Immortalized Human Primary Pancreatic Fibroblasts

Catalog Number: IHPPF001
Product Format: Frozen or T-25 Flask
Cell Number: 500,000 cells/vial

Materials Required

• Immortalized Human Primary Pancreatic Fibroblasts

• Complete Immortalized Pancreatic Fibroblast Growth Medium (IPFGM001)

• Phosphate-buffered saline (1× PBS), sterile (PBS001)

• Cell Detachment Solution

• Alphabiocoat-coated Tissue Culture Flasks (T25)

• 37°C, 5% CO₂ humidified incubator

• Centrifuge (for passaging, if needed)

• Hemocytometer or automated cell counter

• Cryopreservation medium

• Liquid nitrogen storage system (for long-term storage)

Protocol

1. Thawing Frozen Cells

1. Prepare Media: Warm complete fibroblast growth medium to 37°C.

2. Quick Thaw:

   • Remove cryovial from liquid nitrogen.

   • Thaw in a 37°C water bath for 1–2 minutes.

   • Remove and disinfect with 70% ethanol.

3. Transfer Cells:

   • Pipette contents into a 15 mL conical tube.

   • Slowly add 5–10 mL of pre-warmed medium dropwise to minimize osmotic shock.

4. Centrifuge: Spin at 200 × g for 5 minutes.

5. Resuspend & Plate:

   • Aspirate the supernatant.

   • Resuspend in fresh medium.

   • Transfer to an Alphabiocoat-coated T25 flask.

6. Incubate: Place in a 37°C, 5% CO₂ humidified incubator.

2. Routine Maintenance & Passaging

1. Monitor Growth:

   • Observe cells daily under a microscope.

   • Fibroblasts should appear spindle-shaped and reach ~80–90% confluency before passaging.

2. Aspirate Medium: Remove spent medium and rinse gently with 1–2 mL PBS.

3. Detachment:

   • Add 1–2 mL of Cell Detachment Solution.

   • Incubate at 37°C for 2–5 minutes.

   • Monitor under a microscope for detachment.

4. Neutralize:

   • Add 2–3 mL of Neutralization Solution directly to the flask.

5. Cell Collection: Pipette up and down to ensure a single-cell suspension.

6. Centrifuge (Optional): Spin at 200 × g for 5 minutes and resuspend in fresh medium.

7. Seeding: Plate cells at a 1:2 to 1:4 split ratio based on growth rate.

3. Cryopreservation

1. Harvest Cells: Follow steps 2.2–2.5.

2. Count Cells: Use a hemocytometer to determine concentration.

3. Freezing: Resuspend cells in cryopreservation medium at 1–5 × 10⁶ cells/mL.

4. Aliquot: Dispense 1 mL per cryovial.

5. Freeze Slowly:

   • Use an isopropanol freezing container at –80°C overnight.

   • Transfer vials to liquid nitrogen for long-term storage.

4. Quality Control & Troubleshooting

• Contamination Check: Inspect cultures regularly for cloudiness or pH change.

• Morphology: Healthy fibroblasts should maintain spindle-shaped morphology.

  • Rounding may indicate over-confluence or stress.

• Doubling Time: Immortalized lines should proliferate reliably (24–48 hrs typical).

Expected Results

• Adhesion: Within 24 hours

• Proliferation: Doubling time ~24–48 hours

• Cells typically support 20–30 passages before signs of senescence (if not fully immortalized)

Product Usage

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