Human Breast Cancer-Associated Fibroblasts (CAF)

Catalog Number: HB-CAF11
Applications: Cell Assays
Type: Human Primary Cancer-Associated Fibroblasts (CAFs)
Tissue: Breast
Organism: Homo sapiens (Human)
Format: Frozen
Shipping: Frozen (Dry Ice/Liquid Nitrogen)
Storage: Keep frozen in liquid nitrogen until plating

Storage Recommendation

These cultures should be stored in the vapor phase of liquid nitrogen rather than submerged, in order to maintain optimal cell viability.

Handling Information

Unpacking and Storage Instructions

• Inspect all containers for leakage or breakage.

• Remove the frozen cells from the dry ice packaging and immediately store at temperatures below -130°C, preferably in liquid nitrogen vapor, until ready for use.

Culture Conditions

• Complete Medium: The base medium and necessary additives are available from our company.

• Incubation Temperature: 37°C

• Atmosphere: 5% CO₂ in air

Thawing & Initial Culture Procedure

To ensure the highest cell viability, thaw and initiate culture immediately upon receipt.
Do not store at -70°C as this will result in loss of viability.

1. Thaw the vial in a 37°C water bath using gentle agitation (~2 minutes).

2. Keep the O-ring and cap out of the water to prevent contamination.

3. Once thawed, decontaminate the vial by dipping or spraying with 70% ethanol.

4. Perform all remaining steps under strict aseptic conditions.

5. Transfer vial contents to a centrifuge tube containing 9.0 mL of complete culture medium.

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

7. Discard the supernatant.

8. Resuspend the cell pellet in complete medium using the batch-specific dilution ratio.

9. Transfer the suspension to a 75 cm² tissue culture flask.

10. Important: To avoid excessive alkalinity, pre-incubate the flask with medium at 37°C for at least 15 minutes to allow the pH to stabilize (target range: 7.0–7.6).

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

Subculturing Procedure

The following steps apply to a 75 cm² flask. Adjust all volumes proportionally for other vessel sizes.

1. Remove and discard the culture medium.

2. Briefly rinse the cell layer with 1× PBS, then discard the PBS.

3. Add 2.0–3.0 mL of Cell Detachment Solution.

4. Observe under an inverted microscope until the monolayer is dispersed (typically 5–15 minutes).

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

   • If cells are difficult to detach, place the flask at 37°C to assist.

5. Add 6.0–8.0 mL of complete growth medium and gently pipette to aspirate cells.

6. Transfer the cell suspension to a centrifuge tube and spin at 125 × g for 5–10 minutes.

7. Discard the supernatant.

8. Resuspend the cells in fresh serum-free growth medium.

9. Plate appropriate aliquots into new culture vessels.

10. Incubate at 37°C.

• Subcultivation Ratio: 1:2 to 1:6

• Medium Renewal: Every 2–3 days