Immortalized Human Pancreatic Cancer-Associated Fibroblasts (CAF)

Catalog Number: IM06
Product Format: Frozen Vial
Cell Number: 1,000,000 cells/vial
Suggested Medium: CAF Growth Medium (CAF01)

General Information

hTERT-immortalized Human Pancreatic CAFs were generated by infecting primary fibroblasts with hTERT-expressing lentiviral particles and expanded under specialized CAF Growth Medium conditions.
These cells are isolated from human pancreatic tumor tissue and cultured in AlphaBioCoat-coated T75 flasks. After 3–7 days of incubation, cells are 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, alpha-smooth muscle actin

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

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

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

Laboratory Applications

• Tumor-stroma interaction studies

• Adhesion and migration assays

• Immunoprecipitation, Western blot, and PCR

• Immunofluorescence and flow cytometry

• CAF-based pancreatic cancer modeling

Shipping

Frozen vials shipped on dry ice

Post-Thaw Recovery Protocol

1. Thawing & Aseptic Handling

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

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

   • Disinfect vial with 70% ethanol.

   • Proceed under strict aseptic conditions.

2. Recovery & Seeding

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

   • Discard supernatant and resuspend the pellet in fresh CAF medium.

   • Pre-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.

   • Pre-equilibrate growth medium in the incubator for 15 minutes (pH 7.0–7.6).

Subculturing Instructions

Volumes below apply to 75 cm² flasks. Adjust as needed.

1. Remove and discard the culture medium.

2. Rinse the cell layer with 1× PBS.

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

   • Do not tap or shake the flask.

   • For resistant cells, warm or extend incubation.

4. Pre-coat a new flask with AlphaBioCoat. Rinse and discard PBS.

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

6. Plate appropriate aliquots into 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

• Catalog #: CGM5

• Freeze at –80°C overnight in an isopropanol freezing container

• Transfer to liquid nitrogen for long-term storage

Product Usage

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