WO2023120710A1 - Procédé de production de tissu tridimensionnel synthétique, dispositif de production de tissu tridimensionnel synthétique, et corps de tissu tridimensionnel synthétique - Google Patents

Procédé de production de tissu tridimensionnel synthétique, dispositif de production de tissu tridimensionnel synthétique, et corps de tissu tridimensionnel synthétique Download PDF

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Publication number
WO2023120710A1
WO2023120710A1 PCT/JP2022/047671 JP2022047671W WO2023120710A1 WO 2023120710 A1 WO2023120710 A1 WO 2023120710A1 JP 2022047671 W JP2022047671 W JP 2022047671W WO 2023120710 A1 WO2023120710 A1 WO 2023120710A1
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Prior art keywords
hollow
hollow fiber
artificial
holders
culture
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PCT/JP2022/047671
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English (en)
Japanese (ja)
Inventor
昌治 竹内
銘昊 聶
亜衣 田嶋
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国立大学法人東京大学
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Publication of WO2023120710A1 publication Critical patent/WO2023120710A1/fr

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    • 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
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention relates to an artificial three-dimensional tissue manufacturing method, an artificial three-dimensional tissue manufacturing apparatus, and an artificial three-dimensional tissue.
  • Patent Document 1 a culture medium and cell masses are introduced from a supply port of a channel space portion in which a plurality of fibers are arranged, and are accumulated on the outer surface of each fiber with the vicinity of the discharge port of the channel space portion serving as a growth starting point. Culturing is disclosed.
  • Each fiber in Patent Document 1 is hollow or has a groove formed along its longitudinal direction, and has micropores communicating from the outer surface to the inside of the hollow or the inside of the groove.
  • Each fiber of Patent Document 1 removes metabolic waste products from the cell mass through the micropores and supplies at least one of growth factors and nutrient factors to the cell mass through the micropores.
  • the present invention has been made in consideration of the above points, and aims to provide an artificial three-dimensional tissue manufacturing method and an artificial three-dimensional tissue manufacturing apparatus capable of easily manufacturing an oriented large-sized tissue.
  • Another object of the present invention is to provide an oriented large-sized artificial three-dimensional tissue.
  • a hollow fiber having a hollow portion is prepared; and a second culture space located on the opposite side of the culture space in the first direction with respect to the engagement wall, wherein the engagement wall connects the engagement wall to the first culture space.
  • a plurality of holding portions provided in a plane orthogonal to the first direction and penetrating in the direction perpendicular to the first direction; a communicating portion for communicating, wherein the holding portion prepares a pair of the holders for holding the end portions of the hollow fibers in the first direction; holding one end side of the hollow fiber in each of the holders, holding the other end side of the hollow fiber in each of the plurality of holding sections in the other of the pair of holders; Disposing a pair of holders in the first direction with the culture space interposed therebetween, and supplying a pre-cured scaffold material containing cells to the culture space and the second culture space and curing the scaffold material. and culturing the cells while perfusing the hollow portion with a medium.
  • a hollow fiber having a hollow portion and a pair of holders spaced apart in the first direction across a culture space in which a cell-containing scaffold material is disposed are provided.
  • the holder has an engagement wall facing the culture space, and a second culture space located on the opposite side of the engagement wall to the culture space in the first direction with respect to the engagement wall.
  • the engaging wall includes a plurality of holding portions provided at intervals in a plane perpendicular to the first direction and penetrating the engaging wall in the first direction, and the engaging wall.
  • a cell-containing scaffold material culture body and hollow fibers having a hollow portion and provided through the culture body in a direction in which the culture body extends, are provided.
  • An artificial three-dimensional tissue is provided, wherein the hollow portion is a perfusion channel, and the outer peripheral surface of the hollow fiber is a treated surface sterilized with plasma.
  • an oriented large-sized tissue can be easily produced.
  • FIG. 1 is a cross-sectional view of an artificial three-dimensional tissue manufacturing device according to a first embodiment of the present invention
  • FIG. FIG. 4 is an external perspective view of the anchor section viewed from the culture space side.
  • FIG. 4 is a cross-sectional view showing a process related to formation of muscle tissue;
  • FIG. 4 is a cross-sectional view showing steps related to culturing muscle tissue.
  • FIG. 4 is a cross-sectional view of the main body of the culture body;
  • FIG. 4 is a cross-sectional view along the channel of the main body of the culture body.
  • FIG. 4 is a diagram showing a fluorescence image of a cell nucleus in a cross section perpendicular to the X direction of one hollow fiber in the main body.
  • FIG. 8 is a diagram showing the relationship between the distance range from the center of the hollow fiber and the cell nucleus density in the fluorescence image of the cell nucleus in FIG. 7;
  • FIG. 10 is a diagram showing a fluorescence image of muscle tissue in a cross section along the X direction in a hollow fiber; 10 is a diagram showing the relationship between the direction and amount of muscle tissue in the fluorescence image shown in FIG. 9.
  • FIG. 4 is a diagram showing a fluorescence image of muscle tissue T in a cross section perpendicular to the X direction of the hollow fiber.
  • FIG. 4 is a photographic view of a muscle tissue T cultured using nine hollow fibers F in a cross section perpendicular to the X direction of the hollow fibers.
  • FIG. 15 is a diagram showing a fluorescence image of the muscle tissue shown in FIG. 14;
  • FIG. 16 is a diagram showing an enlarged fluorescence image of a portion of the muscle tissue shown in FIG. 15; 17 is a diagram showing a fluorescence image of cell nuclei around the hollow fibers shown in FIG. 16.
  • FIG. FIG. 3 is an external perspective view of muscle tissue formed in an elongated shape.
  • FIG. 3 is a perspective view of a muscle tissue block in which muscle tissue is stacked in 4 rows ⁇ 2 layers.
  • FIG. 10 is an exploded perspective view of the artificial three-dimensional tissue manufacturing device according to the second embodiment of the present invention, before passing the hollow fibers. It is a perspective view which shows one side of the anchor part which concerns on the artificial three-dimensional tissue manufacturing apparatus. It is a perspective view which shows the other of the anchor part which concerns on the artificial three-dimensional tissue manufacturing apparatus. It is a figure which shows the state by which the anchor part was connected.
  • 7 is a graph showing the results of texture analysis of the artificial three-dimensional tissue according to the second embodiment; 4 is a table showing amino acid analysis results of the artificial three-dimensional tissue according to the second embodiment.
  • FIG. 1 Embodiments of an artificial three-dimensional tissue manufacturing method, an artificial three-dimensional tissue manufacturing apparatus, and an artificial three-dimensional tissue structure according to a first embodiment of the present invention will be described below with reference to FIGS. 1 to 19.
  • FIG. It should be noted that each of the embodiments shown below is one aspect of the present invention, does not limit the present invention, and can be arbitrarily changed within the scope of the technical idea of the present invention.
  • FIG. 1 is a cross-sectional view of an artificial three-dimensional tissue manufacturing apparatus 1.
  • the artificial three-dimensional tissue manufacturing apparatus 1 has a support member 10 , a pair of holders 20 , hollow fibers F, and a culture medium supply section 40 .
  • the support member 10 supports the pair of holders 20 with a predetermined distance in the first direction (horizontal direction in FIG. 1).
  • the support member 10 is a rectangular parallelepiped flat plate.
  • the support member 10 has a groove portion 12 on its upper surface 11 .
  • the grooves 12 are spaced apart in the first direction.
  • the groove portion 12 extends in a second direction orthogonal to the first direction (a direction orthogonal to the plane of the paper in FIG. 1).
  • the support member 10 is made of, for example, a soft elastic material such as PDMS (polydimethylsiloxane).
  • the horizontal direction in which the pair of holders 20 are arranged apart from each other is defined as the X direction
  • the horizontal direction orthogonal to the X direction is defined as the Y direction
  • the vertical direction is defined as the vertical direction
  • the direction orthogonal to the X direction and the Y direction is defined as the Z direction.
  • the pair of holders 20 are symmetrical about the center line parallel to the Z direction, the same reference numerals are given to the pair of holders 20 below, and the holder 20 located on the +X side will be described, and the -X side will be described. The description of the holder 20 positioned at is omitted.
  • a culture space 2 is between the pair of holders 20 in the X direction. That is, the pair of holders 20 are spaced apart in the X direction with the culture space 2 interposed therebetween.
  • the holder 20 has a holder body (facing portion) 21 , an anchor portion 22 and a fitting projection 23 .
  • the fitting projection 23 projects downward from the holder body 21 .
  • the fitting projection 23 fits into the groove 12 of the support member 10 from above. By fitting the fitting protrusion 23 into the groove 12 , the holder 20 is supported while being positioned by the support member 10 .
  • FIG. 2 is an external perspective view of the anchor part 22 viewed from the culture space 2 side. As shown in FIG. 2, the anchor portion 22 has a cubic shape. The anchor part 22 has an engaging wall 30 , a side wall 35 and a second culture space 37 .
  • the engagement wall 30 is arranged facing the culture space 2 .
  • the engagement wall 30 has a rectangular plate shape parallel to the YZ plane.
  • the engagement wall 30 has a holding portion 31 and a communicating portion 32 .
  • the holding portion 31 has a circular cross section and penetrates the engaging wall 30 in the X direction.
  • a plurality of holding portions 31 are provided at intervals in the YZ plane.
  • the holding portions 31 are provided in total of nine pieces, three each in the Y direction and the Z direction, centering on the central position of the engaging wall 30 in the YZ plane, with regular intervals therebetween. .
  • a hollow fiber F can be inserted through each of the plurality of holding portions 31 .
  • the ends of the hollow fibers F on the +X side are inserted through each of the plurality of holding portions 31 .
  • the plurality of holding portions 31 hold the outer peripheral surfaces Fb of the hollow fibers F that are inserted therethrough.
  • the communicating portion 32 penetrates the engaging wall 30 in the X direction, which is the thickness direction.
  • the communication part 32 communicates the culture space 2 with a second culture space 37, which will be described later.
  • a plurality of communicating portions 32 are provided at intervals in the YZ plane.
  • a plurality of communicating portions 32 are provided at positions that do not overlap with the holding portion 31 in the YZ plane at intervals.
  • a total of 16 communication portions 32 are provided, four each in the Y direction and the Z direction at regular intervals.
  • the side wall 35 extends from each edge of the rectangular engagement wall 30 to the +X side opposite to the culture space 2 in the X direction. That is, the side wall 35 has a plate shape parallel to the XZ plane or parallel to the XY plane.
  • Each side wall 35 has a plurality of second communication portions 36 .
  • the second communicating portion 36 penetrates the side wall 35 in the thickness direction.
  • the second communication part 36 allows the second culture space 37 and the outside of the holder 20 to communicate with each other.
  • Four second communicating portions 36 are provided on each side wall 35 at regular intervals, for a total of 16 second communicating portions 36 .
  • the second culture space 37 is formed inside the anchor section 22 .
  • the second culture space 37 is a cubic space surrounded by the engaging walls 30 , the side walls 35 and the holder body 21 .
  • the second culture space 37 communicates with the culture space 2 via the communicating portion 32 .
  • the second culture space 37 communicates with the outside of the holder 20 in the Y and Z directions via the second communicating portion 36 .
  • the holder main body 21 is positioned on the +X side, which is the outside of the anchor portion 22 in the X direction.
  • the holder main body 21 faces the engagement wall 30 with the second culture space 37 interposed therebetween in the X direction.
  • the holder main body 21 has an insertion hole 24 , an adhesive portion 25 , a culture medium storage portion 26 and a connection portion 27 .
  • the insertion hole 24 extends in the X direction.
  • the insertion hole 24 opens to the second culture space 37 at the ⁇ X side end and opens to the medium reservoir 26 at the +X side end.
  • the insertion hole 24 is arranged coaxially with the holding portion 31 . Accordingly, there are nine insertion holes 24 in total, three each in the Y direction and the Z direction at regular intervals.
  • a hollow fiber F is inserted through the insertion hole 24 . By arranging the insertion hole 24 and the holding portion 31 coaxially, the hollow fiber F can be inserted across the holding portion 31 and the insertion hole 24 .
  • the bonding portion 25 extends in a direction perpendicular to the X direction (only the bonding portion 25 extending in the Z direction is shown in FIG. 1). The position of the adhesion portion 25 in the X direction is between the second culture space 37 and the medium reservoir portion 26 .
  • the adhesive portion 25 has one end connected to the insertion hole 24 and the other end opened to the outside of the holder main body 21 .
  • the bonding portion 25 is provided for each of the plurality of insertion holes 24 .
  • the bonding portion 25 is filled with an adhesive 25A. By filling the bonding portion 25 with the adhesive 25A, the gap between the hollow fiber F inserted in the insertion hole 24 and the insertion hole 24 can be closed to prevent the culture medium from leaking out of the gap.
  • the medium reservoir 26 is an area in which the medium is reserved.
  • the culture medium reservoir 26 is arranged on the +X side of the insertion hole 24 .
  • the culture medium storage part 26 is formed in a range spanning the plurality of insertion holes 24 . Therefore, all of the plurality of insertion holes 24 open to the culture medium reservoir 26 .
  • the culture medium reservoir 26 in the holder 20 located on the +X side stores the culture medium supplied from the culture medium supply section 40 and before perfusing the hollow portions Fa of the hollow fibers F described later.
  • the medium reservoir 26 in the holder 20 located on the -X side stores the medium after the hollow portion Fa of the hollow fiber F has been perfused.
  • the connecting portion 27 protrudes from the holder main body 21 to the +X side.
  • a pipe 28 is connected to the connecting portion 27 .
  • a hole portion 29 is provided inside the connection portion 27 .
  • the hole portion 29 extends in the X direction, one end of which is open to the culture medium storage portion 26 and the other end of which is open to the +X side end of the connection portion 27 .
  • the above holder 20 is manufactured using, for example, a 3D printer.
  • the material of the holder 20 is not particularly limited as long as it does not adversely affect the three-dimensional structure, and an appropriate material can be appropriately used according to the selected manufacturing method.
  • the medium supply unit 40 supplies the medium.
  • the medium supply unit 40 is, for example, a perfusion pump.
  • the culture medium supplied from the culture medium supply unit 40 is introduced into the holder 20 located on the +X side through the pipe 28 . That is, the connecting portion 27 of the holder 20 located on the +X side constitutes an introducing portion 27A into which the culture medium for perfusing the hollow portion Fa of the hollow fiber F is introduced via the pipe 28 .
  • the connection portion 27 of the holder 20 positioned on the ⁇ X side constitutes a discharge portion 27B through which the culture medium perfused in the hollow portion Fa of the hollow fiber F is discharged through the pipe 28 .
  • a hollow fiber F is a fiber having a hollow portion Fa.
  • the hollow fiber F is not particularly limited as long as it has a semipermeable membrane structure.
  • the inner diameter of the hollow portion Fa is preferably about 20-1000 ⁇ m, more preferably about 50-500 ⁇ m, even more preferably about 50-150 ⁇ m.
  • the thickness of the hollow fiber F is preferably about 10 to 200 ⁇ m.
  • the interval between the hollow fibers F defined by the arrangement of the holding portion 31 and the insertion hole 24 is a minute interval of about several hundred ⁇ m, preferably 20 to 1000 ⁇ m, more preferably 50 to 500 ⁇ m, and even more preferably 50 to 150 ⁇ m. Regular arrangement is preferred.
  • the material of the hollow fiber F is not particularly limited as long as it does not have a detrimental effect on cells, scaffold materials (hydrogel, etc.), and the medium. can be mentioned.
  • the average pore size of the porous hollow fiber membrane is desirably large in consideration of the material exchange property, and is desirably about 0.1 to 5 ⁇ m.
  • the pore size is not limited to this range, and can be appropriately set according to the purpose of use, such as a pore size of less than 0.1 ⁇ m. It is also desirable that the minimum average molecular weight (molecular weight cutoff) of standard molecules that do not effectively diffuse through the membrane is greater than 70 kDa.
  • the outer peripheral surface Fb of the hollow fiber F and the anchor portion 22 are preferably subjected to hydrophilic treatment.
  • hydrophilic treatment By subjecting the outer peripheral surface Fb of the hollow fibers F and the anchor portions 22 to a hydrophilic treatment, when the hollow fibers F are arranged in an array, the inside of the array (between the plurality of hollow fibers F) is filled with a viscous scaffolding material. becomes possible.
  • hydrophilization treatment for the outer peripheral surface Fb of the hollow fiber F and the anchor portion 22 for example, O 2 plasma treatment or Aqua Plasma (registered trademark) treatment can be employed.
  • Aquaplasma (registered trademark) treatment is plasma treatment using water vapor.
  • surface hydrophilization and sterilization of the artificial three-dimensional tissue manufacturing apparatus 1 can be performed.
  • the outer peripheral surface Fb of the hollow fiber F is subjected to a plasma treatment using water vapor. That is, the outer peripheral surface Fb of the hollow fiber F is a plasma-sterilized surface.
  • the outer peripheral surface Fb can be sterilized without causing slack in the hollow fibers F arranged in an array.
  • cells contained in the scaffolding material can be cultured with high viability around the hollow fibers F whose outer peripheral surface Fb is sterilized, and a large oriented tissue can be easily produced. can.
  • a pre-cured scaffold material e.g., hydrogel, etc.
  • the hollow fibers F having the outer peripheral surface Fb previously subjected to the above-described hydrophilic treatment are attached to the holding portions 31 and the insertion holes 24 of the pair of holders 20 as shown in FIG. Insert each.
  • the hollow fibers F pass from the holding portion 31 through the second culture space 37 and communicate with the insertion holes 24 .
  • the end of the hollow fiber F may be located at the end facing the culture medium reservoir 26 in the insertion hole 24 or may protrude into the culture medium reservoir 26 .
  • the number of hollow fibers F to be inserted through the holding portion 31 and the insertion hole 24 can be arbitrarily selected from one to nine.
  • the fitting protrusion 23 is fitted into the groove 12 of the support member 10 from above, and is supported while being positioned at a predetermined distance in the X direction.
  • the hollow fibers F inserted through the holding portion 31 and the insertion hole 24 are fixed to the insertion hole 24 by filling the bonding portion 25 with an adhesive 25A.
  • the culture medium stored in the culture medium reservoir 26 can be prevented from flowing out through the gap between the insertion hole 24 and the hollow fibers F.
  • FIG. 3 is a cross-sectional view showing the steps involved in forming muscle tissue.
  • a mixture of cells C suspended in a scaffolding material for example, hydrogel
  • a scaffolding material for example, hydrogel
  • a scaffold material for example, hydrogel
  • Myoblasts are used as cells C in this embodiment.
  • an extracellular matrix component is used as a scaffold material (eg, hydrogel, etc.) G.
  • a mixture of Matrigel ((registered trademark)) and Collagen is used as the extracellular matrix component.
  • the mixing ratio of Matrigel (registered trademark) and collagen is preferably 20 to 80% Matrigel (registered trademark) and 80 to 20% collagen, and 40 to 60% Matrigel (registered trademark) and 60% collagen. More preferably ⁇ 40%.
  • the pre-hardening scaffolding material eg, hydrogel, etc.
  • the scaffold material for example, hydrogel
  • the outer peripheral surface Fb of the hollow fibers F is subjected to a hydrophilic treatment, it is can also be filled and cured.
  • the culture conditions As an example, it is cultured at a temperature of 37°C for 30 minutes. As a result, a culture B in which a scaffolding material (for example, hydrogel, etc.) G containing cells C is cultured is formed.
  • the culture body B consists of a body part B1 cultured in the culture space 2, an outside culture body B2 located outside the body part B1 in the X direction and cultured in the second culture space 37, the body part B1 and the outside culture. It includes a bound culture B3 that is bound to the body B2 and cultured in the communicating section 32 .
  • the outer culture B2 is held in the second culture space 37 as a holding space.
  • the main body B1 which is cultured in the culture space 2 and contacts the engagement wall 30 from the inside in the X direction, shrinks inward in the X direction without restraint as the scaffolding material (for example, hydrogel, etc.) G shrinks.
  • the scaffolding material for example, hydrogel, etc.
  • the engaging wall 30 engages with the outer culture body B2 from the inside in the X direction and acts as an anchor to suppress the contraction of the outer culture body B2 inward in the X direction.
  • FIG. 4 is a cross-sectional view showing the steps involved in culturing muscle tissue.
  • the culture body B is formed by hardening the scaffolding material (for example, hydrogel) containing the cells C, as shown in FIG. be placed on.
  • the scaffolding material for example, hydrogel
  • FIG. 5 is a cross-sectional view of the body portion B1 in the culture body B.
  • muscle tissue T is formed around hollow fibers F by culturing a scaffolding material (for example, hydrogel, etc.) containing cells C.
  • a scaffolding material for example, hydrogel, etc.
  • Nutrients, oxygen, and the like permeate the porous hollow fiber membrane from the culture medium that perfuses the hollow portion Fa and diffuse into the muscle tissue T, which is the culture body B.
  • metabolic waste products permeate the porous hollow fiber membrane and diffuse into the culture medium.
  • the medium in which the waste products have diffused by perfusing the hollow portion Fa is once stored in the medium storage section 26 in the holder 20 located on the -X side, and then discharged to the culture tank 100 via the discharge section 27B and the pipe 28. be done.
  • the scaffolding material (eg, hydrogel, etc.) G containing cells C before hardening is supplied to the outer periphery of the hollow fiber F, and then the scaffolding material (eg, hydrogel, etc.) G is hardened to form a culture body B.
  • the muscle tissue T can be cultured by forming the muscle tissue T as , and perfusing the medium in the hollow portion Fa. As the culture of the muscle tissue T progresses, the contraction of the body portion B1 increases. However, the engagement wall 30 engages the outer culture body B2 from the inside in the X direction as an anchor, so that the outer culture body B2 contracts. suppressed.
  • FIG. 6 is a cross-sectional view of the main body B1 of the culture body B along the channel.
  • the muscle tissue T is easily oriented along the X direction as shown in FIG.
  • FIG. 7 shows a fluorescence image of cell nuclei in a cross section perpendicular to the X direction of the hollow fiber F in the main body B1 of the muscle tissue T cultured for 4 days in a state where only one hollow fiber F is perfused with the above medium.
  • FIG. 4 is a diagram showing; As shown in FIG. 7, cell nuclei are present at high density in the region near the outer peripheral surface Fb of the hollow fiber F, but cell nuclei are present at low density in the region away from the outer peripheral surface Fb of the hollow fiber F. rice field. From this, it can be concluded that nutrients, oxygen, and the like diffuse from the medium that perfuses the hollow portion Fa into the muscle tissue T formed around the hollow fibers F, and the cells are cultured in a live state. I can judge.
  • FIG. 8 is a diagram showing the relationship between the distance range from the center of the hollow fiber F and the cell nucleus density in the fluorescence image of the cell nucleus in FIG.
  • the distance range from the center of the hollow fiber F includes a ring-shaped range with a radius of 250-300 ⁇ m from the center of the hollow fiber F, a ring-shaped range with a radius of 300-350 ⁇ m from the center of the hollow fiber F, and a ring-shaped range with a radius of 300-350 ⁇ m from the center of the hollow fiber F.
  • a ring-shaped range with a radius of 350-400 ⁇ m, a ring-shaped range with a radius of 400-450 ⁇ m from the center of the hollow fiber F, and a ring-shaped range with a radius of 450-500 ⁇ m from the center of the hollow fiber F are defined.
  • the number of cell nuclei with respect to the area in each ring-shaped range is shown as the cell nucleus density.
  • the distance from the center of the hollow fiber F when the perfusion rate of the medium for perfusing the hollow portion Fa was 0 ⁇ L/min, 15 ⁇ L/min, 100 ⁇ L/min, and 500 ⁇ L/min.
  • the relationship between range and cell nucleus density is shown. As shown in FIG. 8, no significant correlation was observed between the distance range from the center of the hollow fiber F and the cell nucleus density. It was confirmed that the cell nucleus density became higher than the perfusion rate.
  • FIG. 9 shows the body portion B1 of muscle tissue T cultured for 10 days in a state where only one hollow fiber F was perfused with the medium at a perfusion rate of 15 ⁇ L/min.
  • FIG. 4 is a diagram showing a fluorescent image of muscle tissue in a cross section;
  • FIG. 10 is a diagram showing the relationship between the direction and amount of muscle tissue in the fluorescence image shown in FIG. The direction (Direction (°)) in FIG. 10 is 0° in the Y direction and 90° in the X direction.
  • FIG. 11 shows the body portion B1 of the muscle tissue T cultured for 10 days in a state where only one hollow fiber F was perfused with the above medium at a perfusion rate of 500 ⁇ L/min.
  • FIG. 4 is a diagram showing a fluorescent image of muscle tissue in a cross section; 12 is a diagram showing the relationship between the direction and amount of muscle tissue in the fluorescence image shown in FIG. 11.
  • FIG. The direction (Direction (°)) in FIG. 12 is 0° in the Y direction and 90° in the X direction.
  • muscle tissue T could be formed when the medium was perfused at a perfusion rate of 15 ⁇ L/min.
  • a muscle tissue T with a greater thickness could be formed than when the medium was perfused at a perfusion rate of 15 ⁇ L/min.
  • the scaffold material eg, hydrogel, etc.
  • muscle tissue T can be formed when the medium perfusion rate is 15 ⁇ L/min or higher. In this case, even in the range where the medium perfusion rate exceeds 500 ⁇ L/min, not only can the muscle tissue T be formed, but also the muscle tissue T can be formed with a larger film thickness, a higher cell nucleus density, and a greater degree of orientation in the X direction. is assumed.
  • FIG. 13 shows the fluorescence of the muscle tissue T in the cross section perpendicular to the X direction of the hollow fiber F in the main body B1 of the muscle tissue T cultured for 10 days in a state where only one hollow fiber F is perfused with the above medium.
  • FIG. 4 is a diagram showing an image; As shown in FIG. 13, since muscle tissue is differentiated and formed around the hollow fibers F, the muscle tissue T formed around the hollow fibers F is perfused with the hollow portion Fa. It can be determined that nutrients, oxygen, and the like diffuse from the cells, and the cells are cultured in a live state.
  • FIG. 14 is a photograph of the muscle tissue T in the cross section perpendicular to the X direction of the hollow fibers F in the main body B1 of the muscle tissue T cultured for 4 days in the state of perfusion with the medium using the nine hollow fibers F. It is a diagram. As shown in FIG. 14, the muscle tissue T was able to be formed in a size surrounding the nine hollow fibers F. The center-to-center distance of the hollow fibers F was 900 ⁇ m before culturing the muscle tissue T, but the center-to-center distance of the hollow fibers F in the muscle tissue T after culturing was approximately 600 ⁇ m due to contraction accompanying the culture. In addition, the muscle tissue T after culture had a minimum width of approximately 2300 ⁇ m (2.3 mm).
  • the muscle tissue T cultured by the artificial three-dimensional tissue manufacturing method using the artificial three-dimensional tissue manufacturing apparatus 1 of the present embodiment has an orientation and can easily produce a larger tissue than the conventional artificial three-dimensional tissue. I was able to
  • FIG. 15 is a diagram showing a fluorescence image of the muscle tissue in FIG. 14.
  • FIG. 15 muscle tissue was formed around eight hollow fibers F out of the nine hollow fibers F, indicating that the eight hollow fibers F were cultured by perfusion. I can judge.
  • FIG. 16 is a diagram showing a fluorescence image in which a part of the muscle tissue shown in FIG. 15 is enlarged.
  • cell nuclei are present at high density in the region near the outer peripheral surface Fb of the hollow fiber F, but cell nuclei are present at low density in the region away from the outer peripheral surface Fb of the hollow fiber F. . From this fact, nutrients, oxygen, etc. diffuse from the medium perfusing the hollow portion Fa into the muscle tissue T formed around the eight hollow fibers F out of the nine hollow fibers F, and the cells It can be judged that the culture is performed in a living state.
  • FIG. 17 is a diagram showing a fluorescent image of cell nuclei around the hollow fibers F shown in FIG.
  • FIG. 17 similar to the case of using one hollow fiber F, in the region near the outer peripheral surface Fb of the hollow fiber F, cell nuclei exist at a high density, but the outer peripheral surface Fb of the hollow fiber F Cell nuclei were present at a low density in regions away from . From this, it can be concluded that nutrients, oxygen, and the like diffuse from the medium that perfuses the hollow portion Fa into the muscle tissue T formed around the hollow fibers F, and the cells are cultured in a live state. I can judge.
  • FIG. 18 is an external perspective view of an elongated muscle tissue.
  • the muscle tissue shown in FIG. 18 is formed by culturing using four hollow fibers F arranged in two rows in the Y and Z directions. Whereas the muscle tissue described above was approximately 5 mm in length of body portion B1, the muscle tissue shown in FIG. 18 is approximately 4 cm in length of body portion B1.
  • the muscle tissue block TB has a cross-sectional side of 2-2.5 mm. Therefore, the muscle tissue block TB can produce a large tissue on the order of centimeters with a width of 8-10 mm and a height of 4-5 mm. Also, by removing the hollow fibers F from the muscle tissue, it becomes possible to construct a large-sized artificial three-dimensional tissue suitable for meat.
  • a hardened scaffold material for example, hydrogel, etc.
  • Hollow fibers F are passed through G, and the medium is perfused into the hollow portions Fa of the hollow fibers F for culturing. It becomes possible to manufacture to
  • the gap between the hollow fiber F and the insertion hole 24 can be closed to prevent the culture medium from leaking out of the gap.
  • the distance between the pair of holders 20 can be accurately defined. Therefore, in the present embodiment, it is possible to define the length in the X direction of the body portion B1 of the culture body B with high accuracy.
  • the outer peripheral surface Fb of the hollow fibers F and the anchor portion 22 are subjected to a hydrophilic treatment, when the hollow fibers F are arranged in an array, a viscous scaffolding material (for example, hydrogel, etc.) ) can be filled in the array (between a plurality of hollow fibers F), and the muscle tissue T can be easily produced even in an array with narrow gaps between the hollow fibers F.
  • a viscous scaffolding material for example, hydrogel, etc.
  • the artificial three-dimensional tissue structure M can be constructed by removing the pair of holders 20 containing the culture B (muscle tissue T) and the hollow fibers F from the support member 10.
  • the fitting protrusion 23 on one side of the holder 20 is supported by, for example, a fixed portion of the biological model, and the fitting protrusion 23 on the other side of the holder 20 is supported by the movable portion of the biological model. It can be used as an actuator in a biological model by supporting and contracting the culture body B (muscle tissue T) by energizing it.
  • animal-derived cells C having a large contractile force in the muscle tissue T.
  • animal-derived cells C for example, C2C12 cells can be used. The use of C2C12 cells makes it possible to construct actuators without sacrificing animals.
  • the artificial three-dimensional tissue structure N in which the hollow fibers F penetrate the culture body B (muscle tissue T) in the longitudinal direction is formed.
  • the outer peripheral surface Fb of the hollow fiber F is a treated surface sterilized with plasma, the outer peripheral surface Fb is sterilized without causing slack, and the cells contained in the scaffold material survive. It becomes possible to culture efficiently, and it becomes a large-sized tissue with orientation.
  • FIG. 20 to 25 A second embodiment of the present invention will be described with reference to FIGS. 20 to 25.
  • FIG. 20 to 25 the same reference numerals are given to the same configurations as those already described, and redundant descriptions will be omitted.
  • FIG. 20 shows an exploded view of the artificial three-dimensional tissue manufacturing device 101 according to this embodiment.
  • the artificial three-dimensional tissue manufacturing device 101 includes a holder 120 in place of the holder 20 .
  • Each holder 120 has a structure in which the guide 102 is arranged between the anchor portion and the holder body 121 .
  • one holder is provided with an anchor portion 122A, and the other holder is provided with an anchor portion 122B.
  • the anchor portion 122A and the anchor portion 122B are slightly different in shape.
  • FIG. 21 shows the anchor part 122A viewed from the front side facing the culture space.
  • the anchor part 122A has 50 cylindrical holding parts 131A, and is configured so that more hollow fibers can be inserted than in the first embodiment.
  • the holding portions 131A are arranged in a two-dimensional matrix of 5 ⁇ 10, but this is not essential, and by arranging them in another manner such as a honeycomb shape, the per unit cross-sectional area It is also possible to increase the arrangement density of the hollow fibers.
  • the holding portion 131A has a portion with a small inner diameter inside, thereby having a step 202 inside.
  • FIG. 22 shows the anchor part 122B viewed from the front side.
  • the anchor portion 122B also has 50 cylindrical holding portions 131B like the anchor portion 122A.
  • the outer diameter of the holding portion 131B is smaller than the inner diameter of the end opening of the holding portion 131A, and can be inserted into the holding portion 131A. Since the outer diameter of the holding portion 131B is larger than the inner diameter of the step 202 inside the holding portion 131A, it cannot enter deeper than the step 202 .
  • the anchor part 122A and the anchor part 122B are brought close to each other with their front sides facing each other, and the ends of the respective holding parts 131B are inserted into the opposing holding parts 131A. Thereby, the anchor portion 122A and the anchor portion 122B are connected as shown in FIG.
  • the connecting operation when the holding portion 131B enters the holding portion 131A by a certain amount, the end portion of the holding portion 131B abuts against the step 202, so excessive entry is suppressed.
  • a hollow fiber is passed from the rear side of one anchor part through the holes that communicate with the holding parts, and protrudes from the rear side of the other anchor part.
  • the hollow fiber may be inserted from either the anchor portion 122A or the anchor portion 122B.
  • the guides 102 are attached to the anchor portions 122A and 122B while passing the plurality of hollow fibers protruding from the rear side of the anchor portions 122A and 122B through different guides 102 respectively.
  • the attachment method is not particularly limited, and examples include mechanical fitting and adhesion.
  • the guide 102 has an inclined surface 102a, and the width of the space through which the hollow fibers pass gradually narrows as the distance from the anchor portion increases. For this reason, the hollow fibers passing through the guide 102 are bundled into one while gradually narrowing the distance between them and guided to the holder main body 121 .
  • the holder body 121 is fixed to the guide 102, the anchor portion, the guide, and the holder body are integrated to form a pair. becomes the holder 120 of The operation for adjusting the distance between the anchor portions can be performed at any timing after the hollow fibers are passed through the two anchor portions in the connected state. After that, the anchor portions 122A and 122B are inserted into the grooves 10a provided in the support member 10. As shown in FIG. As a result, the holder 120 holding the hollow fibers is attached to the support member 10 to complete the artificial three-dimensional tissue manufacturing apparatus 101, and perfusion and culture can be performed in the same manner as in the first embodiment.
  • the artificial three-dimensional tissue manufacturing apparatus it is possible to produce a larger artificial three-dimensional tissue by increasing the number of hollow fibers. becomes complicated.
  • the hollow fiber passed through one holder may be bent, making it difficult to pass through the other holder, or may be held in a non-corresponding (non-face-to-face) manner.
  • the possibility of having to pass it through a part and need to redo it will increase, and the difficulty of the work will also increase.
  • the anchor portions 122A and 122B are configured to be connectable. Therefore, by separating the anchor portion 122A and the anchor portion 122B after passing the hollow fiber in a connected state, it is possible to significantly suppress the occurrence of the above-described error when passing the hollow fiber. As a result, even if the number of hollow fibers increases, it is possible to prevent the assembly of the artificial three-dimensional structure manufacturing apparatus and the execution of the artificial three-dimensional structure manufacturing method from becoming complicated. With such a configuration, the artificial three-dimensional tissue manufacturing apparatus can be expected to be automatically manufactured by a robot.
  • the number of hollow fibers is not limited to 50 as described above, and it is of course possible to increase the number. Even if the number of anchors increases, the difficulty of assembling the artificial three-dimensional tissue manufacturing device and executing the artificial three-dimensional tissue manufacturing method does not change significantly by passing the hollow fibers while the anchor portions are connected.
  • an engaging wall around the holding portion can be used as a stopper to prevent excessive intrusion during connection.
  • a plurality of hollow fibers are bundled together in the guide 102, but by changing the number and arrangement of slopes provided on the guide, the hollow fibers can be bundled into two or more bundles.
  • the guide may be configured as follows. Such a mode is suitable when the number of hollow fibers increases, for example, 100 or more.
  • the evaluation results of the artificial three-dimensional tissue manufactured using the artificial three-dimensional tissue manufacturing apparatus 101 according to the present embodiment are shown.
  • An artificial three-dimensional tissue was produced using chicken myoblasts using an artificial three-dimensional tissue manufacturing apparatus configured using 50 hollow fibers.
  • Two types of artificial three-dimensional tissues were prepared: a perfused sample cultured for 9 days with perfusion and a non-perfused sample cultured for 9 days without perfusion.
  • the hollow fiber was pulled out and measured, and the weight of the perfused sample was 470 mg.
  • the perfused sample it was confirmed that cell nuclei were present at high density around the hollow fibers F, as in the first embodiment.
  • the tissue was larger than that of the first embodiment, the orientation of the muscle tissue was well aligned in the longitudinal direction of the hollow fiber, and good contraction was observed by energization.
  • the horizontal axis is the elapsed time (seconds), and the vertical axis is the magnitude of the reaction force (g/mm 2 ) that the probe received from the tissue.
  • All perfused samples had a longer time to peak reaction force than non-perfused samples. This indicates that the perfused samples are thicker than the non-perfused samples.
  • the peak values of the reaction force of the perfused samples were all higher than the non-perfused samples. This indicates that the perfused samples have higher elasticity than the non-perfused samples, suggesting a denser tissue.
  • Evaluation 1 indicates that, for example, when an artificial three-dimensional tissue is used for meat, the elasticity of the tissue can be moderately improved by culturing the tissue while perfusion is performed, and the texture can be improved.
  • hollow fibers that branch midway may be used.
  • one holder on one side of the hollow fiber and two or more holders on the other side. can be identified as a pair of holders, and the direction in which the pair of holders are separated can be identified as the first direction.
  • the present invention can be suitably applied to an artificial three-dimensional tissue and its manufacture.

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Abstract

La présente invention comprend les étapes suivantes : préparation de fibres creuses possédant une partie creuse ; préparation d'une paire de supports séparés dans une première direction par un espace de culture, les supports de ladite paire comprenant chacun une paroi de couplage faisant face à l'espace de culture, et un second espace de culture placé à l'opposé de l'espace de culture disposé dans la première direction, vis-à-vis de la paroi de couplage, la paroi de couplage ayant une pluralité de sections de maintien qui traversent la paroi de couplage dans la première direction et qui présentent entre elles des intervalles dans un plan orthogonal à la première direction, et une section de communication qui traverse la paroi de couplage dans la première direction et qui met en communication l'espace de culture et le second espace de culture, les sections de maintien maintenant une partie d'extrémité des fibres creuses dans la première direction ; maintien des fibres creuses dans chacune des sections de maintien de la paire de supports ; placement des supports de la paire, qui maintiennent les fibres creuses, à l'écart l'un de l'autre dans la première direction, l'espace de culture se trouvant entre les deux ; apport à l'espace de culture et au second espace de culture d'un matériau de structuration non durci qui contient des cellules et durcissement du matériau de structuration ; et mise en culture des cellules tout en irriguant les parties creuses à l'aide d'un milieu de culture.
PCT/JP2022/047671 2021-12-23 2022-12-23 Procédé de production de tissu tridimensionnel synthétique, dispositif de production de tissu tridimensionnel synthétique, et corps de tissu tridimensionnel synthétique WO2023120710A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001128660A (ja) * 1999-08-25 2001-05-15 Toyobo Co Ltd 血管網類似構造体を有する細胞培養用モジュール
JP2013507143A (ja) * 2009-10-12 2013-03-04 テルモ ビーシーティー、インコーポレーテッド 中空糸型バイオリアクタの組立方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001128660A (ja) * 1999-08-25 2001-05-15 Toyobo Co Ltd 血管網類似構造体を有する細胞培養用モジュール
JP2013507143A (ja) * 2009-10-12 2013-03-04 テルモ ビーシーティー、インコーポレーテッド 中空糸型バイオリアクタの組立方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BETTAHALLI N.M.S., VICENTE J., MORONI L., HIGUERA G.A., VAN BLITTERSWIJK C.A., WESSLING M., STAMATIALIS D.F.: "Integration of hollow fiber membranes improves nutrient supply in three-dimensional tissue constructs", ACTA BIOMATERIALIA, ELSEVIER, AMSTERDAM, NL, vol. 7, no. 9, 1 September 2011 (2011-09-01), AMSTERDAM, NL, pages 3312 - 3324, XP093073553, ISSN: 1742-7061, DOI: 10.1016/j.actbio.2011.06.012 *
YAMAMOTO, YASUNORI; ITO, AKIRA; JITSUNOBU, HIDEAKI; YAMAGUCHI, KATSUYA; KAWABE, YOSHINORI; MIZUMOTO, HIROSHI; KAMIHIRA, MASAMICHI: "Hollow Fiber Bioreactor Perfusion Culture System for Magnetic Force-Based Skeletal Muscle Tissue Engineering", JOURNAL OF CHEMICAL ENGINEERING OF JAPAN, SOCIETY OF CHEMICAL ENGINEERS, JP, vol. 45, no. 5, 1 January 2012 (2012-01-01), JP , pages 348 - 354, XP009547190, ISSN: 0021-9592, DOI: 10.1252/jcej.11we237 *

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