Ordering Information:

Catalog Number: UBP01

Format: Cryopreserved or proliferating cultures

Quantity: 500,000 cells/vial (custom quantities available)

For Research Use Only (RUO). Not for diagnostic or therapeutic use.

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1. Product Description

Human Umbilical Vein Endothelial Cells (HUVECs) are primary cells isolated from the umbilical vein of human umbilical cords. These cells are widely used in vascular biology, angiogenesis research, drug discovery, and toxicity testing due to their endothelial properties.

2. Cell Characteristics

• Origin: Human umbilical vein
• Cell Type: Primary endothelial cells
• Morphology: Cobblestone monolayer when confluent
• Markers: Positive for CD31 (PECAM-1), von Willebrand Factor (vWF), VE-Cadherin (CD144)
• Growth Properties: Adherent, requires Endothelial Cell Growth Media

3. Culture Conditions

• Basal Medium: Endothelial Cell Growth Medium
• Surface: Universal Coated flasks/plates
• Incubation Conditions: 37°C, 5% CO₂, humidified atmosphere

4. Cell Handling & Subculturing

• Passaging: Universal detachment solution
• Split Ratio: 1:2 to 1:4 (recommended)
• Seeding Density: 5,000–10,000 cells/cm²
• Doubling Time: ~24–48 hours (depending on conditions)

5. Quality Control

• Viability: ≥90% (Trypan Blue exclusion)
• Sterility Test: Negative for bacteria, fungi, mycoplasma
• Functional Assays: Tube formation assay, LDL uptake, NO production

6. Applications

• Angiogenesis & Vascular Biology Studies
• Drug Screening & Toxicity Testing
• Inflammation & Immune Response Research
• Wound Healing & Tissue Engineering

7. Storage & Shipping

• Fresh Cells: Shipped in culture medium at ambient temperature (live delivery)
• Cryopreserved Cells: Shipped in liquid nitrogen (dry ice for transit)
• Storage:
• Fresh Cells: Use within 3–5 days
• Cryopreserved Cells: Store in liquid nitrogen (≤ -150°C)

8. Limitations

• Finite lifespan (~5–10 passages before senescence)
• Sensitive to shear stress and contamination
• Requires specialized growth factors for optimal performance

Protocols for Thawing Cells and Subculture**

A) Pre-coating of T25 Flasks:

To prepare T25 flasks for cell culture, begin by adding 2 ml of Universal Coating Solution into each flask. Ensure that the solution fully covers the entire surface area of the flask. After allowing the coating to sit for 5 minutes, carefully aspirate the excess solution. Rinse the flask thoroughly two times with 1x PBS (phosphate-buffered saline) to remove any remaining coating agent. Once rinsed, the flask is primed and ready for cell seeding.

B) Thawing Frozen Cell Vials:

Thaw the frozen cell vial by placing it in a 37°C water bath. Gently swirl the vial until only a small amount of ice remains visible. At this stage, take a 70% ethanol wipe and use it to disinfect the outer surface of the vial. Proceed to transfer the thawed cells into the pre-coated T25 flask, adding 10 ml of appropriate growth medium. The cells typically adhere and become confluent overnight, making them ready for the next passage.

C) Passage of Cells:

To passage the cells, first rinse the cell layer in the T25 flask twice with 5 ml of 1x PBS at room temperature (RT). Following the rinse, introduce 2 ml of Universal Detachment Solution (also at RT) into the flask. Allow the solution to act for no more than 30 seconds, then gently aspirate to remove excess fluid.

D) Detaching Cells:

Leave the T25 flask containing the cells at either room temperature or 37°C for approximately 1 minute. Most cells will typically begin detaching from the surface within 1 to 2 minutes. To confirm detachment, you can monitor the cells under a microscope. Once you observe that the majority of the cells appear rounded, tap the flask gently against the bench surface. You should see the cells start to move across the flask surface while observing them microscopically.

E) Cell Recovery:

After detaching the cells, add 5 ml of Neutralization Solution to halt the action of the detachment solution. Next, centrifuge the cell suspension at 800RPM for 5 minutes to pellet the cells at the bottom of the tube.

G) Re-suspension and Distribution:

Carefully re-suspend the cell pellet in either 10 or 15 ml of fresh growth medium. Subsequently, distribute 5 ml of this re-suspended cell solution into each of 2 or 3 pre-coated T25 flasks, aiming for a subculture ratio of 1/2 to 1/3.

H) Medium Renewal:

It is recommended to change the growth medium every 2 to 3 days. Under these conditions, the cells should reach confluence within approximately 7 days, especially when split at the 1/3 ratio.

I) Preparing Quiescent Cells: 

To induce quiescence in the cells prior to experimental usage, replace their growth medium with Endothelial Basal Medium containing 0.5% Fetal Bovine Serum (FBS) for a period of about 8 to 12 hours, ensuring they are adequately prepared for subsequent experiments.