IV. Subculturing CnOb
A. Preparing Subculture Reagents
- Remove the Trypsin-EDTA solution (T3924) and Trypsin Inhibitor (T6414) from the -20 °C freezer and thaw overnight in a refrigerator.
- Make sure all the subculture reagents are thawed. Swirl each bottle gently several times to form homogeneous solutions.
- Store all the subculture reagents at 4 °C for future use.
- Aliquot Trypsin/EDTA solution (T3924) and store the unused portion at -20 °C if only a portion of the Trypsin/EDTA (T3924) is needed.
B. Preparing Culture Flask
- Take the Canine Osteoblast Growth Medium (Cn417-500) from the refrigerator. Decontaminate the bottle with 70% alcohol in a sterile hood.
- Pipette 30 mL of Canine Osteoblast Growth Medium (Cn417-500) to a T-175 flask (SIAL1080) (to be used in Section IV C Step 15.)
C. Subculturing CnOb
Trypsinize Cells at Room Temperature. Do Not Warm Any Reagents to 37 °C.
- Remove the medium from culture flasks by aspiration.
- Wash the monolayer of cells with HBSS (H6648) and remove the solution by aspiration.
- Pipette 6 mL of Trypsin/EDTA Solution (T3924) into the T-75 flask (SIAL0641). Rock the flask gently to ensure the solution covers all the cells.
- Remove 5 mL of the solution immediately.
- Re-cap the flask tightly and monitor the trypsinization progress at room temperature under an inverted microscope. It usually takes about 2 to 4 minutes for the cells to become rounded. The cells may not become completely round during the trypsinization and some cells may maintain some processes even though they are loosened from the culture surface.
- 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.
- Pipette 5 mL of Trypsin Inhibitor Solution (T6414) to the flask to inhibit further tryptic activity.
- Transfer the cell suspension from the flask to a 50 mL sterile conical tube.
- Rinse the flask with an additional 5 mL of Trypsin Inhibitor Solution (T6414) and transfer the solution into the same conical tube.
- Examine the T-75 flask (SIAL0641) under a microscope. If there are >20% cells left in the flask, repeat Steps 2-9.
- Centrifuge the conical tube at 220 x g for 5 minutes to pellet the cells.
- Aspirate the supernatant from the tube without disturbing the cell pellet.
- Flick the tip of the conical tube with your finger to loosen the cell pellet.
- Resuspend the cells in 5 mL of Canine Osteoblast Growth Medium (Cn417-500) by gently pipetting the cells to break up the clumps.
- Count the cells with a hemocytometer or cell counter. Inoculate at 10,000 cells per cm2 for rapid growth, or at 5,000 cells per cm2 for regular subculturing.
V. Differentiating CnOb
A. Seeding CnOb for Differentiating Into Canine Osteoblast
- Seed CnOb in the desired format at 10,000 per cm2 in Canine Osteoblast Growth Medium (Cn417-500) following instructions in section IV C.
- Place the cells 37 oC, 5% CO2 humidified incubator.
- Change to Canine Osteoblast Differentiation Medium (Cn417D-250) the following day by removing the growth medium from culture tissue ware by aspiration and adding the appropriate volume of Canine Osteoblast Differentiation Medium (Cn417D-250). Do not allow cells to dry during medium changes.
B. In Vitro Mineralization
- Remove growth medium from culture tissue ware by aspiration. Do not allow cells to dry during medium changes.
- Add the appropriate volume of Canine Osteoblast Differentiation Medium (Cn417D-250).
- Incubate cells in a 37 oC, 5% CO2 humidified incubator in the Canine Osteoblast Differentiation Medium (Cn417D-250).
- Change to fresh Canine Osteoblast Differentiation Medium (Cn417D-250) every other day.
- Dense extracellular matrix will be secreted and mineralized structure formed in 10-14 days.