WO2021029567A1 - Bioadhesive-pretreated apparatus for culturing and testing three-dimensional cellular construct - Google Patents

Bioadhesive-pretreated apparatus for culturing and testing three-dimensional cellular construct Download PDF

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WO2021029567A1
WO2021029567A1 PCT/KR2020/009873 KR2020009873W WO2021029567A1 WO 2021029567 A1 WO2021029567 A1 WO 2021029567A1 KR 2020009873 W KR2020009873 W KR 2020009873W WO 2021029567 A1 WO2021029567 A1 WO 2021029567A1
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adhesive
bio
culture
dimensional cell
tissue
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French (fr)
Korean (ko)
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김미정
장일호
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주식회사 티앤알바이오팹
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins

Definitions

  • the present invention relates to an apparatus for culturing and testing a 3D cell tissue body pretreated with a bio-adhesive, and prevents contraction of a 3D cell culture tissue that occurs when culturing a 3D cell culture tissue in a culture structure such as a transwell insert.
  • a culture and test apparatus that improves reliability when using the cultured three-dimensional cell culture tissue in the field of transplantation, drug efficacy and toxicity evaluation. will be.
  • Cell culture is a technology that collects cells from a living body and cultivates them outside the body.
  • the technology that applies to the efficacy evaluation and toxicity evaluation of drugs or cosmetics using cultured cells is expanding widely.
  • Interest in cell culture as it has been found that it can be used for the treatment of various diseases by differentiating into various tissues of the body such as organs and nerves and transplanting them into the human body or by transplanting them into the human body before differentiation to achieve engraftment and differentiation simultaneously And research is increasing.
  • Transwell is composed of a transwell insert with a microporous membrane at the lower end and a lower chamber that accommodates the transwell insert, and a transwell containing a three-dimensional cell culture structure in which natural polymers and cells are mixed in a lower chamber filled with a liquid medium.
  • a liquid medium is shared by a microporous membrane, thereby culturing cells in a three-dimensional cell culture body.
  • the liquid medium can be easily exchanged by separating the lower chamber and the transwell insert, and cell culture efficiency is high.
  • the present invention is intended to provide a technology for preventing the contraction of the 3D cell cultured tissue that occurs when the 3D cell cultured tissue is cultivated in a support such as a transwell insert.
  • One embodiment of the present invention for achieving the object as described above relates to an apparatus for culturing and testing a three-dimensional cell tissue body pretreated with a bio-adhesive, comprising: a culture structure; A three-dimensional cell culture structure disposed inside the culture structure; And a bio-adhesive disposed at at least a partial interface between the culture structure and the three-dimensional cell culture tissue.
  • the culture structure preferably includes a body, a hole penetrating the body, and a porous membrane formed to cover one end of the hole.
  • the bio-adhesive may include at least one or more of mussel adhesive protein, fibrin glue, gelatin glue, cyanoacrylate-based bio-adhesive, and polyurethane-based bio-adhesive, and preferably, the bio-adhesive is mussel adhesive protein. .
  • the bio-adhesive is disposed between the three-dimensional cell culture structure and the porous membrane of the culture structure.
  • the bioadhesive is disposed between the 3D cell culture structure and the porous membrane, and extends to the side of the 3D cell culture structure along the inner surface of the hole penetrating the body.
  • the lower surface of the 3D cell culture tissue is attached to the porous membrane by a bio-adhesive, and at least some side surfaces or the entire side of the 3D cell culture tissue are attached to the inner side of the hole by the bio-adhesive.
  • the three-dimensional cell culture tissue may be in the form of a gel in which cells or tissues are dispersed in a natural polymer.
  • the cells are stem cells, osteoblasts, myoblasts, tenocytes, neuroblasts, fibroblasts, glioblasts, germ cells ( germcell), hepatocyte, renal cell, sertoli cell, chondrocyte, epithelial cell, cardiovascular cell, keratinocyte, smooth muscle cell ), cardiomyocytes, glial cells, endothelial cells, hormone secreting cells, immune cells, pancreatic islet cells, and neurons selected from the group consisting of It is desirable.
  • the three-dimensional cell culture tissue may include two or more types of cells, but two or more types of cells may be uniformly dispersed, or two or more types of cells may be separated from each other to form a hierarchical structure.
  • the hierarchical structure may be a hierarchical structure having two layers of a dermal layer and an epidermal layer.
  • the porous membrane is preferably a microporous membrane through which the liquid medium can permeate, and is preferably attached to the body through an adhesive or adhesive tape, and the peripheral portion of the porous membrane is fixed between the cover and the body by the cover. desirable.
  • a method for culturing a three-dimensional cell tissue body pretreated with a bio-adhesive including: a body; A hole through the body; And a porous membrane formed to cover one end of the hole; applying a bio-adhesive to the inside of the culture structure including a bio-adhesive, drying the bio-adhesive, and three-dimensional so that at least a portion of the bio-adhesive contacts the bio-adhesive And a three-dimensional cell culture tissue arrangement step of disposing the cell culture tissue body inside the culture structure.
  • the bio-adhesive used at this time includes at least one or more of mussel adhesive protein, fibrin glue, gelatin glue, cyanoacrylate-based bio-adhesive, and polyurethane-based bio-adhesive.
  • bio-adhesive may be disposed between the 3D cell culture structure and the porous membrane, and may extend to the side of the 3D cell culture structure along the inner side of the hole penetrating the body.
  • the lower surface of the 3D cell culture tissue may be attached to the porous membrane by a bio-adhesive, and at least some side surfaces or the entire side of the 3D cell culture tissue may be attached to the inner side of the hole by the bio-adhesive.
  • the three-dimensional cell culture tissue is preferably in the form of a gel in which cells or tissues are dispersed in a natural polymer, and may include one or two or more types of cells.
  • two or more types of cells may be uniformly dispersed, or two or more types of cells may be separated from each other to form a hierarchical structure.
  • the hierarchical structure may be a hierarchical structure having two layers of a dermal layer and an epidermal layer.
  • the apparatus for culturing and testing a 3D cell tissue body pretreated with the bioadhesive of the present invention prevents the change in the shape and structure of the 3D cell culture tissue by preventing contraction of the 3D cell culture tissue cultured in a support such as a transwell insert. Therefore, it can be used to minimize errors between test subjects and improve the reliability of test results when evaluating efficacy and toxicity of drugs or cosmetics.
  • FIG. 1 is a cross-sectional view schematically showing an apparatus for culturing and testing a 3D cell tissue body pretreated with a bio-adhesive according to an embodiment of the present invention.
  • FIG. 2 shows a method of applying a bio-adhesive to a culture and test apparatus of a three-dimensional cell tissue body pretreated with a bio-adhesive of the present invention.
  • FIG. 3 is a cross-sectional view schematically showing an apparatus for culturing and testing a 3D cell tissue body pretreated with a bio-adhesive according to an embodiment of the present invention.
  • test device 100 culture structure
  • membrane 200 bio adhesive
  • the present invention relates to a technology for preventing contraction of a 3D cell cultured tissue that occurs when a 3D cell cultured tissue is cultivated in a culture structure such as a transwell insert.
  • FIG. 1 is a diagram illustrating a culture and testing apparatus 10 of a three-dimensional cell tissue body pretreated with a bio-adhesive according to an embodiment of the present invention.
  • the culture and testing apparatus 10 is Structure 100; A three-dimensional cell culture structure 300 disposed inside the culture structure 100; And a bio-adhesive 200 disposed on at least a partial interface between the culture structure 100 and the 3D cell culture tissue 300.
  • the culture structure 100 As the culture structure 100, a transwell insert or an upper chamber used in a transwell used in the technical field to which the present invention pertains may be used, and the culture structure 100 includes a hole 120 passing through the body 110 and It is preferable to include a porous membrane 130 formed to cover one end of the hole.
  • transwell Commercially available products may be used as the transwell, and for example, products of Corning incorporated may be used.
  • a culture structure having a detachable structure that can be adjusted in height as disclosed in Registration Patent No. 1981053 (registered on May 16, 2019) may be used, but is not limited thereto.
  • the transwell is a culture structure 100; And a lower chamber accommodating the culture structure 100 and the medium, wherein the culture structure 100 is accommodated in the lower chamber, and the medium accommodated in the lower chamber by the porous membrane 130 of the culture structure 100 is By sharing, cell differentiation and proliferation of the three-dimensional cell culture tissue 300 in the culture structure 100 may be achieved.
  • the porous membrane 130 may be a microporous membrane through which the liquid medium is permeable, and the size of the pores is not particularly limited, but may preferably be a microporous membrane in which microscopic pores are formed.
  • the porous membrane 130 may be attached to the body 110 through an adhesive, an adhesive tape, or the like, or the circumference of the porous membrane 130 is fixed between the cover and the body 100 by means such as a cover. It can also be attached.
  • the 3D cell culture structure 300 is generally the upper surface of the porous membrane 130 and the lower inner surface of the hole 120. Attached to and cultured.
  • test bodies there is a problem that a large error between test bodies is generated when performing transplantation using the cultured 3D cell culture body 300 or when evaluating the efficacy, stimulation, and toxicity of drugs or cosmetics.
  • the culture structure 100 and the 3D cell culture structure 300 are arranged by disposing the bio-adhesive 200 on at least some interfaces between the culture structure 100 and the 3D cell culture tissue 300. ) To prevent contraction of the 3D cell culture tissue body 300 by improving the adhesion between.
  • the bio-adhesive 200 used at this time may include at least one or more of mussel adhesive proteins, fibrin glue, gelatin glue, cyanoacrylate-based bio-adhesive, and polyurethane-based bio-adhesives that can be used for bonding biological tissues.
  • mussel adhesive protein may be used, fibrin glue has a weak adhesion problem, gelatin glue has excellent adhesion, but may be toxic by formalin used as a crosslinking agent, and cyanoacrylate-based or polyurethane-based bioadhesive This is because, in the case of a synthetic polymer adhesive such as, it may exhibit problems such as biotoxicity and side effects.
  • the mussel adhesion protein is a protein rich in L-dihydroxylalanine (DOPA), and exhibits strong adhesion to various surfaces such as hydrophilic surfaces, hydrophobic surfaces, and metal surfaces even in an environment with water, does not attack human cells, and immune response Since it does not cause harm to the human body, it is the most preferred material for the bio adhesive 200 used in the present invention.
  • DOPA L-dihydroxylalanine
  • the bioadhesive 200 may be disposed between the 3D cell culture tissue 300 and the porous membrane 130.
  • the lower surface of the 3D cell culture structure 300 is attached to the porous membrane 130 by the bio-adhesive 200, and the side surface is attached to the inner surface of the hole 120.
  • the bio-adhesive 200 is disposed between the three-dimensional cell culture body 300 and the porous membrane 130, and from there, the three-dimensional cell culture along the inner surface of the hole 120 penetrating the body 110 It may be disposed extending to the side of the tissue 300.
  • the lower surface of the 3D cell culture tissue 300 is attached to the porous membrane 130 by the bio-adhesive 200, and at least some side surfaces or the entire side of the hole 120 by the bio-adhesive 200 Can be attached to the inner side.
  • the bio-adhesive 200 may be applied to the culture structure 100 through an injection method, and dried for a predetermined time after application to be prepared.
  • the injection method may be performed manually by an operator, or may be performed by a floating or printing method.
  • a 3D inkjet printing method may be used, and if this method is used, the bio-adhesive 200 can be uniformly distributed, and the nozzle from which the bio-adhesive 200 is discharged does not contact the porous membrane 130. Therefore, there is an advantage of minimizing damage to the porous membrane 130.
  • the three-dimensional cell culture tissue 300 may be in the form of a gel in which cells are dispersed in a natural polymer, and the natural polymer is a natural polymer including collagen, fibrin gel, matrigel, alginate, gelatin, agarose, or at least two or more thereof. It may be a polymer mixture.
  • the type of cells that can be applied to the cells is not particularly limited, and may be animal cells, plant cells, or tissues thereof.
  • the cells are stem cells, osteoblasts, myoblasts, tenocytes, neuroblasts, fibroblasts, glioblasts, Germcells, hepatocytes, renal cells, serolicells, chondrocytes, epithelial cells, cardiovascular cells, keratinocytes, smooth muscle cells muscle cells), cardiomyocytes, glial cells, endothelial cells, hormone secreting cells, immune cells, pancreatic islet cells, and neurons selected from the group consisting of There can be more than one.
  • the three-dimensional cell culture tissue 300 may include only one type of cell or two or more types of cells. In the latter case, two or more types of cells may be uniformly distributed and included, or two or more types of cells may be included. Cells can be separated from each other to form a hierarchical structure.
  • the hierarchical structure refers to a tissue structure composed of at least two or more layers such as skin or respiratory organs. For example, the skin has a hierarchical structure having two layers of a dermal layer and an epidermal layer.
  • the apparatus 10 for culturing and testing a three-dimensional cell tissue body pretreated with a bio-adhesive exhibits a particularly superior effect when forming a skin structure using the same.
  • the remaining surfaces except for the upper surface of the three-dimensional cell culture body 300 composed of the epidermal layer and the remaining dermal layer forming the upper surface are strongly attached to the culture structure 100 by the bio-adhesive 200, so that the porous membrane ( 130) is supplied only to the dermal layer and invasion into the epidermal layer is prevented in advance, so that the epidermal layer on the upper surface can be stably cultured in the air layer.
  • the gap between the side surface of the three-dimensional cell culture structure 300 and the culture structure 100 is not formed due to strong adhesion, cell migration to the gap of the cells constituting the epidermal layer is prevented, so that the epidermal cells are only in a certain position. Can multiply.
  • another embodiment of the present invention relates to a method of manufacturing a 3D cell tissue culture and test apparatus 10 pretreated with a bio-adhesive, and the culture and test apparatus 10 is the same as described above, Description is omitted.
  • the manufacturing method of the apparatus 10 for culturing and testing a 3D cell tissue body pretreated with the bio-adhesive includes a body 110, a hole 120 penetrating the body 110, and one end of the hole 120
  • the step of applying the bio-adhesive is a step of applying the bio-adhesive 200 to the inner side of the culture structure 100, and may be applied on the porous membrane 130 through an injection method.
  • the application of the bio-adhesive 200 may be performed manually by an operator as shown in FIG. 2(a), or may be performed in a 3D printing method as shown in FIGS. 2(b) and 2(c).
  • a 3D inkjet printing method capable of uniformly applying the bio-adhesive 200 while minimizing damage to the porous membrane 130 may be used.
  • the drying step is a step performed to dry the bio-adhesive 200 applied to the culture structure 100, and the bio-adhesive 200 is transformed into a thin thin film through the drying step.
  • the bio-adhesive 200 may exist in the form of a thin film on the porous membrane 130 (FIG. 1), or the upper portion of the porous membrane 130, and extend from thereon.
  • the hole 120 may be present in a form that covers the inner side (FIG. 3).
  • an appropriate amount may be applied to the inner surface of the hole 120 of the porous membrane 130 and the culture structure 100 and dried to form the bioadhesive 200 in the shape as shown in FIG. 3.
  • a gel-like mixture of natural polymers and cells is injected into the culture structure 100 by hand or 3D printing, and cooled to room temperature to obtain a gel-like three-dimensional cell culture structure 300. ) Is placed in the culture structure 100.
  • the 3D cell culture tissue body 300 due to contraction of the 3D cell culture tissue body 300 Since a gap between the and the culture structure 100 does not occur, the shape and shape of the 3D cell culture structure 300 when performing transplantation, the efficacy of cosmetics and drugs, and toxicity evaluation using the cultured 3D cell culture structure 300 It is possible to minimize the occurrence of errors due to the structure, thereby improving the completeness of the implantation and the reliability of the evaluation results.
  • a device for culturing and testing a three-dimensional cell tissue body pretreated with a bio-adhesive according to an embodiment of the present invention is manufactured, and specific actions and effects of the present invention will be described through this.
  • this is presented as a preferred example of the present invention, and the scope of the present invention is not limited according to the embodiment.
  • the mussel adhesive protein was injected into the transwell insert using a 3D inkjet printing method and dried to prepare a transwell insert containing the dried mussel adhesive protein in the form of FIG. 3.
  • a gel in which collagen and fibroblasts are mixed is injected into the transwell insert, and a test device is prepared by inoculating keratinocytes on the upper surface, and then the lower part is in contact with the liquid medium, and the upper part is Cell culture was carried out in an air-liquid interface (ALI) method exposed to air.
  • ALI air-liquid interface
  • KGMTM Gold SingleQuots supp1ements As a liquid medium, KGMTM Gold SingleQuots supp1ements (LONZA) 7 kinds (Hydrocortisone 0.50 mL, Transferrin 0.50 mL, Epinephrine 0.25 mL, GA-1000 0.50 mL, BPE 2.00 mL, hEGF, 0.50 mL) in KBMTM Gold Basal Medium (LONZA) , Insulin 0.50 mL) was added, and Calcium chloride (Sigma-Aldrich), which is known to induce differentiation of keratinocytes, was added to a concentration of 1.8 mM.
  • Cell culture was carried out under conditions of C0 2 5%, 37°C, and 95% relative humidity, and was carried out by assembling two transwell inserts in a 100 mm Petri dish containing 15 mL of liquid medium, but the liquid medium was exchanged every 3 days. Became.
  • a test device was manufactured in the same manner as in Preparation Example except for the mussel adhesive protein injection step, and then cell culture was performed. At this time, the experiment was conducted by varying the concentration of the injected fibroblasts, and the keratinocytes were used at the same concentration of 5 ⁇ 10 5 cells/well in all samples, and 3, 4 and 7 days elapsed after the start of culture. The appearance of the three-dimensional cell culture tissue was photographed at the time point and shown in FIG. 4.
  • test device of Example was manufactured using the same method as in Preparation Example, and the test device of Comparative Example was manufactured using the same method as in Preparation Example except for the injection of mussel adhesive protein, and then cell culture was performed. After days and 10 days, the appearance of the three-dimensional cell culture tissue was photographed and shown in FIG. 5.
  • fibroblasts were injected at a concentration of 1xlO 6 cells/ml and keratinocytes at a concentration of 5 ⁇ 10 5 cells/well.
  • each test sample is fixed in 4% paraformaldehyde at 4°C for 24 hours, washed with PBS for 1 hour, and then separated from the insert, immersed in 30% sucrose, and allowed to stand until the tissue precipitates.
  • an OCT block was prepared using an OCT compound, quenched with liquid nitrogen, and then cryosectioned to prepare a tissue section.
  • tissue sections were stained using Hematoxylin and Eosin stain kit HAE-1 (ScyTek), and photographed using a transmission electron microscope, and then shown in FIG. 6.
  • the three-dimensional cell culture body of the comparative example not treated with the mussel adhesive protein showed that the hierarchical structure of the dermis and the epidermis was collapsed due to cell migration of the keratinocytes constituting the epidermis into the gap with the culture structure. On the other hand, it was confirmed that the hierarchical structure of the dermis and the epidermis was stably maintained in the three-dimensional cell culture tissue of the example treated with the mussel adhesive protein.
  • the mussel adhesive protein strongly forms the adhesion between the culture structure and the 3D cell culture structure, preventing the contraction of the 3D cell culture structure, thus damaging the hierarchical structure due to cell migration to the contracted area, and cell loss in the upper layer. It was confirmed that the product reliability of the cultured three-dimensional cell cultured tissue can be improved because the problems such as are prevented.
  • a mixed reagent in which 300 ⁇ l of KBMTM Gold Basal Medium (LONZA) and 30 ⁇ l of WST-1 reagent are mixed is prepared, and each tissue cultured for 10 days is separated from a transwell insert and transferred to a 24-well plate.
  • the mixed reagent was dispensed only and incubated for 4 hours in an incubator at 37° C., 5% CO 2 , and 95% relative humidity, and the absorbance of the mixed reagent in the well was measured.
  • the absorbance was measured in the wavelength range of 440nm, and Table 1 shows the wavelength value of pure tissue excluding the wavelength value of 650nm, which is the reference wavelength, and the wavelength value of the mixed reagent without tissue.
  • the test apparatus including the three-dimensional cell culture tissue according to the present invention can prevent the contraction of the three-dimensional cell culture tissue that occurs when the three-dimensional cell culture tissue is cultivated in a support such as a transwell insert. Availability exists.

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Abstract

A bioadhesive-pretreated apparatus for culturing and testing a three-dimensional cellular construct according to the present invention prevents, using a bioadhesive, the contraction of a three-dimensional cell culture construct which is cultured in a scaffold such as a Transwell insert, and thus can prevent morphological and structural changes of the three-dimensional cell culture construct. Thus, when the efficacy and toxicity of drugs, cosmetics, or the like are evaluated using the apparatus, errors between specimens can be minimized and the reliability of test results can be improved.

Description

바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치A device for culturing and testing 3D cell tissues pretreated with bio-adhesives
본 발명은 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치에 관한 것으로, 트랜스웰 인서트와 같은 배양 구조체 내에서 3차원 세포 배양 조직체를 배양시킬 때 발생하는 3차원 세포 배양 조직체의 수축을 방지하여, 수축에 의해 발생하는 간극으로의 세포 이동(cell migration)을 예방함으로써 배양 완료된 3차원 세포 배양 조직체를 이식, 약물의 효능 및 독성 평가 분야에 이용할 때의 신뢰도를 향상시킨 배양 및 테스트 장치에 관한 것이다.The present invention relates to an apparatus for culturing and testing a 3D cell tissue body pretreated with a bio-adhesive, and prevents contraction of a 3D cell culture tissue that occurs when culturing a 3D cell culture tissue in a culture structure such as a transwell insert. Thus, by preventing cell migration to the gap caused by contraction, a culture and test apparatus that improves reliability when using the cultured three-dimensional cell culture tissue in the field of transplantation, drug efficacy and toxicity evaluation. will be.
세포배양은 생체로부터 세포를 채취하고, 이를 생체 밖에서 배양시키는 기술로, 배양된 세포를 이용하여 약물이나 화장료 등의 효능 평가, 독성 평가에 적용하는 기술이 널리 확대되고 있고, 배양된 세포를 피부, 장기, 신경 등 신체의 다양한 조직으로 분화시켜 인체에 이식하거나 분화시키기 전 상태에서 인체에 이식시켜 생착 및 분화를 동시에 이루어지게 함으로써 다양한 질병 치료에 활용될 수 있음이 밝혀짐에 따라 세포 배양에 관한 관심 및 연구가 증가하고 있다. Cell culture is a technology that collects cells from a living body and cultivates them outside the body.The technology that applies to the efficacy evaluation and toxicity evaluation of drugs or cosmetics using cultured cells is expanding widely. Interest in cell culture as it has been found that it can be used for the treatment of various diseases by differentiating into various tissues of the body such as organs and nerves and transplanting them into the human body or by transplanting them into the human body before differentiation to achieve engraftment and differentiation simultaneously And research is increasing.
기존에는 평평한 페트리 디쉬의 바닥에 세포를 부착시키고 배양시켜 얻어진 배양체를 이용하여 신호 전달, 세포 분화 등의 연구를 수행하였으나, 실제 세포는 페트리 디쉬의 바닥에 부착되어 있는 것과 같이 하나의 면에 부착되어 치자라는 것이 아니라 주변의 세포와 액체 등에 둘러싸인 3차원 공간에 떠 있는 상태로 존재하기 때문에 2차원 배양 방식으로 얻어진 배양체의 거동은 실제 인체내의 세포 거동과의 오차가 존재한다.Previously, studies on signal transduction and cell differentiation were conducted using cultures obtained by attaching and culturing cells to the bottom of a flat Petri dish, but the actual cells are attached to one side as if attached to the bottom of a Petri dish. Since it is not a gardenia but exists in a state floating in a three-dimensional space surrounded by surrounding cells and liquids, the behavior of the culture obtained by the two-dimensional culture method has an error with that of the actual cells in the human body.
이에, 콜라겐과 같은 천연고분자와 세포가 혼합된 젤 형태의 3차원적 환경에서 세포를 배양하는 방식이 대두되었고, 이와 같은 3차원 배양 방식으로 배양된 배양체는 실제 인체의 조직과 보다 유사한 거동을 나타내는 것이 확인되어, 최근에는 3차원 배양 방식이 널리 사용되고 있다.Accordingly, a method of culturing cells in a gel-like three-dimensional environment in which natural polymers such as collagen and cells are mixed has emerged, and cultures cultured in such a three-dimensional culture method exhibit more similar behavior to the actual human tissue. It has been confirmed that, recently, a three-dimensional culture method has been widely used.
3차원 세포 배양은 일반적으로 트랜스웰을 이용하여 수행된다. 트랜스웰은 하단부가 미세 다공성 멤브레인으로 이루어진 트랜스웰 인서트와, 트랜스웰 인서트를 수용하는 하부 챔버로 구성되어, 액체배지로 채워진 하부 챔버에 천연고분자와 세포가 혼합된 3차원 세포 배양 조직체가 수용된 트랜스웰 인서트를 삽입하면, 미세 다공성 멤브레인에 의해 액체배지가 공유됨으로써 3차원 세포 배양 조직체에서의 세포 배양이 이루어지는 장치이다.Three-dimensional cell culture is generally performed using transwells. Transwell is composed of a transwell insert with a microporous membrane at the lower end and a lower chamber that accommodates the transwell insert, and a transwell containing a three-dimensional cell culture structure in which natural polymers and cells are mixed in a lower chamber filled with a liquid medium. When inserts are inserted, a liquid medium is shared by a microporous membrane, thereby culturing cells in a three-dimensional cell culture body.
이와 같은 장치를 이용하여 세포 배양을 수행하는 경우, 하부 챔버와 트랜스웰 인서트를 분리하여 손쉽게 액체배지를 교환할 수 있고, 세포 배양 효율이 높은 장점이 있다.When cell culture is performed using such an apparatus, the liquid medium can be easily exchanged by separating the lower chamber and the transwell insert, and cell culture efficiency is high.
그러나, 배양 과정에서 세포가 증식 및 분화함에 따라 천연고분자의 수축이 발생하게 되는데, 이와 같은 수축에 의해 3차원 세포 배양 조직체의 구조 및 형태가 불균일해지는 문제가 있다. 뿐만 아니라, 피부와 같이 계층적 구조를 갖는 조직체를 배양하는 경우에는 수축에 의해 발생한 트랜스웰 인서트와의 간극으로 세포 이동(cell migration)이 이루어져 계층 구조가 무너지게 되므로, 이후 배양이 완료된 3차원 세포 배양 조직체를 이용한 효능이나 독성 시험을 수행할 때, 각 시험체간의 오차를 크게 발생시켜 시험의 신뢰도를 저해하는 문제가 있다.However, as the cells proliferate and differentiate in the culture process, contraction of natural polymers occurs, and there is a problem in that the structure and shape of the three-dimensional cell culture tissue are uneven due to such contraction. In addition, in the case of culturing tissues having a hierarchical structure such as skin, cell migration occurs due to the gap with the transwell insert caused by contraction and the hierarchical structure is collapsed. When performing an efficacy or toxicity test using a cultured tissue, there is a problem that the reliability of the test is impaired by causing a large error between each test body.
이에, 3차원 세포 배양 조직체를 상부 챔버와 같은 지지체를 이용하여 배양할 때 발생하는 3차원 세포 배양 조직체의 수축을 방지할 수 있는 기술 개발이 요구된다. Accordingly, there is a need to develop a technology capable of preventing the contraction of the 3D cell culture tissue, which occurs when the 3D cell culture tissue is cultivated using a support such as an upper chamber.
본 발명에서는 3차원 세포 배양 조직체를 트랜스웰 인서트와 같은 지지체 내에서 배양할 때 발생하는 3차원 세포 배양 조직체의 수축을 방지하는 기술을 제공하고자 한다.In the present invention, it is intended to provide a technology for preventing the contraction of the 3D cell cultured tissue that occurs when the 3D cell cultured tissue is cultivated in a support such as a transwell insert.
상술한 바와 같은 목적을 달성하기 위한 본 발명의 일 실시 형태는 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치에 관한 것으로, 배양 구조체; 상기 배양 구조체 내측에 배치되는 3차원 세포 배양 조직체; 및 상기 배양 구조체와 3차원 세포 배양 조직체 사이의 적어도 일부 계면에 배치되는 바이오 접착제;를 포함한다. One embodiment of the present invention for achieving the object as described above relates to an apparatus for culturing and testing a three-dimensional cell tissue body pretreated with a bio-adhesive, comprising: a culture structure; A three-dimensional cell culture structure disposed inside the culture structure; And a bio-adhesive disposed at at least a partial interface between the culture structure and the three-dimensional cell culture tissue.
상기 배양 구조체는, 몸체, 상기 몸체를 관통하는 홀 및 상기 홀의 일단을 덮도록 형성되는 다공성 멤브레인을 포함하는 것이 바람직하다.The culture structure preferably includes a body, a hole penetrating the body, and a porous membrane formed to cover one end of the hole.
상기 바이오 접착제는, 홍합 접착 단백질, 피브린 글루, 젤라틴 글루, 시아노아크릴레이트계 바이오 접착제 및 폴리우레탄계 바이오 접착제 중 적어도 어느 하나 이상을 포함할 수 있으며, 바람직하게는, 상기 바이오 접착제가 홍합 접착 단백질이다.The bio-adhesive may include at least one or more of mussel adhesive protein, fibrin glue, gelatin glue, cyanoacrylate-based bio-adhesive, and polyurethane-based bio-adhesive, and preferably, the bio-adhesive is mussel adhesive protein. .
상기 바이오 접착제는, 3차원 세포 배양 조직체와 상기 배양 구조체의 다공성 멤브레인 사이에 배치된다.The bio-adhesive is disposed between the three-dimensional cell culture structure and the porous membrane of the culture structure.
특히 상기 바이오 접착제는, 3차원 세포 배양 조직체와 다공성 멤브레인 사이에 배치되고, 상기 몸체를 관통하는 홀의 내측면을 따라 3차원 세포 배양 조직체의 측면으로 연장 배치되는 것이 바람직하다.In particular, it is preferable that the bioadhesive is disposed between the 3D cell culture structure and the porous membrane, and extends to the side of the 3D cell culture structure along the inner surface of the hole penetrating the body.
이때 상기 3차원 세포 배양 조직체의 하면은 바이오 접착제에 의해 다공성 멤브레인에 부착되고, 상기 3차원 세포 배양 조직체의 적어도 일부 측면, 혹은 측면 전체가 상기 바이오 접착제에 의해 홀의 내측면에 부착되는 것이 더욱 바람직하다.At this time, it is more preferable that the lower surface of the 3D cell culture tissue is attached to the porous membrane by a bio-adhesive, and at least some side surfaces or the entire side of the 3D cell culture tissue are attached to the inner side of the hole by the bio-adhesive. .
상기 3차원 세포 배양 조직체는, 천연고분자 내에 세포 혹은 조직이 분산된 젤 형태일 수 있다.The three-dimensional cell culture tissue may be in the form of a gel in which cells or tissues are dispersed in a natural polymer.
이때 세포는, 줄기세포(stem cell), 조골세포(osteoblast), 근아세포(myoblast), 건세포(tenocyte), 신경아세포(neuroblast), 섬유아세포(fibroblast), 신경교아세포(glioblast), 배세포(germcell), 간세포(hepatocyte), 신장세포(renal cell), 지대세포(Sertoli cell), 연골세포(chondrocyte), 상피세포(epithelial cell), 심혈관세포, 각질세포(keratinocyte), 평활근세포(smooth muscle cell), 심장근세포(cardiomyocyte), 신경교세포(glial cell), 내피세포(endothelial cell), 호르몬 분비세포, 면역세포, 췌장섬세포(pancreatic islet cell) 및 신경세포(neuron)로 이루어진 군에서 선택된 어느 하나 이상인 것이 바람직하다.At this time, the cells are stem cells, osteoblasts, myoblasts, tenocytes, neuroblasts, fibroblasts, glioblasts, germ cells ( germcell), hepatocyte, renal cell, sertoli cell, chondrocyte, epithelial cell, cardiovascular cell, keratinocyte, smooth muscle cell ), cardiomyocytes, glial cells, endothelial cells, hormone secreting cells, immune cells, pancreatic islet cells, and neurons selected from the group consisting of It is desirable.
또한, 상기 3차원 세포 배양 조직체는, 두 종류 이상의 세포를 포함하되, 두 종류 이상의 세포가 균일하게 분산되거나, 두 종류 이상의 세포가 서로 분리되어 계층적 구조를 형성할 수 있다.In addition, the three-dimensional cell culture tissue may include two or more types of cells, but two or more types of cells may be uniformly dispersed, or two or more types of cells may be separated from each other to form a hierarchical structure.
이때 상기 계층적 구조는, 진피층과 표피층의 두 개의 층을 갖는 계층적 구조일 수 있다.In this case, the hierarchical structure may be a hierarchical structure having two layers of a dermal layer and an epidermal layer.
상기 다공성 멤브레인은, 액채배지가 투과될 수 있는 미세다공성 막인 것이 바람직하고, 접착제 혹은 접착 테이프를 통해 몸체에 부착되는 것이 바람직하며, 커버에 의해 다공성 멤브레인의 둘레부가 커버와 몸체 사이에 고정되는 것이 더욱 바람직하다.The porous membrane is preferably a microporous membrane through which the liquid medium can permeate, and is preferably attached to the body through an adhesive or adhesive tape, and the peripheral portion of the porous membrane is fixed between the cover and the body by the cover. desirable.
본 발명의 다른 실시 형태로, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치의 제조방법을 들 수 있는데, 몸체; 상기 몸체를 관통하는 홀; 및 상기 홀의 일단을 덮도록 형성되는 다공성 멤브레인;을 포함하는 배양 구조체의 내측에 바이오 접착제를 도포하는 바이오 접착제 도포 단계, 상기 바이오 접착제를 건조하는 건조 단계 및 적어도 일부가 상기 바이오 접착제와 맞닿도록 3차원 세포 배양 조직체를 배양 구조체의 내측에 배치하는 3차원 세포 배양 조직체 배치 단계를 포함한다.In another embodiment of the present invention, there may be mentioned a method for culturing a three-dimensional cell tissue body pretreated with a bio-adhesive and a method of manufacturing a test apparatus, including: a body; A hole through the body; And a porous membrane formed to cover one end of the hole; applying a bio-adhesive to the inside of the culture structure including a bio-adhesive, drying the bio-adhesive, and three-dimensional so that at least a portion of the bio-adhesive contacts the bio-adhesive And a three-dimensional cell culture tissue arrangement step of disposing the cell culture tissue body inside the culture structure.
이때 사용되는 상기 바이오 접착제는, 홍합 접착 단백질, 피브린 글루, 젤라틴 글루, 시아노아크릴레이트계 바이오 접착제 및 폴리우레탄계 바이오 접착제 중 적어도 어느 하나 이상을 포함한다.The bio-adhesive used at this time includes at least one or more of mussel adhesive protein, fibrin glue, gelatin glue, cyanoacrylate-based bio-adhesive, and polyurethane-based bio-adhesive.
또한, 상기 바이오 접착제는, 3차원 세포 배양 조직체와 다공성 멤브레인 사이에 배치되되, 상기 몸체를 관통하는 홀의 내측면을 따라 3차원 세포 배양 조직체의 측면으로 연장 배치될 수 있다.In addition, the bio-adhesive may be disposed between the 3D cell culture structure and the porous membrane, and may extend to the side of the 3D cell culture structure along the inner side of the hole penetrating the body.
아울러 상기 3차원 세포 배양 조직체의 하면은, 바이오 접착제에 의해 다공성 멤브레인에 부착되며, 상기 3차원 세포 배양 조직체의 적어도 일부 측면, 혹은 측면 전체가 상기 바이오 접착제에 의해 홀의 내측면에 부착될 수 있다.In addition, the lower surface of the 3D cell culture tissue may be attached to the porous membrane by a bio-adhesive, and at least some side surfaces or the entire side of the 3D cell culture tissue may be attached to the inner side of the hole by the bio-adhesive.
상기 3차원 세포 배양 조직체는, 천연고분자 내에 세포 혹은 조직이 분산된 젤 형태인 것이 바람직하며, 한 종류 혹은 두 종류 이상의 세포를 포함할 수 있다.The three-dimensional cell culture tissue is preferably in the form of a gel in which cells or tissues are dispersed in a natural polymer, and may include one or two or more types of cells.
3차원 세포 배양 조직체가 두 종류 이상의 세포를 포함하는 경우에는, 두 종류 이상의 세포가 균일하게 분산되거나, 두 종류 이상의 세포가 서로 분리되어 계층적 구조를 형성할 수 있다.When the three-dimensional cell culture organization includes two or more types of cells, two or more types of cells may be uniformly dispersed, or two or more types of cells may be separated from each other to form a hierarchical structure.
상기 계층적 구조는, 진피층과 표피층의 두 개의 층을 갖는 계층적 구조일수 있다.The hierarchical structure may be a hierarchical structure having two layers of a dermal layer and an epidermal layer.
본 발명의 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치는 트랜스웰 인서트와 같은 지지체 내에서 배양되는 3차원 세포 배양 조직체의 수축을 방지함으로써 3차원 세포 배양 조직체의 형태 및 구조 변화를 방지할 수 있으므로, 이를 이용하여 약물이나 화장품 등의 효능, 독성 평가시, 시험체간의 오차를 최소화하고 시험 결과의 신뢰성을 향상시킬 수 있다.The apparatus for culturing and testing a 3D cell tissue body pretreated with the bioadhesive of the present invention prevents the change in the shape and structure of the 3D cell culture tissue by preventing contraction of the 3D cell culture tissue cultured in a support such as a transwell insert. Therefore, it can be used to minimize errors between test subjects and improve the reliability of test results when evaluating efficacy and toxicity of drugs or cosmetics.
도 1은 본 발명의 일 실시예에 따른 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치를 개략적으로 도시한 단면도이다.1 is a cross-sectional view schematically showing an apparatus for culturing and testing a 3D cell tissue body pretreated with a bio-adhesive according to an embodiment of the present invention.
도 2는 본 발명의 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치에 바이오 접착제를 도포하는 방법을 도시한 것이다.FIG. 2 shows a method of applying a bio-adhesive to a culture and test apparatus of a three-dimensional cell tissue body pretreated with a bio-adhesive of the present invention.
도 3은 본 발명의 일 실시예에 따른 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치를 개략적으로 도시한 단면도이다.3 is a cross-sectional view schematically showing an apparatus for culturing and testing a 3D cell tissue body pretreated with a bio-adhesive according to an embodiment of the present invention.
도 4, 도 5 및 도 6은 각각 실험예 1, 실험예 2 및 실험예 3의 결과를 도시한 사진이다.4, 5, and 6 are photographs showing the results of Experimental Example 1, Experimental Example 2, and Experimental Example 3, respectively.
[부호의 설명][Explanation of code]
10: 테스트 장치 100: 배양 구조체10: test device 100: culture structure
110: 몸체 120: 홀110: body 120: hole
130: 멤브레인 200: 바이오 접착제130: membrane 200: bio adhesive
300: 3차원 세포 배양 조직체300: three-dimensional cell culture tissue
이하에서 본 발명의 바람직한 실시예를 통해 상세히 설명하기에 앞서, 본 명세서 및 청구범위에 사용된 용어나 단어는 통상적이거나 사전적인 의미로 한정하여 해석되어서는 아니 되며, 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야 함을 밝혀둔다.Before describing in detail through a preferred embodiment of the present invention below, terms or words used in the present specification and claims should not be construed as being limited to a conventional or dictionary meaning, and conforming to the technical idea of the present invention. It must be interpreted as meaning and concept.
본 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함" 한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성요소를 더 포함할 수 있는 것을 의미한다.Throughout the present specification, when a certain part "includes" a certain component, it means that other components may be further included rather than excluding other components unless otherwise stated.
이하에서는, 본 발명의 실시예를 살펴본다. 그러나 본 발명의 범주가 이하의 바람직한 실시예에 한정되는 것은 아니며, 당업자라면 본 발명의 권리범위 내에서 본 명세서에 기재된 내용의 여러 가지 변형된 형태를 실시할 수 있다.Hereinafter, an embodiment of the present invention will be described. However, the scope of the present invention is not limited to the following preferred embodiments, and those skilled in the art can implement various modified forms of the contents described in the present specification within the scope of the present invention.
먼저, 본 발명은 트랜스웰 인서트와 같은 배양 구조체 내에서 3차원 세포 배양 조직체를 배양할 때 발생하는 3차원 세포 배양 조직체의 수축을 방지하는 기술에 관한 것이다.First, the present invention relates to a technology for preventing contraction of a 3D cell cultured tissue that occurs when a 3D cell cultured tissue is cultivated in a culture structure such as a transwell insert.
도 1은 본 발명의 일 실시예에 따른 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치(10)를 도시한 것으로, 도 1을 참조하면, 상기 배양 및 테스트 장치(10)는, 배양 구조체(100); 상기 배양 구조체(100) 내측에 배치되는 3차원 세포 배양 조직체(300); 및 상기 배양 구조체(100)와 3차원 세포 배양 조직체(300)의 적어도 일부 계면에 배치되는 바이오 접착제(200);를 포함한다.1 is a diagram illustrating a culture and testing apparatus 10 of a three-dimensional cell tissue body pretreated with a bio-adhesive according to an embodiment of the present invention. Referring to FIG. 1, the culture and testing apparatus 10 is Structure 100; A three-dimensional cell culture structure 300 disposed inside the culture structure 100; And a bio-adhesive 200 disposed on at least a partial interface between the culture structure 100 and the 3D cell culture tissue 300.
상기 배양 구조체(100)로 본 발명이 속하는 기술분야에서 사용되는 트랜스웰에 사용되는 트랜스웰 인서트 또는 상부 챔버가 사용될 수 있으며, 배양 구조체(100)는 몸체(110)를 관통하는 홀(120) 및 상기 홀의 일단을 덮도록 형성되는 다공성 멤브레인(130)을 포함하는 것이 바람직하다.As the culture structure 100, a transwell insert or an upper chamber used in a transwell used in the technical field to which the present invention pertains may be used, and the culture structure 100 includes a hole 120 passing through the body 110 and It is preferable to include a porous membrane 130 formed to cover one end of the hole.
상기 트랜스웰은 상업적으로 시판되는 제품이 사용될 수 있으며, 예를 들어, Corning incorporated 사의 제품이 사용될 수 있다. 또는, 등록특허 제1981053호(2019.05.16 등록)에 제시된 높이조절이 가능한 분리형 구조의 배양구조체가 사용될 수 있으나, 이에 제한되는 것은 아니다.Commercially available products may be used as the transwell, and for example, products of Corning incorporated may be used. Alternatively, a culture structure having a detachable structure that can be adjusted in height as disclosed in Registration Patent No. 1981053 (registered on May 16, 2019) may be used, but is not limited thereto.
상기 트랜스웰은 배양 구조체(100); 및 상기 배양 구조체(100)와 배지를 수용하는 하부 챔버;로 구성되며, 배양 구조체(100)가 하부 챔버 내에 수용되어, 배양 구조체(100)의 다공성 멤브레인(130)에 의해 하부 챔버에 수용된 배지가 공유됨으로써 배양 구조체(100) 내의 3차원 세포 배양 조직체(300)의 세포 분화 및 증식이 이루어질 수 있다.The transwell is a culture structure 100; And a lower chamber accommodating the culture structure 100 and the medium, wherein the culture structure 100 is accommodated in the lower chamber, and the medium accommodated in the lower chamber by the porous membrane 130 of the culture structure 100 is By sharing, cell differentiation and proliferation of the three-dimensional cell culture tissue 300 in the culture structure 100 may be achieved.
상기 다공성 멤브레인(130)은 액채배지가 투과될 수 있는 미세다공성 막일 수 있으며, 공극의 크기는 특별히 제한되지 않으나, 바람직하게는 마이크로 단위의 공극이 형성된 미세다공성 막일 수 있다. 다공성 멤브레인(130)은 접착제, 접착 테이프 등을 통해 몸체(110)에 부착될 수 있으며, 또는 커버 등의 수단에 의해 다공성 멤브레인(130)의 둘레부가 커버와 몸체(100) 사이에 고정되는 방식으로 부착될 수도 있다.The porous membrane 130 may be a microporous membrane through which the liquid medium is permeable, and the size of the pores is not particularly limited, but may preferably be a microporous membrane in which microscopic pores are formed. The porous membrane 130 may be attached to the body 110 through an adhesive, an adhesive tape, or the like, or the circumference of the porous membrane 130 is fixed between the cover and the body 100 by means such as a cover. It can also be attached.
이와 같은 배양 구조체(100)는 다공성 멤브레인(130)이 하부에 위치하도록 배치되어 사용되므로, 3차원 세포 배양 조직체(300)는 일반적으로 다공성 멤브레인(130)의 상면과 홀(120)의 하부 내측면에 부착되어 배양된다.Since such a culture structure 100 is disposed and used so that the porous membrane 130 is positioned at the bottom, the 3D cell culture structure 300 is generally the upper surface of the porous membrane 130 and the lower inner surface of the hole 120. Attached to and cultured.
그러나, 배양 과정에서 세포의 분화 및 증식이 일어나면서 3차원 세포 배양 조직체(300)가 수축되는 현상이 발생하여 3차원 세포 배양 조직체(300)의 형태를 변형시킬 뿐만 아니라, 3차원 세포 배양 조직체(300)가 진피와 표피를 갖는 피부 조직과 같이 계층적 구조를 가질 경우, 수축에 의해 발생한 배양 구조체(100)와의 간극으로의 세포 이동(cell migration)이 일어나며 조직체의 계층적 구조를 무너뜨린다.However, during the cultivation process, as the differentiation and proliferation of cells occurs, a phenomenon in which the 3D cell culture body 300 contracts occurs, thereby not only changing the shape of the 3D cell culture body 300, but also the 3D cell culture body ( When 300) has a hierarchical structure, such as a skin tissue having a dermis and an epidermis, cell migration occurs to the gap with the culture structure 100 caused by contraction, thereby destroying the hierarchical structure of the tissue.
따라서, 배양이 완료된 3차원 세포 배양 조직체(300)를 이용하여 이식을 수행하거나, 약물이나 화장품의 효능, 자극, 독성 등의 평가를 할 때 시험체간의 오차를 크게 발생시키는 문제가 있다.Therefore, there is a problem that a large error between test bodies is generated when performing transplantation using the cultured 3D cell culture body 300 or when evaluating the efficacy, stimulation, and toxicity of drugs or cosmetics.
본 발명에서는 이러한 문제를 방지하기 위해 배양 구조체(100)와 3차원 세포 배양 조직체(300) 사이의 적어도 일부 계면에 바이오 접착제(200)를 배치시킴으로써 배양 구조체(100)와 3차원 세포 배양 조직체(300) 사이의 부착력을 향상시킴으로써 3차원 세포 배양 조직체(300)의 수축을 방지하고자 한다.In the present invention, in order to prevent such a problem, the culture structure 100 and the 3D cell culture structure 300 are arranged by disposing the bio-adhesive 200 on at least some interfaces between the culture structure 100 and the 3D cell culture tissue 300. ) To prevent contraction of the 3D cell culture tissue body 300 by improving the adhesion between.
이때 사용되는 바이오 접착제(200)로 생체조직 접착에 사용될 수 있는 홍합 접착 단백질, 피브린 글루, 젤라틴 글루, 시아노아크릴레이트계 바이오 접착제 및 폴리우레탄계 바이오 접착제 중 적어도 어느 하나 이상을 포함할 수 있다.The bio-adhesive 200 used at this time may include at least one or more of mussel adhesive proteins, fibrin glue, gelatin glue, cyanoacrylate-based bio-adhesive, and polyurethane-based bio-adhesives that can be used for bonding biological tissues.
바람직하게는 홍합 접착 단백질이 사용될 수 있는데, 피브린 글루는 접착력이 약한 문제가 있고, 젤라틴 글루는 접착성은 우수하나 가교제로 사용되는 포르말린에 의해 독성을 나타낼 수 있으며, 시아노아크릴레이트계나 폴리우레탄계 바이오 접착제와 같은 합성 고분자계 접착제의 경우에는 생체 독성 및 부작용 등의 문제를 나타낼 수 있기 때문이다.Preferably, mussel adhesive protein may be used, fibrin glue has a weak adhesion problem, gelatin glue has excellent adhesion, but may be toxic by formalin used as a crosslinking agent, and cyanoacrylate-based or polyurethane-based bioadhesive This is because, in the case of a synthetic polymer adhesive such as, it may exhibit problems such as biotoxicity and side effects.
다만, 이와 같은 피브린 글루, 젤라틴 글루 및 합성 고분자계 접착제의 문제점들은 다른 기술적 수단을 통해 해결될 수 있으므로, 본 발명에서 사용될 수 있는 바이오 접착제(200)로 이들을 배제하는 것은 아니다.However, such problems of fibrin glue, gelatin glue, and synthetic polymer-based adhesive can be solved through other technical means, and thus the bio-adhesive 200 that can be used in the present invention is not excluded.
상기 홍합 접착 단백질은, 도파(L-dihydroxylalanine, DOPA)가 풍부한 단백질로, 물이 있는 환경에서도 친수성 표면, 소수성 표면, 금속 표면 등 다양한 표면에 강력한 접착력을 나타내고, 인간세포를 공격하지 않으며, 면역반응을 일으키지 않아 인체에 무해하므로, 본 발명에 사용되는 바이오 접착제(200)로 가장 바람직한 물질이다.The mussel adhesion protein is a protein rich in L-dihydroxylalanine (DOPA), and exhibits strong adhesion to various surfaces such as hydrophilic surfaces, hydrophobic surfaces, and metal surfaces even in an environment with water, does not attack human cells, and immune response Since it does not cause harm to the human body, it is the most preferred material for the bio adhesive 200 used in the present invention.
상기 바이오 접착제(200)는 3차원 세포 배양 조직체(300)와 다공성 멤브레인(130) 사이에 배치될 수 있다. 이 경우, 3차원 세포 배양 조직체(300)의 하면은 바이오 접착제(200)에 의해 다공성 멤브레인(130)에 부착되고, 측면은 홀(120)의 내측면에 부착된다.The bioadhesive 200 may be disposed between the 3D cell culture tissue 300 and the porous membrane 130. In this case, the lower surface of the 3D cell culture structure 300 is attached to the porous membrane 130 by the bio-adhesive 200, and the side surface is attached to the inner surface of the hole 120.
또는, 상기 바이오 접착제(200)가 3차원 세포 배양 조직체(300)와 다공성 멤브레인(130) 사이에 배치되고, 여기서부터 몸체(110)를 관통하는 홀(120)의 내측면을 따라 3차원 세포 배양 조직체(300)의 측면으로 연장 배치될 수 있다. 이 경우, 3차원 세포 배양 조직체(300)의 하면은 바이오 접착제(200)에 의해 다공성 멤브레인(130)에 부착되고, 적어도 일부 측면, 혹은 측면 전체가 바이오 접착제(200)에 의해 홀(120)의 내측면에 부착될 수 있다.Alternatively, the bio-adhesive 200 is disposed between the three-dimensional cell culture body 300 and the porous membrane 130, and from there, the three-dimensional cell culture along the inner surface of the hole 120 penetrating the body 110 It may be disposed extending to the side of the tissue 300. In this case, the lower surface of the 3D cell culture tissue 300 is attached to the porous membrane 130 by the bio-adhesive 200, and at least some side surfaces or the entire side of the hole 120 by the bio-adhesive 200 Can be attached to the inner side.
상기 바이오 접착제(200)는 인젝션 방식을 통해 배양 구조체(100)에 도포될 수 있고, 도포 후 소정 시간 동안 건조되어 준비될 수 있다.The bio-adhesive 200 may be applied to the culture structure 100 through an injection method, and dried for a predetermined time after application to be prepared.
상기 인젝션 방식은 작업자에 의해 수작업으로 진행될 수도 있고, 플로팅 혹은 프린팅 방식으로도 진행될 수도 있다. 바람직하게는, 3D 잉크젯 프린팅 방식이 이용될 수 있으며, 이와 같은 방식을 이용하면 바이오 접착제(200)를 균일하게 분포시킬 수 있고, 바이오 접착제(200)가 배출되는 노즐이 다공성 멤브레인(130)에 닿지 않아 다공성 멤브레인(130)의 손상을 최소화 할 수 있는 장점이 있다.The injection method may be performed manually by an operator, or may be performed by a floating or printing method. Preferably, a 3D inkjet printing method may be used, and if this method is used, the bio-adhesive 200 can be uniformly distributed, and the nozzle from which the bio-adhesive 200 is discharged does not contact the porous membrane 130. Therefore, there is an advantage of minimizing damage to the porous membrane 130.
상기 3차원 세포 배양 조직체(300)는 천연고분자 내에 세포가 분산된 젤 형태일 수 있으며, 상기 천연고분자는 콜라겐, 피브린겔, 마트리겔, 알지네이트, 젤라틴, 아가로스 또는 이 중 적어도 둘 이상을 포함한 천연고분자 혼합물일 수 있다.The three-dimensional cell culture tissue 300 may be in the form of a gel in which cells are dispersed in a natural polymer, and the natural polymer is a natural polymer including collagen, fibrin gel, matrigel, alginate, gelatin, agarose, or at least two or more thereof. It may be a polymer mixture.
상기 세포로 적용될 수 있는 세포의 종류는 특별히 한정되지 않으며, 동물세포, 식물세포 또는 이들의 조직일 수 있다. 예를 들어, 상기 세포는 줄기세포(stem cell), 조골세포(osteoblast), 근아세포(myoblast), 건세포(tenocyte), 신경아세포(neuroblast), 섬유아세포(fibroblast), 신경교아세포(glioblast), 배세포(germcell), 간세포(hepatocyte), 신장세포(renal cell), 지대세포(Sertolicell), 연골세포(chondrocyte), 상피세포(epithelial cell), 심혈관세포, 각질세포(keratinocyte), 평활근세포(smooth muscle cell), 심장근세포(cardiomyocyte), 신경교세포(glial cell), 내피세포(endothelial cell), 호르몬 분비세포, 면역세포, 췌장섬세포(pancreatic islet cell) 및 신경세포(neuron)로 이루어진 군에서 선택된 어느 하나 이상일 수 있다.The type of cells that can be applied to the cells is not particularly limited, and may be animal cells, plant cells, or tissues thereof. For example, the cells are stem cells, osteoblasts, myoblasts, tenocytes, neuroblasts, fibroblasts, glioblasts, Germcells, hepatocytes, renal cells, serolicells, chondrocytes, epithelial cells, cardiovascular cells, keratinocytes, smooth muscle cells muscle cells), cardiomyocytes, glial cells, endothelial cells, hormone secreting cells, immune cells, pancreatic islet cells, and neurons selected from the group consisting of There can be more than one.
상기 3차원 세포 배양 조직체(300)는 한 종류의 세포만을 포함할 수도 있고, 두 종류 이상의 세포를 포함할 수도 있는데, 후자의 경우, 두 종류 이상의 세포가 균일하게 분산되어 포함될 수도 있고, 두 종류 이상의 세포가 서로 분리되어 계층적 구조를 형성할 수도 있다. 계층적 구조란, 피부나 호흡기와 같이 적어도 두 개 이상의 층으로 구성되는 조직 구조를 의미하며, 예를 들어, 피부의 경우에는 진피층과 표피층 두 개의 층을 갖는 계층적 구조를 갖는다.The three-dimensional cell culture tissue 300 may include only one type of cell or two or more types of cells. In the latter case, two or more types of cells may be uniformly distributed and included, or two or more types of cells may be included. Cells can be separated from each other to form a hierarchical structure. The hierarchical structure refers to a tissue structure composed of at least two or more layers such as skin or respiratory organs. For example, the skin has a hierarchical structure having two layers of a dermal layer and an epidermal layer.
본 발명의 일 실시예에 따른 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치(10)는, 이를 이용하여 피부 구조체를 형성할 때 특히 더 우수한 효과를 나타낸다. 구체적으로, 상부 표면을 이루는 표피층과 나머지 진피층으로 구성된 3차원 세포 배양 조직체(300)의 상부 표면을 제외한 나머지면은 바이오 접착제(200)에 의해 배양 구조체(100)에 강하게 부착되어 있으므로, 다공성 멤브레인(130)을 통해 유입되는 배양액이 진피층으로만 공급되고 표피층으로 침범하는 것이 미연에 방지되어, 상부 표면의 표피층이 안정적으로 공기층에서 배양될 수 있다.The apparatus 10 for culturing and testing a three-dimensional cell tissue body pretreated with a bio-adhesive according to an embodiment of the present invention 10 exhibits a particularly superior effect when forming a skin structure using the same. Specifically, the remaining surfaces except for the upper surface of the three-dimensional cell culture body 300 composed of the epidermal layer and the remaining dermal layer forming the upper surface are strongly attached to the culture structure 100 by the bio-adhesive 200, so that the porous membrane ( 130) is supplied only to the dermal layer and invasion into the epidermal layer is prevented in advance, so that the epidermal layer on the upper surface can be stably cultured in the air layer.
또한, 강한 접착력에 의해 3차원 세포 배양 조직체(300)의 측면과 배양 구조체(100) 사이의 간극이 형성되지 않으므로, 표피층을 구성하는 세포의 간극으로의 세포 이주가 방지되어 표피 세포가 일정한 위치에서만 증식될 수 있다.In addition, since the gap between the side surface of the three-dimensional cell culture structure 300 and the culture structure 100 is not formed due to strong adhesion, cell migration to the gap of the cells constituting the epidermal layer is prevented, so that the epidermal cells are only in a certain position. Can multiply.
한편, 본 발명의 다른 실시예는 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치(10)의 제조방법에 관한 것으로, 상기 배양 및 테스트 장치(10)는 앞서 설명한 것과 동일하므로, 중복되는 설명은 생략한다.On the other hand, another embodiment of the present invention relates to a method of manufacturing a 3D cell tissue culture and test apparatus 10 pretreated with a bio-adhesive, and the culture and test apparatus 10 is the same as described above, Description is omitted.
상기 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치(10)의 제조방법은, 몸체(110), 상기 몸체(110)를 관통하는 홀(120) 및 상기 홀(120)의 일단을 덮도록 형성되는 다공성 멤브레인(130)을 포함하는 배양 구조체(100)의 내측에 바이오 접착제(200)를 도포하는 바이오 접착제 도포 단계;The manufacturing method of the apparatus 10 for culturing and testing a 3D cell tissue body pretreated with the bio-adhesive includes a body 110, a hole 120 penetrating the body 110, and one end of the hole 120 A bio-adhesive application step of applying the bio-adhesive 200 to the inside of the culture structure 100 including the porous membrane 130 to be formed so as to be formed so as to be;
상기 바이오 접착제(200)를 건조하는 건조 단계; 및 적어도 일부가 상기 바이오 접착제(200)와 맞닿도록 3차원 세포 배양 조직체(300)를 배양 구조체(100) 내측에 배치하는 3차원 세포 배양 조직체 배치 단계;를 포함한다.A drying step of drying the bio-adhesive 200; And a 3D cell culture tissue arrangement step of disposing the 3D cell culture tissue 300 inside the culture structure 100 so that at least a portion thereof contacts the bioadhesive 200.
먼저, 상기 바이오 접착제 도포 단계는, 바이오 접착제(200)를 배양 구조체(100)의 내측에 도포하는 단계로, 인젝션 방식을 통해 다공성 멤브레인(130)상에 도포될 수 있다.First, the step of applying the bio-adhesive is a step of applying the bio-adhesive 200 to the inner side of the culture structure 100, and may be applied on the porous membrane 130 through an injection method.
바이오 접착제(200)의 도포는, 도 2(a)에 도시된 바와 같이 작업자에 의해 수작업으로 진행될 수도 있고, 도 2(b) 및 도 2(c)와 같은 3D 프린팅 방식으로 진행될 수도 있다. 바람직하게는, 다공성 멤브레인(130)의 손상을 최소화하면서 바이오 접착제(200)를 균일하게 도포시킬 수 있는 3D 잉크젯 프린팅 방식이 이용될 수 있다.The application of the bio-adhesive 200 may be performed manually by an operator as shown in FIG. 2(a), or may be performed in a 3D printing method as shown in FIGS. 2(b) and 2(c). Preferably, a 3D inkjet printing method capable of uniformly applying the bio-adhesive 200 while minimizing damage to the porous membrane 130 may be used.
상기 건조 단계는, 배양 구조체(100)에 도포된 바이오 접착제(200)를 건조시키기 위해 진행되는 단계로, 건조 단계를 통해 바이오 접착제(200)가 얇은 박막 형태로 변한다. 이때, 도포된 바이오 접착제(200)의 양에 따라 바이오 접착제(200)가 다공성 멤브레인(130) 상에 박막 형태로 존재할 수 있고(도 1), 또는 다공성 멤브레인(130) 상부와, 여기서부터 연장되어 홀(120) 내측면까지 덮는 형태로 존재할 수도 있다(도 3). 또는 적량을 다공성 멤브레인(130)과 배양 구조체(100)의 홀(120) 내측면에 도포하고 건조하여 바이오 접착제(200)를 도 3과 같은 형태로 형성하는 것도 가능하다.The drying step is a step performed to dry the bio-adhesive 200 applied to the culture structure 100, and the bio-adhesive 200 is transformed into a thin thin film through the drying step. At this time, depending on the amount of the applied bio-adhesive 200, the bio-adhesive 200 may exist in the form of a thin film on the porous membrane 130 (FIG. 1), or the upper portion of the porous membrane 130, and extend from thereon. The hole 120 may be present in a form that covers the inner side (FIG. 3). Alternatively, an appropriate amount may be applied to the inner surface of the hole 120 of the porous membrane 130 and the culture structure 100 and dried to form the bioadhesive 200 in the shape as shown in FIG. 3.
상기 3차원 세포 배양 조직체 배치 단계는, 천연고분자와 세포가 혼합된 겔 형태의 혼합물을 배양 구조체(100) 내에 수작업 혹은 3D 프린팅 방식을 통해 주입하고 상온냉각하여 젤 형태의 3차원 세포 배양 조직체(300)를 배양 구조체(100) 내에 배치하는 단계이다.In the step of arranging the three-dimensional cell culture tissue, a gel-like mixture of natural polymers and cells is injected into the culture structure 100 by hand or 3D printing, and cooled to room temperature to obtain a gel-like three-dimensional cell culture structure 300. ) Is placed in the culture structure 100.
이와 같은 방법을 통해 제조된 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치를 이용하여 배양을 진행하는 경우, 3차원 세포 배양 조직체(300)의 수축으로 인한 3차원 세포 배양 조직체(300)와 배양 구조체(100) 사이의 간극이 발생하지 않아, 배양 완료된 3차원 세포 배양 조직체(300)를 이용한 이식, 화장품 및 약품의 효능, 독성 평가를 진행할 때 3차원 세포 배양 조직체(300)의 형태 및 구조에 의한 오차의 발생을 최소화 할 수 있어, 이식의 완성도 및 평가 결과의 신뢰도를 향상시킬 수 있다.In the case of culturing a 3D cell tissue body prepared by such a method and using a test apparatus for culturing a 3D cell tissue body prepared through such a method, the 3D cell culture tissue body 300 due to contraction of the 3D cell culture tissue body 300 Since a gap between the and the culture structure 100 does not occur, the shape and shape of the 3D cell culture structure 300 when performing transplantation, the efficacy of cosmetics and drugs, and toxicity evaluation using the cultured 3D cell culture structure 300 It is possible to minimize the occurrence of errors due to the structure, thereby improving the completeness of the implantation and the reliability of the evaluation results.
이하, 본 발명의 일 실시예에 따른 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치를 제조하고, 이를 통해 본 발명의 구체적인 작용과 효과를 설명하고자 한다. 다만, 이는 본 발명의 바람직한 예시로서 제시된 것으로, 실시예에 따라 본 발명의 권리범위가 한정되는 것은 아니다.Hereinafter, a device for culturing and testing a three-dimensional cell tissue body pretreated with a bio-adhesive according to an embodiment of the present invention is manufactured, and specific actions and effects of the present invention will be described through this. However, this is presented as a preferred example of the present invention, and the scope of the present invention is not limited according to the embodiment.
[제조예][Production Example]
트랜스웰 인서트에 3D 잉크젯 프린팅 방식을 이용하여 홍합 접착 단백질을 주입하고 건조하여, 도 3과 같은 형태로 건조된 홍합 접착 단백질이 포함된 트랜스웰 인서트를 제조하였다.The mussel adhesive protein was injected into the transwell insert using a 3D inkjet printing method and dried to prepare a transwell insert containing the dried mussel adhesive protein in the form of FIG. 3.
이후, 트랜스웰 인서트에 콜라겐과 섬유아세포(Fibroblast)가 혼합된 겔을 주입하고, 상부 표면에 각질형성세포(Keratinocyte)를 접종하여 테스트 장치를 제조한 뒤, 하부는 액체배지와 접촉시키고, 상부는 공기중에 노출시킨 공기-액체 계면(Air-liquid interface, ALI) 방식으로 세포 배양을 진행하였다.Thereafter, a gel in which collagen and fibroblasts are mixed is injected into the transwell insert, and a test device is prepared by inoculating keratinocytes on the upper surface, and then the lower part is in contact with the liquid medium, and the upper part is Cell culture was carried out in an air-liquid interface (ALI) method exposed to air.
액체배지로는 KBM™ Gold Basal Medium(LONZA)에 KGM™ Gold SingleQuots supp1ements(LONZA) 7종(Hydrocortisone 0.50 mL, Transferrin 0.50 mL, Epinephrine 0.25 mL, GA-1000 0.50 mL, BPE 2.00 mL, hEGF, 0.50 mL, Insulin 0.50 mL)을 추가하고, 각질형성세포의 분화를 유도한다고 알려진 Calcium chloride(Sigma-Aldrich)를 1.8mM의 농도가 되도록 첨가한 것을 사용하였다.As a liquid medium, KGM™ Gold SingleQuots supp1ements (LONZA) 7 kinds (Hydrocortisone 0.50 mL, Transferrin 0.50 mL, Epinephrine 0.25 mL, GA-1000 0.50 mL, BPE 2.00 mL, hEGF, 0.50 mL) in KBM™ Gold Basal Medium (LONZA) , Insulin 0.50 mL) was added, and Calcium chloride (Sigma-Aldrich), which is known to induce differentiation of keratinocytes, was added to a concentration of 1.8 mM.
세포 배양은 C0 2 5%, 37℃, 상대습도 95%의 조건에서 수행되었고, 15mL의 액체배지가 수용된 100mm 페트리디쉬에 두 개의 트랜스웰 인서트를 조립하여 수행되되, 액체배지는 3일 단위로 교환되었다.Cell culture was carried out under conditions of C0 2 5%, 37°C, and 95% relative humidity, and was carried out by assembling two transwell inserts in a 100 mm Petri dish containing 15 mL of liquid medium, but the liquid medium was exchanged every 3 days. Became.
[실험예 1][Experimental Example 1]
홍합 접착 단백질 주입 단계를 제외하고 제조예와 동일한 방식을 이용하여 테스트 장치를 제조한 뒤 세포 배양을 진행하였다. 이때, 주입되는 섬유아세포의 농도를 달리하여 실험을 진행하였고, 각질형성세포는 모든 샘플에서 동일하게 5×lO 5 cells/well의 농도로 사용되었으며, 배양 시작 후 3일, 4일 및 7일 경과 시점에서 3차원 세포 배양 조직체의 외관을 촬영하여 도 4에 도시하였다.A test device was manufactured in the same manner as in Preparation Example except for the mussel adhesive protein injection step, and then cell culture was performed. At this time, the experiment was conducted by varying the concentration of the injected fibroblasts, and the keratinocytes were used at the same concentration of 5×10 5 cells/well in all samples, and 3, 4 and 7 days elapsed after the start of culture. The appearance of the three-dimensional cell culture tissue was photographed at the time point and shown in FIG. 4.
도 4를 참조하면, 주입되는 세포의 농도가 높을수록 3차원 세포 배양 조직체의 수축이 더 빠르게 일어나는 것을 확인할 수 있었으며, 주입되는 세포의 농도가 낮더라도 배양이 어느정도 진행 된 시점에서 3차원 세포 배양 조직체의 수축이 발생하는 것을 확인할 수 있었다.Referring to FIG. 4, it was confirmed that the contraction of the 3D cell cultured tissue body occurred more rapidly as the concentration of the injected cells increased. Even if the concentration of the injected cells was low, the 3D cell culture tissue body of It was confirmed that contraction occurred.
따라서, 종래의 방식을 이용하여 트랜스웰 인서트에 3차원 세포 배양을 진행하는 경우, 수축에 의해 3차원 세포 배양 조직체의 형태가 변화하고, 각 개체별 형태가 불균일하게 형성되므로, 이후 이를 이용하여 약물이나 화장품의 효능, 자극, 독성 등의 실험을 진행하는 경우 실험 결과의 신뢰성을 저하시킬 수 있고, 수축 정도가 심한 경우에는 이와 같은 실험에 사용할 수 없는 문제가 발생함을 확인할 수 있었다.Therefore, when 3D cell culture is performed on a transwell insert using a conventional method, the shape of the 3D cell culture tissue changes due to contraction, and the shape of each individual is non-uniformly formed. In the case of conducting experiments such as efficacy, irritation, and toxicity of cosmetics or cosmetics, the reliability of the experimental results could be degraded, and when the degree of contraction was severe, it could be confirmed that a problem that could not be used for such experiments occurred.
[실험예 2][Experimental Example 2]
제조예와 동일한 방법을 이용하여 실시예의 테스트 장치를 제조하고, 제조예와 동일한 방법을 이용하되 홍합 접착 단백질 주입 단계를 제외하여 비교예의 테스트 장치를 제조한 뒤 세포 배양을 진행하였으며, 배양 초기, 3일 경과 후, 10일 경과 후에 3차원 세포 배양 조직체의 외관을 촬영하여 도 5에 도시하였다.The test device of Example was manufactured using the same method as in Preparation Example, and the test device of Comparative Example was manufactured using the same method as in Preparation Example except for the injection of mussel adhesive protein, and then cell culture was performed. After days and 10 days, the appearance of the three-dimensional cell culture tissue was photographed and shown in FIG. 5.
이때, 각 시험체별로 섬유아세포는 1xlO 6 cells/ml, 각질형성세포는 5×10 5 cells/well의 농도로 주입되었다.At this time, for each test specimen, fibroblasts were injected at a concentration of 1xlO 6 cells/ml and keratinocytes at a concentration of 5×10 5 cells/well.
도 5를 참조하면, 실시예의 경우에는 배양 10일 경과 후에도 홍합 접착 단백질에 의해 3차원 세포 배양 조직체와 트랜스웰 인서트간의 강력한 접착력이 형성되어, 3차원 세포 배양 조직체의 수축이 관찰되지 않았으나, 비교예의 경우에는 3일 경과 후부터 계면에서의 3차원 세포 배양 조직체의 수축이 나타나는 것으로 확인되었다.5, in the case of the Example, strong adhesion between the 3D cell culture tissue and the transwell insert was formed by the mussel adhesive protein even after 10 days of culture, so that the contraction of the 3D cell culture tissue was not observed. In the case, it was confirmed that contraction of the three-dimensional cell culture tissue at the interface appeared after 3 days.
따라서, 본 실험 결과로부터, 배양 구조체인 트랜스웰 인서트와 3차원 세포 배양 조직체 사이에 홍합 접착 단백질을 적용하는 경우, 트랜스웰 인서트와 3차원 세포 배양 조직체간의 접착력이 강력하게 유지되어, 배양 과정에서 3차원 세포 배양 조직체가 수축되지 않고 안정적으로 장기간 동안 배양될 수 있음을 확인할 수 있었다.Therefore, from the results of this experiment, when the mussel adhesive protein is applied between the transwell insert as a culture structure and the 3D cell culture tissue, the adhesion between the transwell insert and the 3D cell culture tissue is strongly maintained. It was confirmed that the dimensional cell cultured tissue could be stably cultured for a long period without shrinking.
[실험예 3][Experimental Example 3]
상기 실험예 2의 시험체 중 10일간 배양된 실시예와 비교예의 시험체를 동결절단(cryosection) 방식을 이용하여 조직 절편으로 제작하고 염색한 뒤 투과전자현미경을 이용하여 관찰하였다.Among the specimens of Experimental Example 2, the specimens of Examples and Comparative Examples, which were cultured for 10 days, were prepared as tissue sections using a cryosection method, stained, and observed using a transmission electron microscope.
보다 구체적으로, 4℃에서 각 시험체를 4% paraformaldehyde에 24시간 고정시키고, PBS로 1시간 동안 세척한 후 인서트에서 조직을 분리하여 30% sucrose에 조직을 담궈 조직이 침전될 때까지 정치시킴으로써 조직을 고정한 뒤, OCT 화합물을 이용하여 OCT 블록을 제조하고, 액체 질소를 이용하여 급랭시킨 후, 동결절단(cryosection)하여 조직 절편을 준비하였다.More specifically, each test sample is fixed in 4% paraformaldehyde at 4°C for 24 hours, washed with PBS for 1 hour, and then separated from the insert, immersed in 30% sucrose, and allowed to stand until the tissue precipitates. After fixation, an OCT block was prepared using an OCT compound, quenched with liquid nitrogen, and then cryosectioned to prepare a tissue section.
다음으로, Hematoxylin and Eosin stain kit HAE-1(ScyTek)를 이용하여 상기 조직 절편의 염색을 수행하고 투과전자현미경을 이용하여 촬영한 뒤 도 6에 도시하였다.Next, the tissue sections were stained using Hematoxylin and Eosin stain kit HAE-1 (ScyTek), and photographed using a transmission electron microscope, and then shown in FIG. 6.
도 6을 참조하면, 홍합 접착 단백질로 처리되지 않은 비교예의 3차원 세포 배양 조직체는, 배양 구조체와의 간극으로 표피를 구성하는 각질형성세포의 세포이동이 이루어져 진피와 표피의 계층 구조가 무너진 것으로 나타난 반면, 홍합 접착 단백질로 처리된 실시예의 3차원 세포 배양 조직체는 진피와 표피의 계층 구조가 안정적으로 유지되는 것으로 확인되었다.6, the three-dimensional cell culture body of the comparative example not treated with the mussel adhesive protein showed that the hierarchical structure of the dermis and the epidermis was collapsed due to cell migration of the keratinocytes constituting the epidermis into the gap with the culture structure. On the other hand, it was confirmed that the hierarchical structure of the dermis and the epidermis was stably maintained in the three-dimensional cell culture tissue of the example treated with the mussel adhesive protein.
즉, 홍합 접착 단백질에 의해 배양 구조체와 3차원 세포 배양 조직체간의 접착력이 강력하게 형성되어, 3차원 세포 배양 조직체의 수축이 방지되므로 수축된 영역으로의 세포 이동에 의한 계층 구조 손상, 상층부의 세포 손실 등의 문제가 방지되므로, 배양 완료된 3차원 세포 배양 조직체의 제품 신뢰도를 향상시킬 수 있음을 확인할 수 있었다.In other words, the mussel adhesive protein strongly forms the adhesion between the culture structure and the 3D cell culture structure, preventing the contraction of the 3D cell culture structure, thus damaging the hierarchical structure due to cell migration to the contracted area, and cell loss in the upper layer. It was confirmed that the product reliability of the cultured three-dimensional cell cultured tissue can be improved because the problems such as are prevented.
[실험예 4][Experimental Example 4]
상기 실험예 2의 시험체 중 10일간 배양된 실시예와 비교예의 바닥면 영역에 존재하는 세포를 배양하여 흡광도를 측정한 뒤 그 결과를 표 1에 기재하였다.Among the test specimens of Experimental Example 2, cells present in the bottom area of the Example and Comparative Example were cultured for 10 days to measure absorbance, and the results are shown in Table 1.
구체적으로, KBM™ Gold Basal Medium(LONZA) 300㎕와 WST-1 시약 30㎕가 혼합된 혼합시약을 준비하고, 10일간 배양이 완료된 각 조직을 트랜스웰 인서트에서 분리하여 24웰 플레이트로 옮겨 바닥면에만 상기 혼합시약을 분주하고, 37℃, 5%C0 2, 상대습도 95%의 인큐베이터에서 4시간 동안 배양한 뒤, 웰 내의 혼합시약의 흡광도를 측정하였다.Specifically, a mixed reagent in which 300 µl of KBM™ Gold Basal Medium (LONZA) and 30 µl of WST-1 reagent are mixed is prepared, and each tissue cultured for 10 days is separated from a transwell insert and transferred to a 24-well plate. The mixed reagent was dispensed only and incubated for 4 hours in an incubator at 37° C., 5% CO 2 , and 95% relative humidity, and the absorbance of the mixed reagent in the well was measured.
흡광도는 440nm의 파장범위에서 측정되었으며, 표 1에는 기준 파장값인 650nm의 파장값과 조직이 없는 혼합시약의 파장값을 제외한 순수 조직의 파장값을 기재하였다.The absorbance was measured in the wavelength range of 440nm, and Table 1 shows the wavelength value of pure tissue excluding the wavelength value of 650nm, which is the reference wavelength, and the wavelength value of the mixed reagent without tissue.
Figure PCTKR2020009873-appb-img-000001
Figure PCTKR2020009873-appb-img-000001
상기 표 1의 실험 결과를 참조하면, 비교예의 경우에는 표피층 전체 면에서의 세포 생존율이 불균일한 것으로 확인되었다. 이는, 3차원 세포 배양 조직체가 수축되어 발생한 간극으로 표피층을 구성하는 세포의 이동이 일어나기 때문에 나타나는 문제로 확인되었다.반면, 실시예의 경우에는 표피층 전체 면에서의 세포 생존율이 균일하게 나타났는데, 이는 홍합 접착 단백질에 의해 3차원 세포 배양 조직체의 수축이 방지되어 세포 이동이 일어나지 않고, 각질형성세포를 분주한 상부층 영역에서만 세포의 분화 및 증식이 일어났기 때문에 나타난 결과로 확인되었다.Referring to the experimental results in Table 1, in the case of the comparative example, it was confirmed that the cell viability on the entire surface of the epidermal layer was non-uniform. This was confirmed as a problem due to the movement of cells constituting the epidermal layer into the gaps caused by contraction of the 3D cell culture tissue body. On the other hand, in the case of the Example, the cell viability was uniform over the entire surface of the epidermal layer, which is mussels. The result was confirmed because the contraction of the three-dimensional cell cultured tissue was prevented by the adhesive protein, so that cell migration did not occur, and the differentiation and proliferation of cells occurred only in the upper layer region where keratinocytes were dispensed.
따라서, 본 실험 결과로부터 홍합 접착 단백질을 사용하여 트랜스웰 인서트에 3차원 세포 배양 조직체를 배양할 경우, 각 제품별 품질이 균일하게 유지되어, 이를 이용하여 약물이나 화장품의 효능, 독성 등의 평가 실험을 수행할 때, 실험 결과의 신뢰성을 높일 수 있음을 알 수 있다.Therefore, from the results of this experiment, when a three-dimensional cell culture tissue is cultivated in a transwell insert using mussel adhesive protein, the quality of each product is maintained uniformly, and the evaluation experiment on the efficacy and toxicity of drugs or cosmetics using this When performing, it can be seen that the reliability of the experimental results can be improved.
본 발명은 상술한 특정의 실시예 및 설명에 한정되지 아니하며, 청구범위에서 청구하는 본 발명의 요지를 벗어남이 없이 당해 발명이 속하는 기술 분야에서 통상의 지식을 가진 자라면 누구든지 다양한 변형 실시가 가능하며, 그와 같은 변형은 본 발명의 보호 범위 내에 있게 된다.The present invention is not limited to the specific embodiments and description described above, and any person with ordinary knowledge in the technical field to which the present invention pertains without departing from the gist of the present invention claimed in the claims can implement various modifications And, such modifications are within the scope of protection of the present invention.
본 발명에 따른 3차원 세포 배양 조직체를 포함하는 테스트 장치는, 3차원 세포 배양 조직체를 트랜스웰 인서트와 같은 지지체 내에서 배양할 때 발생하는 3차원 세포 배양 조직체의 수축을 방지할 수 있으므로, 산업상 이용가능성이 존재한다.The test apparatus including the three-dimensional cell culture tissue according to the present invention can prevent the contraction of the three-dimensional cell culture tissue that occurs when the three-dimensional cell culture tissue is cultivated in a support such as a transwell insert. Availability exists.

Claims (18)

  1. 배양 구조체;Culture construct;
    상기 배양 구조체 내측에 배치되는 3차원 세포 배양 조직체; 및A three-dimensional cell culture structure disposed inside the culture structure; And
    상기 배양 구조체와 3차원 세포 배양 조직체 사이의 적어도 일부 계면에 배치되는 바이오 접착제;를 포함하고, Including; a bio-adhesive disposed on at least a partial interface between the culture structure and the three-dimensional cell culture tissue,
    상기 배양 구조체는, 몸체, 상기 몸체를 관통하는 홀 및 상기 홀의 일단을 덮도록 형성되는 다공성 멤브레인을 포함하는, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치.The culture structure, comprising a body, a hole penetrating the body, and a porous membrane formed to cover one end of the hole, a bio-adhesive pre-treated three-dimensional cell tissue culture and testing apparatus.
  2. 제1항에 있어서, 상기 바이오 접착제는,The method of claim 1, wherein the bio-adhesive,
    홍합 접착 단백질, 피브린 글루, 젤라틴 글루, 시아노아크릴레이트계 바이오 접착제 및 폴리우레탄계 바이오 접착제 중 적어도 어느 하나 이상을 포함하는 것을 특징으로 하는, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치.Mussel adhesive protein, fibrin glue, gelatin glue, cyanoacrylate-based bio-adhesive and polyurethane-based bio-adhesive comprising at least one or more of, characterized in that the bio-adhesive pre-treated three-dimensional cell tissue culture and testing apparatus.
  3. 제2항에 있어서, 상기 바이오 접착제는, The method of claim 2, wherein the bio-adhesive,
    홍합 접착 단백질인 것을 특징으로 하는, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치.A device for culturing and testing a three-dimensional cell tissue body pretreated with a bio-adhesive, characterized in that the mussel adhesive protein.
  4. 제1항에 있어서, 상기 바이오 접착제는,The method of claim 1, wherein the bio-adhesive,
    3차원 세포 배양 조직체와 상기 배양 구조체의 다공성 멤브레인 사이에 배치되는 것을 특징으로 하는, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치.A device for culturing and testing a three-dimensional cell tissue body pretreated with a bio-adhesive, characterized in that it is disposed between the three-dimensional cell culture body and the porous membrane of the culture structure.
  5. 제4항에 있어서, 상기 바이오 접착제는,The method of claim 4, wherein the bio adhesive,
    3차원 세포 배양 조직체와 다공성 멤브레인 사이에 배치되되, 상기 몸체를 관통하는 홀의 내측면을 따라 3차원 세포 배양 조직체의 측면으로 연장 배치되는 것을 특징으로 하는, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치.It is disposed between the three-dimensional cell culture structure and the porous membrane, characterized in that extending to the side of the three-dimensional cell culture structure along the inner side of the hole penetrating the body, the culture of the three-dimensional cell tissue body pretreated with a bio-adhesive And testing device.
  6. 제4항에 있어서, The method of claim 4,
    상기 3차원 세포 배양 조직체의 하면은, 바이오 접착제에 의해 다공성 멤브레인에 부착되며,The lower surface of the three-dimensional cell culture tissue is attached to the porous membrane by a bio-adhesive,
    상기 3차원 세포 배양 조직체의 적어도 일부 측면, 혹은 측면 전체가 상기 바이오 접착제에 의해 홀의 내측면에 부착되는 것을 특징으로 하는, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치.At least a portion of the side surface or the entire side surface of the three-dimensional cell culture structure is attached to the inner side of the hole by the bio-adhesive, a bio-adhesive pre-treated three-dimensional cell tissue culture and testing apparatus.
  7. 제1항에 있어서, 상기 3차원 세포 배양 조직체는,The method of claim 1, wherein the three-dimensional cell culture body,
    천연고분자 내에 세포 혹은 조직이 분산된 젤 형태인 것을 특징으로 하는, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치.A device for culturing and testing a three-dimensional cell tissue body pretreated with a bio-adhesive, characterized in that it is in the form of a gel in which cells or tissues are dispersed in a natural polymer.
  8. 제7항에 있어서, 상기 세포는, The method of claim 7, wherein the cell,
    줄기세포(stem cell), 조골세포(osteoblast), 근아세포(myoblast), 건세포(tenocyte), 신경아세포(neuroblast), 섬유아세포(fibroblast), 신경교아세포(glioblast), 배세포(germcell), 간세포(hepatocyte), 신장세포(renal cell), 지대세포(Sertoli cell), 연골세포(chondrocyte), 상피세포(epithelial cell), 심혈관세포, 각질세포(keratinocyte), 평활근세포(smooth muscle cell), 심장근세포(cardiomyocyte), 신경교세포(glial cell), 내피세포(endothelial cell), 호르몬 분비세포, 면역세포, 췌장섬세포(pancreatic islet cell) 및 신경세포(neuron)로 이루어진 군에서 선택된 어느 하나 이상인 것을 특징으로 하는, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치.Stem cells, osteoblasts, myoblasts, tenocytes, neuroblasts, fibroblasts, glioblasts, germcells, hepatocytes (hepatocyte), renal cell, seroli cell, chondrocyte, epithelial cell, cardiovascular cell, keratinocyte, smooth muscle cell, cardiomyocyte (cardiomyocyte), glial cell, endothelial cell, hormone secreting cell, immune cell, pancreatic islet cell, and neuron, characterized in that at least one selected from the group consisting of , Bio-adhesive pre-treated three-dimensional cell tissue culture and testing device.
  9. 제1항에 있어서, The method of claim 1,
    상기 3차원 세포 배양 조직체(300)는, 두 종류 이상의 세포를 포함하되, The three-dimensional cell culture tissue body 300 includes two or more types of cells,
    두 종류 이상의 세포가 균일하게 분산되거나, 두 종류 이상의 세포가 서로 분리되어 계층적 구조를 형성하는 것을 특징으로 하는, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치.Two or more types of cells are uniformly dispersed, or two or more types of cells are separated from each other to form a hierarchical structure, characterized in that the bio-adhesive pre-treated three-dimensional cell tissue culture and testing apparatus.
  10. 제9항에 있어서, The method of claim 9,
    상기 계층적 구조는, 진피층과 표피층의 두 개의 층을 갖는 계층적 구조인 것을 특징으로 하는, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치.The hierarchical structure, characterized in that the hierarchical structure having two layers of a dermal layer and an epidermal layer, a bio-adhesive pre-treated three-dimensional cell tissue culture and testing apparatus.
  11. 제1항에 있어서, 상기 다공성 멤브레인은,The method of claim 1, wherein the porous membrane,
    액채배지가 투과될 수 있는 미세다공성 막인 것을 특징으로 하는, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치.A device for culturing and testing a three-dimensional cell tissue body pretreated with a bio-adhesive, characterized in that it is a microporous membrane through which the liquid medium is permeable.
  12. 제1항에 있어서, 상기 다공성 멤브레인은,The method of claim 1, wherein the porous membrane,
    접착제 혹은 접착 테이프를 통해 몸체에 부착되는 것을 특징으로 하는, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치.A device for culturing and testing a three-dimensional cell tissue body pretreated with bio-adhesive, characterized in that it is attached to the body through an adhesive or adhesive tape.
  13. 제12항에 있어서,The method of claim 12,
    커버에 의해 다공성 멤브레인의 둘레부가 커버와 몸체 사이에 고정되는 것을 특징으로 하는, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치.A device for culturing and testing a three-dimensional cell tissue body pretreated with a bio-adhesive, characterized in that the circumference of the porous membrane is fixed between the cover and the body by the cover.
  14. 몸체; 상기 몸체를 관통하는 홀; 및 상기 홀의 일단을 덮도록 형성되는 다공성 멤브레인;을 포함하는 배양 구조체의 내측에 바이오 접착제를 도포하는 바이오 접착제 도포 단계, Body; A hole through the body; And a porous membrane formed to cover one end of the hole; applying a bio-adhesive to the inside of the culture structure comprising a bio-adhesive,
    상기 바이오 접착제를 건조하는 건조 단계 및A drying step of drying the bioadhesive and
    적어도 일부가 상기 바이오 접착제와 맞닿도록 3차원 세포 배양 조직체를 배양 구조체의 내측에 배치하는 3차원 세포 배양 조직체 배치 단계를 포함하는, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치의 제조방법.A method for culturing and testing a 3D cell tissue body pretreated with a bio-adhesive comprising the step of arranging a 3D cell culture tissue body on the inside of the culture structure so that at least a portion thereof contacts the bio-adhesive .
  15. 제14항에 있어서, 상기 바이오 접착제는,The method of claim 14, wherein the bio-adhesive,
    홍합 접착 단백질, 피브린 글루, 젤라틴 글루, 시아노아크릴레이트계 바이오 접착제 및 폴리우레탄계 바이오 접착제 중 적어도 어느 하나 이상을 포함하는 것을 특징으로 하는, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치의 제조방법.Mussel adhesive protein, fibrin glue, gelatin glue, cyanoacrylate-based bio-adhesive and polyurethane-based bio-adhesive comprising at least one or more of the bio-adhesive pre-treated three-dimensional cell tissue culture and test device Manufacturing method.
  16. 제14항에 있어서, 상기 바이오 접착제는, The method of claim 14, wherein the bio-adhesive,
    3차원 세포 배양 조직체와 다공성 멤브레인 사이에 배치되되, 상기 몸체를 관통하는 홀의 내측면을 따라 3차원 세포 배양 조직체의 측면으로 연장 배치되는 것을 특징으로 하는, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치의 제조방법.It is disposed between the three-dimensional cell culture structure and the porous membrane, characterized in that extending to the side of the three-dimensional cell culture structure along the inner surface of the hole penetrating the body, the culture of the three-dimensional cell tissue body pretreated with bio-adhesive And a method of manufacturing a test device.
  17. 제14항에 있어서,The method of claim 14,
    상기 3차원 세포 배양 조직체의 하면은, 바이오 접착제에 의해 다공성 멤브레인에 부착되며, The lower surface of the three-dimensional cell culture tissue is attached to the porous membrane by a bio-adhesive,
    상기 3차원 세포 배양 조직체의 적어도 일부 측면, 혹은 측면 전체가 상기 바이오 접착제에 의해 홀의 내측면에 부착되는 것을 특징으로 하는, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치의 제조방법.At least a portion of the side surface or the entire side surface of the 3D cell culture structure is attached to the inner side of the hole by the bio-adhesive. A method for culturing and testing a 3D cell tissue body pretreated with a bio-adhesive.
  18. 제14항에 있어서, 상기 3차원 세포 배양 조직체는,The method of claim 14, wherein the three-dimensional cell culture tissue,
    천연고분자 내에 세포 혹은 조직이 분산된 젤 형태인 것을 특징으로 하는, 바이오 접착제가 전처리된 3차원 세포 조직체의 배양 및 테스트 장치의 제조방법.A method of manufacturing a device for culturing and testing a three-dimensional cell tissue body pretreated with a bio-adhesive, characterized in that it is in the form of a gel in which cells or tissues are dispersed in a natural polymer.
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