Immortalized Human Primary Brain Vascular Fibroblasts (HBVFs)

Catalog Number: IHPBV001
Product Format: Frozen or T-25 Flask
Cell Number: 500,000 cells/vial

Materials Required

• Immortalized HBVFs

• Complete Fibroblast Growth Medium

• 1× PBS (sterile, Ca²⁺/Mg²⁺-free)

• Cell Detachment Solution

• Neutralization Solution

• Tissue culture flasks/plates (T25)

• 37°C, 5% CO₂ humidified incubator

• Centrifuge (for passaging)

• Hemocytometer or automated cell counter

• Cryopreservation medium

• Liquid nitrogen storage system

Protocol

1. Thawing Frozen HBVFs

1. Pre-warm Medium: 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.

   • Spray or wipe the vial with 70% ethanol before opening.

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 cells in 5 mL fresh medium.

   • Transfer to an AlphaBioCoat-coated T25 flask.

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

2. Routine Maintenance & Passaging

1. Monitor Growth:

   • Observe daily under a microscope.

   • Cells should be spindle-shaped and 80–90% confluent before passaging.

2. Medium Change: Replace medium every 2–3 days.

3. Passaging Procedure:

   • Aspirate spent medium.

   • Rinse gently with 2–3 mL PBS (for T25 flask).

   • Add 1 mL Cell Detachment Solution (T25) or 2–3 mL (T75).

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

   • Add 2–3 mL Neutralization Solution.

   • Pipette to disaggregate clumps.

   • Optional: Centrifuge at 200 × g for 5 minutes.

   • Resuspend and split cells at a 1:2 to 1:4 ratio.

   • Use 5–8 mL fresh medium for T75 flask.

3. Cryopreservation

1. Harvest Cells: Follow passaging steps.

2. Count Cells: Adjust to 1–5 × 10⁶ cells/mL in freezing medium.

3. Aliquot: Dispense 1 mL per cryovial.

4. Slow Freeze:

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

   • Transfer to liquid nitrogen for long-term storage.

4. Key Considerations & Troubleshooting

• Morphology Check:
  Healthy HBVFs should be elongated and spindle-shaped.
  Rounding or detachment can indicate stress or over-confluence.

• Avoid Overgrowth:
  Prolonged confluency may induce senescence or differentiation.

• Contamination Control:
  Use sterile techniques and antibiotics as needed.

• Low Passage Use:
  Although immortalized, limit use to ≤30 passages for best performance.

Expected Results

• Adhesion: 4–6 hours post-plating

• Proliferation: Begins within 24–48 hours

• Doubling Time: ~24–36 hours (varies)

• Applications:

  • Blood-brain barrier (BBB) co-culture models

  • Fibrosis research

  • Neuroinflammatory response studies

Product Usage

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