Human Lung Cancer-Associated Fibroblasts (CAF) – Squamous Cell Carcinoma

Catalog Number: HLSCAF01
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 for 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 continued storage is required, the culture should be stored in the liquid nitrogen vapor phase, not at -70°C, as storage at -70°C will result in loss of viability.

Thaw the vial by gentle agitation in a 37°C water bath. To reduce contamination risk, 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 once thawed. Decontaminate by dipping or spraying with 70% ethanol. All steps from this point must be conducted under strict aseptic conditions.

2. Immediately remove the cryoprotective agent. Centrifuge the cell suspension at approximately 125 × g for 5 to 10 minutes. Discard the supernatant and resuspend the pellet in fresh growth medium.

3. Add 6.0 to 8.0 mL of AlphaBioCoat to a T-Flask. After 15 minutes, aspirate and rinse with 8 mL of 1× PBS. Discard the rinse and transfer the cells to the T-Flask.

4. To prevent excessive alkalinity during recovery, pre-incubate the vessel containing the growth medium for at least 15 minutes at 37°C to allow the pH to stabilize (target range: 7.0 to 7.6).

5. Incubate the culture at 37°C in a suitable incubator with 5% CO₂ in air.

Subculturing Procedure

Volumes listed are for 75 cm² flasks. Adjust accordingly for other flask sizes.

Note: Do not agitate or tap the flask while waiting for detachment. For difficult-to-detach cells, place the flask at 37°C to assist dispersal.

1. Remove and discard the old medium. Briefly rinse the cells with 1× PBS to eliminate serum inhibitors.

2. Add 2.0 to 3.0 mL of Cell Detachment Solution and observe under a microscope until cells are dispersed (typically 5–15 minutes).

3. Add 6.0 to 8.0 mL of AlphaBioCoat to a new T-Flask and let sit for 15 minutes. Rinse with 8 mL of 1× PBS and discard.

4. Add 6.0 to 8.0 mL of complete growth medium and 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

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