KR100567655B1 - A medium for culturing a fibroblast, a method of producing a dermis equivalent using the medium and a dermis equivalent produced by the method, a method of producing a skin equivalent using the dermis equivalent and a skin equivalent produced by the method - Google Patents

Abstract

The present invention is a fibroblast culture medium comprising a nutrient source having a defined component supplemented with epidermal growth factor (EGF) and serum, a method for producing a dermal substitute using the medium, and a dermal substitute prepared thereby, Provided are methods of making skin substitutes using dermal substitutes and skin substitutes made thereby.

Fibroblasts, keratinocytes, dermis, epidermis, culture, dermal substitutes, skin substitutes

Description

A medium for fibroblast culture, a method for preparing a dermal substitute using the medium, and a dermal substitute prepared thereby, a method for preparing a skin substitute using the dermal substitute, and a skin substitute produced thereby fibroblast, a method of producing a dermis equivalent using the medium and a dermis equivalent produced by the method, a method of producing a skin equivalent using the dermis equivalent and a skin equivalent produced by the method}

1 is a result of visual observation of a fibrous sheet (A) obtained by the method for producing a dermal substitute of the present invention.

FIG. 2A is a diagram showing that the fibrous sheet obtained by the method for preparing the dermal substitute of the present invention is composed of an extracellular matrix including many fibroblasts and is a dermal-like tissue (hematoxylin & iosin staining).

FIG. 2B is a diagram showing that the fibrous sheet obtained by the method for preparing the dermal substitute of the present invention is composed of an extracellular matrix including many fibroblasts and is a dermal-like tissue (wear and trichrome staining).

2C and 2D are diagrams showing that dermal extracellular matrix components such as type 1 collagen and elastin are diffusely expressed in the fibrous sheet.

3 is a view showing the results of electron microscope observation of the fibrous sheet obtained by the method for producing a dermal substitute of the present invention.

It is a figure which shows the result of staining hematoxylin and iosin of the obtained fibrous sheet.

It is a figure which shows the result of horse-trichrome staining of the obtained fibrous sheet.

It is a figure which shows the result of dyeing the obtained fibrous sheet with type 1 collagen.

It is a figure which shows the result of dyeing the obtained fibrous sheet with elastin.

FIG. 8A shows the skin substitute of the present invention, showing that a multi-layered epidermis with a stratum corneum is formed on the dermal components similar to the natural epidermis in the in vivo state (hematoxylin & iosin staining).

Figure 8b shows the skin substitute of the present invention, showing the results of the horseshoe-trichrome staining.

8C and 8D show skin substitutes of the present invention, which show that keratin 1 and keratin 10 are expressed in the upper base layer except for the lower one or two layers, respectively.

8E, 8F, 8G, 8H, and 8i show the skin substitutes of the present invention, wherein the basement membrane components of involuclin, β4 integrin, laminin 5, 4 and 7 collagen dermal-epidermal junction (DEJ) It is a figure showing what is expressed in

Fig. 9 shows the skin substitute of the present invention and shows the result of observing the obtained human skin substitute with an electron microscope.

Figure 10a is a diagram showing the results of staining with ematoxine and iosin from skin biopsy samples obtained from hairless mice transplanted with a dermal replacement of the present invention.

FIG. 10B is a diagram showing the result of horseson-trichrome staining of a skin biopsy sample obtained from hairless mice transplanted with a dermal replacement of the present invention.

The present invention relates to a medium for fibroblast culture, a method for preparing a dermal substitute using the medium, and a dermal substitute prepared thereby, a method for preparing a skin substitute using the dermal substitute, and a skin substitute prepared thereby. .

The skin consists of the dermis and the epidermis, the upper layer. In order to mimic this skin more closely in physiological conditions in vitro, methods have been developed to reconstruct skin substitutes using a three-dimensional culture system. Conventionally, two methods have been widely used to prepare a skin substitute (Regnier et al. Front Matrix Biol., 9, 4-35, 1981; Bell et al. J. Invest. Dermatol. 81: 2s-10s , 1983). One of them is a method for preparing a skin substitute by culturing keratinocytes on dead de-epidermized dermis (DED). Skin substitutes prepared by this method have the epidermis reconstituted on the DED. Another method is to prepare a skin substitute by culturing keratinocytes on collagen matrix including fibroblasts. The two skin substitutes produced by these methods have a multilayered epidermis with a stratum corneum on the dermal matrix, very similar to the in vivo natural epidermis. Furthermore, culturing keratinocytes on various dermal substrates such as DED in combination with collagen-glycosaminoglycan biopolymers, collagen sponges containing fibroblasts and collagen substrates comprising fibroblasts. Several skin replacement methods have been disclosed (Boyce et al., Skin Pharmacol. , 2, 136-143, 1990; Maruguchi et al., Plast Reconstr. Surg. , 93, 537-544, 1994; Lee et al. , J. Dermatol. Sci. , 23, 132-7, 2000). The skin substitutes prepared by these methods have been useful in a variety of fields, such as skin biology, pharmacology and toxicology, compared to monolayer cultures (Boyce et al., Skin Pharmacol. , 2, 136-143, 1990; Ponec 1991 , Toxicol. In Vitro., 5,597-606, 1991; Gay et al., Toxicol. In Vitro 6, 303-315, 1992). However, all these methods have a problem in that foreign materials such as bovine collagen and dead dermis, which are difficult to obtain and become infectious agents, must be used as the dermal base material.

Dermal fibroblasts provide connective tissue matrix material including collagen, elastin, and other matrix proteins. Monolayer cultures have been studied for a long time to study the biological properties of fibroblasts. However, these systems are not similar to in vivo conditions in terms of the absence of extracellular matrix around fibroblasts (Weber et. Al., J. Invest. Dermatol. , 87, 218-221, 1986).

Accordingly, studies on fibroblast culture methods more similar to in vivo conditions have been conducted. For example, instead of conventional monolayer cultures, it has been found that culturing fibroblasts after confluence more efficiently treat extracellular matrix such as collagen (Booth et al., Biochim. Biophys Acta. , 607, 145-60, 1980; Minor et al., J. Biol. Chem. , 261, 10006-14, 1986). It has also been found that dermal-like substrates are produced extensively when supplemented with ascobic acid over a long period in medium used for culturing fibroblasts after confluence (Grinnell et al., Exp. Cell Res. , 181, 483-491,1989). However, there is a problem that ascorbic acid is very unstable in solution, especially under normal culture conditions at neutral pH and 37 ° C. (Peterkofsky B., Arch. Biochem. Biophys., 152, 318-328, 1972).

Accordingly, the present inventors have completed the present invention, which has been intensively studying a method for efficiently preparing the dermis without using an foreign base material.

Accordingly, it is an object of the present invention to provide a stable medium that can be used to prepare dermal substitutes from fibroblasts without the use of foreign bases.

Another object of the present invention is to provide a method for preparing dermal substitutes from fibroblasts using a stable medium that can be used to prepare dermal substitutes from fibroblasts without the use of foreign bases.                         

Another object of the present invention is to provide a dermal substitute prepared by the method for preparing the dermal substitute.

Another object of the present invention is to provide a method for preparing a skin substitute using the dermal substitute.

It is also an object of the present invention to provide a skin substitute prepared by the method for producing the skin substitute.

The present invention provides a medium for fibroblast culture comprising epidermal growth factor (EGF), and a nutrient source having a defined component supplemented with serum.

In the medium of the invention, epidermal growth factor (EGF) comprises a mammalian epidermal growth factor, preferably a human or mouse epidermal growth factor, most preferably a human epidermal growth factor. In the present invention, epidermal growth factor is preferably included at a concentration of 1 to 50 ng / ml.

In the medium of the present invention, the nutrient source supplies essential nutrients to the cultured cells. These nutrients include energy sources such as glucose, fructose or lactose; Essential and non-essential amino acids; Water soluble (group B, biotin, folic acid, nicotinamide, pantothenic acid, pyridoxine, riboflavin and thiamine) and fat soluble (A, D, E, K and ubiquinone) vitamins; Major inorganic ions such as bircarbonate, calcium, chloride, magnesium, phosphate, potassium and sodium; Trace elements such as As, Co, Cr, Cu, F, Fe, Mn, Mo, Ni, Se, Si, Sn, V, and Zn; Lipids; Buffers such as CO ₂ / HCO ₃ and HEPES; Gases (oxygen and carbon dioxide); And nucleic acid precursors such as adenine, cytidine, hypoxanthine, and thymidine.

There are a variety of commercially available nutrients expected to be useful in embodiments of the present invention. These include commercially available nutrients that supply inorganic salts, energy sources, amino acids, and B-vitamins, for example DMEM (Dulbecco's Modified Eagle's Medium); MEM (Minimium Essential Medium); M199; RPMI 1640; (All available from Flow Laboratories); IsMEM's Modified Dulbecco's Modified Eagle's Medium (EDMEM) (Gibco Labs). MEM and M199 need to be supplemented with phospholipid precursors and non-essential amino acids. Commercially available vitamin floating mixtures that provide additional amino acids, nucleic acids, enzyme cofactors, phospholipid precursors, and inorganic salts include Ham's F12; Ham's 10; NCTC 109; (both available from Flow Laboratories) and NCTC 135 (Irvine Scientific).

DMEM: 1X MEM (Modified) Dulbecco's modified liquid without L-glutamine. 12-332

ingredient mg / L Inorganic salt CaCl ₂ CaCl ₂ ㆍ 2H ₂ O  264.90 Fe (NO ₃ ) ₃ ㆍ 9H ₂ O 0.10 KCl 400.0 MgSO ₄ MgSO ₄ ㆍ 7H ₂ O  200.00 NaCl 6400.00 NaHCO ₃ 3700.00 NaH ₂ PO ₄ ㆍ H ₂ O 125.00 Other ingredients Fructose glucose  4500.0 Lipophosphate 0 Phenolic Red 15.0 Sodium pyruvate 110.0 Aminopterin Hypoxanthine Thymidine

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amino acid L-arginineHCl 84.00 L-cystine 48.00 L-cystine, Na ₂ L-Glutamine Glycine 30.00 L-histidineHClH ₂ O 42.00 L-Isoleucine 104.80 L-Leucine 104.80 L-LysineHCl 146.20 L-methionine 30.00 L-phenylalanine 66.00 L-serine 42.00 L-Threonine 95.20 L-Tryptophan 16.00 L-tyrosine 72.00 L-valine 93.60 Vitamins D-Ca Pantothenate 4.00 Choline chloride 4.00 Folic acid 4.00 i-inositol 7.00 Nicotinamide 4.00 Pyridoxal HCl 4.00 Riboflavin 0.40 Thiamine HCl 4.00

MEM with Hank's salt or Earl's salt added

 ingredient 1x MEM (modified) Earle's Salt Liquid without glutamine Product No. 12-102 mg / L 1x MEM Hank's Salt Liquid without glutamine Product No. 12-132 mg / L Inorganic salt CaCl ₂ , or CaCl ₂ ㆍ 2H ₂ O 264.9 185.5 KCl 400.0 400.0 KH ₃ PO ₄ 60.0 MgSO ₄ , or MgSO ₄ ㆍ 7H ₂ O 200.0 200.0  NaCl 6800.0 8000.0 NaHCO ₃ 2000.0 350.0 NaH ₂ PO ₄ ㆍ H ₂ O 140.0 Na ₂ HPO ₄ 47.5  Hepes Other ingredients glucose 1000.0 1000.0 Phenolic red, Na 17.0 17.0 Sodium Succinate6H ₂ O Succinic acid amino acid L-alanine L-arginineHCl or L-arginineH ₂ O 126.40 126.40  L-aspartamic acid L-cystine or L-cystine, Na ₂ 24.02 24.02  L-glutamic acid  L-Glutamine Glycine L-histidineHClH ₂ O 41.90 41.90  L-Isoleucine 52.50 52.50  L-Leucine 52.50 52.50  L-LysineHCl 73.06 73.06  L-methionine 14.90 14.90  L-phenylalanine 33.02 33.02  L-proline  L-serine  L-Threonine 47.64 47.64  L-Tryptophan 10.20 10.20  L-tyrosine 36.22 36.22  L-valine 46.90 46.90

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Vitamins  D-Ca Pantothenate 1.00 1.00  Choline Bitartrate  Choline chloride 1.00 1.00  Folic acid 1.00 1.00 i-inositol 2.00 2.00 Nicotinamide 1.00 1.00 Pyridoxal.HCl 1.00 1.00 Riboflavin 0.10 0.10  Thiamine.HCl 1.00 1.00

RPMI 1640 Medium: 1X RPMI 1640 Liquid without L-Glutamine Catalog No. 12-602

ingredient mg / L Inorganic salt Ca (NO ₃ ) ₃ .4H ₂ O 100.0 KCl 400.0 MgSO ₄ MgSO ₄ .7H ₂ O 100.0 NaCl 6000.0 NaHCO ₃ 2000.0 Na ₂ HPO ₄ 800.7 HEPES Other Ingredients glucose 2000.0 Glutathione 1.00 Phenolic Red, Na 5.0 Hypoxanthine Aminopterin Thymidine Amino acids L-arginine 200.00 L-arginine. H ₂ O 56.82 L-aspartamic acid 20.00 L-cystine L-cystine, Na ₃  50.00 L-glutamic acid 20.00 L-Glutamine Glycine 10.00 L-histidine 15.00 L-hydroxyproline 20.00

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L-Isoleucine 50.00 L-Leucine 50.00 L-lysine.HCl 40.00 L-methionine 15.00 L-phenylalanine 15.00 L-proline 20.00 L-serine 30.00 L-Threonine 20.00 L-Tryptophan 5.00 L-tyrosine 20.00 L-valine 20.00 Vitamins Biotin 0.20 D-Ca Pantothenate 0.25 Choline chloride 3.00 Folic acid 1.00 i-inositol 35.00 Nicotinamide 1.00 PABA 1.00 Pyridoxine.HCl 1.00 Riboflavin 0.20 Thiamine.HCl 1.00 Vitamin B ₁₂ 0.005

references

 1 Moore, G. E., et al. 1967, JAMA, 199 519

 2 Morton, J. H., 1970, In Vitro, 6 89

Medium 199 (Modified): Liquid containing 1X Medium 199 (Modified) glutamine containing Ear's salt Catalog No. 12-203

ingredient mg / L Inorganic salts CaCl ₂ CaCl ₂ .2H ₂ O 264.9 Fe (NO ₁ ) ₃ .9 H ₂ O 0.10 KCl 400.0 KH ₃ PO ₃ MgSO ₄ MgSO ₃ .7H ₂ O  200.0 NaCl 6800.0 NaHCO ₂ 2200.0 NaH ₃ PO ₄ .H ₂ O Na ₃ HPO ₂ 140.0 Other ingredients Adenine-SO ₄ 10.00 S-adenylic acid 0.20 ATP, Na ₃ 10.00 DL-tocopherol-PO ₄ , Na ₂ 0.01 cholesterol 0.20 2-deoxy-D-ribose 0.50 glucose 1000.00 Glutathione 0.05 Guanine.HCl 0.30 Hypoxanthine 0.30 Phenolic red, Na 17.0 D-ribose 0.50 Sodium acetate 36.71 Thymine 0.30 Twin 80 5.00 Urasin 0.30 Xanthine 0.30

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Amino acids L-alanine 25.00 L-arginine.HCl 70.00 L-aspartamic acid 30.00 L-cysteine.HCl.H ₂ O 0.11 L-cystine 20.00 L-cystine, Na ₂ L-glutamic acid 66.82 L-Glutamine 100.00 Glycine 50.00 L-histidine.HCl.H ₂ O 21.88 L-hydroxyproline 10.00 L-Isoleucine 20.00 L-Leucine 60.00 L-lysine.HCl 70.00 L-methionine 15.00 L-phenylalanine 25.00 L-proline 40.00 L-serine 25.00 L-Threonine 30.00 L-Tryptophan 10.00 L-tyrosine 40.00 L-valine 25.00 Vitamins L-ascorbic acid 0.050 Biotin 0.010 Calciterol 0.100 D-Ca Pantothenate 0.010 Choline chloride 0.500 Folic acid 0.010 i-inositol 0.050 Menadione 0.010 Menadione Na Bisulfide. 3 H ₂ O Niacin 0.025 Nicotinamide 0.025 PABA 0.050 Pyridoxal.HCl 0.025 Pyridoxine.HCl 0.025 Riboflavin 0.010 Thiamine.HCl 0.010 Vitamin A Acetate 0.047

Ham's F-10 Badge

ingredient 1xHam's F10 with glutamine Catalog No. 2-403 mg / L Inorganic salts CaCl2 CaCl2.2H2O 44.1 CuSO4 0.0016 FeSO4 0.456 KCl 285.0 KH2PO4 83.0 MgSO4 MgSO4.7H2O 152.7 NaCl 7400.0 NaHCO3 1200.0 Na2HPO4 156.2 ZnSO4.7H2O 0.0288 Other ingredients glucose 1100.0 Hypoxanthine 4.08 Lipophosphate 0.206 Methyl linoleate Phenolic red, Na 1.24 Putrescine. 2HCl Sodium pyruvate 110.0 Thymidine 0.727 Amino acids L-alanine 8.91 L-arginine.HCl 210.70 L-arginine.H2O 15.01 L-aspartamic acid 13.31 L cysteine.HCl.H2O 35.13 L-glutamic acid 14.71 L-Glutamine 146.20 Glycine 7.51 L-histidine.HCl.H2O 20.96 L-Isoleucine 2.62 L-Leucine 13.12 L-lysine.HCl 29.30 L-methionine 4.48 L-phenylalanine 4.96 L-proline 11.51 L-serine 10.51 L-Threonine 3.57 L-Tryptophan 0.61 L-tyrosine 1.81 L-valine 3.51

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Vitamins Biotin 0.024 D-Ca Pantothenate 0.715 Choline chloride 0.698 Folic acid 1.32 i-inositol 0.541 Nicotinamide 0.611 Pyridoxine.HCl 0.206 Riboflavin 0.376 Thiamine.HCl 1.012 Vitamin B12 1.36

 Ham's F-12 Badge

 ingredient 1X Ham's F12 Liquid with Glutamine Catalog No. 12-423 mg / L Inorganic salts CaCl ₂ CaCl ₂ .2H ₂ O  44.1 CuSO ₄  0.0016 FeSO ₄   0.456  KCl  223.7 KH2PO ₄ MgSO ₄ MgSO ₄ .7H ₂ O   147.9  NaCl  7600.0 NaHCO ₃ 1176.0 Na ₂ HPO ₄  142.0 ZnSO ₄ ㆍ 7H ₂ O  0.863 Other ingredients  glucose 1802.0  Hypoxanthine  4.08 Lipophosphate  0.206 Methyl linoleate  0.088  Phenolic red, Na 1.24 Putrescine2HCl   0.1611 Sodium pyruvate 110.0  Thymidine  0.727

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 Amino acids  L-alanine 8.91  L-arginineHCl 210.70 L-arginineH ₂ O  15.01  L-aspartamic acid  13.31 L cysteine, HCl, H ₂ O  35.13  L-glutamic acid 14.71  L-Glutamine 146.20 Glycine 7.51 L-histidineHClH ₂ O  20.96  L-Isoleucine  3.94  L-Leucine  13.12  L-LysineHCl  36.53  L-methionine  4.48  L-phenylalanine  4.96  L-proline  34.54  L-serine  10.51  L-Threonine  11.91  L-Tryptophan  2.04  L-tyrosine  5.44  L-valine  11.72 Vitamins  Biotin  0.0073  D-Ca Pantothenate   0.238  Choline chloride  13.96  Folic acid 1.32 i-inositol  18.02 Nicotinamide  0.037 PyridoxineHCl 0.062 Riboflavin  0.038  Thiamine HCl  0.337 Vitamin B ₁₂ 1.36

NCTC Badge 109 AND 135

 Evans, V. J .: Bryant, J. C .: Kerr, H. A. & Schilling, E. L.,

 Exp. Cell Res. 36: 439-474, 1964

 Morton, H., In Vitro 6: 89-108, 1970

 ingredient   mg / L  L-alanine  31.48  L-amino-n-butyric acid    5.51  L-arginine HCl  31.16 L-arginine-H ₂ O  9.19  L-aspartamic acid 9.91  L-cystine / 2HCl 13.67 ^A * L-cysteine HClH ₂ O * 289.71 ^A  D-(+) glucosamine-HCl  3.85  L-glutamic acid  8.26  L-glutamine  135.73 Glycine  13.51 L-histidine HClH ₂ O   26.65  L-hydroxyproline  4.09  L-Isoleucine  18.04  L-Leucine   20.44  L-lysine HCl   38.43  L-methionine  4.44  L-ornithine HCl 9.41  L-phenylalanine  16.53  L-proline   6.13  L-serine  10.75  L-taurine   4.18  L-Threonine  18.93  L-Tryptophan 17.50 L-tyrosine, 2Na.2H ₂ O 23.70 ^A  L-valine  25.00  p-aminobenzoic acid 0.125  Ascorbic acid  50.0  d-biotin  0.025 Calciferol (vitamin D2) 0.25  D-Ca Pantothenate  0.025  Choline chloride 1.25  Folic acid  0.025 i-inositol 0.125  Menadione (vitamin K3)  0.025  Niacin (Niacin)  0.0625 Nicotinamide  0.0625 Pyridoxal HCl  0.0625 Pyridoxine HCl   0.0625 Riboflavin  0.025  Thiamine HCl  0.025 Alpha-tocopherol phosphate, 2Na  0.025  Vitamin A 0.25 Vitamin B ₁₂ 10.0  D-glucuronolactone 1.80  Glutathione, monosodium 10.0 Sodium GlucuronateH2O 1.80 * Twin 80 ² 12.50  Ethyl alcohol  40.0B

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 * COA (coenzyme A)  2.50  * DPN (diphosphopyridine nucleotide)   7.00  * FAD (flavin-adenine dinucleotide)  1.00  * TPN (triphosphopyridine nucleotide, Na) (NADP)  1.00  * TPP (Cocarboxylase)  1.00  * UTP (uridine triphosphate, Na)  1.00  Deoxyadenosine  10.00  DeoxycytidineHCl  10.00 Deoxyguanosine  10.00  5-methylcytidine  0.10  Thymidine  10.0 CaCl ₂  200.0  KCl  400.0 MgSO ₄ (anhydride)  100.0  NaCl  6800. NaH ₂ PO ₄ ㆍ H ₂ O   140.0  D-glucose  1000.  Phenolic red, Na salt 20.0 ^C Sodium Acetate3H ₂ O 50.0 NaHCO ₃ 2200

* NCTC 109 and 135 have the same composition except that L-cysteine HCl is omitted in NCTC 135 (Ref. 2). Reference 1, page 466 states that one reason for excluding L-cysteine HCl from the 135 formulation is due to toxicity.

* NCTC 109/135 BASE media (9172 & 9502) exclude (*) ingredients. See Badge Introduction for additional information on basal badges.

^A Reference 2 lists L. cystine 10.49 mg / L, L-cysteine HCl 250 mg / L, L-tyrosine 16.44 mg / L.

Excluded from ^B powder medium.

^C Plaque Assay (2X) Medium excludes phenol red and glutamine and contains all components twice.

* Trademark of ICI America's, Inc.

IMDM

 ingredient Liquid (1x) Catalog No. 78-0415 mg / L Powder (1x) Catalog No. 430-2200 mg / L Inorganic salts CaCl ₂ (anhydride)    165.00  165.00 Fe (NO ₃ ) ₃ ㆍ 9H ₂ O      -    -  KCl   330.00  330.00 KNO ₃   0.076 0.076 MgSO ₄ (anhydride)   97.67 97.67 MgSO ₄ ㆍ 7H ₂ O -    -  NaCl    4505.00  4505.00 NaHCO ₃ 3024.00  - NaH ₂ PO ₄ ㆍ H ₂ O   125.00   125.00 Na ₂ SeO ₃ ㆍ 5H ₂ O       0.0173   0.0173 Other Ingredients  D-glucose 4500.00  4500.00  Phenol red  15.00   15.00  HEPES 5958.00  5958.00 Sodium pyruvate  110.00    110.00  Amino acids  L-alanine  25.00  25.00 L-arginineH ₂ O   28.40  28.40  L-arginineHCl  84.00     84.00  L-aspartamic acid 30.00  30.00  L-cystine -   -  L-cystine / 2HCl 91.24     91.24  L-glutamic acid  75.00   75.00  L-Glutamine 584.00    584.00 Glycine  30.00 30.00 L-histidine HClH ₂ O 42.00     42.00  L-Isoleucine 105.00    105.00  L-Leucine 105.00 105.00  L-lysine HCl 146.00  146.00  L-methionine 30.00 30.00  L-phenylalanine    66.00     66.00  L-proline 40.00     40.00  L-serine 42.00     42.00  L-Threonine 95.00 95.00  L-Tryptophan   16.00     16.00  L-tyrosine - -  L-tyrosine (disodium salt)  104.20    103.79  L-valine 94.00 94.00

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Vitamins  Biotin   0.013 0.013  D-Ca Pantothenate   4.00   4.00  Choline chloride   4.00   4.00  Folic acid   4.00   4.00 i-inositol    7.20   7.20 Nicotinamide    4.00   4.00 Pyridoxal HCl    4.00  4.00 Riboflavin    0.40   0.40  Thiamine HCl    4.00   4.00 Vitamin B ₁₂    0.0l3     0.013

Each of these nutrients can be used with minor component variations, some of which may be acceptable nutrients for use in the media of the invention.

Preferred nutrients used in the medium of the invention are nutrients consisting of 90% to 10% DMEM and 10% to 90%, more preferably DMEM 75% and Ham's F12 25%.

The medium of the present invention may further comprise insulin. In the media of the present invention, insulin is most preferred for economic reasons, but proinsulin, IGF-1 or other insulin like growth factors may be used in place of insulin. In the present invention, insulin should be interpreted to include proinsulin, IGF-1 or other insulin-like growth factors. As used herein, an insulin-like growth factor refers to a composition that is structurally similar to insulin and promotes insulin-like growth factor receptor, and includes, for example, IGF-I and II. In the present invention, insulin is present at a concentration of 0.5-50 μg / ml.

The medium of the present invention may further comprise hydrocortisone. The concentration of hydrocortisone is preferably present at a concentration of 0.04 to 4.0 μg / ml.

In addition, the medium of the present invention may further comprise transferrin. The transferrin is preferably present at a concentration of 0.05-50 μg / ml. In the present invention, the transferrin binds to iron to form an iron-transferrin complex, which is bound to the cell-surface receptor and then introduced into the cell through the coated vesicle. In the cell the iron is released and used or stored as an iron-ferritin complex.

In addition, the medium of the present invention may further include triiodothyronine (T ₃ ). The triiodothyronine is preferably present at a concentration of 0.2 to 200 pM. In the present invention, triiodothyronine (T ₃ ) is not only triiodothyronine (T ₃ ) itself but also a substance which performs substantially the same function as triiodothyronine (T ₃ ) and produces substantially the same result. For example, it is used in the sense including a substance such as thyroxine (T ₄ ).

The medium of the present invention may comprise 1 to 20%, preferably 5 to 10% of serum. The serum is preferably FBS, but is not necessarily limited to these examples.

The medium of the present invention is useful for culturing fibroblasts, preferably dermal fibroblasts. However, the fibroblasts can be cultured using any of the mammalian fibroblasts using the medium of the present invention, as well as from the dermis, fibroblasts present in other parts of the body, such as fibroblasts of the oral mucosa. Can be used. The medium of the present invention is a medium containing only defined nutrients supplemented with serum, and when fibroblasts are cultured after confluency, a dermis having a structure similar to that of natural dermis can be obtained. Therefore, using the medium of the present invention, dermal replacement can be easily produced without using a substrate such as collagen.

Therefore, the present invention is a step of inoculating fibroblasts into a fibroblast culture medium comprising an epidermal growth factor (EGF), and a nutrient source having a defined component supplemented with serum and culturing until after confluency. It provides a method for producing a dermal substitute comprising a.

In the method for preparing the dermal substitute of the present invention, the inoculation amount of the fibroblasts, and the culture conditions may be used conventional inoculum amount and culture conditions used for culturing the fibroblasts, and those skilled in the art can easily be appropriate to the specific experimental purpose Can be used. In addition, the culture time can be terminated when the fibroblasts continue to be cultured even after reaching confluence and judged to have the shape of the dermis using the naked eye or a device. Examples of fibroblasts that can be used in the methods of the present invention include, but are not limited to, fibroblasts and oral mucosa fibroblasts, and any of fibroblasts derived from mammals, preferably humans.

In the method for preparing a dermal substitute of the present invention, the fibroblast culture medium may further comprise insulin.

In the method for preparing a dermal substitute of the present invention, the fibroblast culture medium may further include hydrocortisone.

In the method for preparing a dermal substitute of the present invention, the fibroblast culture medium may further include transferrin.

In addition, in the method for preparing a dermal substitute of the present invention, the fibroblast culture medium may further include triiodotyronine.

In addition, in the method for preparing a dermal substitute of the present invention, the nutrient source in the fibroblast culture medium is preferably 75% DMEM and 25% Ham's F12.

In the method for preparing the dermal substitute of the present invention, the ingredients used in the medium of the present invention, for example, epidermal growth factor, insulin, nutrients, hydrocortisone, transferrin and triiodotyronin The description is as described for the medium of the present invention.

The dermal substitutes produced by the method of making the dermal substitutes of the present invention have the form of fibrous sheets and have a dermal-like structure. Histologically, it consists of an extracellular matrix comprising many fibroblasts, the fibroblasts are non-mentin positive, and the extracellular matrix comprises type I collagen, tropoelastin, and fibril. In microstructure, collagen fibrils are found in the fibrous sheet. Thus, the dermal substitutes produced by the method of the present invention have properties similar to natural dermis in form, composition, and microstructure.

Accordingly, the present invention also provides a dermal substitute prepared by a method of making the dermal substitute of the present invention.

The present invention also provides a method for preparing a skin substitute comprising culturing epithelial cells based on the dermal substitute prepared by the method for preparing the dermal substitute of the present invention.

In the method for producing a skin equivalent of the present invention, the epithelial cells are cells contained in the epidermis, preferably keratinocytes. Cultivation of keratinocytes can be cultured in a medium commonly used for culturing keratinocytes, and in culture conditions, except that the dermal substitute prepared by the method for preparing the dermal substitute of the present invention is used as a base material. have. For example, a medium other than EGF may be used in the medium of the present invention or the medium of the present invention, but is not limited thereto. Culture conditions are, for example, culturing keratinocytes in a submerged state in the medium for a period of time, for example 2-7 days, and then incubating at a gas-liquid interface for a period of time, for example, a week or more. Can be. Such culture can be used by those skilled in the art as appropriately adjusted according to the experimental purpose.

The skin substitute prepared by the method for producing the skin substitute of the present invention is composed of the dermis and the epidermis. Like the in vivo natural epidermis, the skin substitute has a multilayer epidermis with a horny layer formed on the dermis. Keratin 1 and keratin 10 are expressed in the upper basal layer, except for the lower one or two layers. Β4 integrin, laminin 5, 4 and 7 collagen, which are components of the basement membrane, are expressed at the dermal-epidermal junction (DEJ). In microstructure, basement membrane regions such as hemidesmosome, lamina lucida and lamina densa are formed, although not complete. Accordingly, the skin substitutes of the present invention are composed of similar forms, structures, and ingredients as natural skin.

Thus, another embodiment of the present invention provides a skin substitute prepared by a method of making a skin substitute of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example

In this example, human dermal fibroblasts were cultured in the medium of the present invention to prepare human dermal substitutes, and human dermal substitutes were prepared by culturing human keratinocytes on the resulting dermal substitutes.

1. Materials and Methods

(1) Culture of human epidermal keratinocytes and dermal fibroblasts

Epidermal keratinocytes and dermal fibroblasts from human foreskin were isolated and cultured according to known methods (Boyce et al . , J. Invest. Dermatol. , 81, 33s-40s, 1983). In the following examples, secondary passage keratinocytes and fourth passage fibroblasts were used. Keratinocytes were cultured in KGM (Clonetics, USA), fibroblasts were cultured in DMEM containing 10% FBS. In the case of culturing keratinocytes on the dermal substitute, the medium of the present invention or the medium of the present invention without EGF was used. In all the examples of the present invention, the cells were cultured in a 37 ° C., 5% CO ₂ incubator.

(3) production of fibrous sheets

Dermal fibroblasts were seeded at 6 x 10 ⁵ cells / ml in 6 well plastic petri dishes for confluence. 3: 1 mixture of DMEM and Ham's nutrient mixture F12 medium supplemented with 10% FBS and 5 ng / ml epithelial growth factor (EGF) (hereinafter also referred to as EGF medium), 10% FBS, 5 ng / ml epithelial cell growth 3: 1 mixture of DMEM and Ham's nutritional mixture F12 medium supplemented with factor (EGF) and 5 μg / ml insulin (hereinafter also referred to as EGF + insulin medium) or 10% FBS, 5 ng / ml epithelial growth factor (EGF ), A mixture of 3: 1 of DMEM and Ham's nutritional mixture F12 medium supplemented with 5 μg / ml insulin, 0.4 μg / ml hydrocortisone, 5 μg / ml transferrin, and 1 × 10 ⁻¹¹ M triiodothyronine Used as culture medium. Culture medium was changed three times weekly. Incubation after confluence of fibroblasts was maintained for 3 weeks.

As a control, dermal fibroblasts were cultured in a medium containing only 10% FBS without supplements containing EGF, insulin, hydrocortisone, transferrin and triiodotyronine during the same culture period.

(4) reconstitution of human skin equivalent (HSE)

Fibroblasts were cultured in a plastic culture dish until after confluence to produce fibrous sheets. The fibrous sheet was separated from the culture dish using a fine forcep and placed in a polycarbonate filter chamber (3.0 μm Millicell-pc; Millipore Co., Bedford, Mass., USA) in a 6-well culture dish. In two or three layers. Epithelial keratinocytes were seeded on the fibrous sheet at a density of 5 × 10 ⁵ cells / Millicell. After keratinocytes were seeded on the fibrous sheet, the cultures were kept submerged in the medium for 2 days and then at the gas-liquid surface for 2 weeks. During the preculture period, the culture medium was the same as the culture medium used for the production of the fibrous sheet except that EGF was not included.

(5) Histology and immunohistochemistry

The fibrous sheets and human skin substitutes (HSE) were immobilized in 4% formaldehyde and stored in the freezer until processing after making paraffin blocks or placing them in staff frozen OCT loading medium. Sections of the fibrous sheet were stained with hematoxylin and iosin for morphological observation. Collagen fibers were visualized using Masson-trichrome staining. Immunohistochemical studies were performed using the avidin-biotin-peroxidase complex technique (Dako Co., Carpinteria, CA, USA). Expression of the extracellular matrix was examined using antibodies against type 1 collagen (Novotec, Lyon, France), elastin (Novotec, Lyon, France). Epithelial differentiation was observed for keratin 1 (Enzo Diagnostics, New York, NY, USA), keratin 10 (ICN Pharmaceuticals, Aurora, Ohio, USA) and human involucrin (Biomedical Tech, Stoughton, Mass., USA). Investigations were made using antibodies. Basement membrane components include β4 integrins (Serotec, Raleigh, NC, USA), laminin 5 (Chemicon, Temecula, CA, USA), type 4 collagen (Dako Co., Carpinteria, CA, USA) and type 7 collagen (Serotec, Raleigh, NC, USA) was used to investigate.

The specific experimental procedure is as follows.

(a) Paraffin tissue was deparaffinized.

(b) 10 minutes or more was reacted with 3% H ₂ O ₂ in methanol to inhibit the endogenous peroxidase activity.

(c) reacted with a blocker for 10 minutes.

(d) The primary antibody was diluted at the appropriate magnification and reacted for 1 hour at room temperature or overnight at 4 ° C.

(e) Reacted with biotinylated secondary antibody for 10 minutes.

(f) React with streptavidin peroxidase for 10 minutes.

(g) DAB was subjected to color reaction for a proper time using a color developing agent.

(h) Control staining with Maier's hematoxylin.

(i) Encapsulated in permount and observed under a microscope.

(6) electron microscopy

The fibrous sheet and human skin substitute (HSE) were immobilized in 2% paraformaldehyde / 2% glutaraldehyde in 0.1 M cacodylate buffer. Postfixation was performed with 1% (wt / vol) osmium tetraoxide, dehydrated in up to 70% ethanol and placed in EPON812. Ultra-thin sections were collected on a copper grid and stained with uranyl acetate and lead hydroxide. Microscopy and photography were taken with a Philips 410 transmission electron microscope.

Example 1 Production of Fibrous Sheets

In this example, a fibrous sheet obtained by culturing human foreskin dermal fibroblasts in culture medium of the present invention containing supplements such as EGF, insulin, hydrocortisone, transferrin, and triiodotyronine for 3 weeks after confluence was obtained. . As a control, the fibroblasts were cultured in medium without the supplement (see 1 (3) above).

1 is a result of visual observation of the obtained fibrous sheet. As shown in FIG. 1A, fibroblasts continued to grow after confluence to produce a fibrous sheet-like structure. FIG. 1B is a result of the control group, and almost no fibrous sheet could be observed.

It is a figure which shows the result of observing the fragment of the obtained fibrous sheet with hematoxylin and iosin (H & E staining) for the purpose of morphology observation, and optical microscope. The fibrous sheet is composed of an extracellular matrix including many fibroblasts, indicating that the fibrous sheet is a dermal-like tissue.

It is a figure which shows the result of observing the fragment of the obtained fibrous sheet with Marson-trichrome for the purpose of morphological observation, and observing with an optical microscope. In FIG. 2B, the blue stained portion is collagen.

2C and 2D show staining results for dermal extracellular matrix components such as collagen type 1 (Novotec, Lyon, France) and elastin (Novotec, Lyon, France). As shown in FIG. 2C and FIG. 2D, it can be seen that type 1 collagen and elastin are diffusely expressed.

It is a figure which shows the result of observing the said fibrous sheet under an electron microscope. As shown in Fig. 3, collagen fibrils were found in the fibrous sheet.

From the above results, it was found that the fibrous sheet was composed of an extracellular matrix containing many fibroblasts and expressed in the dermal extracellular matrix components such as type 1 collagen and elastin. In addition, microstructurally, collagen fibrils could be found in the fibrous sheet. Thus, the fibrous sheet was found to be human dermal-like tissue.

Example 2 Production of Fibrous Sheets

In the present example, the same medium component used in Example 1, that is, EGF, insulin, hydrocortisone, transferrin, and triiodothyronine, except that EGF or other components except EGF and insulin are not included, the same medium. To prepare fibrous sheets using dermal fibroblasts. Dermal fibroblasts were cultured in medium free of EGF or EGF and insulin as a control (see (3) in the Materials and Methods section).

The resulting fibrous acid sheet is shown in FIGS. 4 to 7. 4 is a diagram showing the results of staining hematoxylin and iosin of the obtained fibrous sheet. It is a figure which shows the result of horse-trichrome staining of the obtained fibrous sheet. It is a figure which shows the result of dyeing the obtained fibrous sheet with type 1 collagen. It is a figure which shows the result of dyeing the obtained fibrous sheet with elastin. 4 to 7, (A) shows the result of staining the fibrous sheet obtained from the control medium, (B) the fibrous sheet obtained from the EGF medium, and (C) the fibrous sheet obtained from the EGF and insulin medium, respectively.

Example 3: Reconstitution of Human Skin Substitute (HSE)

In this example, the human skin substitute was reconstituted by culturing epidermal keratinocytes on the fibrous sheet obtained in Example 1.

As a result, it was found that the human skin substitute is composed of dermis and epidermal components.

FIG. 8A shows the skin substitute of the present invention, showing that a multi-layered epidermis with a stratum corneum is formed on the dermal components similar to the natural epidermis in the in vivo state (hematoxylin & iosin staining).

FIG. 8B is a diagram showing the skin substitute of the present invention, which shows collagen stained blue in the dermis by Masson-trichrome staining.

8C and 8D show skin substitutes of the present invention, which show that keratin 1 and keratin 10 are expressed in the upper base layer except for the lower one or two layers, respectively.

8e, 8f, 8g, 8h and 8i also show the skin substitutes of the present invention, involucrin, β4 integrin, laminin 5, 4 and 7 collagen, which are components of the vesicle membrane, at the dermal-epidermal junction (DEJ). It is a figure which shows what is expressed.

Fig. 9 shows the skin substitute of the present invention and shows the result of observing the obtained human skin substitute with an electron microscope. As shown in FIG. 9, it was found that, although not completely, basement membrane regions such as hemidesmosome, lamina lucida and lamina densa were formed.

From the results of the above examples, human dermal fibroblasts are cultured until after confluence in the medium of the present invention without using a base material such as collagen, which has been conventionally used conventionally, and dermis similar to natural dermis and skin. Substitutes and skin substitutes could be produced.

Example 4 Transplantation of Dermal Replacement into Hairless Mouse Skin

In this embodiment, the dermal replacement obtained by the same method as in Example 1 was implanted into the skin of a hairless mouse (BALB / c nude Mouse, Charles River Japan, Inc.), and the dermal replacement prepared according to the present invention was successfully implanted into the hairless mouse. Investigate if possible.

First, a dermal substitute in the form of a fibrous sheet obtained by culturing human dermal fibroblasts in culture medium of the present invention containing EGF, insulin, hydrocortisone, transferrin, and triiodotyronin for 4 weeks after confluence was obtained. (See (3) in the Materials and Methods section). The dermal replacement thus obtained was dissected in the back of the hairless mice, and then the dermal replacement was inserted and sutured to complete the transplant. Ten days after transplantation, samples were taken from the implanted site. The obtained sample was fixed in formaldehyde, and then paraffin blocks were made and subjected to ematoxylin and iosin (H & E) staining and Masson-trichrome staining.

The results are shown in FIGS. 10A and 10B. Figure 10a is a diagram showing the results of staining with ematoxine and iosin from skin biopsy samples obtained from hairless mice transplanted with a dermal replacement of the present invention. As shown in FIG. 10A, live dermal replacements in hairless mice could be observed. FIG. 10B is a diagram showing the result of horseson-trichrome staining of a skin biopsy sample obtained from hairless mice transplanted with a dermal replacement of the present invention. As shown in FIG. 10B, the dermal replacement stained in blue was clearly observed in hairless mice. In FIGS. 10A and 10B, the portion between the arrows corresponds to the implanted dermal replacement.

From these experimental results, it can be seen that the dermal replacement of the present invention can be successfully implanted in hairless mice. Accordingly, it is believed that the dermal replacement of the present invention may be usefully used for therapeutic purposes in human skin diseases.

According to the medium of the present invention, dermal fibroblasts can be used to prepare dermal replacements by culturing dermal fibroblasts after confluency without using a base material such as collagen.

According to the method for preparing the dermal substitute of the present invention, the dermal substitute can be prepared without using a base material such as collagen. Therefore, it is useful to prepare dermal substitutes safely and safely without the problem of contamination caused by the use of foreign base materials at low cost.

According to the dermal substitute of the present invention, it is superior to the dermal substitute prepared by using a base material (substrate), such as conventional collagen, and high safety because it does not use foreign base material.

According to the method for preparing a skin substitute of the present invention, it is possible to prepare a skin substitute from a dermal substitute prepared without using a base substance such as collagen. Therefore, it is useful to prepare skin substitutes safely and simply without the problem of contamination caused by using foreign base materials at low cost.

According to the skin substitute of the present invention, it is superior to the skin substitute prepared from the dermal substitute prepared using a conventional substance such as collagen, and the safety is high because no foreign base is used.

Claims (19)

Inoculating dermal fibroblasts into a fibroblast culture medium comprising epidermal growth factor (EGF), a nutrient source having defined components supplemented with serum, and insulin, followed by culturing until confluency To make a dermal substitute. Inoculating dermal fibroblasts into a fibroblast culture medium comprising epidermal growth factor (EGF), a nutrient source having defined components supplemented with serum, and insulin, followed by culturing until confluency; And Method for producing a skin substitute comprising culturing keratinocytes using the dermal substitute prepared in the step as a base material. The method of claim 1 or 2, wherein said epidermal growth factor (EGF) in said medium is present at a concentration of 1 to 50 ng / ml. The method of claim 1 or 2, wherein the insulin in the medium is present at a concentration of 0.5 to 50 [mu] g / ml. The method of claim 1 or claim 2, wherein the nutrient is at least one nutrient selected from the group consisting of DMEM, IDMEM, MEM, M199, RPMI 1640, Ham's F12, Ham's F10, NCTC 109, and NCTC 135. Way. The method of claim 1 or 2, wherein the medium further comprises hydrocortisone. The method of claim 6, wherein the concentration of hydrocortisone in the medium is present at a concentration of 0.04 to 4.0 μg / ml. The method of claim 1 or 2, wherein the medium further comprises transferrin. The method of claim 8, wherein said transferrin in said medium is present at a concentration of 0.05-50 μg / ml. The method of claim 1 or 2, wherein the medium further comprises triiodothyronine. The method of claim 10, wherein the triiodothyronine in the medium is present at a concentration of 0.2 to 200 pM. The method of claim 1 or 2, wherein said nutrient is comprised of 75% DMEM and 25% Ham's F12. The method of claim 1 or 2, wherein the serum in the medium is included in the range of 1 to 20%. delete delete delete delete delete delete

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