Human Lung Cancer-Associated Fibroblasts (CAF) Adenocarcinoma

Catalog Number: HLACAF01
Product Format: Frozen Vial
Cell Number: 1,000,000 cells/vial
Suggested Medium: CAF Growth Medium (CAF01)

General Information

Human Lung CAF is isolated from human lung 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 Lung 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 express CAF markers FAP, PDGFR, Vimentin, PDPN, and CD70.
Cells can be expanded for 3–5 passages at a split ratio of 1:2 or 1:3.

Laboratory Applications

Human Lung 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 onward should be conducted 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 and rinse with 8 mL of 1× PBS. Discard the PBS. Transfer cells to an appropriate-size T-Flask.

4. To avoid excessive alkalinity of the medium during recovery, pre-warm the culture vessel with growth medium in an incubator for at least 15 minutes prior to adding the vial contents. This allows the medium to equilibrate to a pH between 7.0 and 7.6.

5. Incubate the culture at 37°C in a suitable incubator. A 5% CO₂ in air atmosphere is recommended.

Subculturing Procedure

Volumes listed are for a 75 cm² flask; adjust proportionally for other vessel sizes. T-75 flasks are recommended.

Note: Do not shake or hit the flask to detach cells. Cells that are difficult to detach may be placed at 37°C to assist dispersal.

1. Remove and discard the culture medium. Rinse the cell layer briefly with 1× PBS to eliminate all serum traces.

2. Add 2.0 to 3.0 mL of Cell Detachment Solution and observe under an inverted microscope until the cell layer is fully dispersed (usually within 5 to 15 minutes).

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

4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gentle pipetting.

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

Complete growth medium supplemented with 5% (v/v) DMSO
Catalog #: CGM5