Immortalized Human Primary Umbilical Vein Endothelial Cells (HUVECs)

Catalog Number: IHPH001
Product Format: Frozen or T-25 Flask
Cell Number: 500,000 cells/vial

Materials Required

• Immortalized HUVECs

• Complete Endothelial Cell Growth Medium

• Alphabiocoat-coated flasks or plates

• Sterile phosphate-buffered saline (PBS)

• Cell Detachment Solution

• Neutralizing Solution

• Tissue culture flasks/plates (T25 or multiwell plates)

• 37°C, 5% CO₂ humidified incubator

• Centrifuge (for passaging)

• Hemocytometer or automated cell counter

• Cryopreservation medium

• Liquid nitrogen storage system

Protocol

1. Thawing Frozen Immortalized HUVECs

1. Prepare Coated Flasks:

   • Coat a T25 flask with 1 mL Alphabiocoat.

   • Incubate for 30 minutes at 37°C or overnight at 4°C.

   • Aspirate excess and rinse with 1× PBS.

2. Warm Medium:

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

3. Quick Thaw:

   • Remove cryovial from liquid nitrogen.

   • Thaw in a 37°C water bath for 1–2 minutes.

4. Dilute Cells:

   • Transfer thawed cell suspension to a 15 mL conical tube.

   • Slowly add 5 mL of warm medium dropwise to reduce osmotic shock.

5. Centrifuge:

   • Spin at 200 × g for 5 minutes.

6. Resuspend & Plate:

   • Aspirate the supernatant.

   • Resuspend cells in fresh medium.

   • Transfer to the coated flask.

7. Incubate:

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

2. Routine Maintenance & Passaging

1. Monitor Growth:

   • Check cells daily under a microscope.

   • Look for cobblestone morphology at confluence.

2. Medium Change:

   • Replace medium every 2–3 days. HUVECs are sensitive to waste buildup.

3. Passaging at 80–90% Confluence:

   • Aspirate medium and rinse gently with PBS.

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

   • Incubate at 37°C for 2–3 minutes.

   • Confirm detachment under a microscope.

   • Add 2–3 mL of Neutralization Solution.

   • Pipette to ensure single-cell suspension.

   • (Optional) Centrifuge at 200 × g for 5 minutes.

   • Resuspend and plate cells at a 1:2 to 1:4 split ratio in fresh Alphabiocoat-coated flasks.

3. Cryopreservation

1. Harvest Cells: Follow passaging procedure.

2. Count Cells: Adjust to 1–5 × 10⁶ cells/mL using a hemocytometer or cell counter.

3. Aliquot: Dispense 1 mL per cryovial.

4. Freeze Slowly:

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

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

4. Key Considerations & Troubleshooting

• ✔ Morphology:
  Cells should maintain cobblestone morphology. Elongated or spindled cells may indicate contamination or stress.

• ✔ Avoid Over-Confluence:
  Prolonged confluency may lead to cell death or detachment.

• ✔ Low Passage Use:
  For optimal results, use cells within 20–30 passages.

• ✔ Contamination Control:
  Use sterile technique and appropriate antibiotics as HUVECs are prone to contamination.

Expected Results

• Adhesion: 4–6 hours

• Proliferation: Begins within 24–48 hours

• Doubling Time: ~24–36 hours

• Typical Applications:

  • Angiogenesis assays

  • Barrier function studies

  • Inflammation modeling

Product Usage

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