Human Liver Cancer-Associated Fibroblasts (CAF)

Catalog Number: HLCAF01
Product Format: Frozen Vial
Cell Number: 1,000,000 cells/vial
Suggested Medium: CAF Growth Medium (CAF01)

General Information

Human Liver CAF is isolated from human liver 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 Liver 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 Liver 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 of the frozen culture is necessary upon arrival, 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 forward 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 volume 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 PBS. Transfer the cells to an appropriate-sized T-Flask.

4. To avoid excessive alkalinity of the medium during recovery, place the culture vessel with growth medium in the incubator for at least 15 minutes before adding the cells. This allows the medium to equilibrate to a pH of 7.0 to 7.6.

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

Subculturing Procedure

Volumes below apply to a 75 cm² flask; adjust volumes for other flask sizes. T-75 flasks are recommended.

Note: Do not agitate the flask to detach cells; this may cause clumping. For difficult detachment, place the flask at 37°C.

1. Remove and discard the spent medium. Briefly rinse the cell layer with 1× PBS to remove residual serum inhibitors.

2. Add 2.0 to 3.0 mL of Cell Detachment Solution. Observe under an inverted microscope until the cell layer disperses (typically 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. Gently pipette to aspirate cells.

5. Dispense appropriate aliquots 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