The cells represent an effective cell model for studying endothelial cell biology including signal transduction and angiogenesis.

The telomerase-immortalized human microvascular endothelium cell line, TIME, was derived from a primary culture of neonatal foreskin microvascular endothelial cells (HMVEC) of the dermis.xa0

The primary HMVECs were immortalized by infection with the retrovirus WZLblast3:hTERT and cultured in complete growth medium containing blasticidin.xa0

The immortalized cells do not undergo growth arrest in culture due to the exogenous hTERT expression.

When plated on Matrigel, TIME cells undergo tubule formation exhibiting capillary-like structures.

Volumes used in this protocol are for 75xa0cm² flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
Note: Subculture when cultures are about 80% confluent.

1. Prior to subculturing, determine the number of flasks needed. Add the appropriate volume of medium to each flask and allow the flasks to equilibrate in a 37°C, 5% CO₂, humidified incubator for at least 30 minutes. If not using vented caps, loosen caps of flasks.
2. Remove and discard spent medium.
3. Briefly rinse the cells with Dulbeccos Phosphate Buffered Saline (D-PBS, ATCC 30-2200) and discard rinse solution.
4. Add 2.0 to 3.0 mL room temperature Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) to the flask. Incubate at 37°C for 5 min (until cells have detached).
5. Neutralize trypsin by adding an equal volume of room temperature 2% FBS in D-PBS.
6. Transfer cells to a centrifuge tube. Rinse the flask with an additional room temperature 2% FBS in D-PBS and pool into centrifuge tube with cells.
7. Centrifuge cells at 250 x g for 10 min at room temperature.
8. Remove supernatant. Resuspend pellet in 6.0 to 8.0 mL Complete Growth Medium.
9. Count cells, and seed 5 x 10³ to 8 x 10³ viable cells/cm² to new culture vessels. Subculture when cells become 80 to 90% confluent, which normally yield approximately 3.0 x 10⁴ viable cells/cm².
10. Incubate cultures at 37°C in a 5% CO₂ humidified incubator.

Note:xa0For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney.

Venetsanakos E, et al. Induction of tubulogenesis in telomerase-immortalized human microvascular endothelial cells by glioblastoma cells. Exp. Cell Res. 273(1):21-33, 2002. PubMed: 11795943

Lagunoff M, et al. De novo infection and serial transmission of Kaposis sarcoma-associated herpesvirus in cultured endothelial cells. J. Virol. 76(5):2440-2448, 2002. PubMed: 11836422

Yi X, et al. Both transcriptional and posttranscriptional mechanisms regulate human telomerase template RNA levels. Mol. Cell. Biol. 19(6): 3989-3997, 1999. PubMed: 10330139

Bodnar AG, et al. Extension of life-span by introduction of telomerase into normal human cells. Science 279: 349-352, 1998. PubMed: 9454332

Caputo JL. Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988

Freshney RI. Culture of Animal Cells: A Manual of Basic Technique, 4th edition. New York: Wiley Liss; 2000. For more information on enzymatic dissociation and subculturing of cell lines see Chapter 10.