Human Epidermal Melanocytes
Proliferating Cultures - Catalog Number: 19-HEM-T > 500,000 viable cells/3T flask
Cryopreserved Cells - Catalog Number: 19-HEM > 500,000 viable cells/vial
HEM are human epidermal melanocytes isolated from foreskin of healthy children and provided as such. Each vial or 25 cm² flask of this product contains >500,000 viable cells that have been cryopreserved at the end of the secondary culture stage. Each batch of cells is performance tested by sub-culturing the cells through multiple passages with HAM’s F10 supplemented with Melanomax (19-GEN-MMAX) in the absence of antibiotics and antimycotics. During this culture period, no contamination by bacteria, yeast, mycoplasma or fungi was detected.
þ Recommended precautions are to be taken when handling human cells, please read the caution box.
Instructions for living cells
Reception of the flasks
Upon reception of the flasks
- Put the culture flask in an incubator at 37°C, 5% CO2 and 100 % humidity.
- 24 hours later, remove excess culture medium. The culture medium is a special formulation for the transportation of living cells and contains Melanomax (19-GEN-MMAX) and InfectoSTOP+ (19-GEN-INSTOP+). The excess medium can be reused for subculturing.
- Alternatively, the cells can be transferred in another culture flask.
The cells are guaranteed to be >70% viable and to have a potential of >10 population doublings when handled according to the directions provided in this document.
Instructions for cryopreserved cells
Storage and Stability
Cryopreserved HEM should arrive frozen on dry ice. If the cells are not to be used immediately, the user should prepare a space for storage of the vial in the vapor phase of a liquid nitrogen freezer. If liquid nitrogen (fluid) enters the vial, it may explode when it is brought later to room temperature. While wearing protective eyewear, gloves, and a laboratory coat, remove the vial from its shipping container and place immediately in the liquid nitrogen freezer. Although the viability of cryopreserved cells decreases with time in storage, useful cultures can usually be established even after 2 years of storage at liquid nitrogen temperatures.
Initiating cultures from cryopreserved cells
We recommend seeding cells recovered from cryopreservation at a density of 5,000 viable cells/cm². For example, four 25 cm² tissue culture flasks can usually be established from one vial containing >500,000 HEM. The procedure given below is a sample protocol for establishing cultures from the contents of one vial.
1. Prepare a beaker of water at 37° C. Place the supplemented culture medium (see step 9) in a 37° C water bath.
2. Remove a vial of HEM from liquid nitrogen storage, taking care to protect hands and eyes.
3. Take out the vial from liquid nitrogen and visually verify the absence of liquid inside the vial. In that case: Loosen the cap on the vial 1/4 turn to release any liquid nitrogen that may be trapped in the threads, dip the vial up to 1/3 of its height in a liquid nitrogen bath and wait until the complete evaporation of the liquid (about 20 min), then re-tighten the cap.
4. Dip the lower half of the vial into the 37° C water to thaw, taking care that any fluid enters. Tip: open the vial under a laminar flow and thaw the content by adding inside a suitable amount of culture medium already brought to 37°C.
5. When the contents of the vial have thawed, wipe the outside of the vial with disinfecting solution and move to a Class II, type A laminar flow culture hood.
6. Open the vial and pipette the suspension up and down with a 1 ml pipette to disperse the cells.
7. Remove 20 µl from the vial and dilute the cell suspension in 20 µl of trypan blue solution.
8. Using a hemacytometer, determine the number of viable cells per ml.
9. Use HAM F10 supplemented with Melanomax (Cat. N° 19-GEN-MMAX), Antibiotics, HEPES 6mM, decomplemented Foetal Bovine Serum 5-10% to dilute the contents of the vial (1 ml) to a density of 25,000 viable cells/ml.
10. Add 5 ml of cell suspension to each 25 cm2 culture flask or put the entire vial content in 50 ml adequate culture medium and transfer to a 175 cm² culture flask.
11. Following inoculation, swirl the medium in the flasks to distribute the cells.
12. Incubate the cultures in a 37° C, 5% CO2/95% air, 100% humidified cell culture incubator. For best results, do not disturb the culture for at least 24 hours after the culture has been initiated.
Upon thawing, the cells are guaranteed to be >70% viable and to have a potential of >10 population doublings when handled according to the directions provided in this document.
- Trypsin/EDTA solution (not provided)
- Trypsin Neutralizer solution (not provided)
- Culture vessels (not provided)
- Sterile pipettes (not provided)
- Sterile 15 ml conical tubes (not provided)
Notes: Damage to cultured HEM can occur during trypsinization. This damage may result from exposure of the cells to the Trypsin/EDTA solution for excessive lengths of time, trypsinization at temperatures higher than 20° C and/or excessive mechanical agitation. Check to make sure that the temperature of trypsinization is appropriate and, if necessary, alter the incubation time of the procedure.
Another common source of damage is centrifugation at excessive g forces. Check to make sure that the speed of the centrifuge is appropriate. One manifestation of cellular damage that may be evident after centrifugation is strings of cells (and debris) that do not pellet in the bottom of the tube. This is due to the presence of DNA from lysed cells in the solution. If this occurs, the cell pellet may be lost upon aspiration of the supernatant containing the DNA strings. In many cases, viable cells can be rescued by pipetting the cells (and DNA) up and down in a 10 ml pipette to shear the DNA, and centrifuging the suspension again to recover the cells.
Maintenance of stock cultures
I. Change the culture medium to fresh supplemented HAM’s F10, 24 to 36 hours after establishing a culture from cryopreserved cells. For subsequent subcultures change the medium 48 hours after establishing the subculture. For best results and highest growth rates, warm the medium to 37° C before use.
II. Change the medium every other day thereafter, until the culture is approximately 80-90% confluent.
III. Once the culture reaches 80-90% confluence, change the medium every 2-3 days.
IV. It is possible to supplement culture medium with 30 nM TPA (phorbol 12-myristate 13-acetate) and/or 2nM cholera toxin to the culture medium.
V. To further accelerate cell growth, one may add to the same culture medium 40µg/ml bovine pituitary extract and/or 0.5-5ng/ml EGF.
Notes: To achieve the highest cell densities, the culture medium should be changed every 2 days as the cultures approach confluence. The number of subcultures (passages) that can be achieved will vary with the starting cell density and the methods employed by individual investigators.
HEM cultures seeded at 5,000 cells/cm² from cryopreserved cells should reach 80% confluence in 7-9 days. In this culture, most of the cells should have a multipolar 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 and should be virtually absent in subsequent cultures.
Caution: Although culture cells or donors have been tested for the absences of various hazardous agents, diagnostic tests are not necessarily 100% accurate. In addition, human cells may harbour other known or unknown agents or organisms which could be harmful to your health or cause fatal illness. The user should treat all human cells as potential pathogens. Wear protective clothing and eyewear. Practice appropriate disposal techniques for potentially pathogenic or biohazardous material.
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