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WO2007074500A1 - Procédé de culture de tissu en trois dimensions, ayant une configuration dans l'organisme vivant - Google Patents

Procédé de culture de tissu en trois dimensions, ayant une configuration dans l'organisme vivant Download PDF

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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
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/JP2005/023735
Other languages
English (en)
Japanese (ja)
Inventor
Ken Nakata
Kunihiko Hosotani
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Osaka NUC
TechnoView Inc
Original Assignee
Osaka University NUC
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 NUC, TechnoView Inc filed Critical Osaka University NUC
Priority to JP2007551819A priority Critical patent/JP4919296B2/ja
Priority to PCT/JP2005/023735 priority patent/WO2007074500A1/fr
Publication of WO2007074500A1 publication Critical patent/WO2007074500A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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.

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  • 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)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Materials For Medical Uses (AREA)

Abstract

Procédé de culture de tissu en trois dimensions ayant une configuration dans l'organisme vivant, selon lequel la culture du tissu peut se faire dans un état et dans une configuration voisins de ceux du tissu à l'intérieur d'un organisme vivant. L'invention concerne un procédé de culture de tissu en trois dimensions ayant une configuration dans l'organisme vivant, ledit procédé comprenant les étapes consistant à : (a1) obtenir par photographie l'image du site malade ou du tissu périphérique du transplant prévu ; (b1) préparer des données de configuration en trois dimensions du site dans l'organisme vivant à partir des informations d'image du site malade ou du tissu périphérique du transplant prévu photographié ; (c1) préparer un moule sur la base des données de configuration en trois dimensions dans l'organisme vivant ; (d1) préparer une ossature ayant la configuration en trois dimensions dans l'organisme vivant en se servant du moule ; (e1) préparer un tissu ayant la configuration en trois dimensions dans l'organisme vivant en introduisant des cellules sur l'ossature ; et (f1) cultiver le tissu.
PCT/JP2005/023735 2005-12-26 2005-12-26 Procédé de culture de tissu en trois dimensions, ayant une configuration dans l'organisme vivant Ceased WO2007074500A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2007551819A JP4919296B2 (ja) 2005-12-26 2005-12-26 生体形状を含んだ三次元組織の培養方法
PCT/JP2005/023735 WO2007074500A1 (fr) 2005-12-26 2005-12-26 Procédé de culture de tissu en trois dimensions, ayant une configuration dans l'organisme vivant

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP2005/023735 WO2007074500A1 (fr) 2005-12-26 2005-12-26 Procédé de culture de tissu en trois dimensions, ayant une configuration dans l'organisme vivant

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WO2017056866A1 (fr) * 2015-09-29 2017-04-06 株式会社カネカ Dispositif de génération d'informations de fabrication de tissu biologique, système de fabrication de tissu biologique et système de calcul des dépenses médicales

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WO2017056866A1 (fr) * 2015-09-29 2017-04-06 株式会社カネカ Dispositif de génération d'informations de fabrication de tissu biologique, système de fabrication de tissu biologique et système de calcul des dépenses médicales
CN108138102A (zh) * 2015-09-29 2018-06-08 株式会社钟化 生物体组织制备信息生成装置、生物体组织制备系统、医疗费计算系统
JPWO2017056866A1 (ja) * 2015-09-29 2018-07-19 株式会社カネカ 生体組織作製情報生成装置、生体組織作製システム、医療費演算システム
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

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JP4919296B2 (ja) 2012-04-18

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