Immortalized Human Primary Brain Vascular Fibroblasts (HBVF)

$3,000.00

Immortalized Human Primary Brain Vascular Fibroblasts (HBVFs) are derived from cerebral vasculature and genetically modified for extended proliferative capacity while retaining key physiological properties. These cells exhibit characteristic fibroblast morphology and maintain expression of vascular-specific markers including collagen I/III, fibronectin, and α-SMA, while testing negative for endothelial (vWF/Factor VIII) and epithelial (cytokeratin 18) contaminants. Certified free of mycoplasma, bacteria, yeast, and fungi, HBVFs demonstrate stable growth for 15+ passages in specialized fibroblast medium when cultured on collagen-coated surfaces at 37°C/5% CO₂. Ideal for studying neurovascular unit dynamics, blood-brain barrier modeling, and cerebrovascular remodeling mechanisms, these cells are supplied cryopreserved at passage 3 with ≥1×10⁶ viable cells/vial and guaranteed doubling times under 48 hours during logarithmic growth phase. HBVFs retain responsiveness to TGF-β and PDGF signaling pathways, making them particularly suitable for investigations of cerebrovascular fibrosis, angiogenesis, and neuroinflammatory processes.

Immortalized Human Primary Brain Vascular Fibroblasts (HBVFs) are derived from cerebral vasculature and genetically modified for extended proliferative capacity while retaining key physiological properties. These cells exhibit characteristic fibroblast morphology and maintain expression of vascular-specific markers including collagen I/III, fibronectin, and α-SMA, while testing negative for endothelial (vWF/Factor VIII) and epithelial (cytokeratin 18) contaminants. Certified free of mycoplasma, bacteria, yeast, and fungi, HBVFs demonstrate stable growth for 15+ passages in specialized fibroblast medium when cultured on collagen-coated surfaces at 37°C/5% CO₂. Ideal for studying neurovascular unit dynamics, blood-brain barrier modeling, and cerebrovascular remodeling mechanisms, these cells are supplied cryopreserved at passage 3 with ≥1×10⁶ viable cells/vial and guaranteed doubling times under 48 hours during logarithmic growth phase. HBVFs retain responsiveness to TGF-β and PDGF signaling pathways, making them particularly suitable for investigations of cerebrovascular fibrosis, angiogenesis, and neuroinflammatory processes.

Protocol for Culturing Immortalized Human Brain Vascular Fibroblasts (HBVFs)

Materials Required:

  • Immortalized HBVFs

  • Complete Fibroblast Growth Medium

  • 1X PBS (sterile, Ca²⁺/Mg²⁺-free)

  • Cell Detachment Solution

  • Neutralization Solution

  • Tissue culture flasks/plates (T25 recommended)

  • 37°C, 5% CO₂ humidified incubator

  • Centrifuge (for passaging)

  • Hemocytometer or automated cell counter

  • Cryopreservation medium (containing DMSO)

  • Liquid nitrogen storage system

1. Thawing Frozen HBVFs

  1. Pre-warm Medium: Warm complete fibroblast growth medium to 37°C.

  2. Quick Thaw:

    • Retrieve cryovial from liquid nitrogen.

    • Thaw in a 37°C water bath (1–2 min) until only a small ice crystal remains.

    • Decontaminate vial with 70% ethanol.

  3. Transfer Cells:

    • Gently pipet cell suspension into a 15 mL conical tube.

    • Slowly dilute with 5–10 mL pre-warmed medium (dropwise to minimize osmotic shock).

  4. Centrifuge: Spin at 200 × g for 5 min to pellet cells.

  5. Resuspend & Plate:

    • Aspirate supernatant and resuspend in 5 mL fresh medium.

    • Transfer to an AlphaBioCoat-coated T25 flask.

    • Incubate at 37°C, 5% CO₂.

2. Routine Maintenance & Passaging

  1. Monitor Growth:

    • Check cells daily (healthy HBVFs exhibit spindle-shaped morphology).

    • Passage at 80–90% confluency (avoid overgrowth).

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

  3. Passaging Steps:

    • Wash cells with PBS (2–3 mL for T25).

    • Add 1 mL Cell Detachment Solution (T25) and incubate at 37°C for 2–5 min (monitor detachment).

    • Neutralize with 2–3 mL Neutralization Solution.

    • Collect cells by gentle pipetting.

    • Centrifuge (optional, 200 × g, 5 min) if residual enzyme removal is needed.

    • Resuspend in fresh medium and split 1:2 to 1:4 into new flasks.

3. Cryopreservation

  1. Harvest Cells: Follow passaging protocol.

  2. Count & Adjust Density: Resuspend at 1–5 × 10⁶ cells/mL in freezing medium.

  3. Aliquot: Transfer 1 mL/vial into cryovials.

  4. Slow Freeze:

    • Use a controlled-rate freezing container at –80°C overnight.

    • Transfer to liquid nitrogen for long-term storage.

4. Key Considerations & Troubleshooting

✔ Morphology Check:

  • Healthy HBVFs: Elongated, spindle-shaped.

  • Unhealthy signs: Rounding, detachment (indicates stress or contamination).
    ✔ Avoid Overgrowth: Prolonged confluency may trigger senescence.
    ✔ Contamination Control: Use antibiotics and sterile techniques (mycoplasma testing recommended).
    ✔ Optimal Passage Range: Use within 20–30 passages for consistent results.

Expected Outcomes

  • Adhesion: Within 4–6 hours.

  • Proliferation: Visible within 24–48 hours.

  • Doubling Time: ~24–36 hours (varies with culture conditions).

  • Typical Applications:

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

    • Fibrosis and extracellular matrix studies

    • Neuroinflammation and cytokine response assays