Medium for culturing normal human epidermal melanocytes

Abstract

Provided is a medium for culturing normal human epidermal melanocytes in vitro. The medium comprises a basal medium for culturing animal cells, Ca.sup.2+ at a final concentration of between about 0.15 mM and about 1.2 mM and Mg.sup.2+ at a final concentration of 1.2 mM and 6 mM or Ca.sup.2+ at a final concentration of between about 0.9 mM and about 1.2 mM and Mg.sup.2+ at a final concentration between about 0.6 mM and 6.0 mM and 0.001 to 0.1% scrum (v/v). The medium can promote both dendrite formation and proliferation of the cells.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a medium suitable for in vitro culturing normal human epidermal melanocyte.

2. Prior Art

In vitro culturing of normal cells derived from human is a key technique for investigation in many fields including medical and pharmaceutical, or development of drug, cosmetics and the like. Culturing of normal human melanocyte in vitro is a useful steps for studying, such as biosynthesis of melanine in human skin or whitening of the skin. In order to culture said cells, known basal media for culturing animal cells such as MCDB153 or M199 containing supplements, for example growth factors, such as epidermal growth factor and fibroblast growth factor, hormones such as insulin and triiodothyronine, carriers such as cholera toxin and transferrin, bovine pituitary extract, bovine hypothalamus extract and fetal bovine serum (FBS).

However, those conventional media cannot provide enough growth without comparatively large amount of serum. In addition, while the morphology of melanocytes in vivo characterized by dendrite formation and ramification, high population of melanocyte cultured in vitro with a conventional media shows quite different such as bipolar morphology. Dendrite formation of in vitro cultured cells can be promoted by adding an agent that increase intracellular levels of cAMP, such as dbcAMP or IBMX. However, these agents cause suppression of cell proliferation (K, Nakazawa et. al., Pigment Cell Research (1993) 6, 406-416).

SUMMARY OF THE INVENTION

An object of the present invention is to provided a medium for in vitro culturing normal human epidermal melanocyte which can promote cell proliferation.

In another object of the present invention is to provide a medium for in vitro culturing normal human epidermal melanocyte which can promote both proliferation and dendrite formation of said cells.

In further object of the present invention is to provide a medium for in vitro culturing normal human epidermal melanocyte which can promote intracellular melanin synthesis of said cells.

According to one aspect of the present invention, there is provided a medium comprising of a basal medium for culturing animal cells, Ca.sup.2+ at a final concentration of between about 0.9 mM and about 1.2 mM and Mg.sup.2+ at a final concentration of between about 0.6 mM and 6 mM. In addition, the present invention also provides a medium useful for culturing normal human epidermal melanocyte, comprising of a basal medium for culturing animal cells, Ca.sup.2+ at a final concentration of between about 0.15 mM and about 1.2 mM and Mg.sup.2+ at a final concentration of between about 1.2 mM and 6 mM. The most preferable medium according to the present invention contains Ca.sup.2+ at a final concentration of between about 0.9 mM and about 1.2 mM and Mg.sup.2+ at a final concentration of between about 1.2 mM and 6 mM.

The medium may further comprise one or more growth factor useful for growth of human melanocyte. The medium of the present invention may still further comprise biological materials such as bovine pituitary extract.

The medium of the present invention may further comprise between about 0.001% and about 0.1% (v/v) of heat inactivated fetal bovine serum.

In another aspect of the present invention, there is provided a method for culturing normal human epidermal melanocyte which contains culturing said cells in the medium of the present invention under cell-culturing conditions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents results of example 1.

FIG. 2 represents results of example 2.

FIG. 3 represents results of example 3.

FIG. 4 is a photomicrograph of normal human epidermal melanocyte cultured in a medium containing 0.15 mM of Ca.sup.2+ and 0.6 mM of Mg.sup.2+ at 7 days after inoculation. Original magnification.times.40.

FIG. 5. is a photomicrograph of normal human epidermal melanocyte cultured in a medium containing 0.15 mM of Ca.sup.2+ and 1.8 mM of Mg.sup.2+ at 7 days after inoculation in a medium. Original magnification.times.40.

FIG. 6 is a photomicrograph of normal human epidermal melanocyte cultured in a medium containing 0.9 mM of Ca.sup.2+ and 0.6 mM of Mg.sup.2+ at 7 days after inoculation. Original magnification.times.40.

FIG. 7 is a photomicrograph of normal human epidermal melanocyte cultured in a medium containing 0.9 mM of Ca.sup.2+ and 1.8 mM of Mg.sup.2+ at 7 days after inoculation. Original magnification.times.40.

FIG. 8 is a photomicrograph of normal human epidermal melanocyte cultured in a medium containing 0.9 mM of Ca.sup.2+ and 6.0 mM of Mg.sup.2+ at 7 days after inoculation. Original magnification.times.40.

FIG. 9 represents results of example 4.

FIG. 10 represents results of example 5.

FIG. 11 represents results of example 6.

FIG. 12 represents results of example 7.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The inventors found that by controlling Ca.sup.2+ and Mg.sup.2+ concentration within a defined range, a basal medium for animal cell culture can be used for in vitro culture of normal human epidermal melanocyte. The medium of the present invention promotes both dendrite formation and proliferation of melanocytes. In addition, melanocyte cultured with the medium of the present invention shows comparatively dark color indicating that the cells can synthesis and deposit comparatively large amount of melanin.

"Basal medium for culturing animal cells" used in the present invention may be any known nutrient medium which is suitable for in vitro culture of normal human epidermal melanocyte. Examples of preferred media include MCDB 153, M199, Eagle's MEM, Dulbecco's modified of Eagle's MEM, HAM F 12, RPMI1640 and the like. Most preferable basal medium is MCDB 153 or modified MCDB 153 which contains enriched amino acid ingredients than standard MCDB 153. In the following table 1 and 2, there are shown the ingredients of standard MCDB 153 and modified MCDB 153. In the present specification, without specific indication, "MCDB 153" represents not only standard MCDB 153 of table 1 but also modified MCDB 153 of table 2. The modified MCDB 153 is identical to the standard MCDB 153 except for containing enriched L-histidine, L-isoleucine, L-methionine HCl, L-phenylalanine, L-tryptophane, and L-tyrosine and supplemented with ethanol amine and phosphoethanol amine.

TABLE 1______________________________________Components of standard MCDB 153:component mol/l mg/l______________________________________L-Alanine 1.0 .times. 10.sup.-4 8.91L-Arginine HCl 1.0 .times. 10.sup.-3 210.7L-Asparagine.H.sub.2 O 1.0 .times. 10.sup.-4 15.01L-Aspartic Acid 3.0 .times. 10.sup.-5 3.99L-Cysteine HCl.H.sub.2 O 2.4 .times. 10.sup.-4 42.04L-Glutamic Acid 1.0 .times. 10.sup.-4 14.71L-Glutamine 6.0 .times. 10.sup.-3 877.2Glycine 1.0 .times. 10.sup.-4 7.51L-Histidine HCl.H.sub.2 O 8.0 .times. 10.sup.-5 16.77L-Isoleucine 1.5 .times. 10.sup.-5 1.968L-Leucine 5.0 .times. 10.sup.-4 65.6L-Lysine HCl 1.0 .times. 10.sup.-4 18.27L-Methionine HCl 3.0 .times. 10.sup.-5 4.476L-Phenylalanine 3.0 .times. 10.sup.-5 4.956L-Proline 3.0 .times. 10.sup.-4 34.53L-Serine 6.0 .times. 10.sup.-4 63.06L-Threonine 1.0 .times. 10.sup.-4 11.91L-Tryptophan 1.5 .times. 10.sup.-5 3.06L-Tyrosine 1.5 .times. 10.sup.-5 2.718L-Valine 3.0 .times. 10.sup.-4 35.13d-Biotin 6.0 .times. 10.sup.-8 0.0146Folic Acid 1.8 .times. 10.sup.-6 0.79DL-alpha-Lipoic Acid 1.0 .times. 10.sup.-6 0.2063Niacinamide 3.0 .times. 10.sup.-7 0.03663D-Pantothenate(Ca) 1.0 .times. 10.sup.-6 0.468Pyridoxine HCl 3.0 .times. 10.sup.-7 0.6171Riboflavin 1.0 .times. 10.sup.-7 0.03764Thiamin HCl 1.0 .times. 10.sup.-6 0.3373Vitamin B.sub.12 3.0 .times. 10.sup.-6 4.07Adenine 1.8 .times. 10.sup.-4 24.32Choline Chloride 1.0 .times. 10.sup.-4 13.96D-Glucose 6.0 .times. 10.sup.-3 1081myo-lnositol 1.0 .times. 10.sup.-4 18.02Putrescine 2HCl 1.0 .times. 10.sup.-6 0.1611Sodium Pyruvate 5.0 .times. 10.sup.-4 55.0Thymidine 3.0 .times. 10.sup.-6 0.7266CaCl.sub.2.2H.sub.2 O 1.5 .times. 10.sup.-4 22.05KCl 1.5 .times. 10.sup.-3 111.83MgCl.sub.2.6H.sub.2 O 6.0 .times. 10.sup.-4 122.0NaCl 1.3 .times. 10.sup.-1 7599Na.sub.2 HPO.sub.4.7H.sub.2 O 2.0 .times. 10.sup.-3 536.2CuSO.sub.4.5H.sub.2 O 1.1 .times. 10.sup.-8 0.00275FeSO.sub.4.7H.sub.2 O 5.0 .times. 10.sup.-6 1.39H.sub.2 SeO.sub.3 3.0 .times. 10.sup.-8 0.003867MnSO.sub.4.5H.sub.2 O 1.0 .times. 10.sup.-9 0.000241Na.sub.2 SiO.sub.3.9H.sub.2 O 5.0 .times. 10.sup.-7 0.1421(NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 O 1.0 .times. 10.sup.-9 0.001236NH.sub.4 VO.sub.3 5.0 .times. 10.sup.-9 0.000585NiCl.sub.2.6H.sub.2 O .sup. 5.0 .times. 10.sup.-10 0.000119SnCl.sub.2.2H.sub.2 O .sup. 5.0 .times. 10.sup.-10 0.000113ZnSO.sub.4.7H.sub.2 O 5.0 .times. 10.sup.-7 0.1438HEPES 2.8 .times. 10.sup.-2 6600NaHCO.sub.3 1.4 .times. 10.sup.-2 1176Sodium Acetate.3H.sub.2 O 3.7 .times. 10.sup.-3 500Phenol Red(Na) 3.3 .times. 10.sup.-6 1.242______________________________________ TABLE 2______________________________________Components of modified MCDB 153:Component mol/l mg/l______________________________________L-Alanine 1.0 .times. 10.sup.-4 8.91L-Arginine HCl 1.0 .times. 10.sup.-3 210.7L-Asparagine.H.sub.2 O 1.0 .times. 10.sup.-4 15.01L-Aspartic Acid 3.0 .times. 10.sup.-5 3.99L-Cysteine HCl.H.sub.2 O 2.4 .times. 10.sup.-4 42.04L-Glutamic Acid 1.0 .times. 10.sup.-4 14.71L-Glutamine 6.0 .times. 10.sup.-3 877.2Glycine 1.0 .times. 10.sup.-4 7.51L-Histidine HCl.H.sub.2 O 3.2 .times. 10.sup.-4 67.08L-Isoleucine 7.7 .times. 10.sup.-4 100.3L-Leucine 5.0 .times. 10.sup.-4 65.6L-Lysine HCl 1.0 .times. 10.sup.-4 18.27L-Methionine HCl 1.2 .times. 10.sup.-4 17.91L-Phenylalanie 1.2 .times. 10.sup.-4 19.82L-Proline 3.0 .times. 10.sup.-4 34.53L-Serine 6.0 .times. 10.sup.-4 63.06L-Threonine 1.0 .times. 10.sup.-4 11.91L-Tryptophan 6.0 .times. 10.sup.-5 12.25L-Tyrosine 9.0 .times. 10.sup.-5 16.31L-Valine 3.0 .times. 10.sup.-4 35.13d-Biotin 6.0 .times. 10.sup.-8 0.0146Folic Acid 1.8 .times. 10.sup.-6 0.79DL-alpha-Lipoic Acid 1.0 .times. 10.sup.-6 0.2063Niacinamide 3.0 .times. 10.sup.-7 0.03663Ethanolamine 1.0 .times. 10.sup.-4 6.108Phosphoethanolamine 1.0 .times. 10.sup.-4 14.106D-Pantothenate(Ca) 1.0 .times. 10.sup.-6 0.468Pyridoxine HCl 3.0 .times. 10.sup.-7 0.06171Riboflavin 1.0 .times. 10.sup.-7 0.03764Thiamin HCl 1.0 .times. 10.sup.-6 0.3373Vitamin B.sub.12 3.0 .times. 10.sup.-6 4.07Adenine 1.8 .times. 10.sup.-4 24.32Choline Chloride 1.0 .times. 10.sup.-4 13.96D-Glucose 6.0 .times. 10.sup.-3 1081myo-lnositol 1.0 .times. 10.sup.-4 18.02Putrescine 2HCl 1.0 .times. 10.sup.-6 0.1611Sodium Pyruvate 5.0 .times. 10.sup.-4 55.0Thymidine 3.0 .times. 10.sup.-6 0.7266CaCl.sub.2.2H.sub.2 O 1.5 .times. 10.sup.-4 22.05KCl 1.5 .times. 10.sup.-3 111.83MgCl.sub.2.6H.sub.2 O 6.0 .times. 10.sup.-4 122.0NaCl 1.3 .times. 10.sup.-1 7599Na.sub.2 HPO.sub.4.7H.sub.2 O 2.0 .times. 10.sup.-3 536.2CuSO.sub.4.5H.sub.2 O 1.1 .times. 10.sup.-8 0.00275FeSO.sub.4.7H.sub.2 O 5.0 .times. 10.sup.-6 1.39H.sub.2 SeO.sub.3 3.0 .times. 10.sup.-8 0.003867MnSO.sub.4.5H.sub.2 O 1.0 .times. 10.sup.-9 0.000241Na.sub.2 SiO.sub.3.9H.sub.2 O 5.0 .times. 10.sup.-7 0.1421(NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 O 1.0 .times. 10.sup.-9 0.001236NH.sub.4 VO.sub.3 5.0 .times. 10.sup.-9 0.000585NiCl.sub.2.6H.sub.2 O .sup. 5.0 .times. 10.sup.-10 0.000119SnCl.sub.2.2H.sub.2 O .sup. 5.0 .times. 10.sup.-10 0.000113ZnSO.sub.4.7H.sub.2 O 5.0 .times. 10.sup.-7 0.1438HEPES 2.8 .times. 10.sup.-2 6600NaHCO.sub.3 1.4 .times. 10.sup.-2 1176Sodium Acetate 3H.sub.2 O 3.7 .times. 10.sup.-3 500Phenol Red(Na) 3.3 .times. 10.sup.-6 1.242______________________________________

According to the present invention, at least one of Ca.sup.2+ and Mg.sup.2+ concentrations is enriched from the basal medium. The final Ca.sup.2+ concentration of the medium of the present invention is in the range between about 0.15 mM and about 1.2 mM, preferably, about 0.9 mM and about 1.2 mM. However, when Ca.sup.2+ concentration is lower than 0.9 mM, final concentration of Mg.sup.2+ is adjusted in the range of between about 1.2 mM and 1.8 mM. The final concentration of Ca.sup.2+ can be adjusted by adding Ca.sup.2+ source such as CaCl.sub.2 into the basal medium.

The final Mg.sup.2+ concentration of the medium of the present invention is in the range between about 0.6 mM and about 6 mM, preferably, about 1.2 mM and about 1.8 mM. However, when Mg.sup.2+ concentration is lower than 1.2 mM, a final concentration of Ca.sup.2+ is adjusted to more than 0.9 mM. The final concentration of Mg.sup.2+ can be adjusted by adding Mg.sup.2+ source such as MgCl.sub.2 and MgSO.sub.4 into the basal medium.

The medium of the present invention may further comprise various supplements that are employed in conventional animal cell in vitro culture, if desired. For example, one or more growth factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), Interleukin-1, transforming growth factor alpha and the like, are preferably contained in the medium.

The medium of the present invention preferably contains comparatively small amount of serum. Examples for serum include that of derived from bovine, horse, porcine and the like. Among them, fetal bovine serum is most preferable because of its commercial availability. In the medium of the present invention, serum may be contained at a final concentration of between about 0.001% and about 1%(v/v), more preferably between about 0.001% and about 0.1% (v/v).

Further, the medium of the present invention may comprise biological materials for example bovine pituitary extract (BPE), bovine hypothalamus extract, bovine cerebrum extract and albumin; insulin, phorbol 12 myristrate 13-acetate(PMA), heparin, hydrocortisone, transferrin, cholera toxin, and triiodothyronine. Further more, the medium may comprise antibiotics such as gentamicin sulfate, amphotericin B, penicillin, mitomycin, kanamycin sulfate and streptomycin. These supplements described above may be added into the medium singly or in mixture as required.

The present invention also provides a method for culturing normal human epidermal melanocyte in a medium of the present invention. According to the method of the present invention, the normal human epidermal melanocyte may be the cells freshly derived from any age and any sexes of human, successively cultured cells of such freshly derived cells, or both of freshly derived and successively cultured cells which are preserved by conventional frozen manner.

As the "cell-culture conditions" of the present invention, any conventional conditions for in vitro cell culturing can be employed and it is well known in the art.

To further illustrate the present invention, and not by way of limitation, the following examples are given.

EXAMPLE OF THE INVENTION

Example 1

Normal human epidermal melanocyte culture was performed in modified MCDB 153 with various Ca.sup.2+ concentrations.

The modified MCDB 153 medium shown in the above table 2 supplemented with the substances shown in following table 3 and fetal bovine serum at a final concentration of 5%(v/v) was used. Final Ca.sup.2+ concentration of the medium was adjusted with CaCl.sub.2.2H.sub.2 O between 0.15 and 1.2 mM. The final Mg.sup.2+ concentration of 0.6 mM is same as conventional MCDB 153.

TABLE 3______________________________________additives concentration (final)______________________________________BPE 0.2% (v/v)Insulin 10 .mu.g/mlPMA 20 ng/mlbFGF 2 ng/mlHeparin 1 .mu.g/mlhydrocortisone 0.5 .mu.g/mltransferrin 1 .mu.g/mlgentamicin sulfate 50 .mu.g/mlamphotericin B 50 ng/ml______________________________________

Normal human epidermal melanocyte obtained from secondarily cultured cells and stored frozen at liquid nitrogen temperature (Cascade Biologics Inc.) were used. The culture of the cells was performed by using 12-well cell culture plate (Corning). After thawing, the cells were inoculated into the respective media at an initial cell density of 5000 cells/cm.sup.2. Cells were incubated at 37.degree. C. in 5% CO.sub.2 humidified incubator. Cultures were fed fresh media every or every other day. After 7 days incubation, cells were harvested with 0.025% trypsin/0.01% EDTA, and cell numbers were counted. Cell numbers were determined with hemocytometer counting chamber. Results are shown in FIG. 1. Improved cell growth was shown with the medium containing 0.6-0.9 mM of Ca.sup.2+.

Example 2

The modified MCDB 153 medium shown in the above table 2 supplemented with the substances shown in above table 3 and fetal bovine serum at a final concentration of 5%(v/v) was used. Final Ca.sup.2+ concentration was adjusted to 0.15 mM (medium 1) and 0.9 mM (medium 2).

Normal human epidermal melanocyte obtained from secondarily cultured cells and stored frozen at liquid nitrogen temperature (Cascade Biologics Inc.) were used. The culture of the cells was performed by using 12-well cell culture plate (Corning). After thawing, the cells were inoculated into the respective media at an initial cell density of 5000 cells/cm.sup.2. Cells were incubated at 37.degree. C. in 5% CO.sub.2 humidified incubator. Cultures were fed fresh media every or every other day.

After 7 days incubation, cells were harvested with 0.025% trypsin/0.01% EDTA, and obtained cell (3rd generation culture) numbers were determined with hemocytometer counting chamber.

Then, the obtained cells were washed and inoculated into freshly prepared respective media at an cell density of 5000 cells/cm.sup.2. The cells were incubated for additional 7 days and then cell numbers of the culture (4th generation culture) were determined as above. Results are shown in FIG. 2.

Improved cell proliferation were observed with the Ca.sup.2+ enriched medium both of 3rd and 4th generation of cultures.

Example 3

In to the media of example 2, containing Ca.sup.2+ at final concentrations of 0.15 mM and 0.9 mM respectively, various amounts of MgCl.sub.2 6H.sub.2 O were added to provide final Mg.sup.2+ concentration from 0.6 to 6 mM. Normal human epidermal melanocyte culture was done with said media according to the procedure of example 1. After 7 days incubation, cells were harvested and cell numbers were determined. The results are shown in FIG. 3.

Cell morphology on light microscope in the different culture conditions were shown in FIGS. 4-8. With a modified MCDB 153 containing 0.15 mM of Ca.sup.2+ and 0.6 mM of Mg.sup.2+, almost all cells were shown to be bipolar morphology, i.e. having only two ramifications per cell (FIG. 4). With a medium enriched only Ca.sup.2+ or Mg.sup.2+, cells were shown to be bipolar morphology, too (FIGS. 5 and 6). In contrast, with media enriched both of Ca.sup.2+ and Mg.sup.2+, high population of the cultured cells were shown to be dendrites (FIGS. 7 and 8). In addition, cells cultured with such media were shown comparatively deep color indicating increased intracellular melanin synthesis and deposition (FIGS. 7 and 8).

Example 4

A basal medium shown in following table 4 supplemented with the substances shown in above table 3 and fetal bovine serum at a final concentration of 5%(v/v) were used. Final Ca.sup.2+ and Mg.sup.2+ concentrations were adjusted according to table 5 respectively.

Normal human epidermal melanocyte were cultured using the above media according to the procedure of example 1. After 5 days incubation, the cells were harvested and cell numbers were determined. The results are shown in FIG. 9.

TABLE 4______________________________________Components of basal medium of example 4Component mg/l______________________________________L-Alanine 4.45DL-.alpha.-Alanine 25L-Arginine HCl 140.0L-Asparagine.H.sub.2 O 7.51L-Aspartic Acid 2.00DL-Aspartic Acid 30L-Cysteine HCl.H.sub.2 O 21.26L-Cystine 10L-Glutamic Acid 7.36DL-Glutamic Acid.H.sub.2 O 75L-Glutamine 488.6Glycine 28.76L-Histidine HCl.H.sub.2 O 43.5L-Isoleucine 50.15DL-Isoleucine 20L-Leucine 32.8DL-Leucine 60L-Lysine HCl 44.14L-Methionine 8.96DL-Methionine 15L-Phenylalanie 9.91DL-Phenylalanine 25L-Proline 32.27Hydroxy-L-Proline 5L-Serine 31.53DL-Serine 25L-Threonine 5.96DL-Threonine 30L-Tryptophan 6.13DL-Tryptophan 10.00L-Tyrosine 28.16L-Valine 17.57DL-Valine 25p-Amino Benzoic acid 0.025Ascorbic Acid 0.025d-Biotin 0.012Folic Acid 0.4DL-.alpha.-Lipoic Acid 0.103Nicotinamide 0.0308Nicotinic Acid 0.0125Ethanolamine 3.05Phosphoethanolamine 7.05D-Pantothenate(Ca) 0.239Pyridoxal.HCl 0.0125Pyridoxine.HCl 0.0434.alpha.-tocopherol phosphate.2Na 0.005Riboflavin 0.376Thiamin HCl 0.174Vitamin A 0.05Vitamin B.sub.12 2.04Vitamin B.sub.2 0.05Vitamin K.sub.3 0.005Adenine sulfate 5Adenine 12.2AMP 0.1ATP-2Na 0.5Cholesterol 0.1Deoxyribose 0.25Choline Chloride 7.23D-Glucose 1041myo-lnositol 9.035Glutathione 0.025Guanine.HCl 0.15Hypoxanthine 0.15Xanthine 0.15Ribose 0.25Thymine 0.15Tween 80 10Uracil 0.15Putrescine.2HCl 0.0806Sodium Pyruvate 27.5Thymidine 0.363CaCl.sub.2.2H.sub.2 O 11.03CaCl.sub.2 70KCl 256MgCl.sub.2.6H.sub.2 O 61MgSO.sub.4.7H.sub.2 O 100NaCl 7800Na.sub.2 HPO.sub.4.7H.sub.2 O 268Na.sub.2 HPO.sub.4.2H.sub.2 O 30KH.sub.2 PO.sub.4 30CuSO.sub.4.5H.sub.2 O 0.00138FeSO.sub.4.7H.sub.2 O 0.695Fe(NO.sub.3).sub.3 0.05H.sub.2 SeO.sub.3 0.00193MnSO.sub.4.5H.sub.2 O 0.000121Na.sub.2 SiO.sub.3.9H.sub.2 O 0.0711(NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 O 0.000618NH.sub.4 VO.sub.3 0.000293NiCl.sub.2.6H.sub.2 O 0.0000595SnCl.sub.2.2H.sub.2 O 0.0000565ZnSO.sub.4.7H.sub.2 O 0.0719HEPES 3336NaHCO.sub.3 1213Sodium Acetate 3H.sub.2 O 250Sodium Acetate 25Phenol Red(Na) 10______________________________________ TABLE 5______________________________________Final concentrations of Ca.sup.2+ and Mg.sup.2+medium No. 1 2 3 4 5 6______________________________________Ca.sup.2+ (mM) 0.95 0.95 0.95 1.1 1.1 1.1Mg.sub.2+ (mM) 0.4 0.7 5.5 0.4 0.7 5.5______________________________________

Example 5

A basal medium shown in following table 6 supplemented with the substances shown in above table 3 and fetal bovine serum at a final concentration of 5%(v/v) was used. Final Ca.sup.2+ and Mg.sup.2+ concentrations were adjusted according to table 7 respectively.

Normal human epidermal melanocyte were cultured using the above media according to the procedure of example 1. After 5 days incubation, the cells were harvested and cell numbers were determined. The results are shown in FIG. 10.

TABLE 6______________________________________Components of basal medium of example 5Component mg/l______________________________________L-Alanine 8.96L-Arginine.HCl 210.9L-Asparagine.H.sub.2 O 15.01L-Aspartic Acid 8.50L-Cysteine HCl.H.sub.2 O 20.01L-Cystine 12.5L-Glutamic Acid 14.71L-Glutamine 511.6Glycine 7.51L-Histidine HCl.H.sub.2 O 45.0L-Isoleucine 51.45L-Leucine 39.3L-Lysine.HCl 23.64L-Methionine 11.20L-Phenylalanie 12.41L-Proline 23.02L-Serine 36.78L-Threonine 7.74L-Tryptophan 6.43L-Tyrosine 9.06L-Valine 19.32d-Biotin 0.019Folic Acid 1.06DL-alpha-Lipoic Acid 0.203Nicotinamide 0.326Ethanolamine 3.05Phosphoethanolamine 7.05D-Pantothenate(Ca) 0.592Pyridoxine.HCl 0.134Riboflavin 0.376Thiamin HCl 0.669Vitamin B.sub.12 2.72Adenine 12.2Choline Chloride 7.33D-Glucose 1091myo-lnositol 9.281Hypoxanthine 2Putrescine.2HCl 0.0806Sodium Pyruvate 82.5Thymidine 0.713CaCl.sub.2.2H.sub.2 O 33.08KCl 198MgCl.sub.2.6H.sub.2 O 61MgSO.sub.4.7H.sub.2 O 76.4NaCl 7500Na.sub.2 HPO.sub.4.7H.sub.2 O 268Na.sub.2 HPO.sub.4 76.9KH.sub.2 PO.sub.4 41.5CuSO.sub.4.5H.sub.2 O 0.00263FeSO.sub.4.7H.sub.2 O 1.112H.sub.2 SeO.sub.3 0.00193MnSO.sub.4.5H.sub.2 O 0.000121Na.sub.2 SiO.sub.3.9H.sub.2 O 0.0711(NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 O 0.000618NH.sub.4 VO.sub.3 0.000293NiCl.sub.2.6H.sub.2 O 0.0000595SnCl.sub.2.2H.sub.2 O 0.0000565ZnSO.sub.4.7H.sub.2 O 0.0863HEPES 3336NaHCO.sub.3 1188Sodium Acetate 3H.sub.2 O 250Phenol Red(Na) 0.621______________________________________ TABLE 7______________________________________Final concentrations of Ca.sup.2+ and Mg.sup.2+medium No. 1 2 3 4 5 6 7______________________________________Ca.sup.2+ (mM) 0.20 0.20 0.20 0.95 0.95 1.15 1.15Mg.sub.2+ (mM) 0.3 1.25 5.5 1.25 5.5 1.25 5.5______________________________________

Example 6

A basal medium shown in following table 8 supplemented with the substances shown in above table 3 and fetal bovine serum at a final concentration of 5%(v/v) were used. Final Ca.sup.2+ and Mg.sup.2+ concentrations were adjusted according to table 9 respectively.

Normal human epidermal melanocyte were cultured using the above media according to the procedure of example 1. After 7 days incubation, the cells were harvested and cell numbers were determined. The results are shown in FIG. 11.

TABLE 8______________________________________Components of basal medium of example 6Component mg/l______________________________________L-Alanine 9L-Arginine.HCl 211L-Asparagine.H.sub.2 O 15.01L-Aspartic Acid 13L-Cystine 25L-Glutamic Acid 14.7L-Glutamine 146Glycine 7.51L-Histidine HCl.H.sub.2 O 23L-Isoleucine 2.6L-Leucine 13L-Lysine.HCl 29L-Methionine 4.48L-Phenylalanie 5L-Proline 11.5L-Serine 10.5L-Threonine 3.57L-Tryptophan 0.6L-Tyrosine 1.81L-Valine 3.5d-Biotin 0.024Folic Acid 1.32DL-.alpha.-Lipoic Acid 0.2Nicotinamide 0.615D-Pantothenate(Ca) 0.715Pyridoxine.HCl 0.206Riboflavin 0.0238Thiamin HCl 1Vitamin B.sub.12 1.36Choline Chloride 0.698D-Glucose 1100myo-lnositol 0.541Hypoxanthine 4Sodium Pyruvate 110Thymidine 0.7CaCl.sub.2.2H.sub.2 O 44.1KCl 285MgSO.sub.4.7H.sub.2 O 152.8NaCl 7400Na.sub.2 HPO.sub.4 153.7KH.sub.2 PO.sub.4 83CuSO.sub.4.5H.sub.2 O 0.0025FeSO.sub.4.7H.sub.2 O 0.834ZnSO.sub.4.7H.sub.2 O 0.0288NaHCO.sub.3 1200______________________________________ TABLE 9______________________________________Final concentrations of Ca.sup.2+ and Mg.sup.2+medium No. 1 2 3 4 5______________________________________Ca.sup.2+ 0.3M) 0.95 0.95 1.15 1.15Mg.sub.2+ 0.65) 0.65 5.5 0.65 5.5______________________________________

Example 7

The modified MCDB153 medium shown in above table 2 supplemented with the substances shown in above table 3 was used. The medium contains 0.15 mM of Ca.sup.2+ and 0.60 mM of Mg.sup.2+. In addition, the medium containing 0.9 mM of Ca.sup.2+ and 2.0 mM of Mg.sup.2+ was prepared. Various amounts of FBS were added into the both of media to yield a final concentration of from 0.001 to 10% (v/v). Normal human epidermal melanocyte were cultured in the above respective media according to the procedure of example 1. After 6 days incubation, the cells were harvested and cell number were determined. The results were shown in FIG. 11.

A maximum proliferation of the cell was obtained With a medium containing 0.01% of FBS. At any concentration of FBS, the cell proliferation obtained with the Ca.sup.2+ or Mg.sup.2+ enriched media was superior to that of conventional media.

Claims

1. A medium for culturing normal human epidermal melanocytes in vitro, comprising a basal medium for culturing animal cells, Ca.sup.2+ at a final concentration of between about 0.9 mM and about 1.2 mM, Mg.sup.2+ at a final concentration of between about 1.2 mM and about 6.0 mM, and serum at a final concentration of between about 0.001% and about 0.1% (v/v).

2. The medium of claim 1, further comprising one or more growth factors useful for growth of normal human epidermal melanocytes.

3. The medium of claim 2, wherein the basal medium for animal cell culture is MCDB 153.

4. The medium of claim 1, further comprising bovine pituitary extract.

5. The medium of claim 4, wherein the basal medium for animal cell culture is MCDB 153.

6. The medium of claim 1, wherein the basal medium for animal cell culture is MCDB 153.

7. A medium for culturing normal human epidermal melanocyte in vitro, comprising a basal medium for culturing animal cells, Ca.sup.2+ at a final concentration of between about 0.15 mM and about 1.2 mM, Mg.sup.2+ at a final concentration of between about 1.2 mM and about 6.0 mM and serum at a concentration of between about 0.001% and about 0.1% (v/v).

8. The medium of claim 7, wherein the final concentration of Ca.sup.2+ is between about 0.9 mM and about 1.2 mM and the final concentration of Mg.sup.2+ is between about 1.2 mM and 6.0 mM.

9. The medium of claim 7, further comprising one or more growth factors useful for growth of normal human epidermal melanocytes.

10. The medium of claim 7, further comprising bovine pituitary extract.

11. The medium of claim 7, wherein the basal medium for animal cell culture is MCDB 153.