hTERT Human Ovarian Cancer-Associated Fibroblasts (CAF)

Catalog Number: HT12
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 after infection with lentiviruses expressing hTERT.
These Human Ovarian CAFs are isolated from ovarian tumor tissue. Cells are cultured in T75 flasks pre-coated with AlphaBioCoat and incubated in CAF Growth Medium for 3–7 days. Cultures are expanded, harvested at passage 1, and cryopreserved. 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: mycoplasma, bacteria, yeast, fungi

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

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

Laboratory Applications

• Cell-cell interaction and adhesion studies

• Western blot, PCR, and immunoprecipitation

• Immunofluorescence and flow cytometry

• Ovarian tumor microenvironment modeling

• Derivation of CAF-based systems for research

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.

   • Keep the cap and O-ring out of the water.

   • Disinfect the vial with 70% ethanol.

   • Proceed under strict aseptic conditions.

2. Recovery & Seeding

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

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

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

   • Rinse with 8 mL of 1× PBS and discard.

   • Plate cells into the prepared flask.

3. Culture Conditions

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

   • To stabilize pH (7.0–7.6), pre-equilibrate the medium in the incubator for at least 15 minutes before use.

Subculturing Instructions

Volumes below are based on a 75 cm² flask. Adjust accordingly for other formats.

1. Remove and discard the culture medium.

2. Rinse cell layer with 1× PBS to remove serum traces.

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

   • Do not shake or hit the flask to avoid clumping.

   • For difficult detachment, extend incubation time or rewarm the flask.

4. Pre-coat a new flask with 6.0–8.0 mL AlphaBioCoat for 15 minutes. Rinse with 8 mL of PBS and discard.

5. Resuspend cells in 6.0–8.0 mL of complete growth medium.

6. Transfer appropriate aliquots to new vessels.

• 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 in isopropanol-based container at –80°C overnight

• Transfer to liquid nitrogen for long-term storage

Product Usage

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