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.
Applications angiogenesis, leukocyte trafficking, wound healing, inflammation, circulation, tumor growth and metastasis.cancer research, endothelium function, cell trafficking, surface molecule interactions, drug screening, toxicological studies for the pharmaceutical and cosmetic industry
Storage Conditions liquid nitrogen vapor phase Derivation Microvascular endothelial cells isolated from human foreskins 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 The cells express von Willebrand's factor (vWF), cell adhesion molecules ICAM-1 and are capable of acetylated LDL uptake. Ref (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)
Comments CRL-3243, HMEC-1, has been well characterized and 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 dermal 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 µg/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). 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℃ 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℃.
Subcultivation Ratio: 1:6 to 1:12 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