Physiological 3D Tissue Model of the Airway Wall and Mucosa Melanie M. Choe Alice A. Tomei
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Transcript of Physiological 3D Tissue Model of the Airway Wall and Mucosa Melanie M. Choe Alice A. Tomei
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)
END