C) for medium aspiration.

5. DMEM VFF medium for VF fibroblasts cell cultures:

   • DMEM high glucose (Millipore Sigma, St. Louis, MO; cat #: D6429-500ml).
   • 10% FBS (Ppeak Serum, Wellington, CO; cat #: PSFB3).
   • 1% Penicillin-streptomycin P/S (Gibco, Invitrogen, Carlsbad, CA; cat #: 15-140-122).
   • 1% MEM–non-essential amino acids solution (Gibco, Life Technologies, cat #: 11140-050).

2.8. Collagen-fibroblast constructs preparation (Day 9)

1. 6-well tissue culture plates (Corning BD Falcon, Tewksbury, MA; cat #: 353046).

2. 6-well plate culture inserts; transparent PET membrane, 6 well, 0.4μm pore size (Corning BD Falcon, Tewksbury, MA; cat #: 353090).

3. 25mm GD/X sterile disposable filters; pore size 0.2μm (GE Healthcare Life Sciences, Whatman, Chicago, IL; cat #: 6901-2502).

4. BD 5 ml syringes (Becton Dickinson, East Rutherford, NJ: cat #: 309646).

5. Sterile glass disposable pipets (5ml and 10ml) (Fisher Scientific, Hampton, NH; cat #: 13-678-27E and 13-678-27F).

6. Disposable Pasteur pipets (Fisher Scientific, Hampton, NH; cat #: 13-678-20C) for medium aspiration.

7. Primary VF fibroblasts, passage P5-6 (500,000 cells per 1ml of collagen matrix).

8. Trypsin for VFF detachment; HyClone Trypsin 0.25% (1x) (GE Healthcare Life Sciences, Whatman, Chicago, IL; cat #: SH30042.02).

9. Collagen matrix preparation:

   • High-concentration rat tail collagen (4mg/ml; 80% final volume Cornick; Bedford, MA; cat#: 354249).
   • 10xPBS (Quality Biological, Gaithersburg, MD; cat #: 119-069-101).
   • HyClone HyPure cell culture grade water (Hyclone, GE Healthcare Life Sciences, Chicago, IL: cat#: SH 3052903).
   • 1N NaOH according for pH adjustment (pH=7.2).
   • 10xDMEM (10% final volume; Millipore Sigma, St. Louis, MO; 02429 −100ml).
   • FBS (10% final volume) (Ppeak Serum, Wellington, CO; cat #: PSFB3).

10. Culture medium for collagen constructs flooding - DMEM basal medium (Subheading 2.3) + supplements.

Supplements:

• 50μg/ml l-Ascorbic acid (Millipore Sigma, St. Louis, MO; cat #: A82902).

• 0.4mM 1-Thioglycerol (MTG; Millipore Sigma, St. Louis, MO; cat #: M6165).

2.9. Conditional FAD medium preparation (Day 11)

1. Primary VF fibroblasts, passage P5-6 cultured in 10cm culture dishes.

2. Sterile glass disposable pipets (5ml and 10ml) (Fisher Scientific, Hampton, NH; cat #: 13-678-27E and 13-678-27F).

3. Disposable Pasteur pipets (Fisher Scientific, Hampton, NH; cat #: 13-678-20C) for medium aspiration.

4. Sterile DPBS without Ca ²⁺ and Mg ²⁺ (Lonza, Morristown, NJ; cat #: 17-512F).

5. 25mm GD/X sterile disposable filters; pore size 0.2μm (GE Healthcare Life Sciences, Whatman, Chicago, IL; cat #: 6901-2502).

6. BD 5 ml syringes (Becton Dickinson, East Rutherford, NJ: cat #: 309646).

7. Flavonoid Adenine Dinucleotide medium (FAD) (Section 2.6).

3. Methods

3.1. Maintenance of hiPS cells

Colonies of hiPS cells are maintained in Matrigel-coated 6-well plates in mTeSR1 medium (2ml/well). Medium is changed every day. For regular maintenance, cells are passaged at 60% confluency with Versene in a ratio 1:6.

3.2. Definitive endoderm (DE) differentiation (days 1-3)

The ideal condition to start differentiation is a 80% confluent plate with expansion of distinct, flattened hiPSC colonies. Cells should be in the Matrigel-coated plate in the mTeSR1 medium.

Day 1

1. Inside the biosafety cabinet mix RPMI medium/Glutamax with Activin A (100ng/ml), Wnt 3a (25ng/ml) and Y-27632 (10μM). Calculations: 2ml of the freshly prepared culture medium per a well; therefore, for a 6-well plate of the cell culture, prepare 12ml of the culture medium.

2. Remove the hiPS cell culture 6-well plate from the tissue culture incubator and place it into the biosafety cabinet.

3. Aspirate mTeSR1 medium, wash the cells twice with RPMI medium/Glutamax (2 ml/well) to remove the rest of the mTeSR1 and add freshly prepared culture medium (Step 1) to the plate (2ml/well). Return the plate to the 37°C 5% CO₂ tissue culture incubator overnight.

Day 2

• 4.Inside the biosafety cabinet mix RPMI medium/Glutamax with Activin A (100ng/ml) and 0.2% FBS. Calculations: 2ml of freshly prepared culture medium per a well; therefore, for a 6-well plate of the cell culture, prepare 12ml of the culture medium.
• 5.Remove the DE cell culture 6-well plate from the tissue culture incubator and place it into the biosafety cabinet.
• 6.Aspirate the old medium and add the freshly prepared culture medium (step 4) to the plate (2ml/well). Return the plate to the 37°C 5% CO₂ tissue culture incubator overnight.

Day 3

• 7.Inside the biosafety cabinet mix medium/Glutamax with Activin A (100ng/ml) and 0.2% FBS. Calculations: 2ml of the freshly prepared culture medium per a well; therefore, for a 6-well plate of the cell culture, prepare 12ml of the culture medium.
• 8.Remove the DE cell culture 6-well plate from the tissue culture incubator and place it into the biosafety cabinet.
• 9.Aspirate the old medium and add the freshly prepared medium (step 7) to the plate (2ml/well). Return the plate to the 37°C 5% CO₂ tissue culture incubator overnight (see Note 1).

AFE differentiation (Days 4-7)

• 10.Inside the biosafety cabinet mix the DMEM/F12/Glutamax medium 500ml with sterile 2% B27 (10ml), sterile 1% N2 (5ml) and sterile 1% Penicillin-streptomycin P/S (5ml) to form the DMEM basal medium. Keep in fridge for one month.

Days 4 – 7

• 11.Inside the biosafety cabinet mix DMEM basal medium (step 10) with ascorbic acid (0.05mg/ml), 1-Thioglycerol (0.4mM), Noggin (200ng/ml) and SB-431542 (10μM). All supplements have to be sterile. Calculations: 2ml of culture medium per a well; therefore, for a 6-well plate of the cell culture, prepare 12ml of culture medium.
• 12.Remove the AFE cell culture 6-well plate (Day 4 of Differentiation) from the tissue culture incubator and place it into the biosafety cabinet.
• 13.Aspirate the old medium and add culture medium (Step 11) to the plate. Return the plate to the 37°C 5% CO₂ tissue culture incubator overnight.
• 14.Continue culturing AFE cells for additional 3 days, by replacing the old culture medium with the freshly prepared culture medium (step 11) every day (see Note 2).

3.4. VBP differentiation (Days 8-10) and collagen–fibroblast construct Preparation (Day 9)

Day 8

• 15.Inside the biosafety cabinet mix DMEM basal medium (step 10) with ascorbic acid (0.05mg/ml), 1-Thioglycerol (0.4mM), FGF2 (250ng/ml), FGF7 (100ng/ml) and FGF10 (100ng/ml). Calculations: 2ml of culture medium per a well; therefore, for a 6-well plate of the cell culture, prepare 12ml of culture medium.
• 16.Remove the VBP cell culture 6-well plate (Day 8 of Differentiation) from the tissue culture incubator and place it into the biosafety cabinet.
• 17.Aspirate the old medium and replace it with the freshly prepared culture medium (step 15). Return the plate to the 37°C 5% CO₂ tissue culture incubator for 48h.
• 18.Next day (Day 9), prepare collagen-fibroblast constructs for VBP reseeding at Day 10)

Day 9

Preparation of collagen-fibroblast constructs

• 19.For collagen-fibroblast construct preparation collect VF primary fibroblasts in DMEM VF fibroblast medium in the tissue culture incubator (primary VFF; passage 5 – 6 in a 10cm dish). For making collagen-fibroblast constructs, the VFF cultures need to be confluent (see Note 3).
• 20.In the biosafety cabinet, mix high concentration rat tail collagen (4mg/ml; 80% final volume) with 10xPBS, HyPure cell culture grade water according to the manufacturer’s instructions.
• 21.Add 10x DMEM (10% final volume; Millipore Sigma, St. Louis, MO; 02429 −100ml)
• 22.Adjust pH = 7.2 with sterile 1N NaOH, check the pH with a pH paper indicator
• 23.Meanwhile detach VFF with trypsin (2 ml/10 cm dish) for 2 min, add 8ml of VFF medium and collect cells into 15ml conical tube. Calculate the cell number: for successful VBP reseeding you need app. 500,000 cells/1ml of collagen matrix. For formation of three collagen-fibroblasts constructs, you need approximately 3 mil VFF.
• 24.Spin down the cell suspension and resuspend the cell pellet in ice cold FBS (10% final volume) and add them to collagen mixture.
• 25.Plate mixture of collagen gel and VF fibroblasts on a cell culture insert, 2ml per a 6-well culture insert.
• 26.Solidify for one hour in a tissue incubator at 5% CO₂, 37°C degrees.
• 27.After one hour, gently detach collagen with a pauster pipette and flood constructs with DMEM basal medium (step 10) mixed with ascorbic acid (0.05mg/ml) and 1-Thioglycerol (0.4mM). Return constructs into an incubator fr at least 24 hours to allow for gel contraction.
• 28.Next day (Day 10), mildly trypsinize VBP and plate them on collagen constructs at high density in 100 μl DMEM basal medium supplemented with ascorbic acid (0.05mg/ml), 1-Thioglycerol (0.4mM) and high concentrations of FGF2 (250ng/ml), FGF10 (100ng/ml) and FGF7 (100ng/ml).

Day 10

Re-plating VBP on collagen-fibroblast constructs

The ideal condition for reseeding VBP on the collagen-fibroblast constructs is to have an 80% confluent plate with expansion of cuboidal VBP. VBP cell cultures should be in the Matrigel-coated plate in DMEM basal medium supplemented with ascorbic acid, 1-Thioglycerol and FGFs (step 15).

• 29.Mix freshly DMEM basal medium (step 10) with ascorbic acid (0.05mg/ml), 1-Thioglycerol (0.4mM), FGF2 (250ng/ml), FGF7 (100ng/ml) and FGF10 (100ng/ml). Calculations: Collect VBP cell cultures from two wells of a 6-well plate, pool together and reseed on the top of one collagen-fibroblast construct (ratio 2:1). VBP cells collected from 6 wells (a 6-well plate) will be used to generate 3 VBP-collagen-fibroblast constructs. Each 3D construct will be submerged in 3ml of the fresh culture medium (1ml in upper chamber and 2ml in lower chamber). 6ml of freshly prepared media for cell collection and 9ml of freshly prepared media for cell reseeding is required; total volume = 15ml of fresh medium for reseeding cells from a 6-well plate.
• 30.Prepare collagen-fibroblast constructs from Day 9 for VBP cell reseeding. Remove 6-well plate with collagen-fibroblast constructs from the tissue culture incubator and place it into the biosafety cabinet. Aspire the old medium from bottom chambers and from inserts with collagen constructs. Remove as much of the medium as possible.
• 31.Leave 6-well plate with transwells/constructs in the biosafety cabinet putting it aside.
• 32.Remove VBP cell culture 6-well plate (Day 10 of VBP differentiation) from the tissue culture incubator and place it into the biosafety cabinet.
• 33.Select two confluent wells to be collected and reseeded. Do not aspire the old medium. Instead, collect and transfer the old medium from the wells into a 15ml conical tube (approximately 4ml). Filter old medium using 0.2 μM filter and add 2ml of freshly prepared medium (step 29). You will have 6ml of collected culture medium total. Add 0.2% of FBS to the medium mixture to neutralize the trypsin activity.
• 34.Meanwhile wash cells in DPBS without Ca²⁺ and Mg²⁺ twice to remove cell debris and briefly digest cells with the warm trypsin (TrypLE express diluted to 1:5 with HBSS) for 5 min. Watch cells carefully, they should start detaching, but need to be still attached to the plate.
• 35.Aspirate the trypsin and add collected culture medium with FBS (step 33) back into the wells (2ml/well). Mechanically detach the cells with a glass pipette (a 5ml pipette) and transfer the cell suspension to a 15ml conical tube. Wash wells to collect all cells with the remaining collected culture medium. Add remaining 2ml of collected culture medium to the first well, wash cells off the plate and then transfer the medium with cells into the second well. Collect the cell suspension from the second well and add it to a 15ml conical tube with the cell suspension.
• 36.Pipette cell suspension in a 15ml conical tube and centrifuge cells briefly (5 min; 1000rpmi). Aspirate gently to remove the supernatant and leave cell pellet on the bottom with approximately 60-100μl of the culture medium.
• 37.Resuspend VBP cells in 60-100μl of the remaining culture medium. Re-plate cells in 2 x 50μl or 2 x 30μl (100μl pipette tip) on top of collagen-fibroblast constructs. Target the center of the construct. Drops need to stay on the construct otherwise cells will escape from the construct and attach to the bottom of the insert. Place the plate with a collagen-fibroblast construct with newly reseeded VBP into the 37°C 5% CO₂ tissue culture incubator and repeat steps 33-37 to reseed all VBP cells.
• 38.After cell reseeding, leave the plate with constructs in a 37°C 5% CO₂ tissue culture incubator for at least 2 hours to allow for cell attachment.

Days 10-12

• 39.After 2 hours, cells should be firmly attached to the collagen matrix. Flood the constructs with freshly prepared medium (step 29). For each insert, add 1ml of media into the upper chamber (an insert with the VBP-collagen-fibroblast construct) and 2 ml into the bottom chamber (well). Return the plate with constructs to the 37°C 5% CO₂ tissue culture incubator for 48h (see Note 4).

3.5. Preparation of FAD and conditional FAD media (Day 11)

Day 11

Prepare FAD medium

• 40.Inside the biosafety cabinet, prepare 100ml of fresh FAD medium: Mix 25ml of DMEM medium high glucose and 75ml of F12 Ham supplement (ratio 1:3) with 2.5 ml FBS (2.5% FBS), 0.4 μg/ml hydrocortisone, 8.4ng /ml cholera toxin, 5 μg/ml of insulin, 24μg/ml adenine, 10ng/ml epidermal growth factor, 1% penicillin-streptomycin P/S. All supplements have to be sterile. Keep FAD medium in the fridge for one week. Prepare fresh FAD medium every week.

Prepare conditional FAD medium

• 41.To prepare conditional FAD medium, you need 60% confluent VF primary fibroblasts in DMEM VF fibroblast medium in the tissue culture incubator (Primary VFF; passage 5 – 6 in a 10cm dish).
• 42.Remove the VFF cell culture dish from the tissue culture incubator and place it into the biosafety cabinet.
• 43.In the biosafety cabinet, aspirate the old VF fibroblast medium from the cell culture of human primary VF fibroblasts and add 10ml of freshly prepared FAD medium (step 40).
• 44.Put the VF fibroblast cell culture into the 37°C 5% CO₂ tissue culture incubator overnight.
• 45.Next day, collect FAD medium from the VF fibroblast cell culture and transfer it into a 15ml conical tube. Filter sterile the collected FAD medium using a 0.2 μM filter and place in a −20°C freezer for a long-term storage. Discard VFF cell culture.

3.6. Inducing of stratification of VBP

Days 12 – 16

Inducing stratification in submerged cell cultures using the conditional FAD medium supplemented with FGFs.

• 46.At day 12, mix freshly conditional FAD medium at a ratio 30:70 (30% conditional FAD from step 45 and 70% fresh FAD from the step 40) and add FGF2 (250ng/ml), FGF7 (100ng/ml) and FGF10 (100ng/ml). Calculations: for three VBP-collagen-fibroblast constructs you need 9ml of freshly prepared conditional FAD medium with FGFs.
• 47.Remove VBP-collagen-fibroblast constructs from the tissue culture incubator and place them into the biosafety cabinet.
• 48.Aspire old medium from upper and lower chambers and add freshly prepared conditional FAD medium with FGFs (step 46) to the plates. Return plates to the 37°C 5% CO₂ tissue culture incubator for 48h.
• 49.Repeat steps 46-48 at day 14 by replacing the old medium with freshly prepared conditional FAD medium with FGFs (step 46). Replace old medium in both chambers (upper and lower chambers). Return the plates to the 37°C 5% CO₂ tissue culture incubator for 48h (see Note 5).

Days 16 - 20

VBP stratification at the Air/Liquid interface in the conditional FAD with FGFs

• 50.At day 16, mix freshly conditional FAD medium at a ratio 30:70 (30% conditional FAD from step 45 and 70% fresh FAD from step 40) and mix it with FGF2 (250ng/ml), FGF7 (100ng/ml) and FGF10 (100ng/ml). Calculations: for three VBP-collagen-fibroblast constructs you need 4.5ml of freshly prepared conditional FAD medium with FGFs.
• 51.Remove VBP collagen-fibroblast constructs from the tissue culture incubator and place them into the biosafety cabinet.
• 52.Aspire old medium from the upper and lower chambers and add freshly prepared medium (step 50) to the lower chamber only (1.5ml per insert/well). Return the plates to the 37°C 5% CO₂ tissue culture incubator for 48h.
• 53.Repeat steps 50-52 at day 18 by replacing the old medium with freshly prepared conditional FAD medium with FGFs (step 50). Replace the old medium in the lower chamber only. Return the plates to the 37°C 5% CO₂ tissue culture incubator for 48h.

Day 20 - 32

VBP stratification at the Air/Liquid interface in regular FAD medium

• 54.Use freshly prepared FAD medium (step 40).
• 55.Remove VBP-collagen-fibroblasts constructs from the tissue culture incubator and place them into the biosafety cabinet.
• 56.Aspire told medium from to lower chamber. Add regular FAD medium to lower chamber only (1.5ml per an insert/well). Return the plates to the 37°C 5% CO₂ tissue culture incubator.
• 57.Culture the plates for additional 12 days, changing regular FAD medium three times a week in the lower (basolateral) chamber only. Cultivation at the A/Li interface can be extended up to three weeks.

Day 32

Initiate the experiments or set the constructs aside for RNA extraction, IF staining and/or flow cytometry cell sorting.

4. Notes

1. if you experience the cell death, wash the detached cells with RPMI/Glutamax medium or DPBS once or two times (2ml/well) and then add freshly prepared culture medium to the plate (2ml/well).

2. Make ascorbic acid stock 1000x solution (50mg/ml). Prepare a 50mg/ml ascorbic acid solution by dissolving 500mg of l-ascorbic acid powder in 10ml of Hyclone water, and then filter sterile with a 0.2 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.

3. VF primary fibroblast cell cultures are cultivated in a 10cm dish and are maintained in DMEM VF fibroblast medium in the 37°C 5% CO₂ tissue culture incubator. Prior to making collagen-fibroblast constructs, expand enough plates for making collagen-fibroblast constructs (Day 9) and FAD conditional medium (Day 11). For collagen-fibroblast construct preparation, VFF cell cultures need to be confluent (80%). One confluent VFF 10cm dish is enough for making three collagen-fibroblast constructs, as it yields usually around 4 mil of cells (you need 500,000 cells per 1ml of collagen matrix). For making FAD conditional medium, we recommend having 50 – 60% confluent plate.

4. If the VBP colonies in two- dimensional conditions (2D) grow slowly and do not cover the whole plate (80% confluency) at Day 10, remove old medium, add freshly prepared medium to the cells (step 15) and return the plate into the 37°C 5% CO₂ tissue culture incubator overnight. Re-plate cells on top of collagen-fibroblast constructs on Day 11 .

5. This step is optional and can be skipped. If VBP respond well to stratification initiate A/Li at day 14 in conditional FAD medium supplemented with FGFs (steps 50-53). Proceed with regular FAD treatment at day 18 (steps 54-57). Maintain VF mucosae in regular FAD medium for two weeks (14 days) with the medium changed three times a week.

Differentiation protocol for hiPSC-derived VF mucosa is summarized in the Figure 1.

Figure 1:

Summary of the differentiation protocol for the hiPSC-derived VF mucosa. Abbreviations: hiPSC, human induced pluripotent stem cells; AFE, Anterior Foregut Endoderm; A/Li, Air/Liquid Interface; DE, Definitive Endoderm; VBP, Vocal Fold Basal Progenitors; VFE, Vocal fold Epithelium.