Primary Dermal Microvascular Endothelial Cells; Normal, Human, Neonatal (HDMVECn) (ATCC^® PCS-110-010^™)

1. Obtain one growth kit from the freezer; make sure that the caps of all components are tight.
2. Thaw the components of the growth kit just prior to adding them to the basal medium. It is necessary to warm the L-glutamine component in a 37°C water bath and shake to dissolve any precipitates prior to adding to the basal medium.
3. Obtain one bottle of Vascular Cell Basal Medium (475 mL) from cold storage. 
4. Decontaminate the external surfaces of all growth kit component vials and the basal medium bottle by spraying them with 70% ethanol.
5. Using aseptic technique, and working in a laminar flow hood or biosafety cabinet, transfer the volume of each growth kit component, as indicated in either Table 1 or 2, to the bottle of basal medium using a separate sterile pipette for each transfer.

Table 1. If using the Microvascular Endothelial Cell Growth Kit-BBE (ATCC^® PCS-110-040), add the indicated volume for each component:

Component	Volume	Final Concentration
Bovine Brain Extract (BBE)	1.0 mL	0.2%
rh EGF	0.5 mL	5 ng/mL
L-glutamine	25.0 mL	10 mM
Heparin sulfate	0.5 mL	0.75 Units/mL
Hydrocortisone hemisuccinate	0.5 mL	1 µg/mL
Fetal Bovine Serum	25.0 mL	5%
Ascorbic acid	0.5 mL	50 µg/mL

 

       Table 2. If using the Microvascular Endothelial Cell Growth Kit-VEGF (ATCC^® PCS-110-041), add the indicated volume for each component:

Component	Volume	Final Concentration
rh VEGF	0.5 mL	5 ng/mL
rh EGF	0.5 mL	5 ng/mL
rh FGF basic	0.5 mL	5 ng/mL
rh IGF-1	0.5 mL	15 ng/mL
L-glutamine	25.0 mL	10 mM
Heparin sulfate	0.5 mL	0.75 Units/mL
Hydrocortisone hemisuccinate	0.5 mL	1 µg/mL
Fetal Bovine Serum	25.0 mL	5%
Ascorbic acid	0.5 mL	50 µg/mL

 

Antimicrobials and phenol red are not required for proliferation but may be added if desired. The recommended volume of either of the optional components (GA solution or PSA solution) to be added to the complete growth media is summarized in Table 3.

Table 3. Addition of Antimicrobials/Antimycotics and Phenol Red (Optional)

Component	Volume	Final Concentration
Gentamicin-Amphotericin B Solution	0.5 mL	Gentamicin: 10 mg/mL Amphotericin B: 0.25 µg/mL
Penicillin-Streptomycin-Amphotericin B Solution	0.5 mL	Penicillin: 10 Units/mL Streptomycin: 10 µg/mL Amphotericin B: 25 ng/mL
Phenol Red	0.5 mL	33 µM



6. Tightly cap the bottle of complete growth medium and swirl the contents gently to assure a homogeneous solution. Do not shake forcefully to avoid foaming. Label and date the bottle.
7. Complete growth media should be stored in the dark at 2°C to 8°C (do not freeze). When stored under these conditions, complete growth media is stable for 30 days.

1. Passage normal HDMVECn cells when culture has reached approximately 80% confluence.
2. Warm both the Trypsin-EDTA for Primary Cells (ATCC® PCS-999-003) and the Trypsin Neutralizing Solution (ATCC PCS-999-004) to room temperature prior to dissociation. Warm complete growth medium to 37°C prior to use with the cells.
3. For each flask, carefully aspirate the spent media without disturbing the monolayer.
4. Rinse the cell layer two times with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual traces of serum. 
5. Add pre-warmed trypsin-EDTA solution (1 to 2 mL for every 25 cm²) to each flask.
6. Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells, and then aspirate the excess fluid off of the monolayer.
7. Observe the cells under the microscope. When the cells pull away from each other and round up (typically within 1 to 2 minutes), remove the flask from the microscope and gently tap it from several sides to promote detachment of the cells from the flask surface.
8. When the majority of cells appear to have detached, quickly add an equal volume of Trypsin Neutralizing Solution (ATCC PCS-999-004) to each flask. Gently pipette or swirl the culture to ensure all of the trypsin-EDTA solution has been neutralized.
9. Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any remaining cells in the flask.
10. Add 3 to 5 mL D-PBS (ATCC 30-2200) to the flask to collect any additional cells that might have been left behind.
11. Transfer the cell/D-PBS suspension to the centrifuge tube containing the trypsin-EDTA-dissociated cells.
12. Repeat steps 10 and 11 as needed until all cells have been collected from the flask.
13. Centrifuge the cells at 150 x g for 3 to 5 minutes. 
14. Aspirate the neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mL fresh, pre-warmed, complete growth medium.
15. Count the cells and seed new flasks at a density of 5,000 cells per cm².
16. Place newly seeded flasks in a 37°C, 5% CO₂, incubator for at least 24 to 48 hours before processing the cells further. Refer to Maintenance for guidelines on feeding.

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