Generation of In vitro Derived Mucus

Mucin Collection Method 
 

Materials and Reagents

1. 0.05% Trypsin-EDTA (Gibco, Catalog number: 25-300-054); Store at -20 °C; Self life: refer expiration date
2. Fetal Bovine Serum (Gibco, Catalog number: 10082147); Store at -20 °C; Self life: refer expiration date
3. Phosphate Buffer Saline (Sigma, Catalog number: 806552)
4. 12mm transwell 0.4um pore polyster membrane insert (Corning, Costar, Catalog number: 3460)
5. Purecol (Advanced Biomatrix, Catalog number: 5005); Store at 4 °C.
6. Prepare complete EpiX medium by adding following components:
   1. 500ml EpiX medium (Propagenix, Catalog number: 27600D); Store at 4 °C.
   2. 100ul Supplement A (Propagenix, Catalog number: 27600A); Store at -20 °C.
   3. Whole content of supplement B vial (Propagenix, Catalog number: 27600B); Store at -20 °C.
   4. 100ul of supplement C (Propagenix, Catalog number: 27600B); Store at -20 °C.
   5. 5ml of penicillin/streptomycin (Gibco, Catalog number:15140-122); Store at -20 °C.
   6. Mix thoroughly by gently swirling. 
   7. Store complete EpiX medium at 2-8 °C.
7. Prepare complete ALI culture medium by adding following components:
   1. 500ml of PneumaCult-ALI Base Medium (Stem cell Tech, Catalog number:05002)
   2. 50ml 10X supplement provided with the kit (Stem cell tech, Catalog number:05003)
   3. 1ml (5 in total) 100X supplement provided with the kit (Stem cell tech, Catalog number:05006)
   4. 500 µl Hydrocortisone solution (Stem cell tech, Catalog number: 07925); Store at -20 °C; Self life: 2 year from the manufacture.
   5. Heparin solution (Stem cell tech, Catalog number: 07980); Store at -20 °C; Self life: refer expiration date
   6. 5 ml Sodium bicarbonate (Thermofisher Scientific, Catalog number:25080094); Store at RT; Self-life: 18 months)
   7. 5 ml penicillin/streptomycin (Gibco, Catalog number:15140-122; Store at -20 °C; Self life: refer expiration date
   8. Mix thoroughly by gently swirling.
   9. Store complete ALI medium at 2-8 °C.
       

Equipment

1.  Cell counter (BECKMAN COULTER, model/catalog number: Vi-CELL XR CELL VIABILITY ANALYZER/ 1042328)
2. Cell culture cabinet (THE BAKER COMPANY, model/catalog number: SG603A-HE CALSS II TYPE A2)
3. Centrifuge (BECKMAN COULTER, model/catalog number: Allegra X-15R)
4. Microscope (OLYMPUS CORPORATION, model/catalog number: CKX31SF)
5. Hemocytometer (Hausser Scientific, model/catalog number: 02-671-51B)

Procedure

A. Cell culture and passaging

1. Prepare the (50µg/ml) purecol solution in PBS. Store the solution at 4 °C.
2. Coat T-75 culture flask with 7ml (50µg/ml) of purecol solution overnight or at least 1hr inside incubator at 37 °C.
3. Place the flask inside the cell culture cabinet and UV-treat the flask for 20-30min.
4. Aspirate the purecol and gently wash the flask with PBS once. 
5. Warm complete Epix medium.
6. Thaw 0.5-1 million basal epithelial cells in water bath until there is a small pellet of ice.
7. Clean vial with 70% ethanol.
8. Remove cells from vial using a serological pipet and transfer it to a 15ml tube.
9. Add 9ml of warm complete EpiX media slowly dropwise.
10. Centrifuge 300 rcf for 5min.
11. Aspirate media and gently resuspend cells in 15ml of complete Epix medium. Transfer the cell suspension to T-75 flask and put it inside the 37 °C incubator.
12. change  media every 48-72 hrs.

B. Air-Liquid Interface culture

1. Add 1 ml (50µg/ml) purecol to each filter insert of each well. Incubate minimum of 1 hr in incubator or overnight at 37 °C.
2. Aspirate the coating solution and place the plate under the UV light for 30 min After irradiation, and wash the filter with PBS once and aspirate the solution.
3. Add 3-4 ml of warm trypsin-EDTA to T-75 flask containing basal epithelial cells.
4. Monitor detachment of cells. Don’t tap the cells. 
5. Add 5 ml FBS to neutralize trypsin. Transfer cells to 15ml tube. Wash the flask with PBS and add that to the collection tube. 
6. Count cells using ViCell or hemocytometer.
7. Suspend cells in complete ALI culture medium.
8. Seed 500ul of containing 250,000 cells/well at the top of 12mm filter insert. 
9. Incubate for 1hr at 337 °C.
10. Add 700-800µl of complete ALI medium at the basal compartment.
11. Change media at the bottom and top of the filter insert every 48 hr.

C. Air-Lift

1. At day 7 check for tight junction formation by aspirating media from the top of the filter insert. If the media is not leaking, the culture is ready to be switched to air-liquid interface culture.
2. Change media at the bottom every 48-72h for 3 weeks by exposing apical part to air.
3. Mucus secretion is observed within 4-5 days of ALI culture.

D. Mucus collection

1. Add 500ul of warm PBS on the top of the filter insert and incubate at 37 °C for 1h.
2. Gently pipet up and down 5-6 times.
3. Collect the mucin and flash freeze the samples at -80 °C if not used immediately.

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