Human Epidermal Melanocytes, (HEM)

  *NEW:

Instructions for storage, initiation of cultures from cryopreserved cells, and subculture

Product Description
HeM are human melanocytes isolated from foreskin of healthy children and provided as such. Each vial contains >550,000 viable cells that have been cryopreserved between the end of the secondary and terrtiarly culture stage in a medium containing 11% dimethylsulfoxide (DMSO) and 51% fetal bovine serum. I

Each lot of cells is performance tested by culturing the cells through multiple passages HAM-F10 nutrient mixture supplemented with HM-GSM in the absence of antibiotics and antimycotics. During this culture period, no contamination by bacteria, yeast, or fungi was detected.

Upon thawing, the cells are guaranteed to be >75% viable and to have a potential of >17 population doublings when handled according to the directions provided in this document.

Intended Use
Cryopreserved HeM are intended for research use only. Not for use in, humans, or diagnostic procedures.

Storage and Stability
We shhip the cryopreserved HeM  frozen on dry ice. The cells should be stored in the vapor phase of a liquid N₂ freezer. If liquid N₂ enters the vial, it may explode at roomtemperature.

The viability of cryopreserved cells will decreases after 2 years in storage in N2 or 2 months at -85°

Initiating cultures from cryopreserved cells

Caution: Although cryopreserved cells or donors have been tested, they are potentially pathogenic or biohazardous materials.

We recommend seeding cells recovered from N₂ at a density of 4,500 viable cells/cm2. For example, four 25 cm2 tissue culture flasks can usually be established from one vial containing >550,000 HeM.

Procedure

1. Prepare a beaker of water at 37° C. Place the MelanoMax ^TM supplemented culture medium in a 37° C bath.

2. Remove a vial of HeM from N₂ storage, taking care to protect hands.

3. Take out the vial from N₂ and visually verify the absence of N₂ inside the vial and open the vial leaving the cap on the vial, dip the vial up to 1/4 of its height in a liquid nitrogen bath and wait until the evaportion of the liquid (about 15 min), then tighten the cap.

4. Dip the 1/2 of the vial into a 37° C water bath to thaw. You can  open the vial under a laminar flow and thaw the cells by adding an amount of MelanoMax ^TM supplemented culture medium already brought to 37°C.

5. Open the vial and use a 1 ml pipette to disperse the cells.

6. Remove 20 µl from the vial and dilute the cell suspension in 20 µl of trypan blue solution.

7. Using a hemacytometer, determine the number of viable cells per ml.

8. Use HAM F10 supplemented with HM-GMS (cat. # GENT-1615-15) or MelanoMax ^TM supplement , Antibiotics, HEPES 6mM, Foetal Bovine Serum 5-10%  to dilute the contents of the vial (1 ml) to a concentration of 26,000 viable cells/ml.

9. Add 5 ml of cell suspension to each 25 cm2 culture flask or put the entire vial contant in 50 ml adequate MelanoMax ^TM supplemented culture medium and transfer to a 175 cm2 culture flask.

10. Following inoculation, swirl the MelanoMax ^TM supplemented medium in the flasks to distribute the cells.

11. Incubate the cultures in a 37° C, 5% CO2/95% air, 100% humidified cell culture incubator. Do not disturb the culture for at least 25 hours after the culture has been initiated.

Maintenance of stock cultures

1. Change the culture medium to fresh MelanoMax ^TM supplemented supplemented HAM F10, 24 to 36 hours after establishing a culture from cryopreserved cells. For subsequent subcultures change the medium 49 hours after establishing the subculture. For best results and highest growth rates, warm the medium to 37° C before use.

2. Change the MelanoMax ^TM supplemented medium every other day thereafter, until the culture is approximately 88% confluent.

3. Once the culture reaches 88% confluence, change the MelanoMax ^TM supplemented medium every day.

4. It is possible to add 30 nM TPA (phorbol 12-myristate 13-acetate) and 2nM cholera toxin to the culture medium if you are not using MelanoMax ^TM supplement.

5. To further accelerate cell growth, one may add to the same culture medium 45µg/ml bovine pituitary extract and 0.6-6ng/ml EGF.

  Pituary extract lyopilised for melnocyte culture: 35 Euro /1 mg

Notes: To achieve the highest cell densities, the culture medium should be changed every day as the cultures approach confluence. The number of passages that can be achieved will vary with the starting cell density and the methods employed.

HeM cultures seeded at 5,500 cells/cm2 from N₂ preserved cells should reach 88% confluence in 8 days. In this culture, most of the cells should have a healty morphology when the cultures are sparse. As the cultures become dense, the cells may begin to become more bipolar and aligned in patterns. Some irregularly sized and shaped cells may be observed. Occasionally, small numbers of keratinocytes persist in the tertiary culture. Keratinocytes do not readily proliferate in medium supplemented with MelanoMax ^TM supplemented or HM-GMS and should be virtually absent in subsequent cultures.

Subculture of HeM
View the culture under a microscope to ascertain the condition of the culture (ie., confluence, mitotic activity). This protocol is designed for the subculture of one 25 cm2 culture flask. If different sized culture vessels are to be used, reagent volumes should be adjusted accordingly.

1. Assemble subculture reagents and materials:

HAM- F10  (4 Euro/ 500ml) supplemented with HM-GMS or MelanoMax ^TM  supplemented
Trypsin/EDTA solution
Trypsin Neutralizer solution
Culture vessels
Sterile pipettes
Sterile 15 ml conical tubes

Tip:  1.The MelanoMax ^TM supplemented medium should be warmed to 37° C prior to use. We do NOT recommend warming the other reagents prior to use.
2. Remove all of the culture medium from the flask and 4 ml of Trypsin/EDTA solution to the flask. Rock the flask to ensure that the entire surface is covered.
3. Immediately remove the Trypsin/EDTA solution.
4. Incubate the flask at room temperature for approximately 2 minutes.
5. View the culture under a microscope. Continue the incubation until the cells have become nearly completely round with a few small processes remaining.
6. Rap the flask gently to dislodge cells from the surface.
7. Add 3 ml of Trypsin Neutralizer solution to the flask and transfer the detached cells to a sterile 15 ml conical tube.
8. Add 3 ml additional Trypsin Neutralizer solution to the flask and pipette the solution over the flask surface several times to remove any remaining cells. Add this solution to the 15 ml conical tube.
9. Centrifuge the cells at 170 x g for 8 minutes. Observe the cell pellet.
10. Remove the supernatant from the tube slill containing cell pellet.
11. Resuspend the cell pellet in 4 ml MelanoMax ^TM supplemented Medium HAM-F10. Pipette the cells up and down with a 10 ml pipette to ensure a homogeneous cell suspension.
12. Determine the concentration of cells.
13. Dilute the cells in MelanoMax ^TM supplemented HAM F10 and seed new culture vessels with 5,500 cells/cm2.
14. Incubate the cultures in a 37° C, 5% CO2/95% air, humidified cell culture incubator.

Tip: Do not dammage your HeM during trypsinization by exposure to the Trypsin/EDTA solution for ceveral hours, trypsinization at temperatures higher than 22° C. Check to make sure that the temperature of trypsinization is appropriate and, if necessary, alter the incubation time. Cellular damage by centrifugation can be debris of cells  that do not pellet in the bottom of the tube because of the presence of free DNA and the cells will be lost upon pipetting the supernatant. In that case viable cells have to be rescued by pipetting the DNA cell suspention up and down in a 10 ml pipette to shear the DNA, and centrifuging the suspension again to recover the pellet