KR100684118B1 - Highly enriched medium and method for culture of hair follicle - Google Patents

Abstract

The present invention relates to a high-concentration medium for culturing hair follicle organs and a method for culturing hair follicle organs using the same, specifically, in amounts of 2,000 to 3,000 mg / l amino acids and 40 to 60 mg / l vitamins, respectively. It relates to a serum-free medium, characterized in that it comprises a culturing method of hair follicle organ comprising the step of culturing the hair follicle organ in the medium. The high-concentration medium of the present invention can cultivate various types of cells contained in the hair follicle organs for a long time and maintain the normal phenotype of the hair follicle organs and the ability to produce hair. It can increase. Moreover, since it is a serum-free medium, there is no problem, such as an infection, and it can apply also to the storage medium of a hair follicle organ.

Description

High concentration medium and culture method for culturing hair follicles {{HIGHLY ENRICHED MEDIUM AND METHOD FOR CULTURE OF HAIR FOLLICLE}             

1 is a culture medium of William medium E (William's E Medium) + ITS + HC and IMDM + ITS + HC and DHGM (Dongguk Hair Growth Media) + ITS + HC as a medium of the present invention over time, It is a graph showing the amount of change in the length of hair,

Figure 2a to 2c is a photograph of the observation of the change in hair length over time when cultured with William medium E + ITS + HC and IMDM + ITS + HC and high concentration medium of the present invention DHGM + ITS + HC ,

Figure 3 shows the change in hair length over time when ITS and HC were incubated with conventional medium such as IMDM, DMEM, DMEM / F-12, F-12, Mc Coy's 5A, RPMI1640 and cultured. Is a graph,

Figure 4 is a graph showing the amount of active alkaline phosphate in hair follicle organs on the 4th and 8th day of culture when incubated with fillpot medium (William medium E + ITS + HC) and DHGM + ITS + HC.

The present invention relates to a high concentration medium and a culture method for culturing hair follicle organs, and more particularly, the amino acid is contained in an amount of 2,000 to 3,000 mg / L, vitamins in an amount of 40 to 60 mg / L, respectively The present invention relates to a serum-free medium and a method for culturing hair follicle organs using the medium.

Hair follicles are pocket-shaped epithelial tissue that wraps around the hair roots. Hair follicles refer to areas where hair is embedded in the skin.

Hair grows actively due to cell division near the hair follicles. If the cell's ability to divide decreases, the hair follicles contract and move to shallower areas of the skin. Normally, the cell divisions are regained, and the generated cell populations move deep into the skin to form new hair follicles, grow hair, and repel old hair. If such a 'hair exchange' does not occur, alopecia is caused. .

Although the optimal treatment for hair loss developed until now is hair transplantation, hair loss is also performed at the initial stage of hair loss with the approval of the FDA on the market, external drug Minoxidil, or Propecia, which is mainly composed of orally administered finasteride. Progress can be slowed (Arca E. et al., Dermatology , 209 (2): 117-25, 2004).

To find an effective substance useful for the treatment of early hair loss, Roh SS et al., J Dermatol Sci ., 30 (1): 43-9, 2002), in vivo experiments, depilated rats, The hair growth phenomenon was observed after application to the skin, Philpott MP et al., British Journal of Dermatology, 127: 600-607, 1992, Philpott MP et al., J Invest. Deramtol, 115: 1152-1155, 2000) has conducted animal experiments to examine the effects of growth factors such as IGF (Insulin-like growth factor), EGF (Epidermal growth factor), and HGF (Hepatocyte growth factor) through in vitro organ culture using rat mustache. Unlike rodents that molt at regular intervals, humans have separate cycles for hair follicles, so the trends for growth factors are not all consistent. The monkey model, on the other hand, has a similar tendency to humans, but the burden of time and experiment costs is too high. In addition, in recent years, in vivo animal experiments have been socially criticized and legally regulated in terms of animal protection, and development of an in vitro toxicity test model to replace animal tests has emerged as a global problem.

Unlike in vivo experiments, in vitro experiments have difficulty in long-term experiments because nutrition or oxygen supply through blood vessels is blocked during cell culture. In addition, in the case of in vitro experiments, it is most important to maintain the function of artificial tissues in a similar environment to maintain them for a long time. .

In vitro hair growth model using hair follicle organ culture was conducted by Kondo S et al., Arch Dermatol Res . 282 (7): 442-5, 1990) by immersion culture in DMEM (Dulbecco's Modified Eagle Medium). There was a study incubated in glass bottles after the addition of 100%, Jindo et al. (Jindo T. et al., Dermatol . 20 (12): 756-62, 1993) increased the oxygen pressure and incubated with RPMI 1640 medium at 31 ° C. In this case, the growth rate and length increase were decreased, but the morphology of the hairballs was smaller than that at 37 ° C.

The medium for culturing hair follicle organs, which has been commonly used since 2000, was developed by Philpott, and 10 µg / ml insulin and 10 µg / ml transferrin in William's medium E. ), 10 μg / ml sodium selenite and 10 μg / ml hydrocortisone are added. In addition, a study was confirmed to increase the amount of ATP synthesis of cells in hair follicle organs when cultured by increasing the concentration of L-glutamine to 4 mM (Kealey T. et al., Skin Pharmacol . 7 (1-2): 41-6, 1994) and studies confirming that hair follicle organs have a longer survival than 37 ° C. when stored at 4 ° C. (Kurata S et al, Dermatol Surg ., 25 (1): 26-9, 1999). In addition, there have been many studies to extend the survival time in the medium by adding various growth factors to the known medium composition, but the composition of the culture medium for hair follicle organ culture that increases the amount of amino acids or vitamins is not known.

Accordingly, the present inventors increased the amount of amino acids and vitamins in the culture medium for existing follicle organs, and cultured serum-free follicle organs containing insulin, transferrin, sodium selenite, hydrocortisone, albumin, trace amounts of copper, iron, zinc, and manganese. The present invention was completed by discovering that the medium can survive cells for a longer time than the conventional medium and maintain the normal phenotype of the hair follicle organs.

Accordingly, it is an object of the present invention to provide a high concentration medium for culturing hair follicle organs, which is capable of culturing cells in a normal form for a longer time than conventional cell culture medium.

Another object of the present invention is to provide a method for culturing hair follicle organs using the medium.

In order to achieve the above object, the present invention, in the medium for culturing hair follicles, amino acids are contained in an amount of 2,000 to 3,000 mg / L, vitamins in an amount of 40 to 60 mg / L, respectively, characterized in that Serum medium is provided.

In addition, the present invention provides a method for culturing hair follicle organs comprising culturing the hair follicle organs in the medium.

Hereinafter, the present invention will be described in more detail.

The high concentration medium of the present invention (hereinafter referred to as DHGM (Dongguk Hair Growth Media)) is conventionally used as a cell culture medium, DMEM (Dulbecco's Modified Eagle Medium), DMEM / F-12, F-12, Mc Coy's 5A, RPMI1640 Occurs in the culture of hair follicle organs with many types of cells because they contain 2 to 5 times higher concentrations of amino acids and vitamins than conventional media such as William's Medium E and IMDMve's Modified Dulbecco's Modification (IMDM). It is characterized by overcoming nutrient deficiency, overcoming oxygen deficiency, and improving the specific functions of low follicular organs following culture.

The high concentration DHGM medium of the present invention is an amino acid of L-arginine, L-asparagine, L-aspartic acid, L-cystine 2HCl, L-isoleucine, L-leucine, and L-lysine HCl are each 30 To 200 mg / l, the aromatic amino acids L-phenylalanine, L-tryptophan, and L-tyrosine of the essential amino acids each containing 30 to 210 mg / l, and the rest of the essential amino acids each containing 50 to 600 mg / l. It is preferable.

In addition, as vitamins, biotin, D-Ca pantothenate, and riboflavin, which are water-soluble vitamin B groups, were 0.02 to 1 mg / l, respectively, and choline chloride, folic acid, niacinamide, pyridoxine HCl, and thiamine HCl were respectively 3 to 16 mg / l. L, i-inositol preferably contains 10 to 15 mg / l, and vitamin B ₁₂ contains 0.02 to 0.03 mg / l. In addition, the medium of the present invention more preferably contains 0.03 to 0.07 mg / L of glutathione, 400 to 600 mg / L of glutamine, and 1,500 to 3,000 mg / L of D-glucose.

In addition, sodium bicarbonate (NaHCO ₃ ) 3,000 to 3,500 mg / l with pH buffering effect, and Hepis (HEPES, N- [2-hydroxyethyl] piperazine-N '-[2-ethanesulfonic acid]) 2,000 to 2,500 mg / l, Ca and Mg, minerals important for cell-to-cell adhesion, at concentrations of 1.0 to 2.0 mM and 0.5 to 1.0 mM, respectively, Cu, Fe, Mn, and Zn, other minerals that are important for cell metabolism It is more preferable to contain at a concentration of 0.25 to 0.7 nM each, and to include sodium chloride at a concentration of 4,000 to 5,000 mg / l in order to adjust the osmotic pressure to 280 to 310 mOsm / kg.

In addition, the medium of the present invention is added hydrocortisone (HC), insulin (I), transferrin (T), and sodium selenite (S) to further extend the culture period of hair follicles For example, the hydrocortisone is 10 to 100 μg / L, insulin is 5 to 20 mg / L, transferrin is 5 to 20 mg / L, and sodium selenite 0.005 to 0.02 mg / L It is preferable to use at the concentration of.

In particular, in the present invention, CaCl ₂ 165 mg / L, CuSO ₄ · 5H ₂ O 0.0001 mg / L, Fe (NO ₃ ) .9H ₂ O 0.0001 mg / L, KCl 330 mg / L, KNO ₃ 0.076 mg / L, MgSO ₄ 98 mg / l, MnCl ₂ · 4H ₂ O 0.0001 mg / l, NaCl 4,800 mg / l, NaHCO ₃ 3,360 mg / l, Na ₂ HPO ₄ 111 mg / l, ZnSO ₄ · 7H ₂ O 0.0002 mg / l, D-glucose 2,000 mg / l, sodium pyruvate 110 mg / l, HEPES 2,383 mg / l, phenol red 15 mg / l, L-alanine 50 mg / l, L-arginine 100 mg / l, L-asparagine 50 Mg / l, L-aspartic acid 60 mg / l, L-cystine.2HCl 182.4 mg / l, L-glutamic acid 150 mg / l, L-glutamine 584 mg / l, L-glycine 60 mg / l, L-histidine 62.1 mg / l, L-isoleucine 210 mg / l, L-leucine 210 mg / l, L-lysine HCl 292 mg / l, L-methionine 60 mg / l, L-phenylalanine 132 mg / l, L-proline 80 mg / l, L-serine 84 mg / l, L-threonine 190 mg / l, L-tryptophan 32 mg / l, L-tyrosine 208 mg / l, L-valine 188 mg / l, biotin 0.026 Mg / l, D-Ca Pantothe Yitrate 0.026 mg / l, choline chloride 8 mg / l, folic acid 16 mg / l, i-inositol 14.40 mg / l, niacinamide 8 mg / l, pyridoxine HCl 8 mg / l, riboflavin 0.8 mg / l, thiamine HCl 3 Preferred is a medium consisting of mg / l, and vitamin B ₁₂ 0.026 mg / l.

The preferred high-concentration DHGM medium has twice the amount of amino acids and vitamins that supply energy for cell metabolism and maintain cell activity, and is less metabolic because it is metabolized to lactic acid than IMDM, which is a general medium. The content of D-glucose is reduced to 2,000 mg / l. The concentration of L-glutamine affecting the amount of ATP synthesis in the amino acid is fixed at 4 mM. In addition, the trace elements include zinc 0.7 nM, iron 0.25 nM, copper 0.4 nM and manganese 0.5 nM, and sodium bicarbonate and HEPES having a pH buffering effect at a concentration of 40 mM and 10 mM, respectively. Final concentrations of calcium and magnesium, which are essential minerals for intercellular adhesion, are 1.5 mM and 0.8 mM, respectively, and contain 4,500 mg / l of sodium chloride to adjust the osmotic pressure to 280 to 310 mOsm / kg.

In addition, 10 mg / L of transferrin is added as an iron source, and 0.01 mg / L of sodium selenite as an inorganic salt, 10 μg / L of hydrocortisone and 10 mg / L of insulin, and 0.2% by weight of albumin are further included.

When using serum-containing media in hair follicle organ cultures, hair follicles have a good initial growth rate but tend to prematurely deteriorate and are susceptible to infection, making them unsuitable for long-term culture of follicular organs (Randall VA. Et al., J Investig). Dermatol Symp Proc. 8 (1): 39-45, 2003) Although it is difficult to analyze the data, the medium of the present invention does not include serum and thus is useful as a medium for culturing and storing hair follicle organs since there is no fear of infection.

In addition, the important pH in cell culture is kept constant by the buffering effect between bicarbonate and the end metabolites. That is, the medium of the present invention may have a higher concentration of amino acids than William medium E, which may cause an increase in pH due to the metabolite ammonia, but the pH is well maintained between 7.2 and 7.5 due to the addition of bicarbonate buffer.

Copper sulfate contained in the culture medium for hair follicle organ culture of the present invention inhibits apoptosis caused by radical ions through Superoxide dismutase (SOD), a copper-dependent enzyme and antioxidant present in hair follicle organs. Other copper-dependent enzymes are known to inhibit both 5α-reductase-1 and -2 in hair follicle organs, promoting natural growth factor synthesis and promoting the synthesis of extracellular matrix. Zinc is also known as an essential mineral for zinc finger transcription factors.

The media of the invention can be used to culture hair follicle organs under conventional cell culture conditions, for example at 37 ° C., preferably in the presence of 5% CO ₂ . In culture, it is preferable to replace the medium with fresh medium every 48 hours.

When the hair follicle organ is cultured using the hair follicle organ culture medium of the present invention, not only increases the length of the hair compared to the existing medium, but also maintains the normal phenotype of the hair follicle organ and the ability to produce hair for a long time (see FIGS. 1 to 4). .

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are not intended to limit the invention only.

Example 1 and Comparative Examples 1 to 7: Hair follicle organ culture and hair length measurement

Vibrissa hair follicle organs of Spague Dawley rats were separated from the skin to remove some of the outer hair roots of the hair and the top of the hair follicles. The isolated hair follicle organs were placed in a 24 well plate and insulin 10 mg / l, transferrin 10 mg / l, sodium selenite 0.01 mg / l, hydrocortisone 0.01 mg / l, and albumin 0.2% by weight (albumin 2 g / l) were added. William μl E and IMDM or 500 μl of DHGM medium of Table 1, respectively, were added thereto, and cultured at 37 ° C. and 5% CO ₂ under 48 hour intervals.

DHGM medium (DHGM + ITS + HC), William medium E (William's E + ITS + HC), and IMDM medium (IMDM + ITS + HC) of the present invention, each with insulin, transferrin, sodium selenite and hydrocortisone added In order to compare the type and total length of hair grown in the above, the length of the hair was measured over time for each medium replacement time of the hair follicle organ cultured by the above method (FIG. 1), and photographed (FIG. 2A to 2C).

In addition, in order to compare the length of the hair grown in the general culture medium having a variety of composition with the length of the hair grown in the medium of the present invention, the general medium is IMDM, DMEM, DMEM / F-12, F-12, Mc Coy's 5A, RPMI1640 Except for using as was measured the length of the hair with time in the same manner as above (Fig. 3).

From the results of FIGS. 1 to 3, when hair follicle organs were cultured in the conventional medium of William medium E and IMDM added with ITS and HC, the hair growth of hair was similar to each other on the ninth day, but William medium E + ITS Hair follicle organs grown in + HC were separated from hair papilla and hair after 9 days due to cell death, and in the case of IMDM + ITS + HC, the length increased continuously up to 11 days. In addition, the media added with ITS and HC in DMEM, DMEM / F-12, F-12, Mc Coy's 5A, RPMI1640 showed less growth than IMDM + ITS + HC, and most of the growth occurred 14 days before culture. I could see it stopped.

In the case of IMDM + ITS + HC and DHGM + ITS + HC of the present invention, the initial growth rate of both media was higher than that of the fillpot medium, but the cell survival after 11 days in the follicle organs cultured in IMDM + ITS + HC On the other hand, there was no length growth, and after 17 days, the hair papilla and the hair were separated and dropped out due to cell death, which tended to enter necrosis as a whole. However, the hair follicle organs cultured in the DHGM + ITS + HC medium of the present invention confirmed that the length growth continued until day 25.

Example 2 and Comparative Example 8: Determination of hair generating ability (amount of alkaline phosphatase)

In order to determine the hair-generating capacity of the hair follicle organs cultured in the medium according to the present invention, the hair follicle organs were cultured in DHGM + ITS + HC medium and fillpot medium in the same manner as in Example 1, and on the 4th and 8th days of the culture, Isolates the dermal papilla in the hair follicle organs, pulverizes the tissue in lysis buffer (SDS, phenylmethylsulfonyl fluoride, ethylendiamine tetraacetic acid, pepstatin A, leupeptin, aprotinin) The amount of phosphatase was measured by a method of measuring fluorescence by attaching a secondary antibody to alkaline phosphatase (FIG. 4).

As can be seen from Figure 4, in the case of DHGM + ITS + HC of the present invention, the amount of alkaline phosphate was significantly superior from the fourth day compared to the existing fillpot medium, and this trend was maintained even on the eighth day of culture. It can be seen that the medium of the invention has an excellent ability to maintain hair generating ability of the hair follicle organ.

 As described above, the high-concentration medium of the present invention can culture various types of cells included in the hair follicle organs for a long time and maintain the normal phenotype and hair-generating ability of the hair follicle organs, thereby maintaining the state of the hair follicle organs during hair transplantation. The engraftment rate can be raised by maintaining favorable. Moreover, since it is a serum-free medium, there is no problem, such as an infection, and it can apply also as a medium for hair follicle organ storage.

Claims (6)

In the medium for hair follicle organ culture, L-arginine, L-asparagine, L-aspartic acid, L-cystine 2HCl, L-isoleucine, L-leucine, and L-lysine HCl each 30 to 200 mg L / Phenylalanine, L-Tryptophan, and L-Tyrosine at 30-210 mg / l, respectively, L-Alanine, L-Glutamic Acid, L-Glycine, L-Histidine, L-Methionine, L-Proline, L-serine, L-threonine and L-valine at 50-600 mg / l respectively, biotin, D-Ca pantothenate, and riboflavin at 0.01-2 mg / l, respectively, choline chloride, folic acid, niacinamide, Serum-free medium comprising pyridoxine HCl and thiamine HCl at 3-16 mg / l, i-inositol at 10-15 mg / l, and vitamin B ₁₂ at 0.02-0.03 mg / l. delete delete The method of claim 1, Glutamine 400-600 mg / l, D-glucose 1,500-3,000 mg / l, sodium bicarbonate (NaHCO ₃ ) 3,000-3,500 mg / l, hepis (HEPES, N- [2-hydroxyethyl] piperazine-N '-[2 -ethanesulfonic acid]) containing 2,000 to 2,500 mg / l, Ca 1.0 to 2.0 mM, Mg 0.5 to 1.0 mM, Cu, Fe, Mn, Zn, 0.25 to 0.7 nM, and sodium chloride 4,000 to 5,000 mg / l, respectively. Serum-free medium characterized by. The method of claim 1,  A serum-free medium characterized by further comprising insulin, transferrin, sodium selenite, hydrocortisone, and albumin. A method of culturing hair follicle organs comprising culturing the hair follicle organs in the medium of any one of claims 1, 4 and 5.

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