Immortalized Human Primary Colonic Fibroblasts

Catalog Number: IHPCF001
Product Format: Frozen or T-25 Flask
Cell Number: 500,000 cells/vial

Materials Required

• Immortalized Human Primary Colonic Fibroblasts

• Complete Immortalized Colonic Fibroblast Growth Medium (ICFGM001)

• 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 until a small ice crystal remains.

   • Disinfect vial with 70% alcohol.

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 supernatant.

   • Resuspend pellet in fresh medium.

   • Transfer cells to an Alphabiocoat-coated T25 flask.

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

2. Routine Maintenance & Passaging

1. Monitor Growth:

   • Observe cells daily under a microscope.

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

2. Aspirate Medium: Remove spent medium and rinse cells gently with 1–2 mL PBS (T25).

3. Detachment: Add 1–2 mL Cell Detachment Solution. Incubate at 37°C for 2–5 minutes.

4. Neutralize: Add 2–3 mL Neutralization Solution directly to the flask.

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

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

7. Reseeding: Seed cells at a 1:2 to 1:4 split ratio depending on proliferation rate.

3. Cryopreservation

1. Harvest Cells: Follow steps 2.2 to 2.5.

2. Count Cells: Use hemocytometer or automated counter to determine cell concentration.

3. Prepare Freezing Medium: Adjust to 1–5 × 10⁶ cells/mL.

4. Aliquot: Dispense 1 mL per cryovial.

5. Freeze Slowly:

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

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

4. Quality Control & Troubleshooting

• Contamination Check: Regularly inspect cultures for cloudiness, color changes, or pH shifts.

• Morphology: Cells should retain a spindle-shaped appearance. Rounding or detachment may signal stress or overgrowth.

• Doubling Time: Track proliferation. Immortalized cells should double every 24–48 hours under optimal conditions.

Expected Results

• Cells should adhere within 24 hours and begin proliferating within 24–48 hours.

• Typical lifespan: 20–30 passages before senescence (if not fully immortalized).

Product Usage

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