Human Metastatic Ovarian Cancer-Associated Fibroblasts (CAF)

Catalog Number: HMOCAF01
Product Format: Frozen Vial
Cell Number: 1,000,000 cells/vial
Suggested Medium: CAF Growth Medium (CAF01)

General Information

Human Metastatic Ovarian CAF is isolated from human metastatic 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 Metastatic 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 Metastatic Ovarian 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 necessary, the cells should be stored in the liquid nitrogen vapor phase and not at -70°C, as storage at -70°C will result in loss of viability.

Thaw the vial by gently agitating it in a 37°C water bath. To minimize 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 as soon as contents are thawed. Decontaminate by dipping or spraying with 70% ethanol. All steps following should be conducted under strict aseptic conditions.

2. Remove the cryoprotective agent 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 a T-Flask for 15 minutes. Aspirate and rinse with 8 mL of 1× PBS. Discard the rinse. Transfer cells to the prepared T-Flask.

4. To avoid excessive alkalinity, pre-incubate the culture vessel containing growth medium for at least 15 minutes at 37°C to allow the medium to stabilize at pH 7.0–7.6.

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

Subculturing Procedure

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

Note: Do not agitate the flask by hitting or shaking. If detachment is difficult, place at 37°C to aid dispersal.

1. Remove and discard the old medium. Rinse the cell layer briefly with 1× PBS to remove serum traces.

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

3. Add 6.0 to 8.0 mL of AlphaBioCoat to the T-Flask and incubate 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 aspirate the cells by gentle pipetting.

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