Human Bronchial Cancer-Associated Fibroblasts (CAF)

Catalog Number: HBCAF54
Product Format: Frozen Vial
Cell Number: 1,000,000 cells/vial
Suggested Medium: CAF Growth Medium (CAF01)

General Information

Human Bronchial CAF is isolated from human bronchial tumor tissue. Cells are grown in T75 tissue culture flasks pre-coated with AlphaBioCoat solution for 30 minutes and incubated in CAF Growth Medium for 3–7 days. Cultures are then expanded. Prior to shipping, cells at passage 1 are detached from flasks and immediately cryo-preserved in vials. Each vial contains at least 1,000,000 cells per ml.

Product Testing

Human Bronchial CAF is tested for negative expression of von Willebrand Factor Expression/Factor VIII, cytokeratin 18, and alpha-smooth muscle actin. Cells are negative for bacteria, yeast, fungi, and mycoplasma. Cells can be expanded for 3–5 passages at a split ratio of 1:2 or 1:3.

Laboratory Applications

Human Bronchial CAF can be used for the assay of cell-cell interaction, adhesion, PCR, Western blot, immunoprecipitation, immunofluorescent flow cytometry, or generating cell derivatives for desired research applications.

Shipping

Frozen Vials on Dry Ice

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in the liquid nitrogen vapor phase and not at -70°C. Storage at -70°C will result in loss of viability.

Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).

Post-Thaw Handling Procedure

1. Remove the vial from the water bath as soon as the contents are thawed and decontaminate by dipping in or spraying with 70% ethanol. All operations from this point on should be carried out under strict aseptic conditions.

2. It is recommended that the cryoprotective agent be removed immediately. Centrifuge the cell suspension at approximately 125 × g for 5 to 10 minutes. Discard the supernatant and resuspend the cell pellet in an appropriate amount of fresh growth medium.

3. Add 6.0 to 8.0 mL of AlphaBioCoat to the T-Flask for 15 minutes. Aspirate the solution after 15 minutes, and rinse with 8 mL of 1× PBS. Discard the 1× PBS. Transfer the cells to an appropriate size T-Flask.

4. It is important to avoid excessive alkalinity of the medium during the recovery of the cells. Prior to adding the vial contents, place the culture vessel containing the growth medium in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).

5. Incubate the culture at 37°C in a suitable incubator. A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.

Subculturing Procedure

Volumes apply to a 75 cm² flask. Adjust proportionally for other vessel sizes. T-75 flasks are recommended for subculturing.

Note: Do not agitate the flask during detachment to avoid clumping. If cells are difficult to detach, place at 37°C to aid dispersal.

1. Remove and discard the culture medium. Briefly rinse the cell layer with 1× PBS to remove traces of serum that contain inhibitors.

2. Add 2.0 to 3.0 mL of Cell Detachment Solution to the flask. Observe cells under an inverted microscope until the cell layer is dispersed (typically within 5 to 15 minutes).

3. Add 6.0 to 8.0 mL of AlphaBioCoat to the T-Flask for 15 minutes. Aspirate the solution and rinse with 8 mL of 1× PBS. Discard the 1× PBS.

4. Add 6.0 to 8.0 mL of complete growth medium. Gently pipette to aspirate cells.

5. Dispense appropriate aliquots of the cell suspension into new culture vessels.

6. Incubate cultures at 37°C.

• Subcultivation Ratio: 1:2 to 1:3

• Medium Renewal: Every 3 to 4 days

Reagents for Cryopreservation

Use complete growth medium supplemented with 5% (v/v) DMSO.
Catalog #: CGM5