hTERT Human Metastatic Ovarian Cancer-Associated Fibroblasts (CAF)

Catalog Number: HT13
Product Format: Frozen Vial
Cell Number: 1,000,000 cells/vial
Suggested Medium: CAF Growth Medium

General Information

Puromycin-resistant hTERT cells were selected from HMOCAF following lentiviral transduction of human telomerase reverse transcriptase (hTERT).
These Human Metastatic Ovarian CAFs were derived from metastatic ovarian tumor tissue, cultured in T75 flasks pre-coated with AlphaBioCoat, and expanded for 3–7 days in CAF Growth Medium. Cells were cryopreserved at passage 1. Each vial contains ≥1,000,000 viable cells.

Product Testing

• Negative for: von Willebrand Factor/Factor VIII, cytokeratin 18, and alpha-smooth muscle actin

• Free from: bacteria, yeast, fungi, and mycoplasma

• CAF Marker Expression: FAP, PDGFR, Vimentin, PDPN, CD70

• Expansion Capacity: 3–5 passages at a 1:2 or 1:3 split ratio

Laboratory Applications

• Tumor-stroma interaction studies

• Cell adhesion and migration assays

• PCR, Western blot, immunoprecipitation

• Immunofluorescence and flow cytometry

• Development of ovarian tumor microenvironment models

Shipping

Frozen vials shipped on dry ice

Post-Thaw Recovery Protocol

1. Thawing & Aseptic Handling

   • Thaw vial in a 37°C water bath for ~2 minutes with gentle agitation.

   • Do not submerge the cap.

   • Spray or dip in 70% ethanol.

   • Perform all following steps under aseptic conditions.

2. Cell Recovery & Seeding

   • Centrifuge at 125 × g for 5–10 minutes.

   • Discard the supernatant and resuspend pellet in fresh CAF Growth Medium.

   • Coat a T-flask with 6–8 mL AlphaBioCoat for 15 minutes.

   • Rinse with 8 mL 1× PBS and discard.

   • Transfer cells into the prepared flask.

3. Culture Conditions

   • Incubate at 37°C in a humidified 5% CO₂ incubator.

   • To ensure stable pH (7.0–7.6), pre-equilibrate growth medium in the incubator for 15 minutes before adding cells.

Subculturing Instructions

Volumes below are for 75 cm² flasks. Adjust for other formats as needed.

1. Remove and discard spent medium.

2. Rinse cells gently with 1× PBS to remove serum inhibitors.

3. Add 2.0–3.0 mL Cell Detachment Solution. Incubate 5–15 minutes at 37°C.

   • Do not tap or agitate the flask.

   • For difficult detachment, rewarm flask or extend incubation.

4. Coat a new flask with AlphaBioCoat (6–8 mL for 15 minutes), rinse with PBS, and discard.

5. Resuspend cells in 6.0–8.0 mL fresh complete medium.

6. Plate appropriate aliquots into new flasks.

• Subcultivation Ratio: 1:2 to 1:3

• Medium Renewal: Every 3 to 4 days

Cryopreservation Protocol

• Use complete growth medium supplemented with 5% (v/v) DMSO

• Cryopreservation Medium Catalog #: CGM5

• Freeze at –80°C overnight in an isopropanol container before transferring to liquid nitrogen

Product Usage

Cells are offered for Research Use Only. Not for Clinical Use.