Immortalized Human Primary Coronary Artery Endothelial Cells

Catalog Number: IHPC002
Product Format: Frozen or T-25 Flask
Cell Number: 500,000 cells/vial

Materials Required

• Immortalized Human Primary Coronary Artery Endothelial Cells

• Complete Endothelial Cell Growth Medium

• Alphabiocoat-coated flasks or plates

• Sterile phosphate-buffered saline (PBS)

• Cell Detachment Solution

• Neutralizing Solution

• Tissue culture flasks or 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 Cells

1. Prepare Coated Flask:

   • Coat a T25 flask with 1 mL of Alphabiocoat.

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

   • Aspirate the excess before use.

2. Rinse:

   • Wash the flask with sterile 1× PBS.

3. Warm Medium:

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

4. Quick Thaw:

   • Remove cryovial from liquid nitrogen.

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

5. Dilute Cells:

   • Transfer contents to a 15 mL tube.

   • Add 5 mL warm medium dropwise to minimize osmotic shock.

6. Centrifuge:

   • Spin at 200 × g for 5 minutes.

7. Resuspend & Plate:

   • Aspirate the supernatant.

   • Resuspend cells in fresh medium.

   • Transfer to a prepared Alphabiocoat-coated flask.

8. Incubate:

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

2. Routine Maintenance & Passaging

1. Monitor Growth:

   • Observe daily for cobblestone morphology at confluence.

2. Medium Change:

   • Replace medium every 2–3 days. Endothelial cells are sensitive to waste accumulation.

3. Passage at 80–90% Confluence:

   • Aspirate medium and rinse with PBS.

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

   • Incubate 2–3 minutes at 37°C.

   • Observe under a microscope for rounding and detachment.

   • Add 2–3 mL Neutralization Solution.

   • Pipette gently to create a single-cell suspension.

   • (Optional) Centrifuge at 200 × g for 5 minutes to remove residual enzyme.

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

3. Cryopreservation

1. Harvest Cells: Follow passaging steps above.

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

3. Aliquot: Dispense 1 mL per cryovial.

4. Freeze Slowly:

   • Use a controlled-rate freezer or isopropanol freezing container.

   • Store at –80°C overnight, then transfer to liquid nitrogen.

4. Key Considerations & Troubleshooting

• ✔ Morphology Check:
  Cells should exhibit a cobblestone monolayer at confluence. Elongated cells may suggest contamination or stress.

• ✔ Avoid Over-Confluency:
  Prolonged contact inhibition can lead to cell death or detachment.

• ✔ Use at Low Passage:
  Use within 20–30 passages for optimal consistency, even if immortalized.

• ✔ Contamination Control:
  Practice strict aseptic technique. Endothelial cells are highly sensitive to microbial contamination.

Expected Results

• Adhesion: 4–6 hours post-plating

• Proliferation: 24–48 hours

• Doubling Time: ~24–36 hours (varies with media and conditions)

Product Usage

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