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
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
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.
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
TM supplemented culture medium already brought
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
TM supplemented culture medium and transfer to a
175 cm2 culture flask.
10. Following inoculation, swirl the
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
of stock cultures
1. Change the culture medium to
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
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
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.
lyopilised for melnocyte culture: 35 Euro /1 mg
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
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
1. Assemble subculture reagents and
HAM- F10 (4 Euro/ 500ml)
supplemented with HM-GMS or MelanoMax
Trypsin Neutralizer solution
Sterile 15 ml conical tubes
TM supplemented medium should be warmed to 37° C
prior to use. We do NOT recommend warming the other reagents prior to
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
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
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
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
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