WO2012153815A1

WO2012153815A1 - Method for producing three-dimensional skin model

Info

Publication number: WO2012153815A1
Application number: PCT/JP2012/062053
Authority: WO
Inventors: 章子山田; 純一細井
Original assignee: 株式会社資生堂
Priority date: 2011-05-12
Filing date: 2012-05-10
Publication date: 2012-11-15
Also published as: JP2012235921A

Abstract

The invention provides a method for producing a three-dimensional skin model, which is a method comprising 1) a step for preparing an epidermis/dermis model wherein fibroblasts and keratinocytes are layered, in succession from the bottom, on a substrate, 2) a step for subjecting precursor adipocytes to culture and differentiation and/or propagation to a specific degree, and 3) a step for placing the epidermis/dermis model on the adipocytes.

Description

Manufacturing method of three-dimensional skin model

The present invention relates to a method for producing a three-dimensional skin model into which fat cells are introduced, and a three-dimensional skin model obtained by the production method.

In the development of cosmetics, it is common to apply a candidate drug directly and evaluate it after artificially bringing the skin of an animal model such as a mouse close to the target skin symptoms instead of human skin. However, the results obtained with animal models are not necessarily applicable to human skin. In recent years, in Europe, there has been a movement to regulate animal experiments with regard to the development of cosmetics from the viewpoint of animal welfare, and it is necessary to establish an in-vitro drug evaluation system that can replace animal experiments.

Conventionally, various groups have been developing three-dimensional skin models that imitate human skin, and some are actually commercially available. Many of these known three-dimensional skin models have a structure in which fibroblasts are laminated on a support such as a collagen gel and keratinocytes are laminated on the fibroblasts (Amano S. et al., Exp. Cell Res., Vol.271, pp.249-262, 2001 and Tsunenga M. et al., Matrix. Biol., Vol.17, pp.603-613, 1998).

Keratinocytes and fibroblasts are the main constituent cells of the epidermis and dermis, respectively. However, since the skin is roughly composed of three layers of epidermis, dermis, and subcutaneous tissue, the above three-dimensional skin model lacks a corresponding structure for subcutaneous tissue mainly composed of fat cells. Become.

In a real skin, adiponectin released from fat cells inhibits IL-6 production from the epidermis (Ajuwon KM, AJP May, 2005, 288 (5) pp.1220-5) and also adipocytes The factor leptin is known to promote collagen production (EzurezT. Et al.,. Biofactors. 2007; 31 (3-4): pp.229-36)). IL-1 released from fibroblasts and keratinocytes inhibits PPARγ (peroxisome proliferator-activated receptor γ) and suppresses the progression of adipocyte differentiation (Suzawa M et al., Nat Cell Biol. Mar) , 2003, 5 (3): pp.224-30), IL-18 released from the epidermis suppresses the expression of adiponectin (Chandrasekar B et al., February 15, 2008, J. Biol. Chem. 283) (7): pp.4200-9). Thus, adipocytes and fibroblasts and keratinocytes are correlated with each other. Therefore, it is considered necessary to introduce fat cells into a conventional skin model in order to obtain a three-dimensional skin model having a structure closer to the skin.

Sugihara H. et al., British Journal Dermatology, Vol. 144, pp. 244-253 discloses a three-dimensional skin model containing adipocytes in addition to keratinocytes and fibroblasts. However, in the obtained three-dimensional skin model, it was not confirmed that keratinocytes and fibroblasts and adipocytes showed interaction. For this reason, this document does not clarify the effect of introducing fat cells into the skin model.

The present invention provides a method for producing a three-dimensional skin model having a structure close to that of human skin and allowing each cell to interact in order to evaluate the effects of candidate drugs such as cosmetics in vitro. The task is to do.

Sugihara H. et al. (Supra) mixed a collagen gel solution and fat cells to form a gel, and similarly poured a mixed solution containing the collagen gel solution and fibroblasts onto the gel to form the gel. A method of creating a three-dimensional skin model by seeding keratinocytes on a new cell layer is adopted. As a result of intensive studies by the present inventor, it is possible to create a three-dimensional skin model in which constituent cells can interact with each other by combining a model corresponding to the epidermis / dermis and an adipocyte separately. As a result, the present invention has been completed.
That is, this application includes the following inventions:
[1] A method of manufacturing a three-dimensional skin model,
1) A step of preparing an epidermis / dermis model in which fibroblasts and keratinocytes are laminated in order from the bottom on a support,
2) culturing preadipocytes to differentiate and / or proliferate to a desired extent;
3) placing the epidermis / dermis model on the fat cells;
Comprising a method,
[2] The method for producing a three-dimensional skin model according to [1], wherein the support is a gel containing collagen and chitosan.
[3] The epidermis / dermis model is formed by seeding fibroblasts on a support and culturing for a predetermined period, then seeding keratinocytes on the fibroblasts, culturing for a predetermined period, and exposing to air. A method for producing a three-dimensional skin model according to [1] or [2],
[4] The method for producing a three-dimensional skin model according to any one of [1] to [3], wherein a medium is interposed between the epidermis / dermis model and fat cells,
[5] A three-dimensional skin model including an epidermis / dermis model and an adipocyte layer,
1) A step of preparing an epidermis / dermis model in which fibroblasts and keratinocytes are laminated in order from the bottom on a support,
2) culturing preadipocytes to differentiate and / or proliferate to a desired extent;
3) placing the epidermis / dermis model on the fat cells;
A three-dimensional skin model obtained by a method comprising
[6] The three-dimensional skin model according to [5], wherein the support is a gel containing collagen and chitosan.
[7] The epidermis / dermis model is formed by seeding fibroblasts on a support and culturing for a predetermined period, then seeding keratinocytes on the fibroblasts, culturing for a predetermined period, and exposing to air. The three-dimensional skin model according to

claim

5 or 6.
[8] The method for producing a three-dimensional skin model according to any one of [5] to [7], wherein a medium is interposed between the epidermis / dermis model and fat cells,
[9] A three-dimensional skin model including an epidermis / dermis model and an adipocyte layer, in which fibroblasts and keratinocytes are layered in order from the bottom on the body, culturing preadipocytes A three-dimensional skin model that is placed on adipocytes that have been differentiated and / or proliferated to a desired degree, and a medium is interposed between the epidermis / dermis model and the adipocytes.

In the three-dimensional skin model obtained by the method of the present invention, the introduction of adipocytes reduces the production level of interleukin-6 (IL-6), promotes the differentiation of keratinocytes, and expresses the collagen gene. Stabilization of the epidermis / dermis model was confirmed, including increased levels. On the other hand, the presence of an epidermis / dermis model also affects adipocytes. For example, the expression level of adipocyte differentiation markers was settled, suggesting the possibility that the differentiation state was stabilized. Furthermore, it has been clarified that the three-dimensional skin model behaves like actual skin when irradiated with ultraviolet rays.

Therefore, according to the present invention, by preparing and combining the epidermis / dermis model and the fat cell layer separately, a three-dimensional skin model closer to the actual human skin configuration in which the constituent cells interact with each other can be obtained. It is done. In addition, by using the three-dimensional skin model, it becomes possible to evaluate the efficacy or toxicity of a drug in vitro without relying on animal experiments.

Furthermore, in the method of the present invention, since the adipocytes are differentiated and induced separately from the preparation of the epidermis / dermis model, the timing of combining the adipocytes with the epidermis / dermis model can be appropriately adjusted according to the purpose of use, It is also possible to apply the obtained three-dimensional skin model to the study of the differentiation / proliferation stage of adipocytes.

FIG. 1 is a diagram showing an example of a procedure for creating a three-dimensional model skin of the present invention. FIG. 2 is a photograph of immunostaining of a three-dimensional skin model with anti-laminin 5 antibody. FIG. 3 is a photograph of immunostaining of a three-dimensional skin model with an anti-transglutaminase antibody. FIG. 4 is an immunostaining photograph of a three-dimensional skin model with an anti-filaggrin antibody. FIG. 5 is a graph showing changes in IL-6 production level due to introduction of adipocytes into the epidermis / dermis model. FIG. 6 is a graph showing changes in the expression of collagen genes due to introduction of adipocytes into the epidermis / dermis model. FIG. 7 is a graph showing changes in the expression of the MMP gene due to the introduction of adipocytes into the epidermis / dermis model. FIG. 8 is a graph showing changes in mRNA levels of adiponectin by combining adipocytes and epidermis / dermis models, measured using quantitative RT-PCR. FIG. 9 is a graph showing changes in the production level of adiponectin by combining adipocytes and the epidermis / dermis model, measured using ELISA.

Epidermis / dermis model The three-dimensional skin model produced by the present invention includes an epidermis / dermis model imitating the epidermis / dermis and an adipocyte layer. As used herein, the term “epidermis / dermis model” means a structure having a structure in which fibroblasts are laminated on a support such as a collagen gel and keratinocytes are laminated on the fibroblasts. To do. A culture medium may be interposed between the epidermis / dermis model and the fat cell layer.

Such epidermis / dermis models are known to those skilled in the art (for example, AmanomanS. Et al., Exp. Cell Res., Vol.271, pp.249-262, 2001 (above) and Tsungega M. et al. , Matrix. Biol., Vol.17, pp.603-613, 1998 (see above)). For example, a mixture of fibroblasts mixed with a support on an insert mesh is seeded. After that, keratinocytes may be seeded thereon, cultured, and exposed to air.

The support used may be a collagen-only gel or a gel containing collagen and chitosan. As described in the previous report (Black AF et al., Tissue Engineering, Vol. 11, 11, 723-733, 2005), it is preferable to use a gel containing collagen and chitosan from the viewpoint of basement membrane structure and dermal fiber formation. In addition, by selecting an epidermis / dermis model having a uniform shape in which the gel is not shrunk, it is possible to prevent variation among lots when a plurality of similar three-dimensional skin models are created.

The epidermis / dermis model contains, for example, fibroblasts in an amount of 1 × 10 ⁴ to 10 ⁸ cells / cm ² , preferably 0.1 to 10 × 10 ⁵ cells / cm ² , and keratinocytes 1 × 10 ² to It may be contained in an amount of 10 ⁶ pieces / cm ² , preferably 1.0 to 10 × 10 ⁴ pieces / cm ² , more preferably about 4 to 8 × 10 ⁴ pieces / cm ² .

The epidermis / dermis model is commercially available and is not particularly limited. For example, a cell culture insert such as TESTSKIN (registered trademark) (TOYOBO) can be used.

The culture of the epidermis / dermis model is carried out using, for example, a culture solution used for normal keratinocyte culture as a culture solution, for example, KG medium, Epilife KG2 (Kurabo), Humedia-KG2 (Kurabo), assay medium (TOYOBO), etc. Can be carried out over 0-14 days. As the medium, other than that, a DMEM medium (GIBCO) or a medium in which 2-0-aD-glucopyranosyl-L-ascorbic acid-containing KGM and DMEM are mixed 1: 1 can be used. The medium may be removed when combining the epidermis / dermis model and the fat cell layer. In another embodiment, the medium for culturing the epidermis / dermis model may be interposed between the epidermis / dermis model and the fat cell layer.

Fibroblasts and keratinocytes may be homologous or heterologous and may be derived from any mammal. Furthermore, the cells constituting the epidermis / dermis model may be those subjected to ultraviolet rays, drugs, or genetic modification. However, the keratinocytes are preferably derived from humans, although not limited thereto, from the viewpoint of bringing the properties of the three-dimensional skin model closer to that of human skin. Further, the ratio of fibroblasts to keratinocytes used is not particularly limited. For example, keratinocytes 4 to 8 × 10 ^{4 to 6} cells, but fibroblasts 1 × 10 ^{5 to 7} cells, preferably keratinocytes 4 Fibroblasts can be 1 × 10 ⁶ cells to ˜8 × 10 ⁵ cells.

Many of the subcutaneous tissues existing under the adipocyte dermis are composed of adipocytes. When the number of fat cells increases or the oil droplets in fat cells accumulate and the cells enlarge, obesity and sagging of the skin occur. Therefore, it is possible to study these symptoms by including fat cells in the three-dimensional skin model. In the three-dimensional skin model created by Sugihara H. et al. (Supra), mature adipocytes were used as adipocytes. In the method of the present invention, preadipocytes are used, and after the cells are cultured and differentiated or proliferated to a desired degree, they are combined with an epidermis / dermis model.

As used herein, “precursor adipocyte” means a cell having the ability to differentiate into a mature adipocyte, and for convenience, means a cell that has matured and has not completely differentiated into an adipocyte. In addition, “mature adipocytes” means a state in which the proliferation is remarkably lower than that of preadipocytes, and oil droplets are considerably accumulated in the cells. The preadipocytes may be the same or different from the cells used in the epidermis / dermis model, and can be derived from any mammal, but the three-dimensional skin approximates the properties of human skin. From the viewpoint of obtaining a model, it is preferably derived from a human. The preadipocytes may be those that have been subjected to ultraviolet rays, drugs, or genetic modifications.

Precursor cells are in a medium containing insulin, indomethacin, dexamethasone, etc., which are factors that induce differentiation into adipocytes (hereinafter referred to as “culture medium for differentiation”) after making cells 100% confluent with high cell density. Differentiate into mature adipocytes by culturing in Differentiation media are commercially available, and human preadipocyte basic media-2 (PBM-2) (CMW) and the like can be used. The degree of differentiation into adipocytes can be confirmed using morphological characteristics of adipocytes, accumulation of oil droplets, and gene expression of differentiation markers as indices. For example, since preadipocytes accumulate oil droplets in the cells as they differentiate, oil droplet formation confirmed by oil red staining may be used as an indicator of differentiation. Adiponectin and PPARγ can be used as differentiation markers.

After differentiation into adipocytes has progressed to some extent, the differentiation medium can be replaced with a proliferation medium or a three-dimensional skin model medium that does not contain a differentiation-inducing factor. In fact, it was confirmed that when the adipocytes on the 7th day of differentiation induction were cultured in the absence of a differentiation-inducing factor, the differentiation rate was slowed but differentiation did not stop (results not shown). Or what mixed the culture medium for differentiation and the culture medium for proliferation can also be used for the further differentiation process. For example, but not limited to, a 1: 1 mixture of differentiation medium: proliferation medium may be utilized to grow and maintain some cells while inducing differentiation of most cells. As used herein, “three-dimensional skin model medium” is a 1: 1 serum-free medium for keratinocytes and Dulbecco's Modified Eagles minimal essential medium supplemented with 10% fetal bovine serum. It is a mixture.

The preadipocytes differentiated to a desired degree may be cultured and proliferated under predetermined culture conditions. Those that have reached 100% confluence can also be combined with the epidermis / dermis model. Note that the number of days of culture leading to confluence varies depending on the preadipocytes used and the growth conditions.

The extent to which the preadipocytes are differentiated and proliferated before being combined with the epidermis / dermis model is determined by the use of the three-dimensional skin model. For example, when investigating the effects of externally applied drugs on the skin on the differentiation state of subcutaneous fat cells, combine fat cells with the epidermis and dermis in an undifferentiated state, and factors derived from fat cells affect the skin. When examining the effects, combine them in a differentiated state.

Three-dimensional skin model Subsequently, a three-dimensional skin model can be created by arranging the epidermis / dermis model on adipocytes appropriately differentiated and proliferated. An example of the production method of the present invention is shown in FIG. The adipocytes and the epidermis / dermis model may be combined indirectly through a medium, or may be physically combined directly without using a medium. From the viewpoint of making it closer to the actual skin configuration, it is preferable that no medium is interposed between the epidermis / dermis model and the fat cells. In addition, when a plurality of similar three-dimensional skin models are created, it is preferable to select a uniform skin shape that does not shrink the gel as the epidermis / dermis model in order to prevent variation between lots.

The three-dimensional skin model obtained by the present invention shows a behavior close to that of actual skin when fat cells are introduced. For example, the production level of the inflammatory cytokine IL-6 derived from the epidermis / dermis model decreases, and the expression level of type I and III collagen genes also increases, so the presence of adipocytes makes the epidermis / dermis model It is thought to have stabilized. Furthermore, in the three-dimensional skin model, an ultraviolet stimulus response similar to that in the living body can be observed.

Next, the present invention will be described more specifically with reference to the following examples.

1) Preparation of epidermis / dermis model 5 mL of 0.7% chitosan solution (0.084M acetic acid) was added to 18 mL of 0.3% collagen solution, and further 2 mL of 2.5% chondroitin sulfate solution was added. 400 μL of the mixture was poured into a 12-well culture insert and lyophilized. Subsequently, sterilized by drying with 70% ethanol and swelled with 1 mL of phosphate buffer (PBS), then PBS was removed, and ⁶ × 10 ⁵ primary cultured dermal fibroblasts were 200 μL of DMEM + 10% fetal bovine serum (medium) ) Was poured into the insert. 1 ml of medium was poured into the well. After culturing at 37 ° C. in a 5% CO ₂ environment for 2 to 3 weeks while changing the medium 3 times a week, the medium was removed, and 5 × 10 ⁶ primary cultured epidermal keratinocytes were seeded in the insert. After culturing in serum-free medium for keratinocytes (KG-2; Kurabo Industries) for 2 to 3 days, remove the upper layer medium, and outside the insert, KG-2 and DMEM + 10% fetal bovine serum in a one-to-one relationship To the mixture, a skin model medium (three-dimensional skin model medium) supplemented with 250 μM ascorbic acid was added. An epidermis / dermis model was cultured for 2 weeks (air exposure) while changing the medium three times a week.

2) Adipocyte differentiation / proliferation Normal human preadipocytes were cultured in a growth medium (L-glutamine, GA-1000, FBS-added PBM-2, CMW) and seeded at 1x10 ⁵ cells in a 12-well plate. . After confirming with a microscope that it was 100% confluent after about 24 hours, the medium was replaced with a differentiation medium (h-INSULIN, INDOMETHACIN, DEXAMETHASONE, IBMX-added PBM-2: CMW), and then cultured for 6 days. Meanwhile, the medium was changed once every two days. Seven days after the replacement with the differentiation medium, the medium was replaced with a medium (mixed medium) in which the differentiation medium and the growth medium were mixed 1: 1, and then cultured for 5 days. Six days after the replacement with the mixed medium, the medium was replaced with a medium for a three-dimensional skin model (KG-2 excluding EGF and DMEM + 10% fetal calf serum mixed 1: 1). After 24 hours, confirm that a large amount of oil droplets accumulated in the fat cells, and without removing the medium for the three-dimensional skin model, place the epidermis / dermis model created in the insert on the fat cells to place the three-dimensional skin. Completed the model.

3) Effect of adipocytes on epidermis / dermis model Various factors such as laminin 5, transglutaminase, filaggrin, IL-6, collagen, MMP are released from the epidermis / dermis model. In order to examine the effect of adipocytes on the epidermis / dermis model, the three-dimensional skin model was subjected to histological analysis.

B) Histological analysis Laminin 5 which is a basement membrane component and transglutaminase and filaggrin which are markers of epidermal cell differentiation were analyzed by immunostaining. The skin model was fixed with acetone, and paraffin sections were prepared. The sections stained by the usual fluorescent immunostaining method were observed under a fluorescence microscope and photographed (FIGS. 2, 3, and 4).

Fig. 2 shows a stained image of laminin 5. The line indicated by the arrow is a basement membrane containing laminin 5, and when there is an adipocyte, the line is clear and continuous, and the basement membrane is formed better than when there is no adipocyte Recognize.

Transglutaminase (FIG. 3) and filaggrin (FIG. 4) are both epidermal differentiation markers. In the presence of adipocytes, the stained images of both are clearer than in the absence, and it can be seen that the epidermal cells are well differentiated to form a stratum corneum close to normal.

B) Evaluation of IL-6 production level IL-6 in the culture supernatant of the formed skin model was quantified using a commercially available ELISA kit (FIG. 5). It can be seen that when adipocytes are incorporated, the amount of IL-6, which is an inflammatory cytokine, decreases, and the skin model is stable, as compared to the case of the skin model alone.

C) Collagen gene expression analysis in the epidermis / dermis model RNA was extracted from the formed skin model and the expression of various collagen genes was quantified by RT-PCR. When adipocytes are incorporated into a skin model, the results of increased expression of type I and type III collagen genes are obtained, indicating that adipocytes activate dermal fibroblasts (FIG. 6).

D) Expression analysis of MMP gene in epidermis / dermis model RNA was extracted from the formed skin model, and expression of various MMP genes was quantified by RT-PCR method. Incorporation of adipocytes into the skin model resulted in a decrease in the expression of the MMP1 gene that degrades the extracellular matrix of the dermis, indicating that adipocytes increased the matrix of the dermis layer (FIG. 7).

4) Effect of epidermis / dermis model on adipocytes Adipocytes release intrinsic factors different from those of the epidermis / dermis model, such as triglycerides, leptin, PPARγ, and adiponectin. The effect of the epidermis / dermis model on adipocytes was examined.

B) Measurement of adiponectin in adipocytes RNA was extracted from adipocytes co-cultured with the skin model or cultured alone, and the expression of the adiponectin gene, an adipocyte differentiation factor, was quantified by RT-PCR (Fig. 8). In combination with the skin model, the expression of the adiponectin gene was reduced. Furthermore, the adiponectin protein in the culture supernatant was measured using a commercially available ELISA kit (FIG. 9). The expression of adiponectin from adipocytes cultured in combination with the skin model was low.

These results indicate that the differentiation state of adipocytes is stabilized by culturing with a skin model.

Claims

A method of manufacturing a three-dimensional skin model,
1) A step of preparing an epidermis / dermis model in which fibroblasts and keratinocytes are laminated in order from the bottom on a support,
2) culturing preadipocytes to differentiate and / or proliferate to a desired extent;
3) placing the epidermis / dermis model on the fat cells;
Comprising a method.
The method for producing a three-dimensional skin model according to claim 1, wherein the support is a gel containing collagen and chitosan.
The epidermis / dermis model is formed by seeding fibroblasts on a support and culturing for a predetermined period, then seeding keratinocytes on the fibroblasts, culturing for a predetermined period, and exposing to air. The manufacturing method of the three-dimensional skin model of Claim 1 or 2.
The method for producing a three-dimensional skin model according to any one of claims 1 to 3, wherein a medium is interposed between the epidermis / dermis model and fat cells.
A three-dimensional skin model including an epidermis / dermis model and an adipocyte layer,
1) A step of preparing an epidermis / dermis model in which fibroblasts and keratinocytes are laminated in order from the bottom on a support,
2) culturing preadipocytes to differentiate and / or proliferate to a desired extent;
3) placing the epidermis / dermis model on the fat cells;
A three-dimensional skin model obtained by a method comprising:
The three-dimensional skin model according to claim 5, wherein the support is a gel containing collagen and chitosan.
The epidermis / dermis model is formed by seeding fibroblasts on a support and culturing for a predetermined period, then seeding keratinocytes on the fibroblasts, culturing for a predetermined period, and exposing to air. The three-dimensional skin model according to claim 5 or 6.
The method for producing a three-dimensional skin model according to any one of claims 5 to 7, wherein a medium is interposed between the epidermis / dermis model and fat cells.
A three-dimensional skin model including an epidermis / dermis model and an adipocyte layer, in which fibroblasts and keratinocytes are laminated on the body in order from the bottom. A three-dimensional skin model that is placed on adipocytes that have been differentiated and / or proliferated to the extent that a medium is interposed between the epidermis / dermis model and adipocytes.