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HULEC-5a; Lung Endothelium; Human (Homo sapiens) Item ID: ATCCRL3244

Permits and Restrictions

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Organism Homo sapiens, human
Tissue lung
Cell Type microvascular endothelium
Product Format frozen
Morphology endothelial-like
Culture Properties adherent
Biosafety Level 2  [Cells contain SV-40 viral DNA sequences]

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
Age newborn
Gender male
Applications Investigating the role of endothelium in infection by virus and bacteria, cancer research, tumor metastastis, endothelium function, cell trafficking, surface molecule interactions
Storage Conditions liquid nitrogen vapor phase
Derivation

Microvascular endothelial cells isolated from human lungs were transfected with pSVT vector,  a pBR-322 based plasmid containing the coding region for Simian virus 40A gene product, large T antigen.
Receptor Expression neonatal FcRn receptor (FcRn) (Goebl N, et al. Neonatal Fc Receptor Mediates Internalization of Fc in Transfected Human Endothelial Cells. Mol. Biol. Cell 19: 5490-5505, 2008. PubMed: 18843053)
Comments CRL-3244, HULEC-5a, has been shown to retain many of the characteristics of endothelial cells. These immortalized cells, because they are a continously renewable source of human endothelial microvascular cells, can be used as a replacement for primary human lung endothelial cells for many research studies.
Complete Growth Medium The base medium for this cell line is MCDB131 (without L-Glutamine). To make the complete growth medium, add the following components to the base medium:
  • 10ng/mL Epidermal Growth Factor (EGF)
  • 1 &microg/mL Hydrocortisone
  • 10 mM Glutamine
  • fetal bovine serum (FBS) to a final concentration of 10%
Subculturing Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. 
  1. Remove and discard culture medium. Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) (ATCC 30-2200) or 0.25% Trypsin – 0.53mM EDTA (ATCC 30-2101) solution to remove all traces of serum which contains trypsin inhibitor. 

  2. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes). 
    3. Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal. 
  3. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. 
  4. Add appropriate aliquots of the cell suspension to new culture vessels. Incubate cultures at 37°C.
Subcultivation Ratio: 1:5 to 1:10 is recommended. Medium Renewal: Do not feed cells with growth medium between subcultures or cells become very tightly attached and will be difficult to disperse with trypsin-EDTA.
Cryopreservation Freeze Medium: Complete growth medium, 92.5%; DMSO, 7.5% Storage Temperature: liquid nitrogen vapor phase
Culture Conditions Temperature: 37°C
STR Profile Amelogenin: X CSF1PO: 11, 12 D13S317: 13, 14 D16S539:  9, 10 D5S818: 11, 13 D7S820: 8, 10 THO1: 6, 7 TPOX: 8, 11 vWA: 16, 17
Name of Depositor Kathryn Kellar, Ph. D.
Year of Origin November 1990
References

Lawrence EC et al. Establishment of immortalized human lung microvascular endothelial cell lines. Chest 108(3) Supplement:126S, 1995.

Ades EW, et al. HMEC-1: Establishment of an Immortalized Human Microvascular Endotheilal Cell Line. J. Invest. Dermatol. 99(6): 683-690, 1992. PubMed: 1361507

Mehta P, et al. Entry and intracellular replication of Mycobacterium tuberculosis in cultured human microvascular endothelial cells. Microb. Pathog. 41: 119-124, 2006. PubMed: 16860530

Goebl N, et al. Neonatal Fc Receptor Mediates Internalization of Fc in Transfected Human Endothelial Cells. Mol. Biol. Cell 19: 5490-5505, 2008. PubMed: 18843053

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