3-D Airway Model Kit Instructions Cat#: PB-502-3D Fig. 1: HBEpC Fig. 2: Crystal Violet staining Fig. 2: Hematoxylin & Eosin Staining I. 3-D Airway Model Kit: Material Cat# Storage Cryopreserved HBEpCa PB-502-3D Liquid N 2 3-D Bronchial Epithelial Cell Growth Medium (GM) PB-511-3D- 4 C 100 Subculture Reagent Mini-Kit PB-090mK -20 C HBSS PB-062-10 -20 C Trypsin/EDTA (T/E) PB-070-10 -20 C Trypsin Neutralization Solution (TNS) PB-080-10 -20 C Millicell PCF inserts PB-013-14 Room Temp. Fixative 1 PB-014-01 Room Temp. Crystal Violet PB-015-01 Room Temp. 3-D Bronchial Epithelial Cell Differentiation Medium (DM) PB-511D-3D- 4 C 150 Fixative 2 PB-016-01 -20 C
II. Procedure A. Grow HBEpC in a T-75 flask Day 1: Thursday 1. Pipette 10 ml of GM into a T-75 flask for HBEpC seeding. 2. Thaw the cells quickly by placing the lower half of the vial in a 37 C water bath for 1 minute. *Do not overthaw the cells, there should still be a small visible ice crystal in the vial. 3. Take the vial out of the water bath and wipe dry. 4. Decontaminate the vial exterior with 70% alcohol in a sterile Biological Safety Cabinet. 5. Remove the vial cap carefully. Do not touch the rim of the vial. 6. Resuspend the cells in the vial by gently pipetting the cells 3 times with a 2 ml pipette. Be careful not to pipette too vigorously as to cause foaming. 7. Pipette the 1 ml cell suspension from the cryovial into the T-75 flask containing 10 ml of GM. 8. Cap the flask and rock gently to evenly distribute the cells. 9. Loosen the cap to allow gas exchange. 10. Place the T-75 flask in a 37 C, 5% CO 2 humidified incubator. For best results, let cells attach overnight without disturbance. Day 2: Friday 1. Change to 15 ml of fresh GM to remove all traces of DMSO. 2. Loosen the cap to allow gas exchange. 3. Place the T-75 flask in a 37 C, 5% CO 2 humidified incubator. 4. Remove the Subculture Reagent Mini Kit from the -20 C freezer and thaw in a refrigerator.
B. Seed inserts with HBEpC Seed inserts when HBEpC in the T-75 flask is 80% confluency. Do not allow the cells to become 100% confluent, as this will trigger differentiation and poor growth of HBEpC. Pre-warm GM and DM to at least room temperature. Do not pre-warm subculture reagents to 37 C. Day 5: Monday 1. Ensure that the HBEpC have reached 80% confluency in the T-75 flask prior to seeding the cells onto inserts. See Fig. 1. 2. Place Millicell inserts in to a 60 mm cell culture dish. A 60 mm cell culture dish can hold up to 6 Millicell inserts. Include 2 additional inserts as Controls for the monitoring of monolayer confluency prior to initiating differentiation. 3. Make sure all the subculture reagents are thawed. Gently swirl each bottle several times to form homogeneous solutions. 4. Remove GM from the T-75 flask by aspiration. 5. Wash the monolayer of cells with 5 ml of HBSS and remove the wash by aspiration. 6. Pipette 8 ml of Trypsin/EDTA Solution into the T-75 flask. Rock the flask gently to ensure the solution covers all the cells and incubate at room temperature for 1.5 minutes. 7. Aspirate Trypsin/EDTA, re-cap the flask. 8. Incubate the flask inside the 37 C incubator until the cells are rounded and show processes, usually after ~1.5 minutes. Observe the trypsinization process under the microscope 9. Release the rounded cells from the culture surface by hitting the side of the flask against your palm until most of the cells are detached. 10. Pipette 5 ml of Trypsin Neutralizing Solution to the flask to inhibit further tryptic activity. 11. Transfer the cell suspension from the flask to a 50 ml sterile conical tube. 12. Wash the flask with 5 ml HBSS to collect the remaining cells and transfer to the same 50 ml conical tube. 13. Centrifuge the cells for 5 minutes at 1000 RPM at room temperature. 14. Aspirate the supernatant, loosen the cell pellet by flicking the tip of the centrifuge tube with a finger, then resuspend cells in 3 ml GM. *Resuspend the cells as a single cell suspension. 15. Count cells and prepare a cell suspension at 5E5 cells/ml in GM. 16. Seed 400 l (2E5 cells) cell suspension evenly* into each insert using a P1000 tip. *Slowly dispense cells at the center of the insert, then around the side of the insert to cover the whole membrane surface. 17. Ensure that the cells are evenly distributed. On a flat surface, gently slide the 60 mm cell culture dish containing inserts in a crisscross shape: once left-right and once forward-backward. 18. Add appropriate amount of GM outside of the inserts* in the 60 mm cell culture dish so that medium levels inside and outside the insert are equal and the cells are submerged. This should be 11 ml. *Make sure no air bubbles are trapped underneath the membrane for the entire duration of the 3- D culture. 19. Place inserts in a humidified incubator at 37 C with 5% CO 2. 20. Grow the cells until they reach 100% confluency. It typically takes 2 days to reach confluency when using early passage cells that have been proliferating well in the preceding week.
21. If the cells are not confluent, change to fresh GM and cultivate for another day* to reach 100% confluency. *If cells do not reach 100% confluency in 3 days, they are not suitable for developing a 3-D Airway Model. How to order: Just seed us your order by Email to: sales@pelobiotech.com or Fax +49 (0)89 517 286 59-88 For further questions just call us under +49 (0)89 517 286 59-0 or send an email to info@pelobiotech.com
C. Check the confluency of HBEpC in inserts Recommended: assess confluency by staining one of the Control Inserts, ensuring that HBEpC are 100% confluent prior to proceeding further with the air lift culture. Day 7: Wednesday C1: Fix cells 1. Place the first Control Insert in a 60 mm petri plate. 2. Aspirate the medium from the inside of the insert. 3. Wash cells in the insert with 400 l PBS, and aspirate the PBS. 4. Add 400 l Fixative 1 to the insert. 5. Fix cells for 5 min at room temperature. 6. Shake off the fixative, wash gently with 400 l PBS, and turn the insert upside down on a paper towel to remove excess liquid. C2: Stain cells 1. Add 300 l 0.05% Crystal violet inside the insert to submerge the cells. 2. Stain fixed cells for 30 min at room temperature. 3. Shake off the crystal violet. 4. Wash gently with tap water until the washed water is clear. 5. Shake off the tap water and turn the insert upside down on a paper towel to remove excess water. 6. The inserts can now be visualized using a light microscope.
D. Establish the 3-D Airway Model Day 7: Wednesday D1: Start cell differentiation 1. Ensure the monolayer is 100% confluent. 2. Replace the GM with 400 l DM inside the inserts. Replace the outside GM with 11 ml DM. Make sure no air bubbles are trapped under the inserts. 3. Place the inserts in the incubator overnight (15 to 16 hrs) to allow cells to form an intracellular adhesion structure. Day 8: Thursday D2: Initiate the 3-D culture of the Airway Model 1. Aspirate all DM from inside the inserts*. *The surface within the inserts should be dry following air lift and should remain dry for the differentiation process. 2. Place 3 inserts in a 60 mm petri dish. 3. Add 3.2 ml DM to the outside of the inserts so that the media touches the bottom of the membrane* (up to the level of the membrane). *Make sure no air bubbles are trapped underneath the membrane for the entire duration of the 3- D air-lift culture. 4. Change the DM 3 times per week if the dish contains 1-3 inserts (Monday-Wednesday-Friday). Change the DM every day if there are more than 4 inserts in the dish. 3-D Airway Models are typically fully established in 21 Days. The cell layers will have a flaky, shiny appearance. 5. Perform Hematoxylin & Eosin staining to confirm establishment of the Airway model. See Fig. 2. 6. Proceed with your 3-D Airway Model experiment.
E. Confirm establishment of the Airway Model by Hematoxylin & Eosin Histochemical Staining Day 28: Wednesday 1. Take the inserts out of the second control insert 60 mm dish and put them inside a dry 10 cm petri plate. 2. Wipe the outside of the insert carefully with a soft tissue to remove any DM (do not touch the membrane area with the cells). 3. Transfer inserts to a 24-well plate. 4. Add 400 µl Fixative 2 to each well and 400 µl Fixative 2 to each insert. 5. Fix cells at 4 C overnight. 6. Remove the Fixative 2 from the inserts with a pipette. 7. Turn the insert upside down and carefully cut the bottom membrane of the insert containing the 3-D Airway Model carefully with sharp-surgical scalpel. Use the following steps as a general guideline: i. Cut half of the circle from point (a) to (b). ii. Leave the section between (b) and (c) uncut. iii. Cut the edge circle between (c) and (a), and immediately hold the insert edge at point (a).* iv. Cut between (b) and (c). *When holding the insert, hold only the very edge to prevent damage to the 3-D model. 8. Carefully transfer the membrane with forceps onto the sponge of the histological cassettes. 9. Proceed to paraffin embedding and H&E staining.