Physiological 3D Tissue Model of the Airway Wall and Mucosa

Melanie M. ChoeAlice A. Tomei

Melody A. Swartz

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Normal human bronchial epithelial cells (NHBEs)

2 x T175

Fetal human lung fibroblasts (HLFs)

4 x T175

70% confluent

70% confluent

• PBS

• trypsin for HLFs

• trypsin for NHBE

• co-culture media

• hemacytometer

• 6 well transwell plate

• at least 12 ml collagen solution (7.25 < pH < 7.3)

• 6 porous PE cylindrical inserts (acid treated to make hydrophilic)

Cells & reagents needed for 6 models

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1. Harvest fibroblasts

(> 6*106 cells)

2. Dissociate pellet in 1 ml co-culture

medium

1 ml

3. Add collagen and co-culture medium to make a final solution of

500,000 cells /ml and 2.5 mg/ml collagen

>12 ml

4. Mix well by gently pipetting up and down

Avoid bubbles

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Place the PE constructs into each transwell insert

Pipette 100 µl HLF suspension in collagen into each insert to create a plug

Incubate (37°C, 5% CO2)

5 min

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Pipette the HLF-collagen suspension to completely fill insert (~2 ml/insert)

Avoid bubblesIncubate

(37°C, 5% CO2)15 min

(collagen turns opaque upon gelation)

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Coat the top of collagen gel with a thin layer of acellular collagen (2.5 mg/ml)

Avoid bubbles

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Gently level the surface of the gel with a cell scraper

Incubate (37°C, 5% CO2)

5 min

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Aspirate excess fluid from the bottom of the well

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Gently pipette 2 ml medium to the bottom chamber

Incubate (37°C, 5% CO2)

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PAUSE POINTThe model can be stored in the incubator for several hours

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2. Dissociate pellet in 1 ml co-culture

medium

1 ml

1. Harvest NHBE

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Pipet 500,000 NHBE cells (200 µl) onto each PE well

Incubate (37°C, 5% CO2)

2 hours

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Every ~20 minutes, examine the wells to ensure the top surface is covered by medium (otherwise pipet extra

medium, ~100µl)

Incubate (37°C, 5% CO2)

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After 2 hours, add medium to cover the surface and submerge the model

Incubate (37°C, 5% CO2)Culture in submersion for at least 7

days

Refresh medium every 2 days

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Check every few days under phase contrast to determine when epithelium

is confluent

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When the epithelium is confluent,

create air-liquid interface (ALI)

Aspirate the medium

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Wash the epithelial surface with warm PBS (~500µl) gently (not directly on the cells)

Aspirate the PBS

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Change medium in the bottom well daily

(maintain level for ALI)

Wash epithelial surface with PBS daily

Culture in air liquid interface for at least 7 days (21 days of optimal epithelial differentiation)

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