Immortalized Human Primary Umbilical Vein Endothelial Cells (HUVEC)

$3,000.00

Immortalized Human Primary Umbilical Vein Endothelial Cells (HUVECs) are vascular cells derived from umbilical veins and genetically modified to maintain proliferative capacity while preserving essential endothelial functions. These cells exhibit characteristic cobblestone morphology and express standard endothelial markers including von Willebrand Factor (vWF), CD31, and VE-cadherin, while testing negative for smooth muscle (α-SMA) and mesenchymal (vimentin) contaminants. Certified free of mycoplasma, bacteria, fungi, and endotoxins, the immortalized HUVECs demonstrate stable growth for 40+ passages in specialized endothelial growth medium when cultured on gelatin-coated surfaces at 37°C/5% CO₂. Particularly valuable for vascular and angiogenesis research, they are ideal for studying endothelial barrier function, inflammatory responses, thrombosis, and vascular permeability mechanisms. Supplied cryopreserved at passage 5 with ≥1×10⁶ viable cells/vial, these cells maintain critical vascular functions including nitric oxide production, LDL uptake, and inducible expression of adhesion molecules (ICAM-1, VCAM-1). Their extended lifespan and preserved angiogenic potential make them excellent for both short-term functional assays and long-term co-culture systems modeling vascular biology, with applications in drug discovery, toxicology screening, and cardiovascular disease research. The cells consistently form tubular structures in Matrigel assays and demonstrate appropriate responsiveness to pro-angiogenic and inflammatory stimuli, providing a reliable model for studying endothelial cell behavior in physiological and pathological conditions.

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Immortalized Human Primary Umbilical Vein Endothelial Cells (HUVECs) are vascular cells derived from umbilical veins and genetically modified to maintain proliferative capacity while preserving essential endothelial functions. These cells exhibit characteristic cobblestone morphology and express standard endothelial markers including von Willebrand Factor (vWF), CD31, and VE-cadherin, while testing negative for smooth muscle (α-SMA) and mesenchymal (vimentin) contaminants. Certified free of mycoplasma, bacteria, fungi, and endotoxins, the immortalized HUVECs demonstrate stable growth for 40+ passages in specialized endothelial growth medium when cultured on gelatin-coated surfaces at 37°C/5% CO₂. Particularly valuable for vascular and angiogenesis research, they are ideal for studying endothelial barrier function, inflammatory responses, thrombosis, and vascular permeability mechanisms. Supplied cryopreserved at passage 5 with ≥1×10⁶ viable cells/vial, these cells maintain critical vascular functions including nitric oxide production, LDL uptake, and inducible expression of adhesion molecules (ICAM-1, VCAM-1). Their extended lifespan and preserved angiogenic potential make them excellent for both short-term functional assays and long-term co-culture systems modeling vascular biology, with applications in drug discovery, toxicology screening, and cardiovascular disease research. The cells consistently form tubular structures in Matrigel assays and demonstrate appropriate responsiveness to pro-angiogenic and inflammatory stimuli, providing a reliable model for studying endothelial cell behavior in physiological and pathological conditions.

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Materials Required

  • Immortalized Human Umbilical Vein Endothelial Cells (HUVECs)

  • Complete Endothelial Cell Growth Medium

  • Alphabiocoat-Coated Flasks or Multiwell Plates

  • Sterile Phosphate-Buffered Saline (1× PBS)

  • Cell Detachment Solution

  • Neutralizing Solution

  • Tissue Culture Flasks or Plates (T25 or multiwell formats)

  • 37°C Humidified Incubator with 5% CO₂

  • Centrifuge (for passaging)

  • Hemocytometer or Automated Cell Counter

  • Cryopreservation Medium

  • Liquid Nitrogen Storage System (for long-term storage)

Protocol

1. Thawing Immortalized HUVECs

  • Prepare Flask: Coat a T25 flask with 1 mL of Alphabiocoat. Incubate for 30 minutes at 37°C or overnight at 4°C. Aspirate excess and rinse once with sterile PBS.

  • Warm Medium: Pre-warm complete endothelial growth medium to 37°C.

  • Thaw Cells: Remove cryovial from liquid nitrogen and rapidly thaw in a 37°C water bath for 1–2 minutes.

  • Dilute Cell Suspension: Transfer contents to a 15 mL conical tube and slowly add 5 mL of warm medium dropwise to reduce osmotic shock.

  • Centrifuge: Spin at 200 × g for 5 minutes to pellet cells.

  • Resuspend & Plate: Remove supernatant, resuspend pellet in fresh growth medium, and transfer to the prepared Alphabiocoat-coated flask.

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

2. Routine Culture Maintenance & Passaging

  • Daily Observation: Inspect cultures microscopically. Healthy HUVECs display cobblestone morphology when confluent.

  • Media Replacement: Change medium every 2–3 days to avoid accumulation of metabolic waste, which HUVECs are sensitive to.

  • Passage Cells at 80–90% Confluence:

    • Aspirate spent medium and rinse with sterile PBS.

    • Add Cell Detachment Solution (1 mL for T25 flasks) and incubate for 2–3 minutes at 37°C.

    • Observe detachment; cells should round up and lift off the surface.

    • Neutralize with 2–3 mL of Neutralizing Solution.

    • Gently pipette to achieve a single-cell suspension.

    • Optional: Centrifuge at 200 × g for 5 minutes to remove any residual enzymes.

    • Resuspend in fresh medium and reseed at a 1:2 to 1:4 split ratio into newly coated flasks.

3. Cryopreservation

  • Harvest Cells: Follow standard passaging procedure to collect cells.

  • Count Cells: Determine viable cell concentration and adjust to 1–5 × 10⁶ cells/mL using cryopreservation medium.

  • Aliquot: Dispense 1 mL per sterile cryovial.

  • Controlled Freezing: Use a controlled-rate freezing container or an isopropanol-based freezing chamber at –80°C overnight.

  • Storage: Transfer vials to liquid nitrogen for long-term storage the next day.

4. Key Considerations & Troubleshooting

  • ✔ Morphology Monitoring: Cells should retain cobblestone morphology. Presence of elongated or spindle-like cells may indicate contamination or phenotypic drift.

  • ✔ Avoid Over-Confluency: Allowing cells to remain confluent too long can lead to detachment or apoptosis.

  • ✔ Low Passage Usage: Although immortalized, it’s best to use cells within 20–30 passages to maintain consistency.

  • ✔ Sterility: HUVECs are sensitive to contamination—use strict aseptic technique and consider antibiotics in culture medium.

Expected Results

  • Attachment: Cells typically adhere within 4–6 hours post-seeding.

  • Proliferation: Active growth observed within 24–48 hours.

  • Doubling Time: Approximately 24–36 hours, depending on media and cell conditions.

  • Applications: Suitable for angiogenesis assays, barrier integrity studies, and vascular inflammation models.