TIME

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. 

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

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.

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

1. Prepare a 25 cm² or a 75 cm² culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents, the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.
2. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
3. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All operations from this point on should be carried out under strict aseptic conditions.
4. Transfer the vial contents to a centrifuge tube containing 9.0 mL of complete culture medium and centrifuge the cell suspension at approximately 125 x g for 5 to 7 minutes.
5. Discard the supernatant and resuspend the cells in fresh growth medium (see the batch-specific information for the recommended dilution ratio). Add this suspension to the prepared culture vessel.
6. Incubate the culture at 37°C in a suitable incubator.
7. A 5% CO₂/95% air atmosphere is recommended if using the medium described on this product sheet.

Volumes used in this protocol are for 75 cm² 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 Dulbecco's 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: For 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.

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

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