JP2022018946A - Method for producing regenerated human skin tissue - Google Patents

Abstract

To provide a method for producing regenerated skin tissue including keratinocytes, melanocytes and fibroblasts.SOLUTION: In a method for producing regenerated human skin tissue including human mature melanocytes, human keratinocytes and human fibroblasts, human adipose tissue stem cells including human melanocyte progenitor cells are cultured with human fibroblasts and human keratinocytes.SELECTED DRAWING: None

Description

The present invention relates to a method for producing regenerated human skin tissue.

As a therapeutic means for skin diseases such as severe burns, skin ulcers, and segmental vitiligo vulgaris, autologous and allogeneic (homogeneous) cultured skin and pigment cell transplantation are performed. Attempts are also being made to use ES cells, iPS cells (Non-Patent Document 1), mesenchymal stem cells (Non-Patent Documents 2 and 3), Muse cells (Non-Patent Documents 4 and 5) and the like for skin regenerative medicine. ..

PLoS One, 2012: 7 (12): e52787 Am. J. Pathol. 173: 803-814,2008 Immunotherapy 4: 1859-1867, 2012 Proc. Natl. Acad. Sci. USA 107: 8639-8643, 2010 Proc. Natl. Acad. Sci. USA 108: 9875-9880, 2011

However, the differentiation of ES cells and iPS cells into epidermal keratinocytes has a low induction rate, and there is a problem of the possibility of tumor formation. Bone marrow mesenchymal cells have problems such as not being easy to collect.
Therefore, it is desired to develop a method for producing a regenerated skin tissue using readily available cells, and the present invention provides a method for producing a regenerated skin tissue containing keratinocytes, melanocytes and fibroblasts.

Therefore, the present inventor has focused on subcutaneous adipose tissue that can be easily collected, and found that, surprisingly, melanosite progenitor cells already exist in adipose tissue stem cells before inducing differentiation. I found. They also found that the melanocyte progenitor cells clearly express the melanocyte-specific marker protein when cultured in a melanocyte-specific medium. Furthermore, they have found that if these melanocyte progenitor cells are cultured together with human fibroblasts and human keratinocytes, regenerated human skin tissue containing human mature melanocytes, human keratinocytes and human fibroblasts can be obtained, and the present invention has been completed. did.

That is, the present invention provides the following [1] to [3].
[1] Regenerated human skin tissue containing human mature melanocytes, human keratinocytes and human fibroblasts, which comprises culturing human adipose tissue stem cells containing human melanosite progenitor cells together with human fibroblasts and human keratinocytes. Manufacturing method.
[2] Human adipose tissue stem cells containing human melanosite progenitor cells are human adipose tissue stem cells containing human melanosite progenitor cells or cells obtained by culturing human adipose tissue stem cells containing human melanosite progenitor cells in a melanosite-specific medium. [1] The method for producing a regenerated human skin tissue according to [1].
[3] The system according to [1] or [2], wherein the culture system performed together with the human fibroblasts and human keratinocytes is a system for culturing human fibroblasts, human keratinocytes and the human adipose tissue stem cells in a collagen gel. A method for producing regenerated human skin tissue.

The adipose tissue-derived stem cells used in the present invention can be collected from 1 g of adipose tissue to about 5 × 10 ³ cells, which is 500 times as much as that of bone marrow tissue-derived stem cells, and are easily available. In addition, since adipose tissue-derived stem cells have low antigenicity and have an immunosuppressive effect, human mature melanocytes induced to differentiate from them also have low antigenicity and can be transplanted into the skin. In addition, the regenerated skin tissue obtained by the present invention has not only human keratinocytes and human mature melanocytes but also fibroblasts and has functions from the epidermis to the basal layer and dermis of the skin, so that various skin diseases and aged skin are present. Can be dealt with.

The results of immunostaining of adipose tissue stem cells are shown. The results of immunostaining of adipose tissue stem cells are shown. The RT-PCR results of adipose tissue stem cells are shown. The RT-PCR results of adipose tissue stem cells are shown. The results of knocking down HMB45 of adipose tissue stem cells by the siRNA method are shown. The results of Western blotting of adipose tissue stem cells are shown. The results of Western blotting of adipose tissue stem cells are shown. The results of immunoelectron microscopy of adipose tissue stem cells are shown. The expression of HMB45 and tyrosinase (TYR) in the tissue after 3D culture is shown.

The present invention comprises culturing human adipose tissue stem cells containing human melanocyte progenitor cells together with human fibroblasts and human keratinocytes, the regenerated human skin containing human mature melanocytes, human keratinocytes and human fibroblasts. It is a method of manufacturing a tissue.

The raw material of the production method of the present invention is human adipose tissue stem cells containing human melanocyte progenitor cells.
Adipose tissue stem cells, which are raw materials, can be collected by treating adipose tissue, for example, subcutaneous adipose tissue with collagenase and then centrifuging to separate stromal vascular cells.
The obtained adipose tissue stem cells are (1) that the cells are CD34, CD44, CD90 and CD105 positive, and (2) that the cells are 3-isobutyl-1-methylxanthine, biotin, pantothenic acid. It may be confirmed that the cells are cultured in the presence of salt, rosiglitazone, dexamesazone, insulin and the like to differentiate into adipocytes.
The adipose tissue stem cells may be derived from animals including humans that do not cause rejection to the patient, but autologous cells are preferable.

The present inventor has found for the first time that human melanocyte progenitor cells are present in human adipose tissue stem cells isolated from easy-to-collect subcutaneous adipose tissue. That is, as shown in Examples below, a) by immunostaining, the melanosite marker proteins MelanA, MATP, HMB45 (precursor melanosomes protein), LAMP1, MITF, TYR, MelEM, and Mel2 are human fats before induction of differentiation. It is expressed in tissue stem cells, b) RT-PCR shows that melanosite transcription factors of MITF, KIT, and PAX3 are expressed in human adipose tissue stem cells before induction of differentiation, and c) human adipose tissue stem cells are expressed by the siRNA method. When HMB45 is knocked down, the expression of HMB45 in these cells disappears, and d) HMB45 and MITF are expressed in human adipose tissue stem cells before induction of differentiation in western blotting, and e) induction of differentiation by immunoelectron microscopy. It was confirmed that HMB45 was expressed in the previous fat cell stem cells.
In the present invention, human adipose tissue stem cells thus containing human melanocyte progenitor cells can be used.

Further, when human adipose tissue stem cells containing human melanosite progenitor cells are cultured in a melanosite-specific medium, the expression of melanosite marker protein is further enhanced. Therefore, in the present invention, human adipose tissue containing the human melanosite progenitor cells. It is more preferable to use human adipose tissue stem cells obtained by culturing stem cells in a melanosite-specific medium. Here, the enhanced expression of the melanocyte marker protein can be confirmed by the immunostaining, RT-PCR, HMB45 knockdown by the siRNA method, Western blotting, and immunoelectron microscopy.
Examples of the melanosite-specific medium include DermaLife (registered trademark) BM (KURABO) of KURABO, and DermaLife (registered trademark) M LifeFactors (L-glutamine 15 mL, epinephrine 0.5 mL, insulin 0.5 mL, StiMel8) as a basal medium. Registered trademark) LifeFactor 5 mL, ascorbic acid 0.5 mL, calcium chloride 1.0 mL, gentamicin / amphotericin 0.5 mL) can be used.
In addition, Wnt3a (R & Dsystems®) 50 ng / ml, Ethanol3 (SIGMA-ALDRICH) 100 nM, SCF (R & Dsystems® 10 ng / mL, cholera toxin (FUJIFILM) 1.66 mg / L A medium supplemented with (Nacalai) 25 μM in ethanol can also be used.

To culture human adipose tissue stem cells in a melanosite-specific medium, for example, seed human adipose tissue stem cells in a petri dish coated with collagen IV (5 μg / mL Nitta gelatin) and use a medium for human adipose tissue stem cells (ADSC-BM, LONZA). ) Is preferably used for culturing for about 3 to 7 days. After that, it is preferable to replace the medium with a melanocyte-specific medium and culture for about 7 to 14 days.

Human adipose tissue stem cells containing human melanocyte progenitor cells are cultured with human fibroblasts and human keratinocytes.
As the human fibroblast, human dermal fibroblast is preferable.
Fibroblasts can be collected from connective tissue, for example, the skin can be cut to a diameter of 5 mm and collected from the dermis component thereof.
As the human keratinocyte, for example, frozen NHEK manufactured by KURABO can be used.

As a means for co-culturing these cells, a three-dimensional culture method is preferable, and from the viewpoint of regenerating skin tissue, it is a system for culturing human fibroblasts, human keratinocytes and the human adipose tissue stem cells in collagen gel. Is more preferable. Furthermore, in the skin tissue, since the epidermis containing melanocytes and keratinocytes is present in the upper layer of the dermis containing fibroblasts via the basal layer, human keratinocytes in which fibroblasts are embedded and cultured in collagen gel. A three-dimensional culture system in which the human adipose tissue stem cells are superposed and cultured is preferable. Here, the collagen gel is preferably type I or type IV collagen gel.
As the culture conditions of the three-dimensional culture method, first, a 6-well plate (IWAKI) and a collagen IV coat of Trans well (5 μg / mL) are applied. Fibroblasts are embedded in collagen I gel at 5.0x10 ⁴ to 1.0x10 ⁵ cells / well on Trans well, seeded, and cultured for about 2 days. After 2 days, human adipose tissue-derived stem cells induced to melanocytes are seeded on the cells at 2.5x10 ⁴ to 5.0x10 ⁴ cells / well. Further, NHEK is seeded on it at 5.0x10 ⁴ to 1.0x10 ⁵ cells / well, and then cultured for about 14 to 15 days. As the medium, DMEM High Glucose (nacalai tesque) containing 10% FBS was used as the lower of 6 well as NHDF Medium (2 ml / well). To the Trans well upper, 500 μL of the above melanocyte-specific medium and 500 μL of HuMedia-KG2 (insulin, hEGF, hydrocortisone, BPE added; kurabo) as NHEK Medium are added. It is preferable to change the medium about once every two days.

Human mature melanocytes are induced in the basal layer of the epidermis by such three-dimensional culture, and the obtained three-dimensional cultured skin is a regenerated human skin tissue containing human mature melanocytes, human keratinocytes and human fibroblasts. Here, the differentiation of human melanocyte progenitor cells into human mature melanocytes can be confirmed by the expression of tyrosinase, that is, an enzyme essential for melanin synthesis.

According to the method of the present invention, easily available human adipose tissue stem cells are used as a raw material, and a regenerated human skin tissue containing human mature melanocytes, human keratinocytes and human fibroblasts can be obtained by a simple operation. Therefore, according to the present invention, it is possible to regenerate the epidermis and hair. Further, by examining the process of the manufacturing method of the present invention, it becomes possible to study the means for treating skin diseases and the means for coping with aged skin.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[Materials and methods]
(1) Collection of human adipose tissue stem cells 1) Subcutaneous adipose tissue was collected and washed with phosphate buffered saline. After soaking in collagenase, collagenase was neutralized in Dulbecco's modified Eagle's medium containing 10% calf serum.
2) The residue was removed through a 40 μm nylon mesh. The precipitate was collected by centrifugation at 1200 rpm for 10 minutes. Adipose tissue stem cells are dissolved again in 5 ml of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, seeded on a culture plate, cultured for several days, and adhered to the bottom surface of the culture plate.
3) It was confirmed by flow cytometry that CD34, CD44, CD90 and CD105 were positive.
4) The obtained cells are seeded and cultured in D-MEM / Ham's F-12 medium containing 10% calf serum, followed by 3% calf serum and 3-isobutyl-1-methylxanthine, biotin, panthenate, The cells were changed to D-MEM / Ham's F-12 medium containing rosiglitazone, dexamesazone, and human insulin, and cultured for 3 days. Next, after changing to the medium from which 3-isobutyl-1-methylxanthine has been removed, the medium is changed every 3 days, and the medium is cultured for about 12 days. It was confirmed that the cells were differentiated into adipocytes by Oil red O staining. It was confirmed by the above steps 3) and 4) that the cells were adipose tissue stem cells.

(2) Human fibroblasts The skin can be cut to a diameter of 5 mm and collected from the dermis component thereof. Specifically, fibroblasts were collected from patient skin with consent and cultured.

(3) Frozen NHEK from KURABO, a human keratinocyte, was used.

(4) Immunostaining HMB45 is multi-stained, and MelanA, MATP, MelEM, MITF, Tyrosinase, and Mel2 are immunostained.
Place the cover glass on the Nunc4well plate and apply collagen IV coating. The induced human adipose tissue-derived stem cells, human adipose tissue-derived stem cells, and melanoma cells were cultured to about 60 to 70%.
Fix at room temperature for 15 minutes using 4% paraformaldehyde. Blocking was performed using 10% Normal Goat Serum and 0.1% saponin or 0.1% Triton / PBS. 2% BSA / PBS plus 0.1% saponin, or 2% BSA / PBS alone as primary antibody Anti-HMB45 antibody (Abcam ab733 Mouse monoclonak IgG) 1/50 dilution and Anti-LAMP1 antibodyMorsome-Lyso ab24170 Rabbit polyclonal IgG) 1/500 diluted, or MelEM_DSHB (added without dilution, Developmental studios hybridoma bank, host mouse) Anti-MITF antibody 206 antibody (ab210546 Abcam Biotechnology, host rabbit) 1/1000, Mel2 (Anti-Melanocyte cell surface antigen antibody, ab128759, Abcam Biotechnology, host mouse) 1/500, MATP (sc-377397, Abcam Biotechnology, host mouse) 1/50 , Anti-Tyrosinase antibody (ab738, Abcam Biotechnology, host mouse) 1/250 is added and the primary antibody reaction is carried out at 4 ° C. over night.
2% BSA / PBS plus 0.1% saponin, or 2% BSA / PBS alone as a secondary antibody Alexa Fluor 594 donkey antibody-mouse IgG (Jackson Immuno Research) 1/300 dilution and Alexa Fluor 488 donkey Anti-rabbit IgG (Jackson Immuno Research) 1/500 dilution is used, or Alexa Fluor 488 Goat anti-mouse IgG (Life technologies) 1/400 dilution and Alexa Fluor 488 Gite The secondary antibody reaction is carried out at room temperature for 1 hour using dilution. After washing, Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA) is dropped and fixed.
After that, it was analyzed and photographed with a Keyence fluorescence microscope (Keyence BZ-X700 fluororescense microscope Osaka).

(5) RT-PCR
A 6-well plate (IWAKI) was coated with collagen IV, and induced human adipose tissue-derived stem cells, human adipose tissue-derived stem cells, and melanoma cells as positive controls were cultured.
2 μg of RNA is converted to cDNA using the RiverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). Use TaqMan Master Mix (Applied Biosystems, Foster City, CA, USA) to rotate 1 μg cDNA using Step One Plus systems (Applied Biosystems) for 50 cycles.
Based on β-actin as an endogenous control, increased expression of HMB45 (Gene Alias: PMEL, Hs00173854_M1), KIT (Hs00174029_M1), MITF (Hs01117294_M1) and PAX3 (Hs00240950_M1) was confirmed.
A comparative cycle threshold (ct) method using 2-ΔΔCt was used to calculate relative mRNA levels.

(6) Human adipose tissue-derived stem cells and human adipose tissue-derived stem cells in which HMB45 was knocked down by the siRNA method were seeded on a 6-well plate (IWAKI) and cultured for 2 days.
siRNA-1 (siRNA ID #: S12861, sense: 5'-GGUUAUCUGGUCAACAAUTt-3'(SEQ ID NO: 1) and antisense: 3'-AUUGUGACCAGAUACCtg-5'(SEQ ID NO: 2)), siRNA-2 , sense: 5'-AGAAGUGUGGUACUACatt-3'(SEQ ID NO: 3) and antisense: 3'-UGUAGUACCCACAACUUCUgt-5' (SEQ ID NO: 4)), siRNA-3 (siRNA ID #: S12860, sen '(SEQ ID NO: 5) and antisense: 3'-UCUUGACAGGCAUGAUAag-5'(SEQ ID NO: 6)) 3 HMB45 siRNAs designed by Silcer select siRNA (Thermo Fisher Scientific, USA).
OptiMem and Lipofectamine RNAi MAX Reagent (Themo Fisher Scientific, USA) were used to introduce 30 pmol SiRNA 1.2.3 with siRNA-Negative control.
After 48 hours, it was confirmed by real-time PCR that the expression of HMB45 was suppressed.

(7) Western blotting A collagen IV coat was applied to a 6-well plate (IWAKI), and induced human adipose tissue-derived stem cells, human adipose tissue-derived stem cells, and melanosite as a positive control were cultured.
Cellular protein recovery is performed using M-PER Mammalian Protein Extension Reagent and 1/100 diluted Protease inhibitor cocktail (P8340, Sigma-Aldrich).
Electrophoresis is performed using 10% Tris-glycine SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to a nitrocellulose membrane.
Dissolve 5% skim milk in Tris-buffered saline and perform blocking. Anti-HMB45 antibody (1: 100; from Santa Cruz Biotechnology) and anti-MITF antibody (1 μg / mL; from Abcam Biotechnology) β-actin monoclonal antibody, Body (1: 2000; Perform the primary antibody reaction overnight. After washing, anti-mouse peroxidase-conjugated secondary antibody (1: 2000, Cell Signaling Technology, USA) and anti-rabbit antibody (USA) and anti-rabbit antibody (1: 2000, Cell Signaling) with antibody at room temperature (1: 2000, Cell Signaling) conduct. SuperSignal West Dura Extended Duration Substrate (Thermo-Scientific, USA) labeled and ImageQuant LAS 4000 system (Cytiva, Tokyo) analysis.

(8) Immunoelectron microscopy A cover glass was placed on the Nunc4well plate and collagen IV coating was applied. The induced human adipose tissue-derived stem cells, human adipose tissue-derived stem cells, and melanoma cells were cultured to about 60 to 70%.
Immobilize with 4% paraformaldehyde buffered with 0.1 M phosphate buffer (pH 7.2) and set at 4 ° C. overnight. The cells were washed 3 times with PBS, soaked in 0.15% glycine in PBS, and embedded in 12% gelatin in 0.1 MPB. The pellet is cut into 1 mm ³ pieces on ice, soaked in 0.1 MPB containing 2.3 M sucrose overnight at 4 ° C., placed in a specimen holder (Leica Microsystems, Vienna, Austria) and stored in liquid nitrogen until use.
Cut to a thickness of 400 nm at -80 ° C with a Leica UC7 / FC7, dipped in a 1: 1 mixture of 2% methylcellulose and 2.3M cellulose, and coated with amino-polytriethoxy silane (Matsunami Glass, Kishiw) glass. Put in.
Then, the primary antibody reaction is carried out overnight at 4 ° C. using mouse anti-HMB45 antibody (1:50, Abcam ab733, Cambridge, UK) on the sections. A secondary antibody reaction is carried out at room temperature for 1 hour using Alexa 488 conjugated donkey anti-mouse IgG (1: 200, Invitrogen Life Technologies, Carlsbad, CA). After nuclear staining with DAPI, the cells were encapsulated in 50% glycerol diluted with distilled water and observed with a BZ-X700 microscope (Keyence, Osaka, Japan).
For the immunoelectron microscope, Leica UC7 / FC7 was used to prepare ultrathin frozen sections with a thickness of up to 70 nm at -120 ° C. 2% methylcellulose and 2.3M sucrose were placed in a 1: 1 mixture and transferred to nickel grid. Sections are rinsed with PBS containing 0.15% glycine, washed with PBS containing 1% BSA, and then primary antibody at 4 ° C. overnight using mouse anti-HMB45 antibody (1: 5, Abcam ab733, Cambridge, UK). Make a reaction.
A secondary antibody reaction was performed using donkey antibody-mouse IgG connected with 12 nm colloidal gold parts (1:40, The Jackson Laboratory, Bar Harbor, ME). Sections were washed with PBS containing 1% GA, encapsulated in a thin layer of 1.8% methylcellulose with 0.4% uranyl acetate (pH 4.0), dried and then Hitachi HT7700 erectron microscope (Hitachi, Tokyo, Tokyo, Tokyo, Tokyo, Japan). It was observed in Japan).

(9) Three-dimensional culture As a collagen gel solution, type1 collagen (3 mg / mL), 5-fold concentration of DME and buffer solution are mixed at a ratio of 7: 2: 1 (purchased from Nitta Gelatin Corp, Osaka), Osaka, Osaka. I used the one.
NHDF (5.0x104-1.0x105cells / well) embedded in collagen gel was seeded in IWAKI's 12-well cell plate transans well coated with collagen IV at a rate of 750 μL / well, cultured at 37 ° C for 30 minutes, and medium was cultivated above and below. put in. Two days later, NHEM or induced human adipose tissue-derived stem cells were overlaid on the NHDF collagen gel at 2.5-5.0x10 ⁴ cells / well, and NHEM was further overlaid on it at 5.0x104-1.0x10 ⁵ cells / well. Sow in.
After culturing for 15 days, the cells are fixed with 4% PFA and embedded in paraffin. After deparaffinization and activation, blocking was performed with normal goat serum (1: 200; from Vector Laboratories).
The primary antibody reaction is carried out at 4 ° C. overnight using primary antibodies against HMB45 (1:25; from Abcam Biotechnology) and TYR (1: 100; from Abcam, Biotechnology).
After washing with PBS, endogenous peroxidase blocking was performed with 0.3% _H2O2 / _methanol for 30 minutes. After washing, a secondary antibody reaction is carried out at room temperature for 1 hour using a goat anti-mouse biotin IgG secondary antibody (1: 300; from Molecular Probe). The reaction is carried out for 60 minutes using Avidin (1: 300; from DAKO) as a labeling reagent.
Then, color is developed with DAB (PBS 50 mL + DAB 500 μL + H ₂ O ₂ 10 μL) for 5 minutes, and nuclear staining is performed with hematoxylin for 1 minute. Wash, dehydrate, permeate, enclose and observe under a microscope.

[result]
(1) (Presence of melanocyte progenitor cells in adipose tissue stem cells)
a) As a result of immunostaining, melanocyte marker proteins, Melan A, MATP, HMB45, LAMP1, MITF, TYR, MelEM, and Mel2, are expressed in adipose tissue stem cells (ADSc) before induction of differentiation, and are melanocyte-based. Its expression is enhanced in the main cells (induced Me) cultured in the culture medium (Fig. 1). And it is particularly remarkable in MITF, which is called the master gene of melanocytes (Fig. 2).

b) As a result of RT-PCR, transcription factors of melanocytes of MITF, KIT, and PAX3 were expressed in adipose tissue stem cells (22851) before induction of differentiation, and the transcription factors of melanocytes cultured in a melanocyte culture medium (induced Me) were expressed. Expression is enhanced (FIGS. 3-1 and 3-2).

c) When HMB45 (precursor melanosome protein) of adipose tissue stem cells (22851 cell) is knocked down by the siRNA method, the expression of HMB45 in the cells is eliminated (FIG. 4).

d) HMB45 and MITF are also expressed in adipose tissue stem cells (22851) before induction of differentiation in western blinking (Fig. 5), and their expression is enhanced in main cells (induced cells) cultured in a melanocyte culture medium (FIG. 5). FIG. 6).

e) HMB45 is also expressed in adipose tissue stem cells (ASC) before induction of differentiation by immunoelectron microscopy, and it is observed that the expression is enhanced in the main cells (stimulated ASC) cultured in a melanocyte culture medium (stimulated ASC). FIG. 7).

(2) In three-dimensional culture, melanocyte precursor cells in fat cell stem cells were induced to differentiate into mature melanocytes. Fat cultured in normal human keratinocytes (NHEK) and normal epidermal melanocytes (NHEM), or NHEK and melanocyte culture medium. When tissue stem cells (induced cells) were three-dimensionally cultured on a collagen gel in which fibroblasts were embedded to create an epidermal model, cells expressing tyrosinase (TYR) and HMB45 in both culture systems were found in the epidermal basal layer. It was observed, and the pigment cell distribution similar to the normal epidermal structure was reproduced (Fig. 8).

Claims (3)

A method for producing regenerated human skin tissue containing human mature melanocytes, human keratinocytes and human fibroblasts, which comprises culturing human adipose tissue stem cells containing human melanosite progenitor cells together with human fibroblasts and human keratinocytes. .. Claimed that the human adipose tissue stem cell containing human melanosite progenitor cell is a human adipose tissue stem cell containing human melanosite progenitor cell or a cell obtained by culturing human adipose tissue stem cell containing human melanosite progenitor cell in a melanosite-specific medium. 1. The method for producing a regenerated human skin tissue according to 1. The regenerated human skin tissue according to claim 1 or 2, wherein the culture system performed together with the human fibroblasts and human keratinocytes is a system for culturing human fibroblasts, human keratinocytes and the human adipose tissue stem cells in a collagen gel. Manufacturing method.

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