KR20210034733A - 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 relates to a co-culture system of a 3-D artificial tissue and monitoring cells, capable of replacing an animal test. The co-culture system of 3-D artificial tissue and monitoring cells of the present invention exhibits functional similarity to living tissues, and thus becomes an optimal test model capable of replacing animal experiments in inflammatory and allergy tests such as cosmetics or pharmaceuticals, and tests to develop drugs for treating inflammation and allergy.

Description

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

The present invention relates to a co-culture system of 3D artificial tissues and monitoring cells for replacement of animal experiments such as cosmetics or pharmaceuticals.

In the US and Japan, leading to the EU, legislation related to alternative testing methods for animal testing and regulations on animal use are spreading in relation to animal welfare. Currently, the National Academy of Toxicology is an internationally emerging animal when evaluating the safety of cosmetics, etc. Guidelines for alternative test methods have been established.

In 2007, as an alternative test method for phototoxicity test, <In vitro 3T3 NRU phototoxicity test> and as an animal substitute test method for skin sensitization test, following guidelines for <local lymph node test method, local lymph assay>, skin corrosion test was conducted in 2009. Alternative tests for Korea, <Transcutaneous Electrical Resistance Test, TER>, <Human-derived artificial skin model test, Human skin model test>, <In vitro barrier test, In vitro membrane barrier test> have been established. There is a demand for research and establishment of guidelines for conventional animal replacement test methods.

As an alternative to the current method for evaluating skin toxicity, there is no research on technology to evaluate the degree of inflammation and allergy in the skin, ocular mucosa, and respiratory system as a system that evaluates only the degree of irritation. It is possible to establish a new animal replacement test method through the evaluation of inflammation and allergic reactivity of macrophages and mast cells in a co-culture system with artificial skin or corneal substitutes by improving the system for evaluation in

Up to now, the techniques for evaluating inflammation and allergy have largely been used for evaluation techniques using animals and evaluation techniques using cells.

Currently, an animal model of atopic dermatitis is most commonly used for the evaluation of skin allergy, and a model that induces atopic dermatitis using NC/Nga mice is the most commonly used, and the spontaneously developed dermatitis model using this is characterized by immunopathological abnormalities in humans. Although it is known that it is very similar to the lesions that occur, the natural incidence rate is less than 50%, the severity and timing of the onset are various, and in terms of cost, it requires a high cost of 100,000 won per animal, and to increase the efficiency of the expression of atopic dermatitis, Hapten And mite antigen (DP, dermatophagoides pteronyssinus allergen; DF, Dermatophagoides farinae allergen). A sadistic method that does not deliberately keep the breeding environment clean is widely used by stripping the administered part three times using 3M tape. In addition, after induction of NC/Nga atopic dermatitis, biochemical analysis and behavioral analysis are performed, and evaluation of atopic dermatitis is currently performed by observation of skin lesions and histopathological examination. Observation of dermatitis-related lesions such as abscess inflammation of the skin, bleeding, eczema, erythema, and dermal peeling caused by administration of the test substance, and immunopathological abnormalities of the lesion skin tissue after H&E staining and toluidine blue staining, and then observation with an optical microscope I am using For behavioral analysis, a method of measuring the number of scratches for 1 hour using a camcorder before autopsy has been adopted.

On the other hand, the evaluation technique using cells is the evaluation method using the mast cell line, and the two immunological abnormalities that appear in atopic dermatitis are increased IgE and functional abnormalities of T lymphocytes, whereas an increase in IgE induces degranulation of mast cells, resulting in hypersensitivity. The immune response is evaluated. Therefore, the degree of allergic reactivity was evaluated by performing an IgE-mediated hexosaminidase assay using the mast cell line, RBL-2H3 cell line. Bone marrow-derived mast cells (BMMC) are also used, and to secure cells, they must be cultured in a special culture medium by removing the inside of the bone after incision in the femur and tibia of the mouse. In addition, an evaluation method using splenocytes is also used, and since IgE, which causes allergic reactions, is secreted by Th2 cytokine, evaluation of cellular immune function is used as an important evaluation index. To this end, after collecting the spleen under aseptic conditions, the spleen cells were cultured in an incubator for 48 hours, and cytokines such as IL-4, IL-1β, IL-6, GM-CSF, and TNF-α secreted in the culture medium were ELISA. It is evaluated as.

To date, allergy evaluation is limited to atopic dermatitis models, and there is a problem in that the experimental animal group is evaluated in an animal model derived by a sadistic experimental method, and high cost is required. Meanwhile, in an in vitro cell model, a mast cell line and a bone marrow-derived mast cell line are fractionally extracted and used directly from the bone marrow.

KR 2001-0070541 A KR 2007-0018220 A

An object of the present invention is to provide a co-cultivation system of three-dimensional artificial tissues and monitoring cells that can replace animal experiments in inflammation and allergy tests such as cosmetics or pharmaceuticals.

The inventors of the present invention have studied intensively on a model that can replace animal experiments in experiments for the development of drugs for inflammation and allergy tests, or drugs for treating inflammation and allergies, such as cosmetics or pharmaceuticals. A co-culture system of artificial tissues 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) Manufacturing step of the upper chamber of the transwell containing a three-dimensional artificial tissue comprising:

(i) Suspension of fibroblasts in type I collagen mixture:

(ii) adding the suspension to the upper chamber of the transwell to solidify, and then adding a culture medium to culture to form a multilayered cell proliferation product;

(iii) coating the upper surface of the multilayered cell growth product obtained by culturing with a mixture of type IV collagen and laminin; And

(iv) culturing after inoculating epithelial cells on the surface of the coated multilayer cell proliferation product,

(2) Preparation step of the lower chamber of the transwell containing the 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 a transwell culture medium, and

(3) Place the upper chamber of the transwell containing the three-dimensional artificial tissue prepared in step (1) at the upper end of the lower chamber of the transwell containing the monitoring cells prepared in step (2), and place the culture medium on the upper side. Filling the chamber lower portion to the upper portion of the three-dimensional artificial tissue to prepare a co-culture system of the three-dimensional artificial tissue and monitoring cells.

In the above production method, the fibroblasts are skin fibroblasts or corneal fibroblasts, preferably, the skin fibroblasts are human-derived fibroblasts, and the corneal fibroblasts are corneal fibroblasts derived from rabbit cornea.

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

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

In the above manufacturing method, as another embodiment for manufacturing a co-culture system for evaluating the inflammatory response, after the step (3), a step of converting macrophages into dermal macrophages by treatment with LPS (lipopolysaccharide) is added. Can be included as.

The transwell plate used in the present invention is widely used in the art, and a commercially available product may be used, and representatively, a product commercially available from Corning Incorporated may be used. Detailed product information can be obtained from the product catalog or website (www.corning.com) provided by Corning Incorporated. It is within the scope of the skill level.

As exemplarily shown in FIG. 10, the transwell plate includes an upper chamber (also referred to as a product catalog or transwell insert in the art) and a medium containing a lower chamber made of a microporous membrane at the lower end of the transwell plate. ), and 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 secretion material, a support that permeates the culture medium but cannot permeate the cells, and the size of the pores of the microporous membrane can be appropriately adopted by those skilled in the art. For example, 0.4 µm to 0.8 Μm.

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

The type I collagen mixture of step (i) is a mixture of an appropriate medium for cell culture and type I collagen, for example, type I collagen (Nitta gelatin), 10-fold concentration of DMEM (Gibco), and reconstitution buffer (2.2 g). A buffer solution in which NaHCO3 and 4.77 g HEPES are dissolved in 100 ml of a 0.05 N NaOH solution) can be prepared by mixing in a ratio of 8:1:1.

The culture medium used in the preparation method is a medium commonly used in the art for cell culture, and the composition of the medium can be modified by a person skilled in the art depending on the type and characteristics of cells, which is within the skill level of those skilled in the art. . For example, a mixed medium of DMEN and F12, a DMEN medium containing 10% FBS, and the like may be used for the implementation of the present invention.

The present invention also provides a co-culture system of three-dimensional artificial tissue and monitoring cells manufactured by the above-described manufacturing method.

The co-culture system of the three-dimensional artificial tissue and monitoring cells of the present invention is not a kind of device or a simple mechanical device, as illustrated exemplarily in FIG. 3, and therefore, for a more appropriate expression, the system is referred to herein as a'system'. Refers to. However, it can be interpreted as a device if it does not impair the essence of the present invention.

The co-culture system of 3D artificial tissue and monitoring cells of the present invention contains a transwell upper chamber containing a three-dimensional artificial tissue, a transwell lower chamber containing a monitoring cell, and a culture medium, wherein the transwell upper chamber is a transwell chamber. Located in the lower chamber of the well, the culture medium is filled from the lower part of the upper chamber to the upper part of the 3D artificial tissue, preferably to the lower part of the upper chamber.

The 3D artificial tissue in the upper chamber of the transwell is formed by the method including steps (i) to (iv), and is a 3D artificial skin tissue or a 3D artificial corneal tissue.

The monitoring cells in the lower chamber of the transwell are macrophages or mast cells, and macrophages are preferable in the case of a co-culture system for an inflammatory response model, and mast cells are preferable in the case of a co-culture system for an allergic reaction model.

Until now, the skin model is a model in which only structural similarity including the epidermal layer and the dermal layer has been secured, and there has been no functional skin model capable of performing the primary defense mechanism. Is a functional skin model containing dermal macrophages and can be used as an evaluation system most similar to biological functions.

Therefore, the co-cultivation system of the three-dimensional artificial tissue and monitoring cells of the present invention can replace the animal test in the test for inflammation and allergy tests, such as cosmetics or pharmaceuticals, or the development of drugs 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 three-dimensional artificial tissue and monitoring cells described above.

The method for evaluating the inflammatory reaction or allergic reaction of the test substance of the present invention includes the step of applying the test substance to the surface of the above-described three-dimensional artificial tissue. FIG. 11 exemplarily shows the application of the test substance to the co-culture system of the present invention to test for inflammation or allergic reactions.

In the present invention, the test substance is any candidate substance for evaluating an inflammatory reaction or allergic reaction to the eye or skin, and is a candidate compound, natural product, extract, for cosmetics or pharmaceuticals for direct or indirect application to the eye or skin. Or it may be a mixture thereof. In addition, the test substance may be a candidate substance for the treatment of an inflammatory reaction or an allergic reaction.

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

The evaluation method of the test substance of the present invention includes (a) the level of formation of vacuoles in the monitoring cells in the cell culture fluid, (b) the level of production of IL-6, IL-10, or both secreted in the cell culture fluid, or (c) The inflammatory response of the test substance can be evaluated by increasing one or more indicators selected from the production levels of iNOS, Cox-2, or both secreted in the cell culture fluid.

The evaluation method of the test substance of the present invention can evaluate the allergic reaction of the test substance through an increase in the activity level of β-hexosaminidase in the cell culture solution.

Currently, as the frequency of inflammatory and allergic diseases increases, research is actively being conducted to search for candidates for treatment and prevention, but the development of a reliable evaluation model for accurate evaluation must be preceded, and in particular, experimental animals with variations between individuals. In order to apply the model to all candidate materials, high cost is required. Therefore, according to the co-culture system of dimensional artificial tissue and monitoring cells of the present invention, large-scale screening with high reproducibility of inflammation and allergic reactions can be made possible by replacing animal experiments conducted for the evaluation of inflammation and allergy of existing cosmetic ingredients. In addition, through this, standardized indicators for the search for therapeutic agents and preventive substances for inflammatory and allergic diseases can be prepared.

The co-cultivation system of the three-dimensional artificial tissue and monitoring cells of the present invention exhibits functional similarity to the biological tissue, and therefore, in the test for the development of drugs for the treatment of inflammation and allergy, such as cosmetics or pharmaceuticals, and animals It becomes the optimal test model that can replace the experiment.

FIG. 1 is a photograph comparing three-dimensional artificial skin tissue, corneal tissue, and living tissue after H&E staining ((A) normal rabbit corneal tissue, (B) artificial corneal tissue of a three-dimensional rabbit, (C) normal Human skin tissue, (D) 3D human artificial skin tissue)
Figure 2 is a photograph showing the results of immunohistochemical staining of 3D artificial skin tissue and corneal tissue (3D artificial skin tissue: A ~ C, 3D artificial corneal tissue: D ~ F; (A) Keratin 5, (B ) Keratin 10, (C) Keratin 3+12, (D) Keratin 5, (E) Keratin 10, (F) Keratin 3/12 Immunostaining photo)
3 is a diagram showing an exemplary structure of a co-culture system of a three-dimensional artificial tissue and a monitoring cell.
4 is a photograph showing the change in the morphology 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)
5 is a graph showing the results of analysis of cytokines (IL-6, IL-10, TNF-α) of RAW264.7 cell lines in an inflammation model.
6 is a gel photograph showing the results of confirming the inflammatory response (iNOS, Cox-2 level) of the RAW264.7 cell line in the inflammatory model.
7 is a gel photograph confirming the characteristics of the dermal macrophage layer of the RAW264.7 cell line in the inflammation model.
8 is a diagram showing the functional similarity of the co-culture system of the present invention with living body skin.
9 is a result of confirming the allergic reactivity induced by DNP-IgE and DNP-BSA in an allergy model.
10 is a diagram showing the structure of a transwell plate.
11 is a diagram illustrating an exemplary application of a test substance to the co-culture system of the present invention in order to test an inflammatory or allergic reaction.

Hereinafter, the present invention will be described in more detail through Preparation Examples, Examples and Test Examples. However, these Preparation 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 3D artificial skin tissue Transwell Top Of the chamber (insert) Produce

A three-dimensional culture method was used in which epidermal cells were inoculated and cultured on the dermal layer made by mixing type 1 collagen with skin fibroblasts. Epidermal cells were cultured in the middle of the air layer and the water layer to differentiate into a middle-layer squamous epithelium and keratinize to form a substantial skin structure.

As a dermal counterpart, the collagen mixture was 8:1 of type I collagen (Nitta gelatin), 10-fold concentration of DMEM (Gibco), and reconstitution buffer (a buffer in which 2.2 g NaHCO3 and 4.77 g HEPES were dissolved in 100 ml of 0.05 N NaOH solution): It was prepared by mixing in a ratio of 1. In order to prevent gelation of collagen, the preparation of the dermal counterpart was carried out at 4°C. 12mm diameter transwell insert

(Transwell; Corning, NY) was prepared. Skin fibroblasts (primarily cultured from human skin tissue) were recovered from the culture dish ^{and suspended in a collagen mixture at a concentration of 1 x 10 6} cells/ml, taking care not to generate air bubbles. 200 μl of the collagen mixture was placed inside the transwell insert, and the collagen mixture was solidified for 1 hour in an incubator at 37°C. A medium in which DMEM:F12 was mixed at 3:1 in the inside and outside of the transwell was adjusted to the water level inside and outside, and cultured for 24 hours. After the dermal counterpart was formed, the medium inside the transwell was removed, and the upper part of the dermal 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 by reacting with 50 μl of a mixture of 0.1 mg/ml of collagen and 30 μg/ml of laminin for 1 hour at 37°C. After removing the coating solution, it was washed 3 times with PBS buffer.

Keratinocytes (primary culture from human skin tissue) were trypsinized, collected from a culture dish, counted, centrifuged, and inoculated at a concentration of ^{5×10 5 cells/well on the coated collagen dermis.} The medium was exchanged every 2-3 days and cultured for 7 days. When the keratinocytes grow evenly on the upper part of the dermal counterpart, remove the medium on the epidermal layer so that the epidermal layer can be cultured on the contact surface of water and air, adjust the height of the dermal counterpart, put the medium outside the transwell insert, and air-dry for 14 days Cultured in the state.

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

A three-dimensional culture method was used in which rabbit cornea-derived epithelial cells (first culture) were inoculated and cultured on the dermal layer made by mixing type 1 collagen with corneal fibroblasts (primary culture from human skin tissue) in the same manner as in Preparation Example 1. . Unlike skin keratinocytes, corneal epithelial cells were maintained in a semi-dry culture so that they did not differentiate and form keratin.

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

(1) H&E dyeing

H&E staining was performed as follows. The tissue accumulated in paraffin was sectioned into 5 μm and attached to the slide, and then the slide was reacted with xylene for 10 minutes to remove paraffin around the tissue. The tissues were hydrated and washed with 100%, 90%, 80%, and 70% ethanol sequentially for 2 minutes each. The nuclei were stained by treatment with hematoxylin (ResGen) for 5 minutes, and the slides were washed with water for 10 minutes, followed by sequential treatment with 70%, 80%, and 90% ethanol for 2 minutes to dehydrate the tissue and eosin (Eosin). , Sigma) was treated for 5 minutes to stain the cytoplasm, washed in 95% ethanol, and then treated with xylene for 2 minutes through 100% ethanol. A drop of a mounting agent (Shandon Synthetic Mountant, Thermo) was added to the tissue part of the slide, and the slide cover was attached to mount it.

The results are shown in FIG. 1. As a result of performing H&E staining of the skin and cornea cultured in the transwell insert, it was confirmed that it showed structural similarity when compared with the structure in which the normal human skin tissue and the rabbit corneal tissue were reconstituted in three dimensions (Fig. 1, (A) normal rabbit corneal tissue, (B) three-dimensionally constructed rabbit artificial corneal tissue, (C) normal human skin tissue, (D) three-dimensionally constructed human artificial skin tissue).

(2) immunohistochemical staining

Immunohistochemical staining was performed as follows. Paraffin was removed from the paraffin cut (5 μm) and hydrated prior to immunohistochemical staining. Staining was performed according to the method provided using ABC kit (Vector Laboratory, CA) and DAB kit (Vector Laboratory, CA). The cut product was counter-stained with Fast Red (Vector Laboratories Inc). The reaction was carried out at the recommended concentrations of the antibody manufacturer against keratin 5, keratin 10, and cytokeratin 3/12 (abCAM).

The results are shown in FIG. 2. As a result of immunohistochemical staining, it was confirmed that keratin 5 expressed in the dividing epithelial cells around the basal layer was strongly expressed in both the 3D artificial skin tissue and the 3D artificial corneal tissue. In particular, it was confirmed that keratin 10, which is expressed only in differentiated skin keratinocytes, is site-specifically expressed in the differentiated stratum corneum of the three-dimensional artificial skin tissue, but not in the three-dimensional artificial corneal tissue. In addition, it was confirmed that keratin 3/12 specifically expressed in the cornea was not expressed in a three-dimensional artificial skin tissue, but was expressed only in a three-dimensional artificial corneal tissue. This confirms that the reconstructed models of Preparation Examples 1 and 2 have actual skin and corneal characteristics.

< Manufacturing example 4> monitoring Cell line selection and culture

(1) inflammation model monitoring Cell line selection

As the inflammation model monitoring cell line, a mouse macrophage cell line, RAW264.7 cell line, was selected. The RAW264.7 cell line, a mouse macrophage cell line, was cultured in a culture solution containing 10% FBS in DMEM, and the culture solution was exchanged once every 2-3 days. When the cells became confluent, they were reacted in 0.25% trypsin-EDTA for 10 minutes, separated into single cells, and subcultured at 1:20.

(2) allergy model monitoring Cell line selection

As the allergy model monitoring cell line, the RBL-2H3 cell line, a mouse mast cell line, was selected. The mouse mast cell line, RBL-2H3 cell line, was cultured in a culture solution containing 10% FBS in DMEM, and the culture solution was exchanged every 2-3 days. When the cells became confluent, they were reacted in 0.25% trypsin-EDTA for 10 minutes, separated into single cells, and subcultured at 1:20.

< Manufacturing example 5> monitoring Cell line analysis method

(One) ELSIA law Cytokine analysis using (IL-6, IL-10, TNF alpha)

Cytokines secreted into the cell culture fluid were quantified by ELISA (R&D systems, Cat no DY406, DY417, DY410).

First, a capturing antibody (IL-6, R&D systems, 840171; IL-10, R&D systems, 840125; TNF alpha, R&D systems, 840143) was added to a 96-well plate and allowed to react for 1 day at room temperature, followed by washing buffer 3 times. Washed. Here, 1% BSA solution was blocked for 1 hour to prepare a culture solution and a reaction. After adding 100 μl of each culture solution, it was reacted for 2 hours. After the reaction for 2 hours, after washing three times with a washing buffer, it was reacted with 100 μl of detection antibodies (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 a washing buffer, 100 μl of streptavidin-HRP was added and reacted for 20 minutes. After the reaction, the reaction was washed three times with a washing buffer, and the same amount of reagents A and B were mixed, 100 μl each, color development, 10 nM NaOH was added, and the reaction was stopped, and the measured value at 450 nM was quantified according to the standard value.

(2) β- Hexosaminidase Assay (β- hexosaminidase Allergic action evaluation through assay)

Using the mouse mast cell RBL-2H3 cell line, the degree of measurement of the activity of β-hexosaminidase secreted into the culture medium through the degranulation of mast cells induced by Anti-DNP-IgE was evaluated as an allergic reaction. After culturing 5x10 ⁵ rat RBL-2H3 cells in a 12-well plate, about 3 hours later, DNP-IgE, an inflammatory substance, was treated with 722 ng/ml, and after 16 hours, twice with phosphate-buffered saline (PBS). After washing, the cell culture solution was replaced with EBSS containing 1% BSA. Thereafter, DNP-BSA 150 ng/ml was treated for 30 minutes to induce degranulation, and only the cell culture solution was taken separately to measure the activity of β-hexosaminidase. Fluorescence was measured at 405 nm (Passante et al. 2009).

(3) Western Blotting Used iNOS And Cox-2 level assessment

For Western blotting, iNOS and Cox-2 (Cell Signaling Technology, cat No 2977, 4842), CD 163 (Abcam, ab111250) were 10%, bcl-xl (Cell Signaling Technology, cat No 2764) and IL- 10 (R&D systems, cat no MAB519), Arginases I (Santa Cruz, cat No sc-18354) were transferred to nitrocellulose paper after performing 15% SDS-PAGE. The transferred membrane was blocked for 1 hour in 0.1% TBST solution containing 5% non-fat dry milk, and then incubated with the corresponding antibody for 2 hours.

After washing three times for 10 minutes with 0.1% TBST solution, it was reacted with the secondary antibody for 1 hour. It was analyzed by reacting with millipore's ECL solution (Chemilucent ECL Detection System cat No 2600) washed three times for 10 minutes with 0.1% TBST solution.

< Manufacturing example 6> 3D artificial organization monitoring Preparation of cell line co-culture system

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

The lower chamber of the 12-well transwell in which the 3D artificial skin tissue and the 3D artificial corneal tissue produced from the insert of the 12-well transwell were ^{inoculated 4 hours ago with 2x10 5 cells in DMEM containing 10% FBS in each RAW264.7 cell line.} It was placed inside and cultured. DMEM containing 50 μl of 10% FBS was added to each of the 3D artificial skin tissue and the 3D artificial corneal tissue, which had been cultured in a semi-dry state.

The co-culture system of the 3D artificial tissue and the monitoring cell line thus prepared shows the structure as shown in FIG. 3.

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

The three-dimensional artificial skin tissue and artificial corneal tissue produced in a 12-well transwell insert were each ^{inoculated with 2x10 5} cells in DMEM containing 10% FBS for 4 hours before the RBL-2H3 cell line was placed inside the lower chamber of the 12w well transwell. Placed and incubated. DMEM containing 50 μl of 10% FBS was added to each of the 3D artificial skin tissue and the artificial corneal tissue, which had been cultured in a semi-dry state.

The co-culture system of the 3D artificial tissue and the monitoring cell line thus prepared shows the structure as shown in FIG. 3.

The skin is a major organ that senses various stimuli and transmits it to the nervous system, and it can be said to be an important part of the human body that interacts with the external environment. Mast cells, which are widely distributed in connective tissues, are mainly involved in the pathophysiology of skin diseases such as urticaria, psoriasis, atopic dermatitis, and wound healing. These mast cells are activated immunologically or non-immunologically to promote the secretion of mediators such as histamine, infiltrating many inflammatory cells in the activated site of mast cells, causing vasodilation, swelling, and tissue damage. Currently, most allergic reactions are experiments using animals, so a co-culture system for allergic reaction models was established in order to establish an in vitro test method under similar conditions to the living body while replacing the animal test.

[ Example ]

< Example 1> 3D artificial organization monitoring Confirmation of inflammatory response of cell line co-culture system

To confirm the inflammatory response of the co-culture system of the 3D artificial tissue and the monitoring cell line prepared in Preparation Example 6, LPS (Sigma, cat No L6893) was added to each of the 3D artificial skin tissue and the artificial corneal tissue, respectively, at 05 μg/ml. Treatment at the concentration of for 24 hours induces an inflammatory reaction.

(1) Observation of morphological changes

The effect of the immune activity of the RAW264.7 cell line induced by LPS was first confirmed by the change in the morphology of the cell. After LPS treatment, the shape of the RAW264.7 cell line in the cell culture medium of the lower chamber of the transwell is shown in Fig. 4 (Fig. 4, +3D skin: co-culture system of 3D artificial skin tissue and monitoring cells; +3D cornea: 3 Dimensional artificial corneal tissue and monitoring cell co-culture system) It was confirmed that the formation of vacuoles indicating phagocytosis, which is used as an indicator of activation of macrophages, is increased in the cytoplasm of the RAW264.7 cell line by treatment with LPS. Became.

(2) Identification of secreted cytokines in cell culture fluid

In the co-culture system, IL-6, IL-10, and TNF-alpha secreted in the cell culture fluid of the RAW264.7 cell line were quantified by ELISA as described in Preparation Example 5 to evaluate the immune activity effect. The results are shown in FIG. 5. As a result, it was confirmed that IL-6 and IL-10 were secreted by LPS stimulation in the same manner as in the RAW 264.7 cell line. However, it was found that TNF-alpha was increased in the 3D artificial tissue model, so it did not respond to the stimulation of LPS.

(3) iNOS And confirmation of inflammatory reactivity through Cox-2

By the method described in Preparation Example 5, the RAW264.7 cell line was immunologically activated in the co-culture system, and the expression levels of iNOS and Cox-2 were measured. The results are shown in FIG. 6. As a result, it was confirmed that the expression levels of iNOS and Cox-2 decreased when co-cultured with 3D artificial tissues.

Inflammation is a defense action of the host that is rapidly activated through various mechanisms of action to protect against physical action, chemical substance, bacterial infection, etc. when any organic change is applied to a living body or tissue. 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 biological defense mechanism of the skin acts even in the co-culture system adopting a three-dimensional artificial tissue model to reduce the degree of inflammatory reaction, and this means that the co-culture system of the present invention works as a mechanism close to the actual living body model, and thus, It means that it can be provided as an experimental model similar to a living body model.

(4) With LPS Activated RAW264 .7 Characterization of cell lines

By the method described in Preparation Example 5, after immunological activity of the RAW264.7 cell line in the co-culture system, the expression levels of Arg1, iNOS, and IL-10 were measured. The results are shown in FIG. 7.

As shown in FIG. 7, it was confirmed that the RAW264.7 cell line, which has undergone immunological activity with LPS, expresses iNOS that mediates the inflammatory response, but simultaneously induces the expression of Arginase I and IL-10, which can induce an anti-inflammatory response at the same time. .

These cytokine changes confirmed in the co-culture system confirmed that RAW264.7 macrophages were converted into skin-specific dermal-type macrophages by the immune activation action by LPS.

In fact, the skin is not only the main organ that senses various stimuli and transmits it to the nervous system, but is also an internal immune organ that is responsible for primary protection from external stimuli and harmful substances. For this, it includes cells that neutralize the skin's own defense mechanisms and immune-inducing substances that invade from the outside. The cells responsible for its representative function are dendritic cells and dermal macrophages derived from bone marrow, and in human skin, dendritic cells are mainly Langerhans cells in the epidermis, and the other is the dermal macrophages in the dermis. It is a phagocyte.

Currently, artificial skin model technology consists of only the epidermal layer consisting of stratum corneum and the dermal layer, which is connective tissue, so that structurally similar skin layers can be reconstructed, but there is a limitation in that functional reconstruction is impossible.

In this experiment, as a result of analyzing the characteristics of the RAW 264.7 cell line immune-activated by LPS by Western blotting, it was confirmed that the change was similar to that of dermal macrophages.

In establishing a skin model as the primary intrinsic immune organ, co-culturing the RAW 264.7 cell line or investigating the inflammatory response of the sample in the state of immunity activation with LPS is not considered in the conventional skin model. It means that it is the best model that can replace animal experiments by considering the immune response. Therefore, the LPS-treated RAW264.7 cell line becomes a model that can replace the dermal macrophage layer.

That is, it was confirmed that the co-culture system of the present invention exhibits functional similarity with living body skin, and FIG. 8 is attached to aid in understanding the functional similarity.

< Example 2> 3D artificial organization monitoring Confirmation of allergic reaction of cell line co-culture system

In order to confirm the allergic reaction of the co-culture system of the 3D artificial tissue and the monitoring cell line prepared in Preparation Example 6, DNP-IgE was added to each of the 3D artificial skin tissue and the artificial corneal tissue at a concentration of 722 ng/ml for 16 hours. After treatment, DNPBSA was treated at 150 ng/ml for 30 minutes to induce degranulation of mast cells, and the activity of β-hexosaminidase released by degranulation was investigated as described in Preparation Example 5. The results are shown in FIG. 9. As a result, the activity of β-hexosaminidase identified in RBL-2H3 was observed in the co-culture system employing 3D artificial skin tissue, but no allergic reaction was induced in the co-culture system adopting 3D artificial corneal skin tissue.

Claims (14)

A method of manufacturing a co-culture system of three-dimensional artificial tissue and monitoring cells comprising:
(1) Manufacturing step of the upper chamber of the transwell containing a three-dimensional artificial tissue comprising:
(i) suspending skin fibroblasts or corneal fibroblasts in a type I collagen mixture,
(ii) adding the suspension to the upper chamber of the transwell to solidify, and then adding a culture medium to culture to form a multilayered cell proliferation product,
(iii) coating the upper surface of the multilayered cell growth product obtained by culturing with a mixture of type IV collagen and laminin, and
(iv) culturing after inoculating epithelial cells on the surface of the coated multilayer cell proliferation product;
(2) Preparation step of the lower chamber of the transwell containing the 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 a transwell culture medium;
(3) Place the upper chamber of the transwell containing the three-dimensional artificial tissue prepared in step (1) at the upper end of the lower chamber of the transwell containing macrophages or mast cells prepared in step (2), and culture Filling the medium from the lower portion of the upper chamber to the upper portion of the three-dimensional artificial tissue to prepare a co-culture system of the three-dimensional artificial tissue and monitoring cells. The method of claim 1, wherein the skin fibroblasts are human-derived fibroblasts, and the corneal fibroblasts are rabbit corneal-derived corneal fibroblasts. The method of claim 1, wherein the epithelial cells are keratinocytes or corneal-derived epithelial cells. The method of claim 1, wherein the macrophage is a mouse macrophage cell line RAW264.7 cell line. The method according to claim 4, wherein after the step (3), the macrophages are converted into dermal macrophages by LPS (lipopolysaccharide) treatment. The method of claim 1, wherein the mast cells are RBL-2H3 cell lines which are murine mast cell lines. The method of claim 1, wherein the bottom surface of the upper chamber is composed of a microporous membrane. As a co-culture system of macrophages or mast cells, and three-dimensional artificial tissues,
A lower chamber containing macrophages or mast cells in a transwell culture medium;
Transwell upper chamber comprising a multi-layered cell proliferative layer formed by solidifying a type 1 collagen mixture and a fibroblast suspension, a coating layer formed by coating a mixture of type IV collagen and laminin on the multi-layered cell proliferation, and an epithelial cell layer over the coating ; And
It is composed of culture medium,
The lower chamber and the transwell upper chamber are connected to the lower chamber with 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 upper chamber to the upper end of the epithelial cell. Cells or mast cells, and a co-culture system of a three-dimensional artificial tissue. The system of claim 8, wherein the co-culture system of macrophages or mast cells, and three-dimensional artificial tissues for evaluating inflammatory or allergic reactions. A method for evaluating an inflammatory reaction or allergic reaction of a test substance comprising the step of applying the test substance to the surface of the epithelial cell layer in the upper chamber of the transwell of the macrophage or mast cell of claim 8, and a three-dimensional artificial tissue co-culture system. The method of claim 10, wherein the test substance is evaluated for inflammatory or allergic reactions to the cornea or skin. The method of claim 10, wherein the inflammatory response of the test substance is evaluated by increasing one or more of the following indicators:
(a) the level of formation of vacuoles in macrophages or mast cells in the cell culture solution,
(b) the level of production of IL-6, IL-10, or both secreted in the cell culture fluid, or
(c) The production level of iNOS, Cox-2, or both secreted in the cell culture fluid. The method of claim 10, wherein the allergic reaction of the test substance is evaluated by increasing the activity level of β-hexosaminidase in the cell culture solution. The method of claim 11, wherein the test substance is a candidate substance for treatment of inflammation or allergy.

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