Immortalized Human Primary Bronchial Fibroblasts

Catalog Number: IHPBF001
Product Format: Frozen or T-25 Flask
Cell Number: 500,000 cells/vial

Materials Required

• Immortalized Human Primary Bronchial Fibroblasts

• Complete Immortalized Bronchial Fibroblast Growth Medium (IBFGM001)

• 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 from water bath and disinfect the vial with 70% ethanol.

3. Transfer Cells:

   • Pipette the contents into a 15 mL conical tube.

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

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

5. Resuspend & Plate:

   • Aspirate supernatant.

   • Resuspend in fresh medium.

   • Transfer cells into an Alphabiocoat-coated T25 flask.

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

2. Routine Maintenance & Passaging

1. Monitor Growth:

   • Inspect 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 gently rinse cells with 1–2 mL of PBS.

3. Detachment:

   • Add 1–2 mL of Cell Detachment Solution.

   • Incubate at 37°C for 2–5 minutes. Monitor under microscope for detachment.

4. Neutralization:

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

5. Cell Collection:

   • Pipette up and down to ensure a single-cell suspension.

6. Centrifuge (Optional):

   • If needed, 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, depending on growth rate.

3. Cryopreservation

1. Harvest Cells: Follow Steps 2.2 to 2.5.

2. Count Cells: Use a hemocytometer or automated counter.

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

4. Aliquot: Dispense 1 mL per cryovial.

5. Freeze Slowly:

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

   • Transfer to liquid nitrogen for long-term storage.

4. Quality Control & Troubleshooting

• Contamination Control: Inspect regularly for signs of bacterial or fungal contamination (e.g., turbidity, color change).

• Morphology: Healthy fibroblasts are elongated and spindle-shaped.

  • Rounding or detachment may indicate over-confluency or stress.

• Doubling Time: Monitor growth rates; immortalized fibroblasts typically double every 24–48 hours.

Expected Results

• Cells should adhere within 24 hours and proliferate within 24–48 hours.

• Cells are typically passaged 20–30 times before senescence, if not fully immortalized.

Product Usage

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