Method for manufacturing a subcutaneous fat layer model

Abstract

To provide a technology that can easily and stably provide a subcutaneous fat model having a well-developed subcutaneous fat layer and uniformly containing mature adipocytes. [Solution] A method for producing a subcutaneous fat model, comprising the step of culturing adipose stem cells or adipose progenitor cells by shaking them in a culture insert having a liquid-permeable membrane with a pore diameter of 3 μm or more at the bottom surface using a shaker, thereby producing a subcutaneous fat layer uniformly containing mature adipocytes.

Description

[Technical Field]

[0001] The present invention relates to a subcutaneous fat layer model having a well-developed subcutaneous fat layer and uniformly containing mature adipocytes, a method for producing the same, and a full-thickness artificial skin model and a method for producing the same. [Background technology]

[0002] Recent advancements in tissue engineering technology have been remarkable, with progress being made in developing tissue models that artificially mimic living tissues. These artificially created tissue models are useful not only for organ replacement in regenerative medicine but also in drug discovery and in evaluating the efficacy and safety of cosmetics and health foods. There is a growing expectation for the development of tissue construction technologies that more accurately reproduce the structure and function of living tissues. In particular, autologous cultured epidermis, created by culturing epidermal stem cells in vitro to reconstruct tissue with a structure similar to living epidermal tissue, is rapidly being put into practical use as a regenerative medicine product. In recent years, research has progressed on more advanced artificial skin models, such as cultured skin models with a dermis layer, in addition to cultured epidermis. These models are used in medical settings for wound closure in burns and skin ulcers, and in cosmetic and pharmaceutical development as tools to predict the skin irritation and toxicity of test substances and to evaluate their efficacy without using experimental animals (Patent Document 1, etc.).

[0003] Today, cultured epidermal models and cultured skin models with dermal layers are commercially available from many companies both domestically and internationally, making it possible to perform safety evaluation tests, such as skin irritation tests, in vitro. On the other hand, living skin has a layer of fat beneath the dermis, formed by a cluster of mature adipocytes. This fat layer enhances skin elasticity, mitigates physical stimuli from the outside world, and the stored fat serves as an energy source, metabolizing and producing the energy needed for activity. Furthermore, the fat layer is important for maintaining body temperature because it is a poor conductor of heat. In recent years, it has also been reported that factors secreted from adipocytes (such as adiponectin) and extracellular vesicles (exosomes) act on dermal and epidermal cells, promoting regeneration. For these reasons, there is a need for the development of tissue models that artificially mimic subcutaneous fat, and furthermore, there is a need for the development of full-thickness artificial skin models with a subcutaneous fat layer that more accurately reflect the structure of living skin. These models are expected to be applied to transplant medicine in the field of regenerative medicine, as well as to testing tools in cosmetics and drug discovery research.

[0004] However, while methods for inducing differentiation from adipose stem cells or adipose progenitor cells to mature adipocytes using planar culture in petri dishes have been established, the development of techniques for uniformly inducing the differentiation of mature adipocytes throughout the tissue under three-dimensional culture conditions is still in progress. To date, skin substitutes with a subcutaneous fat layer in addition to the epidermis and dermis have been reported, with the subcutaneous fat layer being formed by inducing the differentiation of adipose-derived stem cells (ASCs) in collagen hydrogel (Non-Patent Literature 1). However, with this method, the cell density of the subcutaneous fat layer is sparse compared to living skin, and a sufficiently developed subcutaneous fat layer is not formed. On the other hand, there have been reports of studies incorporating excised adipose tissue itself into cultured skin models to create a subcutaneous fat layer (Non-Patent Literature 2). However, this method requires the collection of subcutaneous adipose tissue each time a cultured skin model is created, which is highly invasive and has low stability between tissue lots. Therefore, it is not a practical method for use as a tool for screening various compounds or dermatological research.

[0005] Furthermore, models that mimic the adipose layer using a porous material of collagen-glycosaminoglycan-chitosan (Non-Patent Literature 3) and silk gel (Non-Patent Literature 4) have been reported. While the structure of these models in tissue sections appears similar to that of the adipose layer, they are not constructed from mature adipocytes and are therefore insufficient for use as subcutaneous adipose models. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2018-102186 [Non-patent literature]

[0007] [Non-Patent Document 1] Acta Biomater. 2021 Oct 15:134:215-227. doi: 10.1016 / j.actbio.2021.07.033. Epub 2021 Jul 21.Bio-engineering a prevascularized human tri-layered skin substitute containing a hypodermis [Non-Patent Document 2] Front Bioeng Biotechnol. 2020 May 8:8:388. doi: 10.3389 / fbioe.2020.00388. eCollection 2020.Improvement of a Three-Layered in vitro Skin Model for Topical Application of Irritating Substances [Non-Patent Document 3] PLoS One. 2013 Jun 4;8(6):e66284. doi: 10.1371 / journal.pone.0066284. Print 2013. Reciprocal interactions between breast tumor and its adipose microenvironment based on a 3D adipose equivalent model [Non-Patent Document 4] Macromol Biosci. 2012 Dec;12(12):1627-36. doi: 10.1002 / mabi.201200262. Epub 2012 Nov 19.In vitro 3D full-thickness skin-equivalent tissue model using silk and collagen biomaterials [Overview of the project] [Problems that the invention aims to solve]

[0008] Accordingly, in view of the above-described circumstances, the object of the present invention is to provide a technology that can easily and stably provide a subcutaneous fat model having a subcutaneous fat layer that uniformly contains mature adipocytes and is well-developed. [Means for solving the problem]

[0009] To solve the above problems, the inventors investigated the culture conditions for differentiating adipose stem cells by three-dimensional culture. As a result, they succeeded in developing a subcutaneous fat model that uniformly contains mature adipocytes and has a well-developed subcutaneous fat layer by combining a culture insert with a large pore diameter and shaking culture. Furthermore, by reproducing the dermis and epidermis on top of this subcutaneous fat model, it became possible to create a full-thickness artificial skin model containing all three layers of skin from stem cells, which had been impossible in previous studies. Moreover, it was confirmed that this full-thickness model has the color tone of living skin without the addition of pigments, etc., and has elasticity equivalent to living skin. The present invention is completed based on these findings.

[0010] In other words, the present invention encompasses the following inventions. (1) A method for producing a subcutaneous fat model, comprising the step of culturing adipose stem cells or adipose progenitor cells by shaking them in a culture insert having a liquid-permeable membrane with a pore diameter of 3 μm or more at the bottom using a shaker, thereby producing a subcutaneous fat layer uniformly containing mature adipocytes. (2) The method according to (1), wherein the shaker is a reciprocating shaker or a seesaw shaker. (3) The method according to (1), wherein the shaking speed in the shaking culture is 20 to 35 rpm. (4) A subcutaneous fat model having a subcutaneous fat layer uniformly containing mature adipocytes, obtained by any of the methods described in (1) to (3). (5) A method for preparing a full-thickness artificial skin model, comprising the following steps. (a) A process to produce a subcutaneous fat layer uniformly containing mature adipocytes by shaking adipose stem cells or adipocyte progenitor cells in a culture insert having a liquid-permeable membrane with a pore diameter of 3 μm or more at the bottom using a shaker. (b) A step to create the dermis and epidermis on top of the subcutaneous fat layer created in step (a). (6) The method for producing the artificial full-thickness skin model according to (5), wherein the production of the dermis and epidermis is performed by three-dimensional culturing of dermal stem cells or progenitor cells and epidermal stem cells or progenitor cells on the subcutaneous fat layer in the culture insert. (7) The method according to (5), wherein the stem cells or progenitor cells are immortalized stem cells or progenitor cells. (8) An artificial full-thickness skin model in which the dermis and epidermis are layered on a subcutaneous fat layer uniformly containing mature adipocytes, having the elasticity and skin color of living skin, obtained by the method according to any one of (5) to (7). (9) A method for evaluating the cell communication between adipocytes and epidermal cells or dermal cells by a test substance, which comprises contacting the test substance with the artificial full-thickness skin model according to (8) and measuring the change in the secreted substances from the adipocytes in the subcutaneous fat layer of the model. (10) A method for screening a substance having a slimming effect through transdermal absorption, which comprises contacting the test substance with the artificial full-thickness skin model according to (8) and measuring the change in the adipocytes in the subcutaneous fat layer of the model. [[Effect of the Invention]]

[0011] According to the present invention, a subcutaneous fat model uniformly containing mature adipocytes and having a sufficiently developed subcutaneous fat layer is provided. According to the present invention, an artificial full-thickness skin model having a dermis and an epidermis on the subcutaneous fat layer of this subcutaneous fat model is also provided. By using the artificial full-thickness skin model of the present invention, it becomes possible to evaluate the cell communication between adipocytes and epidermal cells or dermal cells and to screen substances having a slimming effect through transdermal absorption. [[Brief Description of the Drawings]]

[0012] [Figure 1] FIG. 1 shows a production flow of a subcutaneous fat model using adipocytes recovered by centrifugation. [Figure 2]Figure 2 shows photographs of horizontally oriented cells in each layer (upper layer, middle layer, lower layer) of adipose tissue induced to differentiate from adipose stem cells using a culture insert. [Figure 3] Figure 3 shows shakers (reciprocating shaker, seesaw shaker) used for shaking culture to prepare a subcutaneous fat model. [Figure 4] Figure 4 shows a bright-field image and an Oil Red O stained image of a tissue section induced to differentiate from adipose stem cells by combining shaking culture with a culture insert. [Figure 5] Figure 5 shows culture images of epidermal stem cells and dermal stem cells obtained by sorting using FACS from epidermal cells and dermal cells used to prepare a full-thickness artificial skin model. [Figure 6] Figure 6 shows the manufacturing process flow of the full-thickness artificial skin model of the present invention. [Figure 7] Figure 7 shows the appearance of artificial skin models (two-layer, full-thickness). [Figure 8] Figure 8 shows an Oil Red O stained image of a tissue section of the full-thickness artificial skin model of the present invention.

Embodiments for Carrying Out the Invention

[0013] 1. Method for manufacturing a subcutaneous fat model, subcutaneous fat model The method for manufacturing a subcutaneous fat model of the present invention includes a step of subjecting adipose stem cells or adipose progenitor cells to shaking culture using a shaker with a culture insert having a liquid-permeable membrane with a pore diameter of 3 μm or more on the bottom surface to produce a subcutaneous fat layer (which may be simply referred to as "fat layer" in this specification) that uniformly contains mature adipocytes.

[0014] In the present invention, adipose stem cells refer to stem cells contained in adipose tissue that are capable of differentiating into mature adipocytes (white adipocytes, brown adipocytes) that have lipid droplets in their cytoplasm. Adipose stem cells are positive for CD90, CD73, and CD105, and negative for CD31 and CD45 in flow cytometry. In the present invention, adipocyte precursor cells capable of differentiating into adipocytes can be used instead of adipose stem cells. The origin of adipose stem cells or adipocyte precursor cells is not particularly limited as long as they are mammals, and examples include humans, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, cattle, and horses, but human origin is preferred.

[0015] Furthermore, adipose-derived stem cells or adipose-derived progenitor cells may be immortalized cells that maintain stem cell properties while proliferating indefinitely. The immortalization method is not limited as long as it immortalizes cultured cells without inducing cell death, but examples include introducing an immortalization gene into adipose-derived stem cells or adipose-derived progenitor cells. Here, "immortalization gene" refers to a gene that immortalizes cells and gives them the ability to proliferate indefinitely, and examples include the telomerase reverse transcriptase (TERT) gene, genes that regulate telomerase expression or activity (e.g., Myc gene, Ras gene, etc.), and viral genes (SV40T, HPV E6-E7, EBV, etc.), but the telomerase reverse transcriptase (TERT) gene is preferred, and the human telomerase reverse transcriptase (hTERT) gene is more preferred.

[0016] Adipose stem cells or adipose progenitor cells are cultured in a culture insert having a liquid-permeable membrane at its bottom. The liquid-permeable membrane of the culture insert is not particularly limited as long as it can support cells to adhere to or be fixed on, on which cells can proliferate, and can serve as a support, but examples include membranes made of polycarbonate, polyethylene terephthalate, and polystyrene. The membrane may also be coated with an extracellular matrix such as collagen, laminin, or fibronectin, or with a substance that aids cell adhesion such as poly-L-lysine.

[0017] The pore diameter of the liquid-permeable membrane at the bottom of the culture insert described above should be 3 μm or larger, but 3 to 10 μm is preferred, and 3 to 8 μm is more preferred.

[0018] Examples of shakers used for shaking culture of adipose stem cells or adipose progenitor cells include reciprocating, seesaw, swirling, and figure-eight types, with reciprocating and seesaw types being preferred. The shaking speed for shaking culture using the above shaker is preferably in the range of 20 to 35 rpm. By shaking culture at the above shaking speed, uniformly matured adipocytes are formed in all layers of the adipose tissue.

[0019] In one embodiment of the present invention, a collagen solution containing adipose stem cells or adipose progenitor cells is added to the culture insert and cultured to produce a collagen gel embedded with adipose stem cells or adipose progenitor cells. The amount of adipose stem cells or adipose progenitor cells added to the collagen solution is not particularly limited, but is 50 × 10 4 ~2000×10 4 Cells / mL is preferred, 200 × 10 4 ~1000×10 4 Cells / mL is more preferable.

[0020] Next, the outside of the culture insert is filled with cell proliferation medium, and adipose stem cells or adipose progenitor cells are cultured to proliferate. The proliferation culture is carried out for, for example, 1 to 6 days, preferably 2 to 4 days. During this time, the medium may be changed as appropriate. Whether the cells that have proliferated in the culture insert are in a confluent state can be determined by obtaining a bright-field image of the gel.

[0021] Next, the culture medium inside and outside the culture insert is changed from cell proliferation medium to cell differentiation medium, and the adipose stem cells or adipose progenitor cells are further cultured to induce differentiation into mature adipocytes. Differentiation induction is carried out for, for example, 10 to 20 days, preferably 12 to 14 days.

[0022] The culture medium used for the proliferation culture and differentiation induction described above can be any medium commonly used for the proliferation, differentiation, and maturation of adipose progenitor cells (adipose stem cells, mesenchymal stromal cells, mesenchymal stem cells) or adipocytes. Furthermore, the conditions and procedures for the culture method can be carried out according to the conditions and procedures that are standard in the relevant art. For example, basic media containing components necessary for cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids) can be used. Specifically, examples include Dulbecco's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM / F-12), Glasgow Minimum Essential Medium (Glasgow MEM), and Hank's balanced salt solution. During differentiation induction, one or more adipocyte differentiation-inducing factors such as dexamethasone (DEX), isobutylmethylxanthine (IBMX), indomethacin (IDMM), insulin (Ins), and biotin may be added. Basic fibroblast growth factor (bFGF) and / or leukocyte migration inhibitory factor (LIF) may also be added as growth factors. Furthermore, if necessary, the culture medium may contain epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27 supplement, N2 supplement, ITS supplement, antibiotics, etc.

[0023] Commercially available culture media include Invitrogen's mesenchymal stem cell basal medium, Sanko Junyaku's mesenchymal stem cell basal medium, TOYOBO's MF medium, and Sigma's Hank's balanced salt solution.

[0024] 2. Method for manufacturing a full-thickness artificial skin model, full-thickness artificial skin model The present invention provides a method for producing a full-thickness artificial skin model, comprising: (a) culturing adipose stem cells or adipose progenitor cells by shaking them in a culture insert having a liquid-permeable membrane with a pore diameter of 3 μm or more at its bottom surface using a shaker to produce a subcutaneous fat layer uniformly containing mature adipocytes; and (b) producing a dermis and epidermis on top of the subcutaneous fat layer produced in step (a).

[0025] Step (a) is carried out according to the method for preparing the subcutaneous fat model described in the previous section.

[0026] In step (b), the dermis and epidermis are prepared on top of the subcutaneous fat layer prepared in step (a). The preparation of the epidermis and dermis can be carried out in accordance with the standard three-dimensional cultured skin preparation method commonly used in this field, in which epidermal keratinocytes and fibroblasts are cultured and differentiated to reconstruct the skin tissue. Here, reconstruction of skin tissue means forming an epidermal layer on top of the dermis containing fibroblasts, by layering epidermal keratinocytes through exposure to air.

[0027] Commercially available epidermal keratinocytes and fibroblasts can be used as the epidermal keratinocytes and fibroblasts. In particular, it is preferable to use primary cultured epidermal keratinocytes and fibroblasts. Stem cells and progenitor cells isolated from these cells using FACS or similar methods, with the expression of markers that evaluate the undifferentiated state of stem cells as an indicator, can also be used. The origin of the epidermal keratinocytes and fibroblasts is not particularly limited as long as they are mammals, for example, humans, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, cattle, horses, etc., but it is preferable that they be human.

[0028] In addition, epidermal keratinocytes and fibroblasts may be immortalized cells that can proliferate infinitely while maintaining their stem cell properties. The method of immortalization is not limited as long as it is a method for immortalizing cultured cells and not inducing cell death. Examples include methods of introducing immortalizing genes into primary cultured epidermal keratinocytes and fibroblasts. Here, the "immortalizing gene" refers to a gene that immortalizes cells and confers the ability to proliferate infinitely. Examples include the telomerase reverse transcriptase (TERT) gene, genes that regulate the expression or activity of telomerase (such as the Myc gene, Ras gene, etc.), and viral genes (SV40T, HPV E6-E7, EBV, etc.). Among them, the telomerase reverse transcriptase (TERT) gene is preferred, and the human telomerase reverse transcriptase (hTERT) gene is more preferred.

[0029] The production of three-dimensional cultured skin consists of a proliferation culture step and a differentiation induction step. In a preferred embodiment of the present invention, the proliferation culture step is preferably carried out using a collagen gel, a collagen sponge, or a cell-free dermis from which epithelial cells and fibroblasts have been removed as a support, and fibroblasts may be appropriately incorporated into these supports.

[0030] In one embodiment, dermal stem cells are added to a collagen solution and cultured to form a dermal layer in which the dermal stem cells are embedded in the collagen gel. Then, epidermal stem cells are seeded thereon and further cultured until the epidermal stem cells become confluent to form an epidermal layer. The number of epidermal stem cells to be added is not particularly limited, but is preferably 15×10 4 ~120×10 4 cells / cm 2 and more preferably 30×10 4 ~90×10 4 cells / cm 2 The proliferation culture is carried out, for example, for 1 to 6 days, preferably for 2 to 4 days. During this period, the medium may be appropriately exchanged. Whether the proliferated epidermal stem cells are in a confluent state can be confirmed using a cell staining reagent such as the CnT-ST-100 stain kit (manufactured by CELLnTEC).

[0031] The cell proliferation medium described above is not particularly limited, as long as it is a basic medium suitable for the proliferation and subculturing of epidermal keratinocytes, for example, it is preferably a serum-free, low-calcium concentration basic medium. Commercially available media such as MCDB153 medium (Sigma), HuMedia-KG2 (Kurabo), serum-free medium for normal human epidermal keratinocytes (DS Pharma Biomedical), and Keratinocyte-SFM (Thermo Fisher Scientific) can be used. The above medium may also contain growth factors such as basic fibroblast growth factor (bFGF), leukocyte migration inhibitory factor (LIF), and stem cell factor (SCF). Furthermore, to increase the growth rate, the medium may contain, as needed, epidermal growth factor (EGF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, bovine serum albumin (BSA), L-glutamine, fibronectin, progesterone, selenite, B27 supplement, N2 supplement, and ITS supplement. Antibiotics may also be added as needed. The calcium concentration of the cell growth medium is preferably about 0.03 to 0.15 mM.

[0032] Next, in the differentiation induction process, the culture medium is changed to a cell differentiation medium and differentiation induction culture is performed for about 8 to 10 days. For the last 1 to 2 days, the entire model is exposed to air (atmosphere) to induce the final differentiation of the stratified epidermal keratinocytes.

[0033] The cell differentiation medium described above is not particularly limited as long as it is a basic medium suitable for inducing differentiation into epidermal keratinocytes, but commercially available media such as CnT-Prime 3D Barrier Culture Medium (CELLnTEC) can be used. Furthermore, the calcium concentration of the cell differentiation medium is preferably about 1.2 to 3.0 mM.

[0034] The culture temperature for proliferation and differentiation induction varies depending on the cell origin, but for human-derived cells, for example, 30-40°C is preferred, and 36-38°C is more preferred. The CO2 gas concentration is also preferred, for example, about 1-10%, and about 2-5% is more preferred.

[0035] The full-thickness artificial skin model of the present invention, manufactured through these processes, has a three-layer structure in which the dermis and epidermis are superimposed on a subcutaneous fat layer uniformly containing mature fat cells. Because it possesses the elasticity and skin color of living skin, it has a high degree of realism in appearance, texture, and touch.

[0036] 3. How to use the full-thickness artificial skin model The full-thickness artificial skin model of the present invention can be used as an alternative to animal testing to evaluate and screen the efficacy and safety of cosmetics and pharmaceuticals. For example, by contacting the full-thickness artificial skin model of the present invention with a test substance and measuring changes in secretions from adipocytes in the subcutaneous fat layer of the model, intercellular communication between adipocytes and epidermal or dermal cells by the test substance can be evaluated. Examples of secretions from adipocytes include exosomes, lipokines, and adiponectin. For example, exosomes are intercellular communication substances, and a test substance that can increase the amount of exosome secretion can be evaluated as effective in anti-inflammatory, anti-aging, and skin barrier improvement by acting on epidermal or dermal cells via exosomes. Furthermore, by contacting the full-thickness artificial skin model with a test substance and measuring changes in adipocytes in the subcutaneous fat layer of the model, substances with slimming effects via transdermal absorption can be screened. In this case, the evaluation can be made more accurate by using the full-thickness artificial skin model of the present invention, which is not in contact with the test substance, as a control and comparing the measurement results. Changes in adipocytes in the subcutaneous fat layer include changes in their number, morphology, distribution, localization, migration, and disappearance, as well as changes in the expression levels of specific adipocyte genes (e.g., differentiation marker genes such as the peroxisome proliferator-activated receptor-γ (PPARγ) gene). For example, if a decrease or disappearance of adipocytes or a decrease in the thickness of the subcutaneous fat layer is used as an indicator, the test substance can be screened as a candidate for slimming agents and anti-obesity agents. The measurement of changes in adipocytes is not particularly limited and can be performed, for example, by oil red staining or by microscopic observation of changes in the number and morphology of adipocytes. Furthermore, the test substance can be administered, for example, by administering or applying the test substance from the top of a full-thickness artificial skin model, or by adding the test substance to the culture medium outside a culture insert.

[0037] The test substances are primarily ingredients usable in cosmetics and / or pharmaceuticals, and may include, for example, mixtures containing multiple compounds such as extracts of animal or plant tissues or microbial cultures, and purified standards therefrom; naturally occurring molecules (e.g., amino acids, peptides, oligopeptides, polypeptides, proteins, nucleic acids, lipids, steroids, glycoproteins, proteoglycans, etc.); synthetic analogs or derivatives of naturally occurring molecules (e.g., peptide mimics, etc.); and molecules that do not occur naturally (e.g., low-molecular-weight organic compounds produced using combinatorial chemistry techniques, etc.); as well as mixtures thereof. Furthermore, a single test substance may be tested independently, or a mixture of several candidate test substances (including libraries, etc.) may be tested. Examples of libraries containing multiple test substances include synthetic compound libraries and peptide libraries. [Examples]

[0038] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples.

[0039] (Example 1) Investigation of a method for creating a subcutaneous fat model <cell> In the following experimental examples, immortalized human adipose-derived stem cells (ASCs) (DS Pharma) and normal human preadipocytes (DS Pharma) were used as cells for creating a subcutaneous fat model. Immortalization was performed by creating a vector into which genes encoding telomerase reverse transcriptase (TERT), CDK4 (cyclin-dependent kinase 4), and cyclin D1 were inserted. This vector was then introduced into the human preadipocytes by transfection, and single cells were obtained from the immortalized cell population by single-cell cloning. The obtained immortalized human adipose progenitor cells were differentiated using commercially available differentiation induction medium (Kurabo), and single clone lines of immortalized human preadipocytes were obtained based on the appearance of the cells (degree of lipid droplet formation) and used to create a subcutaneous fat model.

[0040] <culture medium> Growth media were prepared by adding 25 mM HEPES, 3% GLUTAMAX-1 (GIBCO), 1% Insulin Transferrin Selenium Ethanolamine Solution (GIBCO), 1% fetal bovine serum (SIGMA) inactivated by heating at 56°C for 30 minutes, 10 ng / mL FGF-basic (PEPROTECH), and 0.4 μg / mL hydrocortison (WAKO) to 50% αMEM (SIGMA) and 50% DMEM (SIGMA).

[0041] The differentiation induction medium was prepared by adding 1 μM dexamethason (SIGMA), 0.5 mM 3-isobutyl-1-methylxanthine (SIGMA), 0.2 mM indomethacin (SIGMA), 10% fetal bovine serum (SIGMA), 10 μg / mL insulin (SIGMA), and 33 μM biotin (SIGMA) to DMEM (Nacalai).

[0042] (Experiment 1) Creation of a subcutaneous fat model using adipocytes recovered by centrifugation Figure 1 shows the method for creating a subcutaneous fat model using adipocytes recovered by centrifugation. First, the human adipocyte-derived stem cells described above were cultured in a culture dish using the growth medium described above until they reached confluence. After the cells reached confluence, differentiation induction was performed for 14 days using the differentiation induction medium described above. Mature adipocytes in which lipid droplet formation was confirmed by differentiation induction were physically detached by pipetting. The obtained cells were subjected to sucrose density gradient centrifugation, and the fraction containing a large number of mature adipocytes was collected.

[0043] Next, using the commercially available collagen preparation reagent Cellmatrix Type IA (manufactured by Nitta Gelatin Co., Ltd.), mature adipocytes were suspended in a collagen gel and incubated at 37°C for 30 minutes to create tissue containing mature adipocytes within the collagen gel, which was then used as a subcutaneous fat model.

[0044] Frozen sections were prepared from the constructed model and stained with oil red to visualize the localization of mature adipocytes within the tissue. As a result, mature adipocytes were found scattered throughout the tissue, but as previously known, this method resulted in large lipid droplets, a small number of mature cells, and sparse density, making it insufficient as a model of well-developed subcutaneous fat. The same result was observed with subcutaneous adipose tissue prepared in the same manner using immortalized human preadipocytes.

[0045] (Experiment 2) Creation of a subcutaneous fat model using differentiated adipocytes with culture inserts (1) Preparation of collagen gel embedded with adipose stem cells Cultured human adipose-derived stem cells were harvested by trypsin treatment, and the cell density was 300 × 10⁶. 4 The cells were suspended in collagen solution to a concentration of individual cells / mL. The collagen solution was poured into a culture insert (pore diameter 0.4 μm), and incubated at 37°C for 30 minutes to create a collagen gel embedded with adipose stem cells.

[0046] (2) Differentiation into adipocytes Next, the inside and outside of the culture insert were filled with the growth medium described above, and growth culture was performed for 3 days. After that, the inside and outside of the culture insert were replaced with the differentiation induction medium described above, and differentiation into mature adipocytes was induced for 14 days.

[0047] Figure 2 shows the results of horizontal imaging of cells in each layer (upper, middle, and lower tissue layers) after tissue differentiation induction was removed from the culture insert. In the upper tissue layer, which is in contact with the culture medium inside the insert, mature adipocytes with a high degree of maturity and large lipid droplets were observed due to differentiation induction. On the other hand, in the middle tissue layer inside the gel, almost no mature adipocytes were observed, and the number of cells was also small. In the lower tissue layer, near the bottom of the insert, although some mature cells were present, the density was low and differentiation was insufficient. The same results were observed for subcutaneous adipose tissue prepared in the same manner as above using immortalized human preadipocytes.

[0048] (Experiment 3) Creation of a subcutaneous fat model by combining culture insert with shaking culture using a shaker. Similar to Experiment 2, a collagen gel containing adipose stem cells was prepared using a culture insert. Then, during the proliferation culture period and the differentiation induction period, the culture plate was shaken using a shaker as shown in Figure 3 to create a subcutaneous adipose model. A reciprocating shaker (Nisshin Rika Co., Ltd.) and a seesaw-type shaker (Nisshin Rika Co., Ltd.) that shakes horizontally were used, and the shaking speed was 30 rpm.

[0049] Furthermore, various culture inserts with different pore diameters were investigated. The culture inserts used had pore diameters of 0.4 μm, 1.0 μm, 3.0 μm, and 8.0 μm.

[0050] After culturing, the tissue was removed from the culture insert, and horizontal cell images were taken of each layer (upper, middle, and lower layers of tissue). The average size of adipocytes (average of the maximum cell diameter) was then calculated. The results are shown in Table 1.

[0051] [Table 1]

[0052] As shown in Table 1, under conditions without the use of a shaker, large, mature adipocytes were observed in the upper tissue layer regardless of the pore diameter of the culture insert used, but maturation was insufficient in the middle and lower tissue layers. On the other hand, culture using a shaker showed improved maturation of adipocytes in the middle and lower tissue layers of the culture insert. In particular, under conditions combining a pore diameter of 3.0 μm or larger with shaking culture using a shaker, uniformly enhanced adipocyte maturation was shown in all layers (Table 1, bolded section).

[0053] Next, for one sample (a combination of a pore diameter of 3.0 μm and a seesaw-type shaker) in which lipid droplet formation was enhanced by shaking culture using a shaker, the tissue was fixed with 4% PFA, and frozen sections perpendicular to the tissue were prepared. Figure 4 shows bright-field images and oil-red stained images of the tissue sections. Compared with the subcutaneous fat model (conventional model) prepared in Experiment 2, it was observed that mature adipocytes filled the entire tissue from the upper to the lower layers, indicating that the subcutaneous fat model prepared by this method has a structure similar to that of living subcutaneous fat. The same result was obtained in a subcutaneous fat model prepared in the same manner as above using immortalized human preadipocytes.

[0054] (Example 2) Investigation of shaking conditions Subcutaneous fat models were created using 3.0 μm culture inserts by varying the shaking speed of each shaker. Since the seesaw-type shaker has an upper limit of 40 rpm and the reciprocating shaker has an upper limit of 100 rpm, horizontal cell images of the differentiated tissue were captured when the shaking speed was increased by 5 rpm increments from 0 rpm to the upper limit of each shaker, and the average cell size of adipocytes was measured. The results are shown in Table 2.

[0055] [Table 2]

[0056] As shown in Table 2, adipocyte maturation was insufficient under conditions without shaking. On the other hand, increasing the shaking speed tended to improve adipocyte maturation. In particular, under conditions of 20 rpm to 35 rpm, the formation of well-developed adipocytes was confirmed in all layers (Table 2, bolded area).

[0057] On the other hand, when the shaking speed exceeded 40 rpm, the fluidity of the liquid surface increased, causing the upper layer of adipocytes to detach, resulting in a tendency for the density and average size of the upper layer of adipocytes to decrease. Furthermore, when the shaking speed of the reciprocating shaker exceeded 85 rpm, the culture medium leaked out of the insert, making the test impossible.

[0058] From the above, it was shown that the most suitable shaker speed for a combination of culture inserts with large pore diameters and shaker-based culture was 20 rpm to 35 rpm. This result was also observed in a subcutaneous fat model prepared in the same manner as above using immortalized human preadipocytes.

[0059] (Example 3) Preparation of a full-thickness artificial skin model with a subcutaneous fat layer In this study, we attempted to create a full-thickness artificial skin model by combining a culture insert with a pore diameter of 3.0 μm, which was deemed suitable in Experiment 3 of Example 1, with shaking culture (30 rpm) using a shaker, and then constructing the epidermal and dermal layers on top of it. In this example, epidermal and dermal stem cells isolated from commercially available epidermal keratinocytes and fibroblasts were used to construct the epidermal and dermal layers. Specifically, for the epidermal cells, we used commercially available normal human epidermal keratinocytes (Kurabo Industries Ltd.) maintained in HuMedia-KG2 medium (Kurabo Industries Ltd.), and for the dermal cells, we used commercially available normal human fibroblasts (Kurabo Industries Ltd.) maintained in DMEM medium (Nacalai Tesque Corporation) containing 10% FBS. These cells were cultured in a 10 cm dish, harvested with trypsin-EDTA, and then immunostained with the stem cell marker anti-CD271 antibody (Origene Corporation). From the stained cells, the CD271-positive cell fraction was isolated using FACS Melody (Becton Dickinson, Inc.), and epidermal stem cells and dermal stem cells were obtained. Figure 5 shows the cultured images of epidermal stem cells and dermal stem cells obtained by FACS.

[0060] Furthermore, we introduced immortalization genes into commercially available normal human epidermal keratinocytes or normal human fibroblasts to establish cell lines that proliferate indefinitely while maintaining stem cell properties. To establish the immortalized cell lines, we created vectors into which genes encoding three genes—telomerase reverse transcriptase (TERT) gene (Genbank number: Nucleotide NM_198253.2), cyclin-dependent kinase 4 (CDK4) gene (Genbank number: Nucleotide NM_000075.3), and cyclin D1 (CCND1) gene—were inserted. After introducing these vectors into each cell by transfection, single cells were obtained from the immortalized cell population by single-cell cloning. For the acquired immortalized human epidermal keratinocytes, the expression levels of differentiation markers (FLG, IVL) when differentiated with 3 mM calcium chloride were used as indicators. For immortalized human fibroblasts, the expression level of the collagen gene (COL1A1) when differentiated with TGFβ was used as an indicator. Single clone lines of immortalized epidermal stem cells and immortalized dermal stem cells with high differentiation potential were obtained and used to create a full-thickness model of artificial skin.

[0061] Figure 6 shows the procedure for creating a full-thickness model of artificial skin with a subcutaneous fat layer. To create the dermis and epidermis layers of the full-thickness model, a concentration of dermal stem cells separated by FACS was added to the upper part of the subcutaneous fat model prepared in Example 1 (Experiment 3). 4 A collagen solution adjusted to a concentration of individual cells / mL was poured in and incubated at 37°C for 30 minutes to form an artificial dermis layer consisting of collagen gel embedded with dermal stem cells. Subsequently, epidermal stem cells separated by FACS were placed on the surface of the dermis layer in a 100 × 10⁶ layer. 4Individual cells / mL were seeded, and the outer and inner layers of the culture insert were replaced with HuMedia-KG2 medium. Culture was continued until the epidermal stem cells reached confluence. Subsequently, the medium was replaced with CnT-Prime 3D Barrier Culture Medium (CELLnTEC), a commercially available keratinocyte differentiation medium, and cultured for 24 hours. After 24 hours, the medium inside the culture insert was removed, exposing the model to the outside air for 8 days. For comparison, a conventional artificial skin model (two-layer model) consisting of an epidermis and dermis without a subcutaneous fat layer was used to form the epidermis and dermis using a similar method under the condition of not having a subcutaneous fat layer.

[0062] After photographing the appearance of the fabricated artificial skin model, the tissue was fixed with 4% PFA. Frozen sections of the fixed tissue were prepared, and the condition of the tissue was examined by oil red staining.

[0063] Figure 7 shows the appearance of the cultured tissue. Compared to the conventional epidermal and dermal layer model (two-layer model), the model with a subcutaneous fat layer (full-layer model) showed a three-layer structure. Furthermore, the fabricated full-layer model had a color tone that resembled living skin, demonstrating a higher level of realism in its appearance.

[0064] Frozen tissue sections were prepared from the constructed full-thickness model and stained with oil red. Figure 8 shows the results of the oil red staining. As a result, it was confirmed that this model is a tissue with a three-layer structure, similar to living skin tissue, with an epidermal layer, a dermal layer, and a fatty layer filled with mature adipocytes beneath it.

[0065] (Example 4) Evaluation of flexibility of a full-thickness model of artificial skin In developing artificial skin, it is necessary to reproduce not only its appearance and structure, but also its skin properties such as elasticity. This study evaluated the effect of adding a subcutaneous fat model to the elasticity of artificial skin. Elasticity was evaluated using a Cutometer DUAL MPA580 (Courage+Khazaka), which can evaluate the elasticity and flexibility of skin. A probe was pressed against the cheek area of ​​living human skin, an artificial skin model without a subcutaneous fat layer (2-layer model), and an artificial skin model with a subcutaneous fat layer (full-layer model). Negative pressure was applied to the skin through the opening of the probe, and the displacement of the skin shape was measured by suction and release of the negative pressure. Among the obtained values, the R7 value, which represents the elasticity of the skin, was used as an index for evaluation.

[0066] The results are shown in Table 3. The relative elasticity of artificial skin models (2-layer and full-layer) was calculated, with the R7 value of living skin (cheek area) set to 1.

[0067] [Table 3]

[0068] As shown in Table 3, the two-layer model without a fat layer exhibited lower elasticity compared to biological skin. On the other hand, the elasticity of the full-layer model with a fat layer was very close to that of biological skin. This indicates that the full-layer model of artificial skin with a fat layer reproduces not only its appearance but also its feel and elasticity, just like real skin. Based on the above, the full-layer model of artificial skin of the present invention reproduces the realism of biological skin and is expected to have applications in future cosmetic development and drug discovery, as well as as a covering material for humanoid robots and a regenerative medicine product. [Industrial applicability]

[0069] The subcutaneous fat layer model and the full-thickness artificial skin model of the present invention can be used in fields such as the development of cosmetics and pharmaceuticals, drug discovery research, regenerative medicine as transplantation materials, and the development of humanoid robots as covering materials.

Claims

1. A method for producing a subcutaneous fat model, comprising the step of culturing adipose stem cells or adipose progenitor cells by shaking them in a culture insert having a liquid-permeable membrane with a pore diameter of 3 μm or more at the bottom using a shaker, thereby producing a subcutaneous fat layer uniformly containing mature adipocytes.

2. The method according to claim 1, wherein the shaker is a reciprocating shaker or a seesaw shaker.

3. The method according to claim 1, wherein the shaking rate in the shaking culture is 20 to 35 rpm.

4. A subcutaneous fat model having a subcutaneous fat layer uniformly containing mature adipocytes, obtained by the method according to any one of claims 1 to 3.

5. A method for creating a full-thickness artificial skin model, including the following steps. (a) A step of culturing adipose stem cells or adipose progenitor cells by shaking them in a culture insert having a liquid-permeable membrane with a pore diameter of 3 μm or more at the bottom using a shaker, thereby producing a subcutaneous fat layer uniformly containing mature adipocytes. (b) A step of creating the dermis and epidermis on top of the subcutaneous fat layer created in step (a).

6. The method for producing a full-thickness artificial skin model according to claim 5, wherein the dermal layer and epidermal layer are produced by three-dimensionally culturing dermal stem cells or progenitor cells and epidermal stem cells or progenitor cells on the subcutaneous fat layer within a culture insert.

7. The method according to claim 5, wherein the stem cells or progenitor cells are immortalized stem cells or progenitor cells.

8. A full-thickness artificial skin model obtained by the method according to any one of claims 5 to 7, wherein the dermis and epidermis layers are superimposed on a subcutaneous fat layer uniformly containing mature adipocytes, and the model has the elasticity and skin color of living skin.

9. A method for evaluating intercellular communication between adipocytes and epidermal or dermal cells mediated by a test substance, characterized by contacting a full-thickness artificial skin model described in claim 8 with the test substance and measuring changes in secretions from adipocytes in the subcutaneous fat layer of the model.

10. A method for screening substances having a slimming effect via transdermal absorption, characterized by contacting a test substance with a full-thickness artificial skin model described in claim 8 and measuring changes in adipocytes in the subcutaneous fat layer of the model.

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