WO2007074500A1 - Method of culturing three-dimensional tissue including living organism configuration - Google Patents

Method of culturing three-dimensional tissue including living organism configuration Download PDF

Info

Publication number
WO2007074500A1
WO2007074500A1 PCT/JP2005/023735 JP2005023735W WO2007074500A1 WO 2007074500 A1 WO2007074500 A1 WO 2007074500A1 JP 2005023735 W JP2005023735 W JP 2005023735W WO 2007074500 A1 WO2007074500 A1 WO 2007074500A1
Authority
WO
WIPO (PCT)
Prior art keywords
tissue
dimensional
living body
culturing
load
Prior art date
Application number
PCT/JP2005/023735
Other languages
French (fr)
Japanese (ja)
Inventor
Ken Nakata
Kunihiko Hosotani
Original Assignee
Osaka University
Technoview, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Osaka University, Technoview, Inc. filed Critical Osaka University
Priority to PCT/JP2005/023735 priority Critical patent/WO2007074500A1/en
Priority to JP2007551819A priority patent/JP4919296B2/en
Publication of WO2007074500A1 publication Critical patent/WO2007074500A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • 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

Definitions

  • the present invention relates to a method for culturing a three-dimensional tissue including a living body shape, and more particularly, to a three-dimensional tissue including a living body shape in which the tissue can be cultured in a shape or state approximate to the tissue in the living body.
  • the present invention relates to a tissue culture method.
  • the present invention provides a method for culturing a three-dimensional tissue including a living body shape capable of culturing a tissue in a shape and state similar to those in a living body.
  • the purpose is to provide.
  • the method for culturing a three-dimensional tissue including a living body shape according to the first invention comprises the following steps (al) to (f 1).
  • the method for cultivating a three-dimensional tissue including a living body shape comprises the following steps (a2) to (e2).
  • Step of obtaining an image of a lesion site or surrounding tissue to be transplanted by imaging Step of generating three-dimensional body shape data of the site from the image information of the taken lesion site or surrounding tissue to be transplanted
  • the method for cultivating a three-dimensional tissue including a living body shape according to the third invention comprises the following steps (a3) to (d3).
  • a vertical load stimulus and / or a transverse shear stress stimulus can be applied.
  • a three-dimensional living body shape produced based on image information of a lesion site or a surrounding tissue to be transplanted is used. Since the tissue is cultured, the tissue can be cultured in a shape and state that approximates the tissue in the living body, and the ability to improve the adaptability of the cultured body to the living body can be improved.
  • the tissue is cultured using a culture container prepared based on the three-dimensional living body shape data. Therefore, the tissue can be cultured in a shape and state approximate to the tissue in the living body, and the compatibility with the living body at the time of transplantation of the cultured body can be improved.
  • the same stimulus as the mechanical stimulus generated in the tissue in vivo on the ground can be applied to tissues in culture, and cells can be cultured in a state close to the mechanical environment of tissues in the living body. This makes it possible to efficiently repair and mature living tissues.
  • FIG. 1 is an exploded perspective view showing an example of a saddle type used in the method for culturing a three-dimensional tissue including a living body shape of the present invention.
  • FIG. 2 An upper mold of the same type is shown, (a) is a perspective view seen from the bottom side, (b) is a perspective view seen from the top side, and (c) is a main part seen from the bottom side. It is a perspective view.
  • FIG. 3 shows a lower mold of the same type, in which (a) is a perspective view seen from the upper surface side, and (b) is a perspective view seen from the lower surface side.
  • FIG. 4 is a perspective view in which a part of the same type assembled state is broken.
  • FIG. 5 is an overall explanatory view showing a biomechanical stimulus loading device.
  • FIG. 6 is an explanatory diagram of a main part of the biomechanical stimulation load device.
  • FIG. 7 is an explanatory diagram of a main part of the biomechanical stimulation load device.
  • C culture tissue consisting of cells and scaffolds
  • the method for culturing a three-dimensional tissue including a living body shape of the present invention includes a tissue constituting a human body such as a joint tissue such as a knee, a crotch, an elbow, and a shoulder, and a cartilage tissue such as a nose and an ear, such as a joint.
  • the present invention relates to a method for culturing bone and cartilage constituting a tissue in a shape and state approximate to tissue in a living body, and includes the following steps (al) to (fl).
  • a tissue having a three-dimensional living body shape composed of cells and scaffolds is produced in substantially one step using a saddle type.
  • An image of the lesion site or the surrounding tissue to be transplanted is taken in the surrounding site or the tissue to be transplanted (for example, in the case of a patient having a lesion in the joint, the patient's
  • the [] part) is obtained by MRI (.magnetic resonance imaging) imaging or CT (computerized tornography) imaging, and an image of the site is obtained.
  • MRI magnetic resonance imaging
  • CT computerized tornography
  • the lesion site or surrounding tissue to be transplanted is obtained.
  • the three-dimensional living body shape data of the part is created by using the computer aided design (3D CAD) by processing the image information.
  • 3D CAD computer aided design
  • the three-dimensional living body shape data of the healthy opposite side of the patient can be used.
  • the three-dimensional body shape data of the lesion site obtained in this way or the surrounding tissue to be transplanted is cut using a three-dimensional CAM (computer aided manufactu ring). Deliver to the machine and make a saddle made of synthetic resin or metal suitable for the living body.
  • a three-dimensional CAM computer aided manufactu ring
  • Figs. 1 to 4 show a saddle 6 produced through the above-described steps.
  • This saddle type 6 is for the meniscus, which is the cartilage tissue of the knee joint, and forms a cavity with a meniscus shape between the upper die 61 and the lower die 62.
  • a convex portion 61a corresponding to the shape of the meniscus is formed, and a concave portion 62a corresponding to the shape of the meniscus is formed on the lower mold 62 side.
  • the shape of the cavity part is collagen having a three-dimensional biological shape produced in a later process, It can be formed larger or smaller than the shape of the actual living tissue of interest in accordance with the characteristics of the scaffolds such as alginate and polylactic acid (shrinkage / expansion characteristics after fabrication).
  • Upper mold 61 and lower mold 62 are formed with bolt holes 61b and 62b at the four corners and fastened with Bonoleto 63 so that a substantially sealed cavity having a meniscus shape can be formed.
  • knock holes 61c and 62c are formed at two locations and the knock 64 is inserted, thereby positioning the upper mold 61 and the lower mold 62.
  • the upper mold 61 is formed with a gel injection hole 6 Id and a vent hole 61 e communicating with the cavity.
  • collagen in a fluidized state is passed through the gel injection hole 61d into the cavity formed by the upper mold 61 and the lower mold 62, which are fastened and integrated with the saddle mold 6 and the bolt 63. After injection, it is cured to produce a collagen scaffold with a three-dimensional biological shape.
  • appropriate cells such as the patient's own autologous cells are introduced into the scaffold thus obtained, and a tissue having a three-dimensional biological shape is cultured.
  • the tissue can be cultured in a general culture vessel.
  • a culture vessel prepared based on the three-dimensional biological shape data for example, a bone and / or a joint part is used.
  • 3D biological shape data of cartilage is transferred to a cutting machine using 3D CAM (computer aided manufacturing), and is performed using a culture vessel made of synthetic resin or metal material suitable for the living body. Can do.
  • the tissue can be cultured in a shape and state more similar to those in the living body.
  • the culture vessel prepared based on the three-dimensional living body shape data does not use a tissue other than the culture of the tissue having the three-dimensional living body shape composed of the cells and the scaffold, specifically, the saddle type 6 described above.
  • the present invention can also be applied to culture of a tissue composed of cells and scaffolds prepared in the above.
  • tissue culture can be performed in a general culture vessel under no stimulation, but if necessary, the tissue composed of cells and scaffolds can be subjected to vertical load loading stimulation and / or By applying a shear stress stimulus in the lateral direction, a stimulus similar to the mechanical stimulus generated in the tissue in vivo on the ground is applied to the tissue in culture. You can.
  • cells can be cultured in a state that approximates the mechanical environment of the tissue in the living body, so that it becomes possible to create a differentiated and mature tissue that can withstand the mechanical load after transplantation, and repair the tissue of the living body. , Maturation can be done efficiently.
  • the vertical load load stimulus and the Z or transverse shear stress stimulus are applied using a biomechanical stimulus load device as previously proposed by the applicant in PCT / JP2005 / 11045. It can be carried out.
  • the biomechanical stimulation load device includes a mechanical stimulation load device 2 and a culture vessel 3 installed in a carbon dioxide incubator 1, and a control computer 4. It is composed of and.
  • All operations of the biomechanical stimulation load device can be performed from outside the carbon dioxide incubator 1, which enables culturing for a long time while maintaining the sterilization state in the carbon dioxide incubator 1. Can do.
  • the carbon dioxide incubator 1 is sterilized and used in an environment in which temperature, humidity, oxygen, carbon dioxide, nitrogen partial pressure, etc. are controlled.
  • the mechanical stimulation load device 2 installed in the carbon dioxide incubator 1 includes a load-loading piston 5 and a piston moving up and down that supports the load-loading piston 5 so as to be movable in a vertical direction within a predetermined range.
  • the piston By driving the stage elevating mechanism 22 in a predetermined cycle by the control computer 4, the piston
  • the weight of the load-loading piston 5 is the culture in the culture vessel 3 (an organization composed of cells and scaffolds; the same applies hereinafter). .) It is configured to take C.
  • a stage elevating mechanism 22 for moving the piston vertical movement stage 21 in the vertical direction is connected to, for example, an electric actuator 22a and the electric actuator 22a, and the piston vertical movement stage 21 is suspended. It consists of the lower wire 22b. Then, by operating the electric actuator 22a in accordance with a command from the control computer 4, the piston vertical movement stage 21 can be moved vertically along the guide member 20 via the wire 22b.
  • the load-loading piston 5 is supported through the piston mounting stage 23 mounted on the piston vertical movement stage 21.
  • the piston vertical movement stage 21 is for mounting the piston mounting stage 23, so that the load-loading piston 5 can freely move in the vertical direction at the center.
  • the hole 21a is formed.
  • the piston loading stage 23 may be omitted, and the load loading piston 5 may be directly supported on the piston vertical movement stage 21.
  • the load-loading piston 5 has a pressurizing portion 51 having a shape corresponding to the culture C at the lower end portion and a guide shaft portion 52 that is fitted into a hole portion 23a formed in the piston mounting stage 23 at the intermediate portion.
  • a large-diameter portion 53 is formed on the upper portion, and a heavy load attaching portion 54 for attaching an additional load heavy load 55 is formed on the upper end portion.
  • the load load stimulus in the vertical direction by the load-loading piston 5 can freely operate the load cycle and load time by operating the control computer 4.
  • the additional load heavy load 55 can be attached to the heavy load attachment portion 54 as necessary, and its weight can be freely set, so that the vertical load load stimulus applied to the culture C is large. The thickness can be easily adjusted.
  • the shape of the pressurizing portion 51 of the load-loading piston 5 can be any shape corresponding to the culture C, and the pressurizing portion 51 having a shape corresponding to the culture C can be separated. It is also possible to form the member and attach it to the lower end of the load-loading piston 5.
  • the load loading piston 5 can be supported on the piston mounting stage 23 (or the piston vertical movement stage 21) so as not to rotate when moving in the vertical direction.
  • the hole portion 23a formed in the piston mounting stage 23 is formed in a polygonal shape, and the guide shaft portion 52 is formed to have a polygonal cross-sectional shape adapted to the polygonal hole portion 23a. To do.
  • the culture vessel 3 is detachably attached to the culture vessel fixing stage 24 of the mechanical stimulation load device 2.
  • a culture vessel (for injection) 32 and a culture vessel (for discharge) 33 are connected to the culture vessel 3 as necessary, and the injection and discharge of the culture medium are controlled by the control computer 4. Say it with a word.
  • a shear stress loading mechanism 26 for moving or vibrating the culture vessel 3 in a horizontal plane is provided.
  • a shear stress stage 25 is arranged on the culture vessel fixing stage 24 so as to be movable in a horizontal plane with respect to the culture vessel fixing stage 24, and the culture vessel 3 is mounted on the shear stress stage 25.
  • the shear stress stage 25 is moved or vibrated in the horizontal plane by the shear stress loading mechanism 26.
  • the direction of movement or vibration in the horizontal plane is not limited to one direction, and can be any direction such as two directions in the X and Y directions and circular motion.
  • the shear stress loading mechanism 26 for example, an electric actuator, a moving mechanism or a vibration mechanism combining a permanent magnet and an electromagnetic stone can be used, and the driving of the shear stress loading mechanism 26 is performed by the control computer 4. Try to control.
  • the shear stress load mechanism 26 moves or vibrates the culture vessel 3 in the horizontal plane, thereby applying the shear stress stimulus in the oblique direction.
  • an internal culture vessel 30 having an arbitrary shape suitable for the culture body C is installed, and the load-loading piston 5 of the internal culture vessel 30 is installed.
  • a receiving group 31 of a structure containing cultured cells, cultured tissues or cultured cells is mounted at a position opposite to the pressurizing unit 51, and a cultured cell, cultured tissue or cultured cell as a cultured body C is placed on the receiving group 31. It can be made to mount a structure including.
  • FIG. 7 shows an example of a biomechanical stimulus loading device that is preferably used for culturing meniscus, articular lip, joint disc, and the like.
  • the meniscus, labial lip, joint disc, etc. are subjected to load stress stimulation by osteochondral rather than being stressed in vivo with hardness similar to bone.
  • the load-load stimulating part X has a structure similar to a predetermined shape (for example, knee joint) of a living body, and the culture body C is placed in the culture-body load-load stimulating part X, so that the bone cartilage (for example, the knee joint) To reproduce the load-induced stimulus by bone cartilage.
  • the culture vessel 3 was filled with the culture medium L, and a recess 30a having a shape corresponding to the culture body C, which was made of a synthetic resin of a biocompatible material similar to femoral bone cartilage was formed. Install the lower culture vessel 30A, and place the culture C on it.
  • the pressurization part 51 of the load-loading piston 5 is provided with an upper culture container 30B having a convex part 30b having a shape corresponding to the culture C so as to face the lower culture container 30A. Do it.
  • a culture body obtained by culturing a tissue composed of cells and a scaffold has a shape and a state approximate to a tissue in the living body. Since it is cultured, the compatibility with the living body at the time of transplantation of the culture is improved.
  • the method for culturing a three-dimensional tissue including a living body shape of the present invention has been described based on the examples thereof, but the present invention is not limited to the configurations described in the above examples. The configuration can be changed as appropriate without departing from the scope of the invention.
  • the method for culturing a three-dimensional tissue including a living body shape of the present invention can culture a tissue in a shape and state similar to the tissue in the living body, joint tissues such as knees, crotch, elbows, shoulders, etc. It is preferably used for culturing tissues constituting the human body, such as cartilage tissues such as nose and ears, for example, bone and soft bones constituting joint tissues, in a shape and a state approximating those in the living body. it can.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Dermatology (AREA)
  • Transplantation (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Botany (AREA)
  • Materials For Medical Uses (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

A method of culturing a three-dimensional tissue including living organism configuration in which tissue culturing can be performed in the state and configuration approximate to those of tissue within living organism. There is provided a method of culturing a three-dimensional tissue including living organism configuration, comprising the steps of: (a1) obtaining by photographing the image of diseased site or peripheral tissue of transplant planned; (b1) preparing three-dimensional living organism configuration data of the site from the image information on the photographed diseased site or peripheral tissue of transplant planned; (c1) preparing a mold on the basis of the three-dimensional living organism configuration data; (d1) preparing a scaffold with three-dimensional living organism configuration by the use of the mold; (e1) preparing a tissue with three-dimensional living organism configuration by introduction of cells on the scaffold; and (f1) culturing the tissue.

Description

明 細 書  Specification
生体形状を含んだ三次元組織の培養方法  Method for culturing three-dimensional tissue including biological shape
技術分野  Technical field
[0001] 本発明は、生体形状を含んだ三次元組織の培養方法に関し、特に、生体内の組織 に近似した形状や状態で組織を培養することができるようにした生体形状を含んだ三 次元組織の培養方法に関するものである。  TECHNICAL FIELD [0001] The present invention relates to a method for culturing a three-dimensional tissue including a living body shape, and more particularly, to a three-dimensional tissue including a living body shape in which the tissue can be cultured in a shape or state approximate to the tissue in the living body. The present invention relates to a tissue culture method.
背景技術  Background art
[0002] 近年、例えば、関節部に病変をもつ患者の治療として、患者本人の残された健常な 軟骨細胞を採取し、体外に取り出し、軟骨細胞を分離し、体外培養にて増殖し、この 自家培養軟骨細胞を元の患者に移植する細胞移植治療方法が、欧米等で行われる よつになった。  [0002] In recent years, for example, as a treatment for a patient having a lesion in a joint, healthy chondrocytes left by the patient are collected, taken out of the body, separated, and proliferated in an in vitro culture. Cell transplantation treatment methods in which autologous cultured chondrocytes are transplanted to the original patient have been performed in Europe and the United States.
この方法は、軟骨細胞の培養を体外で行うことができる反面、生体内の組織に近似 した形状や状態で軟骨細胞の培養を行うものではなレ、ため、病変部位部と形状や性 状が一致しない問題点があった。  Although this method can cultivate chondrocytes outside the body, it does not cultivate chondrocytes in a shape or state that approximates tissue in the living body. There was a problem that did not match.
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0003] 本発明は、上記従来の細胞の培養方法の有する問題点に鑑み、生体内の組織に 近似した形状や状態で組織を培養することができる生体形状を含んだ三次元組織の 培養方法を提供することを目的とする。  [0003] In view of the problems of the above conventional cell culture methods, the present invention provides a method for culturing a three-dimensional tissue including a living body shape capable of culturing a tissue in a shape and state similar to those in a living body. The purpose is to provide.
課題を解決するための手段  Means for solving the problem
[0004] 上記目的を達成するため、本第 1発明の生体形状を含んだ三次元組織の培養方 法は、下記（al)〜（f 1)の工程からなることを特徴とする。  In order to achieve the above object, the method for culturing a three-dimensional tissue including a living body shape according to the first invention comprises the following steps (al) to (f 1).
(al)病変部位あるいは移植する予定の周囲組織の画像を撮影により得る工程  (al) A process of obtaining an image of the lesion site or surrounding tissue to be transplanted by imaging
(bl)前記撮影した病変部位あるいは移植する予定の周囲組織の画像情報から当該 部位の三次元生体形状データを作製する工程  (bl) A step of creating three-dimensional body shape data of the site from the imaged lesion site or image information of the surrounding tissue to be transplanted
(cl)前記三次元生体形状データに基づいて铸型を作製する工程  (cl) A step of producing a saddle shape based on the three-dimensional biological shape data
(dl)前記铸型を用いて三次元生体形状をもつ足場を作製する工程 (el)前記足場に細胞を導入して三次元生体形状をもつ組織を作成する工程 (fl)前記組織を培養する工程 (dl) A step of producing a scaffold having a three-dimensional living body shape using the saddle type (el) a step of creating a tissue having a three-dimensional biological shape by introducing cells into the scaffold (fl) a step of culturing the tissue
[0005] また、同じ目的を達成するため、本第 2発明の生体形状を含んだ三次元組織の培 養方法は、下記（a2)〜（e2)の工程からなることを特徴とする。 [0005] In order to achieve the same object, the method for cultivating a three-dimensional tissue including a living body shape according to the second invention comprises the following steps (a2) to (e2).
(a2)病変部位あるいは移植する予定の周囲組織の画像を撮影により得る工程 (b2)前記撮影した病変部位あるいは移植する予定の周囲組織の画像情報から当該 部位の三次元生体形状データを作製する工程  (a2) Step of obtaining an image of a lesion site or surrounding tissue to be transplanted by imaging (b2) Step of generating three-dimensional body shape data of the site from the image information of the taken lesion site or surrounding tissue to be transplanted
(c2)前記三次元生体形状データに基づいて铸型を作製する工程  (c2) A step of producing a saddle shape based on the three-dimensional living body shape data
(d2)前記錡型を用いて細胞と足場とからなる三次元生体形状をもつ組織を作製す る工程  (d2) Step of producing a tissue having a three-dimensional living body shape composed of cells and scaffolds using the saddle type
(e2)前記組織を培養する工程  (e2) culturing the tissue
[0006] また、同じ目的を達成するため、本第 3発明の生体形状を含んだ三次元組織の培 養方法は、下記（a3)〜（d3)の工程からなることを特徴とする。 [0006] In order to achieve the same object, the method for cultivating a three-dimensional tissue including a living body shape according to the third invention comprises the following steps (a3) to (d3).
(a3)病変部位あるいは移植する予定の周囲組織の画像を撮影により得る工程 (b3)前記撮影した病変部位あるいは移植する予定の周囲組織の画像情報から当該 部位の三次元生体形状データを作製する工程  (a3) Step of obtaining an image of a lesion site or surrounding tissue to be transplanted by imaging (b3) Step of creating three-dimensional body shape data of the site from the imaged image of the lesion site or surrounding tissue to be transplanted
(c3)前記三次元生体形状データに基づいて培養容器を作製する工程  (c3) A step of producing a culture vessel based on the three-dimensional biological shape data
(d3)前記培養容器を用いて細胞と足場とからなる組織を培養する工程  (d3) a step of culturing a tissue composed of cells and a scaffold using the culture vessel
[0007] この場合において、前記組織の培養時に、鉛直方向の荷重負荷刺激及び/又は 横方向の剪断応力刺激を付与することができる。 [0007] In this case, when the tissue is cultured, a vertical load stimulus and / or a transverse shear stress stimulus can be applied.
発明の効果  The invention's effect
[0008] 本第 1及び第 2発明の生体形状を含んだ三次元組織の培養方法によれば、病変部 位あるいは移植する予定の周囲組織の画像情報に基づいて作製した三次元生体形 状をもつ組織を培養するようにしているため、生体内の組織に近似した形状や状態 で組織の培養を行うことができ、培養体の移植時の生体に対する適合性を向上する こと力 Sできる。  [0008] According to the method for culturing a three-dimensional tissue including a living body shape according to the first and second inventions, a three-dimensional living body shape produced based on image information of a lesion site or a surrounding tissue to be transplanted is used. Since the tissue is cultured, the tissue can be cultured in a shape and state that approximates the tissue in the living body, and the ability to improve the adaptability of the cultured body to the living body can be improved.
[0009] また、本第 3発明の生体形状を含んだ三次元組織の培養方法によれば、組織の培 養を、三次元生体形状データに基づいて作製した培養容器を用いて行うようにして いるため、生体内の組織に近似した形状や状態で組織の培養を行うことができ、培 養体の移植時の生体に対する適合性を向上することができる。 [0009] In addition, according to the method for culturing a three-dimensional tissue including a living body shape of the third invention, the tissue is cultured using a culture container prepared based on the three-dimensional living body shape data. Therefore, the tissue can be cultured in a shape and state approximate to the tissue in the living body, and the compatibility with the living body at the time of transplantation of the cultured body can be improved.
[0010] また、前記組織の培養時に、鉛直方向の荷重負荷刺激及び/又は横方向の剪断 応力刺激を付与することにより、地上における生体内で組織に生じている力学的な 刺激と同様の刺激を培養中の組織に付与することができ、細胞を生体内の組織の力 学的な環境に近似した状態で培養することができるため、移植後の力学負荷に耐え 得る分化、成熟した組織の作成が可能となり、生体の組織の修復、成熟を効率的に 行うことができる。  [0010] Further, during the culture of the tissue, by applying a vertical load load stimulus and / or a shear stress stimulus in the lateral direction, the same stimulus as the mechanical stimulus generated in the tissue in vivo on the ground Can be applied to tissues in culture, and cells can be cultured in a state close to the mechanical environment of tissues in the living body. This makes it possible to efficiently repair and mature living tissues.
図面の簡単な説明  Brief Description of Drawings
[0011] [図 1]本発明の生体形状を含んだ三次元組織の培養方法に用いる铸型の一例を示 す分解した状態の斜視図である。  FIG. 1 is an exploded perspective view showing an example of a saddle type used in the method for culturing a three-dimensional tissue including a living body shape of the present invention.
[図 2]同铸型の上型を示し、（a)は底面側から見た斜視図、（b)は上面側から見た斜 視図、（c)は底面側から見た要部の斜視図である。  [Fig. 2] An upper mold of the same type is shown, (a) is a perspective view seen from the bottom side, (b) is a perspective view seen from the top side, and (c) is a main part seen from the bottom side. It is a perspective view.
[図 3]同铸型の下型を示し、（a)は上面側から見た斜視図、（b)は下面側から見た斜 視図である。  FIG. 3 shows a lower mold of the same type, in which (a) is a perspective view seen from the upper surface side, and (b) is a perspective view seen from the lower surface side.
[図 4]同铸型の組み立てた状態の一部を破断した斜視図である。  FIG. 4 is a perspective view in which a part of the same type assembled state is broken.
[図 5]生体力学的刺激負荷装置を示す全体説明図である。  FIG. 5 is an overall explanatory view showing a biomechanical stimulus loading device.
[図 6]同生体力学的刺激負荷装置の要部の説明図である。  FIG. 6 is an explanatory diagram of a main part of the biomechanical stimulation load device.
[図 7]同生体力学的刺激負荷装置の要部の説明図である。  FIG. 7 is an explanatory diagram of a main part of the biomechanical stimulation load device.
符号の説明  Explanation of symbols
[0012] 1 炭酸ガスインキュベーター [0012] 1 Carbon dioxide incubator
2 力学的刺激負荷装置  2 Mechanical stimulation load device
20 ガイド部材  20 Guide member
21 ピストン上下移動用ステージ  21 Piston vertical movement stage
21a 孔部  21a hole
22 ステージ昇降機構  22 Stage lifting mechanism
22a 電動ァクチユエ一ター  22a Electric actuator
22b ワイヤー 3 ピストン載置用ステージ22b wire 3 Piston mounting stage
3a 孔部3a hole
3b ピストン載置用ステージ駆動機構4 培養容器固定ステージ3b Piston mounting stage drive mechanism 4 Culture vessel fixed stage
5 剪断応力ステージ5 Shear stress stage
6 剪断応力負荷機構6 Shear stress loading mechanism
7 追加荷重重鎮上下移動用ステージ8 追加荷重重鎮載置用ステージ8b 追加荷重重鎮載置用ステージ 培養容器7 Additional load heavy lifting stage 8 Additional load heavy lifting stage 8b Additional load heavy lifting stage Culture vessel
0 内部培養容器0 Internal culture vessel
0A 下部培養容器0A Lower culture vessel
0a 凹部0a Recess
0B 上部培養容器0B Upper culture vessel
0b 凸部0b Convex
1 受容基1 acceptor group
2 培地槽 (注入用）2 Medium tank (for injection)
3 培地槽 (排出用） 3 Medium tank (for discharge)
制御用コンピューター  Control computer
荷重負荷用ピストン Piston for load application
a 荷重負荷用ピストンa Load piston
b 荷重負荷用ピストンb Piston for load
1 加圧体1 Pressurized body
1 A 人工大腿骨骨軟骨1 A artificial femur bone cartilage
2 ガイド軸部2 Guide shaft
3 大径部3 Large diameter part
4 重鎮装着部4 Heavyweight wearing part
5 追加荷重重鎮 61 上型 5 Additional load 61 Upper mold
61a 凸部  61a Convex
61b ボルト孔  61b Bolt hole
61c ノック穴  61c dowel hole
6 Id ゲル注入孔  6 Id gel injection hole
61e αΜ¾孑し  61e αΜ¾ 孑 し
62 下型  62 Lower mold
62a 凹部  62a recess
62b ボルト孔  62b Bolt hole
62c ノック穴  62c dowel hole
63 ボノレト  63 Bonoreto
64 ノック  64 Knock
C 培養体 (細胞と足場とからなる組織）  C culture (tissue consisting of cells and scaffolds)
L 培養液  L culture medium
X 培養体荷重負荷刺激部  X culture load stimulator
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0013] 以下、本発明の生体形状を含んだ三次元組織の培養方法の実施の形態を説明す る。 [0013] Hereinafter, an embodiment of a method for culturing a three-dimensional tissue including a living body shape of the present invention will be described.
実施例 1  Example 1
[0014] 本発明の生体形状を含んだ三次元組織の培養方法は、膝、股、肘、肩等の関節組 織、鼻、耳等の軟骨組織等の人体を構成する組織、例えば、関節組織を構成する骨 や軟骨を、生体内の組織に近似した形状や状態で、培養する方法に関するものであ り、下記（al)〜（fl)の工程からなる。  [0014] The method for culturing a three-dimensional tissue including a living body shape of the present invention includes a tissue constituting a human body such as a joint tissue such as a knee, a crotch, an elbow, and a shoulder, and a cartilage tissue such as a nose and an ear, such as a joint. The present invention relates to a method for culturing bone and cartilage constituting a tissue in a shape and state approximate to tissue in a living body, and includes the following steps (al) to (fl).
(al)病変部位あるいは移植する予定の周囲組織、例えば、関節部の画像を撮影に より得る工程  (al) A process to obtain images of the lesion site or surrounding tissue to be transplanted, such as joints, by imaging
(bl)前記撮影した病変部位あるいは移植する予定の周囲組織の画像情報から当該 部位の三次元生体形状データを作製する工程 (cl)前記三次元生体形状データに基づいて铸型を作製する工程 (bl) A step of creating three-dimensional body shape data of the site of the imaged lesion or the surrounding tissue to be transplanted (cl) A step of producing a saddle shape based on the three-dimensional biological shape data
(dl)前記铸型を用いて三次元生体形状をもつ足場を作製する工程 (この足場は、 細胞の足場となるもので、コラーゲン、アルジネート、ポリ乳酸等の材料を用いて作製 すること力 Sできる。）  (dl) A step of producing a scaffold having a three-dimensional living body shape using the saddle type (this scaffold serves as a scaffold for cells and can be produced using materials such as collagen, alginate, polylactic acid S it can.)
(el)前記足場に細胞を導入して三次元生体形状をもつ組織を作成する工程 (fl)前記組織を培養する工程  (el) A step of creating a tissue having a three-dimensional biological shape by introducing cells into the scaffold (fl) A step of culturing the tissue
[0015] この場合において、上記（dl)及び（el)の工程を、铸型を用いて細胞と足場とから なる三次元生体形状をもつ組織を実質的に 1工程で作製するようにすることもできる [0015] In this case, in the steps (dl) and (el) described above, a tissue having a three-dimensional living body shape composed of cells and scaffolds is produced in substantially one step using a saddle type. Can also
[0016] そして、病変部位あるいは移植する予定の周囲組織の画像の撮影は、病変部位あ るいは移植する予定の周囲組織 (例えば、関節部に病変をもつ患者の場合には患者 本人の関貧 []部)を、 MRI (.magnetic resonance imaging)撮影や CT (computerized torn ography)撮影することにより、当該部位の画像を得るもので、このようにして撮影した 病変部位あるいは移植する予定の周囲組織の画像情報をコンピューターで処理する ことにより、具体的には、三次元 CAD (computer aided design)を用いて、当該部位（ 関節部の骨及び/又は軟骨）の三次元生体形状データを作製する。 [0016] An image of the lesion site or the surrounding tissue to be transplanted is taken in the surrounding site or the tissue to be transplanted (for example, in the case of a patient having a lesion in the joint, the patient's The [] part) is obtained by MRI (.magnetic resonance imaging) imaging or CT (computerized tornography) imaging, and an image of the site is obtained. In this way, the lesion site or surrounding tissue to be transplanted is obtained. Specifically, the three-dimensional living body shape data of the part (the bone and / or the cartilage of the joint part) is created by using the computer aided design (3D CAD) by processing the image information.
なお、例えば、患者の関節部等の損傷が著しい場合等には、患者の健常な反対側 の部位の三次元生体形状データを利用することもできる。  Note that, for example, when the patient's joint or the like is significantly damaged, the three-dimensional living body shape data of the healthy opposite side of the patient can be used.
[0017] このようにして得た病変部位あるいは移植する予定の周囲組織（関節部の骨及び /又は軟骨）の三次元生体形状データを、三次元 CAM (computer aided manufactu ring)を用いて切削加工機に受け渡し、生体に適合した合成樹脂又は金属製の铸型 を作製する。  [0017] The three-dimensional body shape data of the lesion site obtained in this way or the surrounding tissue to be transplanted (joint bone and / or cartilage) is cut using a three-dimensional CAM (computer aided manufactu ring). Deliver to the machine and make a saddle made of synthetic resin or metal suitable for the living body.
[0018] 図 1〜図 4は、上記の工程を経て作製された铸型 6を示す。  [0018] Figs. 1 to 4 show a saddle 6 produced through the above-described steps.
この铸型 6は、膝関節部の軟骨組織である半月板用の錡型で、上型 61と下型 62と の間に半月板の形状をした空洞部を形成するため、上型 61側に半月板の形状に対 応した凸部 61aを、下型 62側に半月板の形状に対応した凹部 62aを、それぞれ形成 するようにしたものである。  This saddle type 6 is for the meniscus, which is the cartilage tissue of the knee joint, and forms a cavity with a meniscus shape between the upper die 61 and the lower die 62. In addition, a convex portion 61a corresponding to the shape of the meniscus is formed, and a concave portion 62a corresponding to the shape of the meniscus is formed on the lower mold 62 side.
ここで、空洞部の形状は、後工程で作製される三次元生体形状をもつコラーゲン、 アルジネート、ポリ乳酸等の足場の特性 (作製後の収縮 ·膨脹特性)等に合わせて、 対象となる実際の生体の組織の形状よりも大きく形成したり、小さく形成することがで きる。 Here, the shape of the cavity part is collagen having a three-dimensional biological shape produced in a later process, It can be formed larger or smaller than the shape of the actual living tissue of interest in accordance with the characteristics of the scaffolds such as alginate and polylactic acid (shrinkage / expansion characteristics after fabrication).
上型 61と下型 62とは、半月板の形状をした実質的に密閉された空洞部を形成する ことができるように、四隅にボルト孔 61b、 62bを形成し、ボノレト 63にて締結、一体化 できるようにするとともに、 2箇所にノック穴 61c、 62cを形成してノック 64を嵌入するこ とにより、上型 61と下型 62との位置決めを行うようにする。  Upper mold 61 and lower mold 62 are formed with bolt holes 61b and 62b at the four corners and fastened with Bonoleto 63 so that a substantially sealed cavity having a meniscus shape can be formed. In addition to being able to be integrated, knock holes 61c and 62c are formed at two locations and the knock 64 is inserted, thereby positioning the upper mold 61 and the lower mold 62.
また、上型 61には、空洞部に連通するゲル注入孔 6 Id及び通気孔 61 eを形成する ようにする。  Further, the upper mold 61 is formed with a gel injection hole 6 Id and a vent hole 61 e communicating with the cavity.
[0019] この铸型 6を用レ、、ボルト 63にて締結、一体化した上型 61と下型 62とによって形成 された空洞部内に、ゲル注入孔 61dを通して、例えば、流動状態のコラーゲンを注入 した後に硬化させ、三次元生体形状をもつコラーゲン製の足場を作製する。  [0019] For example, collagen in a fluidized state is passed through the gel injection hole 61d into the cavity formed by the upper mold 61 and the lower mold 62, which are fastened and integrated with the saddle mold 6 and the bolt 63. After injection, it is cured to produce a collagen scaffold with a three-dimensional biological shape.
[0020] このようにして得た足場に、例えば、患者本人の自家細胞等の適当な細胞を導入し 、三次元生体形状をもつ組織を培養する。  [0020] For example, appropriate cells such as the patient's own autologous cells are introduced into the scaffold thus obtained, and a tissue having a three-dimensional biological shape is cultured.
[0021] ここで、組織の培養は、一般的な培養容器で行うことができるが、必要に応じて、三 次元生体形状データに基づいて作製した培養容器、例えば、関節部の骨及び/又 は軟骨の三次元生体形状データを、三次元 CAM (computer aided manufacturing) を用いて切削加工機に受け渡し、生体に適合した合成樹脂又は金属材料で作製し た培養容器を用いて行うようにすることができる。  Here, the tissue can be cultured in a general culture vessel. If necessary, a culture vessel prepared based on the three-dimensional biological shape data, for example, a bone and / or a joint part is used. 3D biological shape data of cartilage is transferred to a cutting machine using 3D CAM (computer aided manufacturing), and is performed using a culture vessel made of synthetic resin or metal material suitable for the living body. Can do.
これにより、生体内の組織に一層近似した形状や状態で組織の培養を行うことがで きる。  As a result, the tissue can be cultured in a shape and state more similar to those in the living body.
なお、三次元生体形状データに基づいて作製した培養容器は、上記の細胞と足場 とからなる三次元生体形状をもつ組織の培養以外の組織、具体的には、上記の錡型 6を用いずに作製された細胞と足場とからなる組織の培養にも適用することができる。  Note that the culture vessel prepared based on the three-dimensional living body shape data does not use a tissue other than the culture of the tissue having the three-dimensional living body shape composed of the cells and the scaffold, specifically, the saddle type 6 described above. The present invention can also be applied to culture of a tissue composed of cells and scaffolds prepared in the above.
[0022] また、組織の培養は、一般的な培養容器で無刺激の下で行うことができるが、必要 に応じて、細胞と足場とからなる組織に、鉛直方向の荷重負荷刺激及び/又は横方 向の剪断応力刺激を付与して行うようにすることにより、地上における生体内で組織 に生じている力学的な刺激と同様の刺激を培養中の組織に付与して行うようにするこ とができる。 [0022] In addition, tissue culture can be performed in a general culture vessel under no stimulation, but if necessary, the tissue composed of cells and scaffolds can be subjected to vertical load loading stimulation and / or By applying a shear stress stimulus in the lateral direction, a stimulus similar to the mechanical stimulus generated in the tissue in vivo on the ground is applied to the tissue in culture. You can.
これにより、細胞を生体内の組織の力学的な環境に近似した状態で培養することが できるため、移植後の力学負荷に耐え得る分化、成熟した組織の作成可能となり、生 体の組織の修復、成熟を効率的に行うことができる。  As a result, cells can be cultured in a state that approximates the mechanical environment of the tissue in the living body, so that it becomes possible to create a differentiated and mature tissue that can withstand the mechanical load after transplantation, and repair the tissue of the living body. , Maturation can be done efficiently.
[0023] ところで、鉛直方向の荷重負荷刺激及び Z又は横方向の剪断応力刺激の付与は 、本件出願人が先に PCT/JP2005/11045において提案したような、生体力学的 刺激負荷装置を用いて行うことができる。  [0023] By the way, the vertical load load stimulus and the Z or transverse shear stress stimulus are applied using a biomechanical stimulus load device as previously proposed by the applicant in PCT / JP2005 / 11045. It can be carried out.
この生体力学的刺激負荷装置は、具体的には、図 5〜図 6に示すように、炭酸ガス インキュベーター 1内に設置される力学刺激負荷装置 2及び培養容器 3と、制御用コ ンピューター 4とで構成されてレ、る。  Specifically, as shown in FIGS. 5 to 6, the biomechanical stimulation load device includes a mechanical stimulation load device 2 and a culture vessel 3 installed in a carbon dioxide incubator 1, and a control computer 4. It is composed of and.
なお、生体力学的刺激負荷装置の操作は、すべて炭酸ガスインキュベーター 1の 外から行えるようにし、これにより、炭酸ガスインキュベーター 1内の滅菌状態を保つ て長時間に亘つての培養を可能にすることができる。  All operations of the biomechanical stimulation load device can be performed from outside the carbon dioxide incubator 1, which enables culturing for a long time while maintaining the sterilization state in the carbon dioxide incubator 1. Can do.
[0024] 炭酸ガスインキュベーター 1は、その内部を滅菌し、温度、湿度、酸素、二酸化炭素 、窒素分圧等を制御した環境で使用するようにする。  The carbon dioxide incubator 1 is sterilized and used in an environment in which temperature, humidity, oxygen, carbon dioxide, nitrogen partial pressure, etc. are controlled.
[0025] この炭酸ガスインキュベーター 1内に設置される力学刺激負荷装置 2は、荷重負荷 用ピストン 5と、この荷重負荷用ピストン 5を所定の範囲内で上下方向に移動可能に 支持するピストン上下移動用ステージ 21と、このピストン上下移動用ステージ 21を上 下方向に移動させるためのステージ昇降機構 22とを備え、制御用コンピューター 4に よってステージ昇降機構 22を所定のサイクルで駆動することにより、ピストン上下移 動用ステージ 21を上下方向に移動させ、ピストン上下移動用ステージ 21の降下時 に、荷重負荷用ピストン 5の重量が培養容器 3内の培養体 (細胞と足場とからなる組 織。以下同じ。）Cにかかるように構成している。  [0025] The mechanical stimulation load device 2 installed in the carbon dioxide incubator 1 includes a load-loading piston 5 and a piston moving up and down that supports the load-loading piston 5 so as to be movable in a vertical direction within a predetermined range. Stage 21 and a stage elevating mechanism 22 for moving the piston vertical movement stage 21 up and down. By driving the stage elevating mechanism 22 in a predetermined cycle by the control computer 4, the piston When the stage 21 for vertical movement is moved in the vertical direction and the stage 21 for vertical movement of the piston is lowered, the weight of the load-loading piston 5 is the culture in the culture vessel 3 (an organization composed of cells and scaffolds; the same applies hereinafter). .) It is configured to take C.
これにより、地上における生体内で組織に生じている鉛直方向の荷重負荷刺激と 同様の刺激を培養体 Cに付与することができるようにしている。  This makes it possible to apply a stimulus similar to the vertical load applied to the tissue in the living body on the ground to the culture C.
[0026] ピストン上下移動用ステージ 21を上下方向に移動させるためのステージ昇降機構 22は、例えば、電動ァクチユエ一ター 22aと、この電動ァクチユエ一ター 22aに接続 され、ピストン上下移動用ステージ 21を吊り下げるワイヤー 22bとからなる。 そして、制御用コンピューター 4の指令で電動ァクチユエ一ター 22aを操作すること により、ピストン上下移動用ステージ 21をワイヤー 22bを介して、ガイド部材 20に沿つ て上下方向に移動できるようにしてレ、る。 [0026] A stage elevating mechanism 22 for moving the piston vertical movement stage 21 in the vertical direction is connected to, for example, an electric actuator 22a and the electric actuator 22a, and the piston vertical movement stage 21 is suspended. It consists of the lower wire 22b. Then, by operating the electric actuator 22a in accordance with a command from the control computer 4, the piston vertical movement stage 21 can be moved vertically along the guide member 20 via the wire 22b. The
[0027] ところで、本実施例においては、ピストン上下移動用ステージ 21に載置したピストン 載置用ステージ 23を介して、荷重負荷用ピストン 5を支持するようにしている。 Incidentally, in this embodiment, the load-loading piston 5 is supported through the piston mounting stage 23 mounted on the piston vertical movement stage 21.
この場合、ピストン上下移動用ステージ 21は、ピストン載置用ステージ 23を載置す るためのものであるので、中央部に荷重負荷用ピストン 5が自由に上下方向に移動で きるようにするための孔部 21aを形成するようにする。  In this case, the piston vertical movement stage 21 is for mounting the piston mounting stage 23, so that the load-loading piston 5 can freely move in the vertical direction at the center. The hole 21a is formed.
これにより、種々の荷重負荷用ピストン 5の支持を簡易に行うことができる。 なお、ピストン載置用ステージ 23を省略して、ピストン上下移動用ステージ 21に荷 重負荷用ピストン 5を直接支持するようにすることもできる。  Thereby, various pistons 5 for load loading can be easily supported. The piston loading stage 23 may be omitted, and the load loading piston 5 may be directly supported on the piston vertical movement stage 21.
[0028] 荷重負荷用ピストン 5は、下端部に培養体 Cに対応した形状の加圧部 51を、中間 部にピストン載置用ステージ 23に形成した孔部 23aに嵌挿されるガイド軸部 52を、そ の上部に大径部 53を、上端部に追加荷重重鎮 55を装着するための重鎮装着部 54 を形成するようにする。 [0028] The load-loading piston 5 has a pressurizing portion 51 having a shape corresponding to the culture C at the lower end portion and a guide shaft portion 52 that is fitted into a hole portion 23a formed in the piston mounting stage 23 at the intermediate portion. A large-diameter portion 53 is formed on the upper portion, and a heavy load attaching portion 54 for attaching an additional load heavy load 55 is formed on the upper end portion.
これにより、図 6 (a)に示すように、ピストン載置用ステージ 23に荷重負荷用ピストン 5を支持した状態から、ピストン上下移動用ステージ 21を下方向に移動させ、図 6 (b)に示すように、ピストン上下移動用ステージ 21の降下時に、荷重負荷用ピストン 5 の大径部 53によるピストン載置用ステージ 23への支持が解除されるようにして、荷重 負荷用ピストン 5の重量が培養容器 3内の培養体 Cに直接力かるようにする。  As a result, as shown in FIG. 6 (a), the piston vertical movement stage 21 is moved downward from the state in which the piston 5 for load loading is supported on the piston mounting stage 23, and FIG. As shown in the figure, when the piston vertical movement stage 21 is lowered, the load-loading piston 5 is not supported by the piston mounting stage 23 by the large-diameter portion 53 of the load-loading piston 5 so that the weight of the load-loading piston 5 is reduced. Apply force directly to the culture C in the culture vessel 3.
その後、ピストン上下移動用ステージ 21を上方向に移動させることによって、図 6 (a )に示すように、ピストン載置用ステージ 23に荷重負荷用ピストン 5を支持した状態に 復帰させる。  Thereafter, the piston vertical movement stage 21 is moved upward to return the piston loading stage 23 to the state in which the load loading piston 5 is supported, as shown in FIG.
この荷重負荷用ピストン 5による鉛直方向の荷重負荷刺激は、制御用コンピュータ 一 4の操作で荷重サイクル、荷重時間を自由に操作できる。  The load load stimulus in the vertical direction by the load-loading piston 5 can freely operate the load cycle and load time by operating the control computer 4.
また、追加荷重重鎮 55は、必要に応じて、重鎮装着部 54に装着することができ、ま た、その重さも自由に設定できることから、培養体 Cに付与する鉛直方向の荷重負荷 刺激の大きさを簡易に調節することができる。 [0029] また、荷重負荷用ピストン 5の加圧部 51の形状は、培養体 Cに対応した任意の形状 とすることができ、さらに、培養体 Cに対応した形状の加圧部 51を別部材で形成し、 荷重負荷用ピストン 5の下端部に装着するようにすることもできる。 In addition, the additional load heavy load 55 can be attached to the heavy load attachment portion 54 as necessary, and its weight can be freely set, so that the vertical load load stimulus applied to the culture C is large. The thickness can be easily adjusted. [0029] In addition, the shape of the pressurizing portion 51 of the load-loading piston 5 can be any shape corresponding to the culture C, and the pressurizing portion 51 having a shape corresponding to the culture C can be separated. It is also possible to form the member and attach it to the lower end of the load-loading piston 5.
これにより、地上における生体内で組織に生じている鉛直方向の荷重負荷刺激と 同様の刺激を培養体 Cに正確に付与することができる。  As a result, a stimulus similar to the vertical load applied to the tissue in vivo on the ground can be accurately applied to the culture C.
[0030] また、荷重負荷用ピストン 5が、上下方向に移動する際に、回転しないようにピストン 載置用ステージ 23 (又はピストン上下移動用ステージ 21)に支持するようにすること ができる。  [0030] Further, the load loading piston 5 can be supported on the piston mounting stage 23 (or the piston vertical movement stage 21) so as not to rotate when moving in the vertical direction.
具体的には、ピストン載置用ステージ 23に形成した孔部 23aを多角形に形成し、ガ イド軸部 52をこの多角形の孔部 23aに適合した多角形の断面形状を有するように形 成する。  Specifically, the hole portion 23a formed in the piston mounting stage 23 is formed in a polygonal shape, and the guide shaft portion 52 is formed to have a polygonal cross-sectional shape adapted to the polygonal hole portion 23a. To do.
これにより、荷重負荷用ピストン 5に培養体 Cに対応した形状（円形以外）の加圧部 51を装着するようにした場合等でも、地上における生体内で組織に生じている鉛直 方向の荷重負荷刺激と同様の刺激を培養体 Cに正確に付与することができる。  As a result, even when the pressurizing part 51 having a shape (other than a circle) corresponding to the culture C is attached to the load-loading piston 5, the vertical load load generated in the tissue in vivo on the ground A stimulus similar to the stimulus can be accurately applied to the culture C.
[0031] 培養容器 3は、力学刺激負荷装置 2の培養容器固定ステージ 24に着脱可能に装 着するようにする。 [0031] The culture vessel 3 is detachably attached to the culture vessel fixing stage 24 of the mechanical stimulation load device 2.
これにより、種々の培養容器 3の装着を簡易に行うことができる。  Thereby, various culture vessels 3 can be easily attached.
また、培養容器 3には、必要に応じて、培地槽 (注入用） 32と、培地槽 (排出用） 33 とを接続し、培地の注入及び排出を制御用コンピューター 4によって制御するように することちでさる。  In addition, a culture vessel (for injection) 32 and a culture vessel (for discharge) 33 are connected to the culture vessel 3 as necessary, and the injection and discharge of the culture medium are controlled by the control computer 4. Say it with a word.
[0032] また、培養容器 3を水平面内で移動又は振動させる剪断応力負荷機構 26を設ける ようにしている。  [0032] Further, a shear stress loading mechanism 26 for moving or vibrating the culture vessel 3 in a horizontal plane is provided.
具体的には、培養容器固定ステージ 24上に、培養容器固定ステージ 24に対して 水平面内で移動可能に剪断応力ステージ 25を配設し、この剪断応力ステージ 25上 に培養容器 3を装着するようにするとともに、剪断応力ステージ 25を、剪断応力負荷 機構 26により水平面内で移動又は振動させるようにする。  Specifically, a shear stress stage 25 is arranged on the culture vessel fixing stage 24 so as to be movable in a horizontal plane with respect to the culture vessel fixing stage 24, and the culture vessel 3 is mounted on the shear stress stage 25. In addition, the shear stress stage 25 is moved or vibrated in the horizontal plane by the shear stress loading mechanism 26.
なお、水平面内で移動又は振動の方向は、 1方向に限定されず、 X、 Y方向の 2方 向や円運動等、任意の方向とすることができる。 剪断応力負荷機構 26としては、例えば、電動ァクチユエ一ターや永久磁石と電磁 石を組み合わせた移動機構又は振動機構を用いることができ、この剪断応力負荷機 構 26の駆動は、制御用コンピューター 4によって制御するようにする。 Note that the direction of movement or vibration in the horizontal plane is not limited to one direction, and can be any direction such as two directions in the X and Y directions and circular motion. As the shear stress loading mechanism 26, for example, an electric actuator, a moving mechanism or a vibration mechanism combining a permanent magnet and an electromagnetic stone can be used, and the driving of the shear stress loading mechanism 26 is performed by the control computer 4. Try to control.
これにより、地上における生体内で組織に生じている横方向の剪断応力刺激、例え ば、膝関節に屈曲、伸展の運動時に加わる刺激と同様の刺激を培養体 Cに付与する こと力 Sできる。  As a result, it is possible to apply force S to the culture body C in the same manner as a stimulus applied in the lateral direction to the shear stress generated in the tissue in the living body, for example, a stimulus applied to the knee joint during flexion and extension.
この場合、荷重負荷用ピストン 5による培養容器 3内の培養体 Cへの鉛直方向の荷 重負  In this case, the vertical load on the culture body C in the culture vessel 3 by the load loading piston 5
荷刺激の付与方法を調整しながら、剪断応力負荷機構 26によって培養容器 3を水 平面内で移動又は振動させることにより、斜め方向の剪断応力刺激を付与するように することちでさる。  By adjusting the method of applying the load stimulus, the shear stress load mechanism 26 moves or vibrates the culture vessel 3 in the horizontal plane, thereby applying the shear stress stimulus in the oblique direction.
[0033] 培養容器 3内には、図 6 (b)に示すように、培養体 Cに適合した任意の形状の内部 培養容器 30を設置し、この内部培養容器 30の荷重負荷用ピストン 5の加圧部 51と 相対する位置に培養細胞、培養組織又は培養細胞を含む構造体の受容基 31を装 着し、受容基 31の上に培養体 Cとしての培養細胞、培養組織又は培養細胞を含む 構造体を載置するようにすることができる。  In the culture vessel 3, as shown in FIG. 6 (b), an internal culture vessel 30 having an arbitrary shape suitable for the culture body C is installed, and the load-loading piston 5 of the internal culture vessel 30 is installed. A receiving group 31 of a structure containing cultured cells, cultured tissues or cultured cells is mounted at a position opposite to the pressurizing unit 51, and a cultured cell, cultured tissue or cultured cell as a cultured body C is placed on the receiving group 31. It can be made to mount a structure including.
[0034] 図 7に、半月板、関節唇、関節円板等の培養に好適に用いられる生体力学的刺激 負荷装置の一例を示す。 [0034] FIG. 7 shows an example of a biomechanical stimulus loading device that is preferably used for culturing meniscus, articular lip, joint disc, and the like.
半月板、関節唇、関節円板等は、骨に類似した硬度で生体内で負荷刺激を受けて いるのではなぐ骨軟骨で荷重負荷刺激を受けているため、その組織の培養を行う 培養体荷重負荷刺激部 Xが、生体の所定の形状 (例えば、膝関節）に類似した構造 となるようにし、この培養体荷重負荷刺激部 Xに培養体 Cを入れて、骨軟骨（例えば、 膝関節の骨軟骨）による荷重負荷刺激を再現するようにする。  The meniscus, labial lip, joint disc, etc. are subjected to load stress stimulation by osteochondral rather than being stressed in vivo with hardness similar to bone. The load-load stimulating part X has a structure similar to a predetermined shape (for example, knee joint) of a living body, and the culture body C is placed in the culture-body load-load stimulating part X, so that the bone cartilage (for example, the knee joint) To reproduce the load-induced stimulus by bone cartilage.
具体的には、培養容器 3内に、培養液 Lを満たすとともに、大腿骨骨軟骨に類似し た生体適合材料の合成樹脂で製作した、培養体 Cに対応した形状の凹部 30aを形 成した下部培養容器 30Aを設置し、その上に培養体 Cを載置するようにする。  Specifically, the culture vessel 3 was filled with the culture medium L, and a recess 30a having a shape corresponding to the culture body C, which was made of a synthetic resin of a biocompatible material similar to femoral bone cartilage was formed. Install the lower culture vessel 30A, and place the culture C on it.
一方、荷重負荷用ピストン 5の加圧部 51には、下部培養容器 30Aに対向するよう に、培養体 Cに対応した形状の凸部 30bを形成した上部培養容器 30Bを設置するよ うにする。 On the other hand, the pressurization part 51 of the load-loading piston 5 is provided with an upper culture container 30B having a convex part 30b having a shape corresponding to the culture C so as to face the lower culture container 30A. Do it.
これにより、細胞外組織であるマトリックスに囲まれた状態での細胞に力かるカを容 易に再現することができるとともに、培養体 Cを生体内の組織の力学的な環境に近似 した状態で培養することができるため、培養体の移植時の生体に対する適合性を向 上すること力 sできる。  As a result, it is possible to easily reproduce the force that is applied to the cells surrounded by the matrix that is the extracellular tissue, and the culture C is approximated to the mechanical environment of the tissue in the living body. Since it can be cultured, it can improve the adaptability of the culture to the living body at the time of transplantation.
[0035] 本発明の生体形状を含んだ三次元組織の培養方法によれば、細胞と足場とからな る組織を培養して得た培養体は、生体内の組織に近似した形状や状態で培養され たものとなるため、培養体の移植時の生体に対する適合性が向上する。  [0035] According to the method for culturing a three-dimensional tissue including a living body shape of the present invention, a culture body obtained by culturing a tissue composed of cells and a scaffold has a shape and a state approximate to a tissue in the living body. Since it is cultured, the compatibility with the living body at the time of transplantation of the culture is improved.
また、三次元生体形状データに基づいて作製した培養容器を用いて得た培養体は 、生体内の組織に近似した形状や状態で培養されたものとなるため、培養体の移植 時の生体に対する適合性が向上する。  In addition, since a culture obtained using a culture vessel prepared based on three-dimensional biological shape data is cultured in a shape and state that approximates the tissue in the living body, Compatibility is improved.
[0036] 以上、本発明の生体形状を含んだ三次元組織の培養方法について、その実施例 に基づいて説明したが、本発明は上記実施例に記載した構成に限定されるものでは なぐその趣旨を逸脱しない範囲において適宜その構成を変更することができるもの である。  As described above, the method for culturing a three-dimensional tissue including a living body shape of the present invention has been described based on the examples thereof, but the present invention is not limited to the configurations described in the above examples. The configuration can be changed as appropriate without departing from the scope of the invention.
産業上の利用可能性  Industrial applicability
[0037] 本発明の生体形状を含んだ三次元組織の培養方法は、生体内の組織に近似した 形状や状態で組織を培養することができることから、膝、股、肘、肩等の関節組織、鼻 、耳等の軟骨組織等の人体を構成する組織、例えば、関節組織を構成する骨ゃ軟 骨を、生体内の組織に近似した形状や状態で、培養するために好適に用いることが できる。 [0037] Since the method for culturing a three-dimensional tissue including a living body shape of the present invention can culture a tissue in a shape and state similar to the tissue in the living body, joint tissues such as knees, crotch, elbows, shoulders, etc. It is preferably used for culturing tissues constituting the human body, such as cartilage tissues such as nose and ears, for example, bone and soft bones constituting joint tissues, in a shape and a state approximating those in the living body. it can.

Claims

請求の範囲 The scope of the claims
[1] 下記（al)〜（f 1)の工程からなることを特徴とする生体形状を含んだ三次元組織の 培養方法。  [1] A method for culturing a three-dimensional tissue containing a living body shape comprising the following steps (al) to (f1):
(al)病変部位あるいは移植する予定の周囲組織の画像を撮影により得る工程  (al) A process of obtaining an image of the lesion site or surrounding tissue to be transplanted by imaging
(bl)前記撮影した病変部位あるいは移植する予定の周囲組織の画像情報から当該 部位の三次元生体形状データを作製する工程  (bl) A step of creating three-dimensional body shape data of the site from the imaged lesion site or image information of the surrounding tissue to be transplanted
(cl)前記三次元生体形状データに基づいて铸型を作製する工程  (cl) A step of producing a saddle shape based on the three-dimensional biological shape data
(dl)前記铸型を用いて三次元生体形状をもつ足場を作製する工程  (dl) A step of producing a scaffold having a three-dimensional living body shape using the saddle type
(el)前記足場に細胞を導入して三次元生体形状をもつ組織を作成する工程  (el) A step of creating a tissue having a three-dimensional biological shape by introducing cells into the scaffold
(fl)前記組織を培養する工程  (fl) Step of culturing the tissue
[2] 下記（a2)〜（e2)の工程からなることを特徴とする生体形状を含んだ三次元組織の 培養方法。 [2] A method for culturing a three-dimensional tissue including a living body, comprising the following steps (a2) to (e2):
(a2)病変部位あるいは移植する予定の周囲組織の画像を撮影により得る工程  (a2) Step of obtaining an image of a lesion site or surrounding tissue to be transplanted by imaging
(b2)前記撮影した病変部位あるいは移植する予定の周囲組織の画像情報から当該 部位の三次元生体形状データを作製する工程  (b2) A step of creating three-dimensional living body shape data of the site from the imaged lesion site or surrounding tissue to be transplanted
(c2)前記三次元生体形状データに基づいて铸型を作製する工程  (c2) A step of producing a saddle shape based on the three-dimensional living body shape data
(d2)前記錡型を用いて細胞と足場とからなる三次元生体形状をもつ組織を作製す る工程  (d2) Step of producing a tissue having a three-dimensional living body shape composed of cells and scaffolds using the saddle type
(e2)前記組織を培養する工程  (e2) culturing the tissue
[3] 下記（a3)〜（d3)の工程からなることを特徴とする生体形状を含んだ三次元組織の 培養方法。 [3] A method for culturing a three-dimensional tissue containing a living body shape, comprising the following steps (a3) to (d3):
(a3)病変部位あるいは移植する予定の周囲組織の画像を撮影により得る工程  (a3) Step of obtaining an image of a lesion site or surrounding tissue to be transplanted by imaging
(b3)前記撮影した病変部位あるいは移植する予定の周囲組織の画像情報から当該 部位の三次元生体形状データを作製する工程  (b3) Step of creating three-dimensional living body shape data of the imaged lesion site or the surrounding tissue to be transplanted from the image information
(c3)前記三次元生体形状データに基づいて培養容器を作製する工程  (c3) A step of producing a culture vessel based on the three-dimensional biological shape data
(d3)前記培養容器を用いて細胞と足場とからなる組織を培養する工程  (d3) a step of culturing a tissue composed of cells and a scaffold using the culture vessel
[4] 前記組織の培養時に、鉛直方向の荷重負荷刺激及び/又は横方向の剪断応力 刺激を付与することを特徴とする請求項 1、 2又は 3記載の生体形状を含んだ三次元 組織の培養方法。 [4] The three-dimensional body shape including a living body shape according to any one of claims 1, 2 and 3, wherein a vertical load stress stimulus and / or a transverse shear stress stimulus is applied when the tissue is cultured. Tissue culture method.
PCT/JP2005/023735 2005-12-26 2005-12-26 Method of culturing three-dimensional tissue including living organism configuration WO2007074500A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/JP2005/023735 WO2007074500A1 (en) 2005-12-26 2005-12-26 Method of culturing three-dimensional tissue including living organism configuration
JP2007551819A JP4919296B2 (en) 2005-12-26 2005-12-26 Method for culturing three-dimensional tissue including biological shape

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP2005/023735 WO2007074500A1 (en) 2005-12-26 2005-12-26 Method of culturing three-dimensional tissue including living organism configuration

Publications (1)

Publication Number Publication Date
WO2007074500A1 true WO2007074500A1 (en) 2007-07-05

Family

ID=38217739

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2005/023735 WO2007074500A1 (en) 2005-12-26 2005-12-26 Method of culturing three-dimensional tissue including living organism configuration

Country Status (2)

Country Link
JP (1) JP4919296B2 (en)
WO (1) WO2007074500A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011523355A (en) * 2008-05-07 2011-08-11 ユーシーエル ビジネス ピーエルシー Biomimetic cell scaffold
WO2017056866A1 (en) * 2015-09-29 2017-04-06 株式会社カネカ Biological tissue fabrication information generating device, biological tissue fabrication system, and medical expense calculation system

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003510108A (en) * 1999-06-08 2003-03-18 ウニヴェルシテートクリニクム フライブルグ Biological joint structures
JP2003144139A (en) * 2001-11-15 2003-05-20 Inst Of Physical & Chemical Res Method for bioadhesion of cell to surface three- dimensional structure
JP2005342112A (en) * 2004-06-01 2005-12-15 Ikuo Morita Artificial tissue body and manufacturing method thereof

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2610821B1 (en) * 1987-02-13 1989-06-09 Hennson Int METHOD FOR TAKING MEDICAL IMPRESSION AND DEVICE FOR IMPLEMENTING SAME
JPH0910253A (en) * 1995-06-30 1997-01-14 Matsushita Electric Ind Co Ltd Manufacture of custom-made hearing aid and hearing aid manufacturing device
JPH0910241A (en) * 1995-06-30 1997-01-14 Matsushita Electric Ind Co Ltd Tooth form molding method and manufacturing device therefor
JP3522394B2 (en) * 1995-07-04 2004-04-26 デジタルプロセス株式会社 Tooth prosthesis design and manufacturing equipment
EP1144596A2 (en) * 1998-12-11 2001-10-17 Advance Tissue Sciences Inc. Application of shear flow stress to smooth muscle cells for the production of implantable structures
ATE374003T1 (en) * 2000-02-15 2007-10-15 Royce Medical Co CAST ORTHOPEDIC DEVICE
AU2002216206B2 (en) * 2000-12-22 2007-10-04 Keele University Culturing tissue using magnetically generated mechanical stresses
JP2004529740A (en) * 2001-06-15 2004-09-30 ジョーンズ ホプキンズ シンガポール ピーティーイー リミテッド Biofunctional fiber
JP2004008536A (en) * 2002-06-07 2004-01-15 Olympus Corp Manufacturing system for biological tissue prosthesis
JP4359528B2 (en) * 2004-04-15 2009-11-04 太美雄 大前 Artificial tooth root and artificial tooth root manufacturing system

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003510108A (en) * 1999-06-08 2003-03-18 ウニヴェルシテートクリニクム フライブルグ Biological joint structures
JP2003144139A (en) * 2001-11-15 2003-05-20 Inst Of Physical & Chemical Res Method for bioadhesion of cell to surface three- dimensional structure
JP2005342112A (en) * 2004-06-01 2005-12-15 Ikuo Morita Artificial tissue body and manufacturing method thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MORITA I. ET AL.: "Insatsu Gijutsu o Mochiita Mousai Kekkan no Sakusei to sono Oyo", JAPANESE JOURNAL OF THROMBOSIS AND HEMOSTASIS, vol. 16, no. 5, 1 October 2005 (2005-10-01), pages 495, XP003014166 *
MORITA I.: "Seihan to Hikari Shokubai o Katsuyo shita Mosai Kekkan no Saisei", EXPECTED MATERIALS FOR THE FUTURE, vol. 5, no. 6, 10 June 2005 (2005-06-10), pages 15 - 19, XP003014167 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011523355A (en) * 2008-05-07 2011-08-11 ユーシーエル ビジネス ピーエルシー Biomimetic cell scaffold
WO2017056866A1 (en) * 2015-09-29 2017-04-06 株式会社カネカ Biological tissue fabrication information generating device, biological tissue fabrication system, and medical expense calculation system
CN108138102A (en) * 2015-09-29 2018-06-08 株式会社钟化 Bio-tissue prepares information generation device, bio-tissue preparation system, payment for medical care computing system
JPWO2017056866A1 (en) * 2015-09-29 2018-07-19 株式会社カネカ Biological tissue production information generation device, biological tissue production system, medical cost calculation system
US11746315B2 (en) 2015-09-29 2023-09-05 Kaneka Corporation Biological tissue fabrication information generating device, biological tissue fabrication system, and medical expense calculation system

Also Published As

Publication number Publication date
JPWO2007074500A1 (en) 2009-06-04
JP4919296B2 (en) 2012-04-18

Similar Documents

Publication Publication Date Title
Yan et al. A review of 3D printing technology for medical applications
WO2005123905A1 (en) Cell culturing method by biomechanical stimulus load and its device
Ho et al. A review on 3D printed bioimplants
Li et al. Three‐dimensional printing: The potential technology widely used in medical fields
Lee et al. Printing of three-dimensional tissue analogs for regenerative medicine
Ghosh et al. Addressing unmet clinical needs with 3D printing technologies
Raman et al. Stereolithographic 3D bioprinting for biomedical applications
Kumar et al. 3D printing technology for biomedical practice: a review
US20020106625A1 (en) Bioreactor for generating functional cartilaginous tissue
CN102395329B (en) Complex and uses thereof
Datta et al. Bone tissue bioprinting for craniofacial reconstruction
Zhang et al. 3D printing method for bone tissue engineering scaffold
WO2001068800A1 (en) Bioreactor for generating functional cartilaginous tissue
GB2395927A (en) Producing artificial bones by laser sintering
Banoriya et al. Modern trends in rapid prototyping for biomedical applications
CN102451048B (en) Complex-shaped gel-metal composite prosthesis and manufacturing method thereof
Javaid et al. 3D printing applications towards the required challenge of stem cells printing
CN103948457A (en) Method for constructing regenerated nerve vascularized bones, cartilages, joints or body surface organs
WO2007074500A1 (en) Method of culturing three-dimensional tissue including living organism configuration
Zhang Biomedical engineering for health research and development.
Das et al. Future of Bioprinting in Healthcare: A Review
Fanucci et al. Bioprinting: Prospects, considerations and challenges for application in South African clinical environments
KR20210003110A (en) 3D bioprinting of medical devices through free-form reversible embedding
CN103992947A (en) Loading device and method for pressure stress of skeletal cells in porous titanium alloy
Jeong et al. Bioreactor mimicking knee-joint movement for the regeneration of tissue-engineered cartilage

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2007551819

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 05842244

Country of ref document: EP

Kind code of ref document: A1