KR101431883B1 - A co-culture system of 3-dimensional artificial tissue and monitering cells to substitute for animal test, and manufacturing method thereof - Google Patents

Abstract

The present invention provides a co-culture system of 3-dimensional artificial tissue and monitoring cells that can replace animal experiments. The three-dimensional artificial tissue and monitoring cell co-culture system of the present invention exhibits functional similarity with a living tissue. Therefore, in a test for the development of a drug for inflammation and allergy test, inflammation and allergy treatment of cosmetics or medicines, It becomes an optimal test model to replace the experiment.

Description

Technical Field [0001] The present invention relates to a three-dimensional artificial tissue and monitoring cell co-culture system for replacing an animal test, and a method for manufacturing the same,

TECHNICAL FIELD [0001] The present invention relates to a three-dimensional artificial tissue and a co-culture system for monitoring cells for replacing animal experiments such as cosmetics or medicines.

In the United States and Japan, the EU and other countries have enacted regulations related to animal welfare legislation related to alternative testing methods for animal experiments and regulations on animal use. Currently, the National Institute of Toxicological Research Guidelines for alternative testing were established.

Following the Guidelines for In vitro 3T3 NRU Phototoxicity Test as an alternative to the phototoxicity test in 2007 and Guideline for local lymph assay as an alternative to the skin sensitization test, the 2009 Skin Corrosion Test Transcutaneous Electrical Resistance Test, TER, Human Skin Model Test, In vitro Membrane Test, and In vitro Membrane Barrier Test were established as alternatives to the test. Studies and guidelines for alternative animal testing methods are required.

Currently, there is no research on evaluation of inflammation and allergy induction of skin, mucous membranes and respiratory system. It is a system that only evaluates degree of irritation. , It is possible to establish a new animal replacement test method by evaluating inflammation and allergic reactivity of macrophages and mast cells in a co-culture system with artificial skin or corneal substitute.

To date, evaluation techniques using animals and cells have been used to evaluate inflammation and allergy.

In order to evaluate skin allergy, atopic dermatitis animal model is most commonly used, and atopic dermatitis induced model using NC / Nga mouse is the most commonly used model. Spontaneously developed dermatitis model shows that immunopathological abnormalities occur in humans However, in order to increase the expression efficiency of atopic dermatitis, Hapten is known to be very similar to the lesion, but the natural induction rate is less than 50%, the incidence and timing are variously difficult, And mite antigen (DP, dermatophagoides pteronyssinus allergen; DF, Dermatophagoides farinae allergen) are stripped three times with 3M tape or the like, or a purgatory method which does not intentionally keep the breeding environment clean is widely used. In addition, biochemical analysis and behavioral analysis are performed after induction of NC / Nga atopic dermatitis, and evaluation of atopic dermatitis is currently performed by skin gross lesion observation and histopathological examination. Observation of dermatitis related lesions such as agranulocytic inflammation, hemorrhage, eczema, erythema and scaling of skin caused by administration of test substance and observation of immunopathological abnormality of lesional skin tissue by H & E staining and Toluidine blue staining followed by optical microscopy . A method of measuring the number of scratches for 1 hour by using a pre-autopsy camcorder for behavioral analysis is adopted.

On the other hand, the evaluation technique using the cell is used an evaluation method using an obesity cell line. Two immunological abnormalities in atopic dermatitis are an increase in IgE and a functional abnormality of T lymphocyte. An increase in IgE induces degranulation of mast cells, The immune response is assessed. Thus, the degree of allergic reactivity is evaluated by performing an IgE-mediated hexosaminidase assay using an RBL-2H3 cell line. Bone marrow-derived mast cells (BMMC) are also used. In order to secure the cells, the bone must be cultured in a special culture medium by expelling the inside of the bone after femoral and tibial incision of the mouse. In addition, splenocyte-based assays are also used, and IgE, which is a cause of allergic reactions, is secreted by Th2 cytokine, so evaluation of cellular immune function is used as an important evaluation index. For this purpose, spleen cells were harvested from aseptic conditions and cultured in an incubator for 48 hours. The cytokines such as IL-4, IL-1β, IL-6, GM-CSF and TNF- .

To date, allergic evaluation is limited to atopic dermatitis models, and there is a problem of evaluating an experimental animal group in an animal model derived from a sadistic experimental method, and a high cost is also required. On the other hand, in an in vitro cell model, a mature cell line and a bone marrow-derived mast cell line are directly extracted from a bone marrow.

Korean Patent Publication No. 2001-0070541 (published on July 25, 2001) Korean Patent Publication No. 10-2007-0018220 (published on February 14, 2007) Korean Patent Publication No. 10-2007-0098973 (published October 8, 2007) Korean Patent Publication No. 10-2009-0094664 (published on September 8, 2009)

The present invention provides a three-dimensional artificial tissue and a co-culture system for monitoring cells that can replace animal tests in inflammation and allergy testing of cosmetics or medicines.

The inventors of the present invention have made intensive studies on a model that can replace an animal experiment in an experiment for the development of a drug for inflammation and allergy test, inflammation and allergy treatment of cosmetics or medicines, A co-culture system of artificial tissue and monitoring cells was completed.

The present invention provides a method for producing a co-culture system of three dimensional artificial tissue and monitoring cells comprising:

(1) a step of preparing a transwell upper chamber containing a three-dimensional artificial tissue comprising:

(i) suspending fibroblasts in a type I collagen mixture:

(ii) adding the suspension to a transwell upper chamber to solidify the suspension, and then culturing the suspension in a culture medium to form a multilayered cell growth plant;

(iii) coating the upper surface of the multi-layered cell proliferation obtained by the culture with a mixture of type IV collagen and laminin; And

(iv) inoculating and culturing epithelial cells on the surface of the coated multi-layer cell culture plant,

(2) preparing a transwell lower chamber containing monitoring cells comprising:

(v) culturing the monitoring cells in a culture medium; And

(vi) inoculating and culturing the cultured monitoring cells in a lower chamber containing transwell culture medium, and

(3) placing the transwell upper chamber containing the three-dimensional artificial tissue prepared in the step (1) at the upper end in the transwell lower chamber containing the monitoring cells prepared in the step (2) Filling the chamber bottom to the upper end of the three-dimensional artificial tissue to manufacture a co-culture system of three-dimensional artificial tissue and monitoring cells.

In the above production method, the fibroblast is a dermal fibroblast or a corneal fibroblast. Preferably, the dermal fibroblast is a human-derived fibroblast, and the corneal fibroblast is a corneal fibroblast derived from a rabbit cornea.

In the above production method, the epithelial cells of step (iv) are keratinocytes or epithelial cells derived from the cornea.

In the above production method, the monitoring cell is a macrophage or a mast cell. Preferably, the macrophage is RAW264.7 cell line (KCLB No. 40071), which is a mouse macrophage cell line, and the mast cell is a mouse obesity cell line RBL-2H3 cell line ACTT, CRL-2256 (TM)). In particular, in the model for assessing the inflammatory response, macrophages are used as monitoring cells and mast cells are used as monitoring cells in a model for evaluating allergic reactions.

As another embodiment for producing a co-culture system for evaluating an inflammatory response in the above production method, after step (3), a step of treating LPS (lipopolysaccharide) to convert macrophages into dermal macrophages is added As shown in FIG.

The transwell plate used in the present invention is widely used in the art, and commercially available products can be used. Typically, products commercially available from Corning Incorporated can be used. Detailed information about the product can be obtained from the product catalog or website (www.corning.com) provided by Corning Incorporated. In fact, information and usage of the transwell plate can be obtained from a person skilled in the art It is within the skill level.

As illustrated in FIG. 10, the transwell plate has an upper chamber (also referred to as a product catalog or a transwell insert in the art) having a lower end made of a microporous membrane and a lower chamber ), The upper chamber is located inside the lower chamber, and the two chambers share the culture medium. Here, the microporous membrane is a cellular material such as a cell secretory substance, a culture medium permeates through the medium but is not permeable to cells. The size of the microporous membrane can be suitably selected by those skilled in the art. For example, Mu m.

The microporous membrane constituting the bottom surface of the transwell upper chamber is a permeable support capable of supporting cell culture and growth thereon, and may be a membrane made of materials conventionally used in the art. For example, polycarbonate , Polyester, collagen-coated polytetrafluoroethylene, or the like can be used as the microporous membrane.

The type I collagen mixture of step (i) is a mixture of type I collagen and a suitable medium for cell culture. For example, I type collagen (Nitta gelatin), DMEM (Gibco) at 10 times concentration and reconstitution buffer NaHCO ₃ and 4.77 g HEPES in 100 ml of 0.05 N NaOH solution) at a ratio of 8: 1: 1.

The culture medium used in the above production method is a culture medium commonly used in the art for culturing cells. The composition of the culture medium can be modified by those skilled in the art depending on the type and characteristics of the cells, and it is within the technical skill of those skilled in the art . For example, a mixed medium of DMEN and F12, DMEN medium containing 10% FBS and the like can be used for the implementation of the present invention.

The present invention also provides a co-culture system of 3-dimensional artificial tissue and monitoring cells produced by the above-described production method.

The three-dimensional artificial tissue and monitoring cell co-culture system of the present invention can not be regarded as a kind of device or simple mechanical device as exemplarily shown in FIG. 3, and therefore, as a 'system' Quot; However, if it does not impair the essence of the present invention, it can be interpreted as a device.

The co-culture system of three-dimensional artificial tissue and monitoring cells of the present invention comprises a transwell upper chamber containing three-dimensional artificial tissue, a transwell lower chamber containing monitoring cells and a culture medium, wherein the transwell upper chamber is a trans- The culture medium is filled from the lower chamber end to the upper end of the three-dimensional artificial tissue, and is preferably filled to the lower end of the upper chamber.

The three-dimensional artificial tissue in the transwell upper chamber is formed by a method including the steps (i) to (iv), and is a three-dimensional artificial skin tissue or a three-dimensional artificial cornea tissue.

Monitoring cells in the transwell lower chamber are macrophages or mast cells, macrophages are preferred for co-culture systems for inflammatory response models, and mast cells are preferred for co-culture systems for allergic response models.

Until now, the skin model has only the structural similarity including only the epidermal layer and the dermal layer, and there has been no functional skin model capable of performing the primary defense mechanism. As a result, the co- culturing system including the three- Is a functional skin model containing dermis macrophages and can be used as an evaluation system that is most similar to biological function.

Therefore, the three-dimensional artificial tissue and monitoring cell co-culture system of the present invention can be substituted for animal experiments in experiments for the development of drugs for inflammation and allergy testing of cosmetics or medicines, and for the treatment of inflammation and allergy.

The present invention also provides a method for evaluating an inflammatory reaction or an allergic reaction of a test substance using the co-culture system of the above-described three-dimensional artificial tissue and monitoring cells.

The method for evaluating an inflammatory reaction or an allergic reaction of a test substance of the present invention comprises the step of applying a test substance to the above-mentioned three-dimensional artificial tissue surface. Figure 11 illustrates, by way of example, the application of a test substance to the co-culture system of the present invention for testing an inflammation or an allergic reaction.

In the present invention, the test substance is an arbitrary candidate substance for evaluating an inflammatory reaction or an allergic reaction to the eye or the skin, and is a candidate compound of cosmetics or pharmaceuticals for application directly or indirectly to the eye or skin, natural products, Or a mixture thereof. In addition, the test substance may be a candidate substance for the treatment of an inflammatory reaction or an allergic reaction.

Therefore, the evaluation method of the inflammatory reaction or the allergic reaction of the test substance of the present invention includes a method of evaluating an inflammatory reaction or an allergic reaction to the cornea or skin of a test substance.

(B) the level of secretion of secreted IL-6, IL-10, or both in the cell culture medium, or (c) the level of secretion of IL- The inflammatory response of the test substance can be assessed through an increase in one or more indicators selected from the level of production of secreted iNOS, Cox-2, or both, in the cell culture.

The evaluation method of the test substance of the present invention can evaluate the allergic reaction of the test substance through an increase of the activity level of? -Hexosaminidase in the cell culture medium.

Currently, as the incidence of inflammation and allergic diseases increases, studies are being actively pursued for the therapeutic agent and the candidate for prophylaxis. However, a reliable evaluation model for accurate evaluation should be preceded, and in particular, High cost is required to apply the model to all candidate materials. Therefore, according to the co-culture system of the artificial tissue and the monitoring cell of the present invention, large-scale screening with high reproducibility of inflammation and allergic reaction can be performed by replacing animal experiments conducted for the evaluation of inflammation and allergy of existing cosmetic ingredients. It also provides a standardized indicator for the search for therapeutic agents and preventive agents for inflammation and allergic diseases.

The three-dimensional artificial tissue and monitoring cell co-culture system of the present invention exhibits functional similarity with a living tissue. Therefore, in a test for the development of a drug for inflammation and allergy test, inflammation and allergy treatment of cosmetics or medicines, It becomes an optimal test model to replace the experiment.

FIG. 1 is a photograph of H & E staining of three-dimensional artificial skin tissue, corneal tissue and living tissue ((A) normal rabbit corneal tissue, (B) three- dimensional rabbit artificial corneal tissue, Human skin tissue, and (D) a three-dimensionally constructed human skin tissue).
FIG. 2 is a photograph showing the results of immunohistochemical staining of three-dimensional artificial skin tissue and corneal tissue (three-dimensional artificial skin tissue: A to C, three-dimensional artificial corneal tissue: D to F; (A) Keratin 10, (C) keratin 3 + 12, (D) keratin 5, (E) keratin 10, and (F) keratin 3/12.
FIG. 3 is a diagram showing an exemplary structure of a co-culture system of three-dimensional artificial tissue and monitoring cells.
FIG. 4 is a photograph showing the morphological change of the RAW264.7 cell line in the inflammation model (+ 3D skin: co-culture system of 3D artificial skin tissue and monitoring cells; + 3D cornea: co-culture of 3D artificial corneal tissue and monitoring cells system).
FIG. 5 is a graph showing the cytokine (IL-6, IL-10, TNF-alpha analysis results of RAW264.7 cell line in the inflammation model.
FIG. 6 is a photograph showing the result of confirming the inflammatory reaction (iNOS, Cox-2 level) of the RAW264.7 cell line in the inflammation model.
Fig. 7 is a gel photograph showing the characteristics of the RAW264.7 cell line to the dermal macrophage layer in the inflammation model.
Fig. 8 is a diagram showing the functional similarity of the co-cultivation system of the present invention with the vital skin.
FIG. 9 shows the results of confirming allergic reactions induced by DNP-IgE and DNP-BSA in an allergic model.
10 is a view showing a structure of a transwell plate.
Figure 11 is an exemplary diagram illustrating the application of a test substance to the co-culture system of the present invention to test an inflammation or an allergic reaction.

Hereinafter, the present invention will be described in more detail through Production Examples, Examples and Test Examples. However, these production examples, examples and test examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

[Manufacturing Example]

< Manufacturing example  1> Contains 3-D artificial skin tissue Transwell  Top Of the chamber (insert)  Produce

We used a three - dimensional culture method in which epidermal cells were inoculated on a dermal layer formed by mixing type 1 collagen with dermal fibroblast. The epidermal cells were cultured in the middle of the air layer and the water layer, thereby differentiating into the middle layer squamous epithelium and keratinized to form a substantial skin structure.

As a dermis counterpart, the collagen mixture was diluted with I-type collagen (Nitta gelatin), DMEM (Gibco) at a concentration of 10 times, and reconstitution buffer (buffer containing 2.2 g NaHCO ₃ and 4.77 g HEPES dissolved in 100 ml of 0.05 N NaOH solution) : 1. To prevent gelation of the collagen, the preparation of the dermis counterpart was carried out at 4 캜. A 12 mm diameter transwell insert (Transwell; Corning, NY.) Was prepared. Skin fibroblasts (primary cultures from human skin tissue) were recovered from the culture dish and suspended in collagen mixture at a concentration of 1 x 10 ⁶ cells / ml taking care not to bubble. 200 [mu] l of collagen mixture was placed inside the transwell insert, and the collagen mixture was solidified for 1 hour at 37 [deg.] C in an incubator. The culture medium in which DMEM: F12 was mixed at a ratio of 3: 1 to the inside and outside of the transwell was set at the inside and outside water levels, and cultured for 24 hours. After the dermis counterpart was formed, the medium in the transwell was removed, and the upper part of the dermis counterpart was coated with type IV collagen and laminin, which are components of the basement membrane, to facilitate adhesion of keratinocytes. The surface was coated with 50 μl of collagen 0.1 mg / ml and 30 μg / ml of laminin at 37 ° C for 1 hour. After the coating solution was removed, it was washed three times with PBS buffer solution.

The keratinocytes (primary culture from human skin tissue) were trypsinized, recovered from the culture dish, counted, centrifuged and inoculated at ⁵ × 10 ⁵ cells / well on coated collagen dermis. The medium was changed every 2-3 days and cultured for 7 days. If keratinocytes are evenly distributed on the upper part of the dermis counterpart, the medium on the skin layer is removed so that the skin layer is cultured on the contact surface of water and air, and the medium is placed outside the transwell insert by adjusting the height of the dermis counterpart, Lt; / RTI &gt;

< Manufacturing example  2> 3-dimensional artificial corneal tissue Transwell  Top Of the chamber (insert)  Produce

A three-dimensional culture method in which rabbit cornea-derived epithelial cells (primary culture) were inoculated on a dermal layer made by mixing 1 type collagen with corneal fibroblast (primary culture from human skin tissue) in the same manner as in Preparation Example 1 was used . Unlike the keratinocytes, the cultured corneal epithelial cells were maintained in a semi-dry state to prevent keratinization.

< Manufacturing example  3> Confirm structural similarity between 3D artificial tissue and living tissue

(1) H & E dyeing

H & E staining was performed as follows. The paraffin-embedded tissue was sectioned at 5 μm and attached to the slide. The slide was then reacted with xylene for 10 minutes to remove the paraffin around the tissue. 100%, 90%, 80% and 70% ethanol for 2 minutes in order to hydrate and wash the tissue. The nuclei were stained with Hematoxylin (ResGen) for 5 minutes, the slides were washed for 10 minutes, and the cells were dehydrated by sequential treatment with 70%, 80%, and 90% ethanol for 2 minutes. Eosin , Sigma) for 5 minutes to stain the cytoplasm, followed by washing with 95% ethanol and treatment with xylene over 100% ethanol for 2 minutes. A drop of a mounting material (Shandon Synthetic Mountant, Thermo) was dropped on the tissue portion of the slide, and then the slide cover was attached to the slide.

The results are shown in Fig. H & E staining of skin and cornea cultured in transwell inserts showed structural similarity when compared to normal reconstructed human skin tissue and rabbit corneal tissue (Fig. 1, (A) normal rabbit corneal tissue, (B) three dimensional rabbit artificial cornea tissue, (C) normal human skin tissue, and (D) three-dimensional human tissue.

(2) Immunohistochemical staining

Immunohistochemical staining was performed as follows. Paraffin was removed from paraffin cuts (5 μm) and hydrated prior to immunohistochemical staining. Dyeing was performed according to the method provided using ABC kit (Vector Laboratory, CA) and DAB kit (Vector Laboratory, CA). The cuts were counter stained with Fast Red (Vector Laboratories Inc.). Keratin 5, keratin 10, and cytocharratin 3/12 (abCAM) as recommended by the manufacturer of the antibody.

The results are shown in Fig. Immunohistochemical staining revealed that keratin 5, expressed in intact epithelial cells around the basal layer, is strongly expressed in three - dimensional artificial skin tissue and three - dimensional artificial corneal tissue. In particular, keratin 10, which is expressed only in differentiated keratinocytes, is expressed locally in the differentiated stratum corneum of 3-dimensional artificial skin tissue but not in 3-dimensional artificial corneal tissue. In addition, keratin 3/12 specifically expressed in the cornea was not expressed in the 3-dimensional artificial skin tissue but was expressed only in the 3-dimensional artificial corneal tissue. This confirms that the reconstructed models of Preparation Examples 1 and 2 have real skin and corneal characteristics.

< Manufacturing example  4> monitoring  Selection and culture of cell line

(1) Selection of an inflammatory model monitoring cell line

RAW264.7 cell line, a mouse macrophage cell line, was selected as an inflammatory model monitoring cell line. RAW264.7 cell line, a mouse macrophage cell line, was cultured in DMEM with 10% FBS, and the culture medium was changed once every 2-3 days. When the cells became confluent, they were incubated with 0.25% trypsin-EDTA for 10 minutes to separate into single cells and subculture at 1:20.

(2) Allergy model monitoring  Selection of cell line

RBL-2H3 cell line, a mouse obesity cell line, was selected as an allergic model monitoring cell line. The mouse obesity cell line, RBL-2H3, was cultured in DMEM with 10% FBS and the culture medium was changed once every 2-3 days. When the cells became confluent, they were incubated with 0.25% trypsin-EDTA for 10 minutes to separate into single cells and subculture at 1:20.

< Manufacturing example  5> monitoring  Method of analysis of cell line

(1) Assessment of inflammation through cytokine analysis (IL-6, IL-10, TNF alpha) using ELSIA

The cytokine secreted into the cell culture was quantified by ELISA (R & D systems, Cat No. DY406, DY417, DY410).

After incubation for 1 day at room temperature in a 96-well plate, the cells were washed 3 times with a washing buffer (pH 7.0) And washed. The cells were blocked with 1% BSA solution for 1 hour to prepare a culture solution and a reaction solution. 100 μl of each culture was added and reacted for 2 hours. After incubation for 2 hours, the cells were washed three times with washing buffer and reacted with 100 μl of detection antibody (IL-6, R & D systems, 840172; IL-10, R & D systems, 840126; TNF alpha, R & D systems, 840144) for 2 hours. After washing three times with Wash Buffer, add 100 μl of streptavidin-HRP and incubate for 20 minutes. After the reaction, the cells were washed three times with Wash Buffer, and 100 μl of the reagents A and B were mixed with each other. The cells were incubated for 20 minutes, and the reaction was terminated by addition of 10 nM NaOH.

(2) β- Hexosamidine  The assay method (β- hexosaminidase assay ) To evaluate allergic effects

The degree of the activity of β-hexosaminidase secreted by the anti-DNP-IgE-induced manganese deagglomerization in the rat mast cell, RBL-2H3 cell line, was evaluated as an allergic reaction. After 5x10 ⁵ RBL-2H3 cells were cultured in 12-well plates, DNP-IgE, which is an inflammation inducer, was treated with 72.2 ng / ml for about 3 hours, and after 16 hours with phosphate buffer buffer (PBS) After washing, the cell culture was replaced with EBSS containing 1% BSA. Then, DNP-BSA 150 ng / ml was treated for 30 minutes to induce degranulation, and the activity of β-hexosaminidase was measured by taking only the cell culture solution separately. Fluorescence was measured at 405 nm (Passante et al. 2009).

(3) Western Blasting  Used iNOS  And Cox -2 level evaluation

For Western blotting, 10% of iNOS and Cox-2 (Cell Signaling Technology, Cat No 2977, 4842) and CD 163 (Abcam, ab111250) 10 (R & D systems, cat no MAB519) and Arginases I (Santa Cruz, Cat No sc-18354) were transferred to nitrocellulose paper after 15% SDS-PAGE. The transferred membrane was blocked with 0.1% TBST solution containing 5% non-fat dry milk for 1 hour and incubated with the antibody for 2 hours.

After washing three times for 10 minutes with 0.1% TBST solution, the cells were allowed to react with the secondary antibody for 1 hour. The cells were incubated with ECL solution (Chemilucent ECL Detection System cat No 2600), which was washed three times for 10 minutes with 0.1% TBST solution.

< Manufacturing example  6> 3D artificial tissue and monitoring  Manufacture of co-culture system of cell lines

(1) Manufacture of co-cultivation system for inflammation model

The 3-dimensional artificial skin tissue and the 3-dimensional artificial corneal tissue prepared in the inserts of the 12-well transwell were respectively transferred to a 12-well transwell lower chamber in which RAW 264.7 cells were inoculated 4 hours before into 2x10 ⁵ cells in DMEM containing 10% And cultured. DMEM containing 50 μl of 10% FBS was added to the cultured three-dimensional artificial skin tissue and the three-dimensional artificial cornea tissue in semi-dry state, respectively.

The thus produced three-dimensional artificial tissue and the co-culture system of the monitoring cell line exhibit the structure as shown in Fig.

(2) Manufacture of co-culture system for allergy model

The 3-dimensional artificial skin tissue and artificial corneal tissue prepared in 12-well transwell inserts were placed in a 12-well Transwell lower chamber in which RBL-2H3 cell line was inoculated 4 hours before with 2x10 ⁵ cells in DMEM containing 10% FBS And cultured. 50 μl of DMEM supplemented with 10% FBS was added to the cultured three-dimensional artificial skin tissue and artificial corneal tissue, respectively.

The thus produced three-dimensional artificial tissue and the co-culture system of the monitoring cell line exhibit the structure as shown in Fig.

Skin is a major organ that senses a variety of stimuli and transmits them to the nervous system. It is an important part of the human body that interacts with the external environment. Mast cells, which are mainly distributed in connective tissues, are involved in the pathophysiology of skin diseases such as urticaria, psoriasis, atopic dermatitis and wound healing. These mast cells are immunologically or nonimmunologically activated and promote the secretion of mediators such as histamine, which causes many inflammatory cells to infiltrate into the area where mast cells are activated, resulting in vasodilatation, edema and tissue damage. Because allergic reactions are mostly animal experiments, we have established a co-culture system for an allergic reaction model in order to establish an in vitro experiment in similar conditions to living animals, replacing animal experiments.

[Example]

Example 1 Identification of Inflammatory Response of Co-culture System of 3-D Artificial Tissue and Monitoring Cell Lines

To confirm the inflammatory response of the co-culture system of the 3-dimensional artificial tissue and the monitoring cell line prepared in Preparation Example 6, LPS (Sigma, Cat No L6893) was added to the 3-dimensional artificial skin tissue and the artificial corneal tissue at 0.5 μg / ml For 24 hours to induce an inflammatory response.

(1) Observation of morphological change

The effect of LPS - induced RAW264.7 cell line was first confirmed by morphological changes of the cell. Figure 4 shows the morphology of the RAW264.7 cell line in the cell culture medium of the transwell lower chamber after LPS treatment (Fig. 4, + 3D skin: co-culture system of 3D artificial dermal tissue and monitoring cells; + 3D cornea: 3 Dimensional artificial corneal tissue and monitoring cell co-culture system). It was confirmed that the treatment of LPS resulted in an increase in the formation of vacuole indicating phagocytosis, which is used as an activation index of macrophages, in the cytoplasm of RAW264.7 cell line.

(2) Identification of Secreted Cytokines in Cell Culture Medium

IL-6, IL-10 and TNF-alpha secreted in the cell culture medium of RAW264.7 cell line in the co-culture system were quantified by ELISA as described in Production Example 5, and the effect of the immune activity was evaluated. The results are shown in Fig. As a result, it was confirmed that IL-6 and IL-10 were secreted by LPS stimulation as in RAW 264.7 cell line. However, TNF-alpha was increased in the three-dimensional artificial tissue model, indicating that LPS did not respond to stimulation.

(3) Identification of inflammatory response through iNOS and Cox-2

The expression level of iNOS and Cox-2 was measured after immune activation of the RAW264.7 cell line in the co-culture system by the method described in Production Example 5. The results are shown in Fig. As a result, it was confirmed that the degree of expression of iNOS and Cox-2 decreased when co-cultured with 3-dimensional artificial tissue.

Inflammation is a defense mechanism of the host which is rapidly activated through various mechanisms of action to protect against any chemical changes such as physical action, chemical substance, bacterial infection, etc. in the living body or tissue. In the inflammation process in the body, inflammatory factors such as nitric oxide (NO) and prostaglandin E2 (PGE2) are formed by inducible NO synthase (iNOS) and cyclooxygenase (Cox-2).

This means that the biochemical defense system of the skin also works in a co-culture system employing a three-dimensional artificial tissue model to reduce the degree of inflammatory reaction. This means that the co-culture system of the present invention operates with a mechanism close to an actual living body model, It can be provided as a similar experimental model to a living body model.

(4) Characterization of RAW264.7 cell line activated by LPS

The expression level of Arg1, iNOS, and IL-10 was measured after immune activation of the RAW264.7 cell line in the co-culture system by the method described in Production Example 5. The results are shown in Fig.

As shown in FIG. 7, it was confirmed that RAW264.7 cell line, which was immunologically activated by LPS, simultaneously expressed iNOS that mediates the inflammatory response but simultaneously induced Arginase I and IL-10 capable of inducing an anti-inflammatory reaction .

Changes in these cytokines identified in the co-culture system could be confirmed by RAW264.7 macrophages to be converted into dermal-type macrophages that are specific for the skin by LPS-induced immune activation.

Actual skin is not only a major organ that senses a variety of stimuli and transmits them to the nervous system, but also is an immune system responsible for primary protection from external stimuli and harmful substances. To this end, the skin contains cells that neutralize the immune inducing substance before and during the defense period of the skin itself. The dendritic cells are mainly dendritic cells and bone marrow-derived dendritic cells. The dendritic cells in the human skin are mainly langerhans cells present in the epidermis and the other are dermis cells in the dermis Phagocytes.

Currently, the artificial skin modeling technique consists of only the epidermal layer consisting of keratinocyte layer and the dermal layer, which is a connective tissue, so that it is possible to reconstruct a structurally similar skin layer, but there is a limitation that functional reconstruction is impossible.

In this experiment, the characteristics of the RAW 264.7 cell line immunoprecipitated by LPS were analyzed by western blotting, and it was confirmed that the cells were similar to the dermal macrophages.

This is because the RAW 264.7 cell line is co-cultured in establishing the skin model as the primary endogenous immunological organ, or the inflammatory reaction of the sample under immunological activation with LPS is examined in the in vivo skin This means that it is the best model to replace the animal experiment considering the immune response. Therefore, the RAW264.7 cell line treated with LPS is a model that can replace the dermis macrophage layer.

That is, it has been confirmed that the co-culture system of the present invention exhibits functional similarity with the vital skin, and FIG. 8 is attached to help understand the functional similarity.

<Example 2> Confirmation of allergic reaction in co-culture system of 3-dimensional artificial tissue and monitoring cell line

In order to confirm the allergic reaction of the co-culture system of the 3-dimensional artificial tissue and the monitoring cell line prepared in Preparation Example 6, DNP-IgE was added to the 3-dimensional artificial skin tissue and artificial corneal tissue at a concentration of 72.2 ng / Treated with DNP-BSA at 150 ng / ml for 30 minutes to induce degranulation of mast cells, and the activity of? -Hexosaminidase liberated by degranulation was examined as described in Production Example 5. The results are shown in Fig. As a result, the activity of β-hexosaminidase identified in RBL-2H3 was observed in a co-culture system using 3D artificial skin tissue, but no allergic reaction was induced in a co-culture system using 3D artificial corneal skin tissue.

Claims (17)

A method for preparing a macrophage or mast cell, and a co-culture system of three-dimensional artificial tissue, comprising:
(1) a step of preparing a transwell upper chamber containing a three-dimensional artificial tissue comprising:
(i) suspending fibroblasts in a type I collagen mixture,
(ii) adding the suspension to a transwell upper chamber to solidify the suspension, and then culturing the suspension in a culture medium to form a multilayered cell-
(iii) coating the upper surface of the multi-layered cell proliferation obtained by the above culture with a mixture of type IV collagen and laminin, and
(iv) inoculating and culturing epithelial cells on the surface of the coated multi-layer cell culture plant;
(2) a transwell lower chamber containing macrophages or mast cells comprising:
(v) culturing macrophages or mast cells in a culture medium, and
(vi) inoculating and culturing the cultured macrophages or mast cells in a lower chamber containing a transwell culture medium;
(3) placing the transwell upper chamber containing the three-dimensional artificial tissue prepared in the step (1) at the upper end in the transwell lower chamber containing the macrophages or the mast cells prepared in the step (2) Preparing a co-culture system of macrophages or mast cells, and a three-dimensional artificial tissue by filling the culture medium from the lower chamber end to the upper end of the three-dimensional artificial tissue. The method according to claim 1, wherein the fibroblasts are dermal fibroblasts or corneal fibroblasts. The method according to claim 2, wherein the dermal fibroblast is a human-derived fibroblast, and the corneal fibroblast is a corneal fibroblast derived from rabbit cornea. The method according to claim 1, wherein the epithelial cells are keratinocytes or corneal epithelial cells. delete The method according to claim 1, wherein the macrophage is a mouse macrophage cell line RAW264.7 cell line. 7. The method according to claim 6, wherein after step (3), macrophages are converted to dermis macrophages by LPS (lipopolysaccharide) treatment. delete The method according to claim 1, wherein the mast cell is a mouse obesity cell line RBL-2H3 cell line. The method of claim 1, wherein the bottom surface of the upper chamber comprises a microporous membrane. As a co-culture system of macrophages or mast cells and three-dimensional artificial tissue,
A lower chamber containing macrophages or mast cells in a transwell culture medium;
A trans-well upper chamber containing a layer of a type 1 collagen mixture and a fibroblast suspension solidified and formed, a coating layer formed by coating with a mixture of type IV collagen and a laminin on top of a multi-layer cell growth plant, ; And
Lt; / RTI &gt; culture medium,
Wherein the lower chamber and the upper transwell chamber are connected to the lower chamber by a connection support fixed to the upper chamber so that the transwell upper chamber is located at the upper end of the lower chamber and the culture medium is filled from the lower end of the upper chamber to the upper end of the epithelial cell, Cell or mast cell, and co-culture system of three-dimensional artificial tissue. 12. The co-culture system of claim 11, wherein the macrophage or mast cell for evaluation of an inflammatory or allergic reaction, and a three-dimensional artificial tissue. The macrophages or mast cells of claim 11, and the three-dimensional artificial tissue A method of evaluating an inflammatory or allergic response of a test substance comprising applying a test substance to a surface of an epithelial cell layer in a transwell upper chamber of a co-culture system. 14. The method according to claim 13, wherein the test substance is evaluated for an inflammatory reaction or an allergic reaction to the cornea or skin. 14. The method of claim 13, wherein the inflammatory response of the test substance is assessed through an increase in one or more of the following indicators:
(a) the formation level of macrophages or mast cells in cell culture medium,
(b) production levels of secreted IL-6, IL-10, or both in the cell culture medium, or
(c) Production levels of secreted iNOS, Cox-2, or both, in cell culture medium. 14. The method according to claim 13, wherein the allergic response of the test substance is assessed by increasing the activity level of? -Hexosaminidase in the cell culture broth. 14. The method of claim 13, wherein the test substance is a therapeutic candidate for inflammation or allergy.

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