HUV-EC-C [HUVEC]

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.

The base medium for this cell line is F-12K Medium (ATCC 30-2004). To make the complete growth medium, add the following components to 450 mL of the base medium:

• 5 mL of a 10 mg/mL stock heparin solution (prepared from Sigma catalog #H3393) for a final concentration of 0.1 mg/mL heparin in complete growth medium 
  • Dissolve 1 g Heparin in 100 mL basal F-12K  and filter to make a 10 mg/mL stock solution
• 50 mL fetal bovine serum (FBS; ATCC 30-2020)
• 500 μL of 30 mg/mL Endothelial Cell Growth Supplement (ECGS)

Note: Because of limited stability, the ECGS should be added to an aliquot of the above culture medium fresh prior to seeding or performing fluid changes. Complete media supplemented with the below item expires 7 days after preparation.

To prepare 30 mg/mL ECGS stock solution, aseptically combine:

• 15 mg Corning™ Endothelial Cell Growth Supplement – ECGS (ECGS; Fisher Scientific cat# CB-40006)
• 500 µL F-12K (ATCC 30-2004)

Store in working aliquots at -20°C. ECGS is stable for 1 month when prepared and stored as directed. Do not repeat freeze/thaw.

To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C.  Storage at -70°C will result in loss of viability.

1. 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).
2. 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 of the operations from this point on should be carried out under strict aseptic conditions.
3. Transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium and centrifuge at approximately 150-400 x g for 8-12 minutes.  Discard the supernatant and resuspend the cells with recommended growth media (see the specific lot information on the Certificate of Analysis for culture recommended dilution ratio) and dispense into a 25 cm² or a 75 cm² culture flask as recommended on the Certificate of Analysis) at a seeding density of 2.0 x 10⁴ to 4.0 x 10⁴ viable cells/cm².
4. Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio). and dispense into a new culture flask at a seeding density of 2.0 x 10⁴ to 4.0 x 10⁴ viable cells/cm². It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6). 
5. Incubate the culture at 37°C in a suitable incubator.  A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet

Comments:
CRL-1730 is a slow growing cell line that has a roughly. 
Estimated doubling time of 5 to 6 days. 

Volumes are given for a 75 cm² flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes. Corning T-75 flasks (catalog #430641U) are recommended for subculturing this product.

Note: A high quality ECGS prepared from bovine neural tissue (BD Biosciences catalog # 354006 or equivalent) should be used to propagate CRL-1730. It is best to initiate the cells with the highest recommended concentration of ECGS. Moderate to heavy debris and numerous floating cells may be routinely observed in cultures of HUV-EC-C cells. Retain the floating cells by gentle centrifugation and add back to the adherent population.

Cultures should be fully fluid changed every 48 hours. The cells should only be allowed to go 72 hours without fluid changing when the density is less than 50% confluent. Perform full fluid changes rather than media additions.

This cell line produces a lot of floaters and debris especially at higher densities. Cells detach before completely filling in to 100% confluence. It is recommended to subculture the cells when 80 to 90% confluent to avoid excessive floaters. Floating cells are viable and if pronounced, they should be spun down and reseeded back into the growing culture.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with D-PBS (ATCC 30-2200)  to remove all traces of serum which contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution (ATCC 30-2101) 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°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.

Subculture Restrictions:
Subculture at <90% confluence

Note: This cell line produces a lot of floaters and debris especially at higher densities. Cells detach before completely filling in to 100% confluence. lt is recommended to subculture the cells when 80 to 90% confluent to avoid excessive floaters. Floating cells are viable and if pronounced, they should be spun down and reseeded back into the growing culture.

Subculturing Seeding Density: 8.0 x 10³ to 3.0 x 10⁴ viable cells/cm²

Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:3 is recommended

Medium Renewal: Two to three times per week

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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