Human Ovarian Cancer-Associated Fibroblasts (CAF)

Catalog Number: HOCAF01
Product Format: Frozen Vial
Cell Number: 1,000,000 cells/vial
Suggested Medium: CAF Growth Medium (CAF01)

General Information

Human Ovarian CAF is isolated from human ovarian 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 Ovarian 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 Ovarian 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 storage is necessary upon arrival, it should be 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 minimize 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 thawed and decontaminate with 70% ethanol. All further operations should be under strict aseptic conditions.

2. Immediately remove the cryoprotective agent. Centrifuge the cell suspension at 125 × g for 5–10 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 and incubate for 15 minutes. Aspirate, rinse with 8 mL of 1× PBS, and discard. Transfer cells to the prepared T-Flask.

4. To stabilize the pH (7.0 to 7.6), place the growth medium in the incubator for 15 minutes prior to adding cells.

5. Incubate the culture at 37°C in a 5% CO₂ in air atmosphere.

Subculturing Procedure

Volumes are for a 75 cm² flask; adjust as needed for different vessel sizes. T-75 flasks are recommended.

Note: Do not shake or hit the flask during detachment to prevent clumping. For difficult detachment, place at 37°C.

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

2. Add 2.0 to 3.0 mL of Cell Detachment Solution and monitor under a microscope until the layer is dispersed (typically 5–15 minutes).

3. Add 6.0 to 8.0 mL of AlphaBioCoat, incubate 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. Plate 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

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