WO2021086058A1 - Tissu artificiel ou substituts organiques préparés à l'aide d'une impression de cellules en trois dimensions et leur procédé de préparation - Google Patents

Tissu artificiel ou substituts organiques préparés à l'aide d'une impression de cellules en trois dimensions et leur procédé de préparation Download PDF

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WO2021086058A1
WO2021086058A1 PCT/KR2020/014918 KR2020014918W WO2021086058A1 WO 2021086058 A1 WO2021086058 A1 WO 2021086058A1 KR 2020014918 W KR2020014918 W KR 2020014918W WO 2021086058 A1 WO2021086058 A1 WO 2021086058A1
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cells
tissue
membrane
organ
cell
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PCT/KR2020/014918
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English (en)
Korean (ko)
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조동우
김병수
조원우
안민준
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포항공과대학교 산학협력단
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Publication of WO2021086058A1 publication Critical patent/WO2021086058A1/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
    • A61L27/3886—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 comprising two or more cell types
    • 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
    • A61L27/14—Macromolecular materials
    • 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
    • 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
    • A61L27/3895—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 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • 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
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/10—Processes of additive manufacturing
    • B29C64/106—Processes of additive manufacturing using only liquids or viscous materials, e.g. depositing a continuous bead of viscous material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y70/00—Materials specially adapted for additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y80/00—Products made by additive manufacturing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
    • 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

Definitions

  • the present invention relates to an artificial tissue or organ analogue manufactured using 3D cell printing and a method for manufacturing the same, and to a tissue or organ analogue having a significantly reduced cell necrosis rate, and a method for manufacturing the same.
  • the supply of organs that can be transplanted is not increasing.
  • 3D printing technology which encloses and prints living cells together in bio-ink, is capable of producing living artificial tissues or organs, and is raising expectations in the field of regenerative medicine.
  • the cells of artificial tissues or organs produced by the current cell printing technology are supplied with nutrients by diffusion between the bioink and the external environment, and the degree of diffusion is known to be about several hundred micrometers.
  • the artificial tissue patch 2 manufactured by 3D cell printing technology is combined and laminated to the support 12 having the first connecting part 11 in the form of a column made of synthetic polymer.
  • Artificial tissue or organ analogues were developed ( Figure 1). Artificial tissues or organ analogues made by such a manufacturing method can produce a 3D structure with significantly lower mechanical strength than the existing manufacturing method by reducing the use of synthetic polymers compared to the 3D structure that can be manufactured by the conventional method (Fig. 5a and Fig. 5b).
  • Fig. 5a and Fig. 5b by forming a gap (3) between the artificial tissue patches (2) so that nutrients can be supplied even in a volume of several cm, necrosis of cells due to hypoxia does not occur (Fig. 4), depending on the cells and bio-ink to be enclosed. It can be applied to various tissues or organs, and it can be applied for more effective regeneration by using several bio-inks.
  • the present invention manufactures a tissue or organ analogue in a bonding and stacking method using a three-dimensional cell printing technology, so that an alternative three-dimensional artificial tissue or organ that does not cause necrosis of cells even though it is a structure having a large volume. I want to provide.
  • Fig. 1 shows a schematic diagram of a method of manufacturing an artificial tissue or an artificial organ by combining and stacking using a three-dimensional cell printing technology according to an embodiment of the present invention.
  • 1 is a schematic diagram showing a process of manufacturing a 3D artificial tissue or organ using a spraying type 3D cell printing technology, after melting a synthetic polymer at a high temperature, a support 1 having a first coupling part 11 ) And, after fabricating the membrane 21 in the form of a mesh (concentric circle with a connection), printing the cell part 23 in an appropriate pattern with bio-ink containing living cells on the membrane to patch artificial tissue (2) After manufacturing, by sequentially stacking the prepared artificial tissue patch (2) on the first coupling portion (11) of the support (1), nutrients and oxygen will be supplied through the gap (3) between the artificial tissue patch (2).
  • it is possible to manufacture an alternative three-dimensional artificial tissue or organ in which cell necrosis due to hypoxia is prevented.
  • a gap and/or a gap through which nutrients can be supplied to the cells inside are required.
  • the following two 3D printing methods were applied as an example to secure such gaps and/or gaps.
  • the synthetic polymer is melted at a high temperature and sprayed on the surface of the rotating cylinder to form a ring-shaped support part 12, and then the rotation is stopped, and the synthetic polymer is sprayed in the axial direction of the cylinder again to form a columnar first coupling part 11
  • a support body 1 with a gap was produced (FIGS. 2A to 2C).
  • a ring-shaped support part 12 is placed on the floor so that the columnar first coupling part 11 faces upward, and several artificial tissue patches 2 are combined.
  • a synthetic polymer is sprayed onto the plate to produce a mesh-shaped membrane 21 and a second coupling part 22 (Figs. 2D to 2F), and bio-ink containing cells is sprayed thereon.
  • the mesh membrane 21 can be deformed according to mechanical strength and fabrication form.
  • the membrane 21 is coupled with the first coupling portion 11 of the support 1 through the second coupling portion 22, and the second coupling portion has a ring shape into which the first coupling portion 11 can be inserted.
  • the height of the second coupling portion 22 was produced by laminating to an appropriate height in consideration of the height of the bioink sprayed on the membrane 21 (the height of the cell portion 23) and the size of the required gap 3 (Fig. 3a to 3c).
  • several bio-inks containing cells can be used, and functional artificial tissue patches (2) can be manufactured by spraying each bio-ink on the membrane in an appropriate pattern.
  • the support 1 and the plurality of artificial tissue patches 2 thus produced may be combined to be manufactured as a three-dimensional artificial tissue or organ analogue.
  • An example of the present invention is a support (1) having a first coupling portion (11), and the first coupling portion (11) of the support (1) is coupled so as to be spaced apart from each other to form a gap (3) It relates to a tissue or organ analogue comprising a plurality of artificial tissue patches (2).
  • the support (1) when a plurality of artificial tissue patches (2) are stacked and combined, so that the artificial tissue patch (2) can be combined and fixed, the first combination It may be provided with a part (11).
  • the support 1 may further include a support 12 supporting the first coupling part 11.
  • the support part 12 may be formed to correspond to the shape of the artificial tissue patch 2.
  • the first coupling portion 11 extends to a predetermined length perpendicular to the plane formed by the support portion 12, and the second coupling portion 22 of the artificial tissue patch 2 is coupled thereto, so that a plurality of artificial tissue patches (2) may be to be stacked. At least two of the first coupling portions 11 may be formed to be more stably coupled with the plurality of artificial tissue patches 2.
  • the artificial tissue patch 2 according to an embodiment of the present invention, a membrane 21 on which bio-ink can be printed; A second coupling portion 22 capable of being coupled with the first coupling portion 11 of the support 1; And a cell portion 23 in which bioink including cells is printed on the membrane 21 by a three-dimensional printing method.
  • the membrane 21 is a portion on which bio-ink can be printed, and the cell portion 23 can be formed by printing bio-ink on the membrane 21 by a three-dimensional printing method.
  • the membrane 21 may be formed in a planar shape so that a plurality of the membranes 21 may be stacked on each other.
  • the membrane 21 may be printed by a three-dimensional printing method, and may be printed by spraying a melt of a thermoplastic resin.
  • the height and width (width) of the printing line may be adjusted to set the height and width (width) of the membrane 21.
  • the height of the membrane 21 may be set as desired, and the height of the membrane 21 may be set to adjust the spacing of the gap 3 formed by stacking a plurality of membranes 21 spaced apart. As the height of the membrane 21 decreases, the size of the gap 3 when the membranes 21 are stacked apart from each other increases, and as the height of the membrane 21 increases, the membranes 21 may be stacked apart from each other. The size of the gap 3 at the time can be reduced.
  • the height of the membrane 21 is 50 to 500um, 50 to 450um, 50 to 400um, 50 to 350um, 50 to 300um, 50 to 250um, 50 to 200um, 100 to 500um, 100 to 450um, 100 to 400um , 100-350um, 100-300um, 100-250um, 100-200um, 150-500um, 150-450um, 150-400um, 150-350um, 150-300um, 150-250um, 150-200um, 200-500um, 200 To 450um, 200 to 400um, 200 to 350um, 200 to 300um, or 200 to 250um, for example, 200um.
  • the membrane 21 may itself have the pores 213, and when printing the membrane, the pores 213 of the membrane may be formed by printing to have an empty space.
  • the porosity (%) of the membrane 21 is 40 to 80%, 40 to 70%, 40 to 65%, 50 to 80%, 50 to 75%, 50 to 70%, 50 to 65%, 55 to 80% , 55 to 75%, 55 to 70%, or 55 to 65%.
  • a tissue or organ analog may have high elasticity.
  • Branches can provide tissue or organ analogues.
  • the modulus of elasticity of the artificial tissue or organ analog according to an embodiment of the present invention is 0.5 to 1 N/mm, 0.5 to 0.9 N/mm, 0.5 to 0.8 N/mm, 0.5 to 0.7 N/mm, 0.6 to 1 N/mm, It may be 0.6 to 0.9 N/mm, 0.6 to 0.8 N/mm, or 0.6 to 0.7 N/mm.
  • the porosity of the membrane can be adjusted by adjusting the width (width) of the printing line when printing the membrane, and specifically, the thinner the width of the printing line and the wider the gap between the printing lines, the higher the porosity of the membrane. The thicker the width of the film and the narrower the gap between the printing lines, the lower the porosity of the membrane.
  • the width (width) of the printing line when printing the membrane 21 is 100 to 500um, 100 to 450um, 100 to 400um, 100 to 350um, 100 to 300um, 150 to 500um, 150 to 450um, 150 to 400um , 150-350um, 150-300um, 200-500um, 200-450um, 200-400um, 200-350um, 200-300um, 250-500um, 250-450um, 250-400um, 250-350um, 250-300um, 300 To 500um, 300 to 450um, 300 to 400um, or 300 to 350um.
  • the membrane 21 may be formed in various shapes depending on the purpose, for example, may be formed in a shape suitable for a site to which the tissue or organ analog of the present invention is to be implanted.
  • the membrane 21 includes at least two or more concentric ring portions 211 having different diameters, and a connection portion 212 for interconnecting the at least two or more concentric ring portions 211 Can be.
  • the connecting portion 212 connects the circumference of the ring portion 211 from the center of the concentric ring portion 211 and extends in a radial direction to interconnect the ring portions 211 so that the membrane 21 is meshed. It may be formed to have a shape or a radial shape.
  • the membrane 21 may have a shape as shown in FIG. 2E.
  • the membrane 21 may have a void 213 formed by a difference in diameter between the at least two ring portions 211.
  • the second coupling portion 22 is a connecting ring portion formed on the membrane 21 so that the artificial tissue patch 2 can be bonded to and stacked with the first coupling portion 11 of the support 1.
  • the second coupling portion 22 is attached to the first coupling portion 11 It may be formed in a ring shape so that it can be fitted.
  • the second coupling portions 22 may be formed in the same number as the first coupling portions 11 formed on the support 1, and for example, at least two or more may be formed.
  • the height of the second coupling part 22 may be set according to the purpose, and the second coupling part 22 is used to adjust the gap 3 formed by spacedly stacking a plurality of membranes 21.
  • the height of the second coupling portion 22 may be set. Specifically, the height of the second coupling portion 22 is set to be greater than the sum of the heights of the membrane 21 and the cell portion 23, so that when a plurality of membranes 21 are stacked, the membranes 21 are spaced apart from each other.
  • the gap 3 can be formed.
  • the gaps 3 may increase as the distances between the membranes 21 increase, and as the height of the second coupling portion 22 decreases, the gaps between the membranes 21 become closer.
  • the gap 3 can be made small.
  • the height of the second coupling part 22 may be 1 to 3mm, 1 to 2.5mm, 1 to 2mm, 1.5 to 3mm, 1.5 to 2.5mm, 1.5 to 2mm, 2 to 3mm, or 2 to 2.5mm.
  • the support 1, the membrane 21, and/or the second coupling part 22 may be formed of a thermoplastic resin material.
  • the thermoplastic resin is polycaprolactone, poly(lactate-co-glycolate, PLGA), polylactic acid (PLA), polyurethane (polyurethane, PU) , Poly(lactide-co-caprolactone, PLCL), polydioxanone (PDO), polystyrene (PS), polyethylene glycol (poly(ethylene glycol), PEG) , Poly(vinyl acetate), PVA), polypropylene glycol (PPG), polyacrylamide (PAAm), polyglycolic acid (PGA), polymethylmetha It may be one or more selected from the group consisting of clylate (polymethylmethacrylate, PMMA), polyhydroxybutyrate (PHB), and polyvinylpyrrolidone (PVP).
  • the cell part 23 is formed by printing bio-ink containing cells on the membrane 21 by a three-dimensional printing method.
  • the bioink may include cells, wherein the cells include adipocytes, chondrocytes, hepatocytes, lung cells, heart cells, cardiovascular cells, bone cells, blood cells, vascular endothelial cells, bone marrow cells, pancreatic cells, kidney cells, Ovarian cells, testis cells, myocytes, skin cells, keratinocytes, fibroblasts, endocrine gland cells, thyroid cells, thyroid epithelial cells, vesicle side cells, adrenal cells, adrenal cortical cells, nerve cells, neural stem cells, retinal cells, corneal cells Corneal epithelial cells, corneal endothelial cells, corneal stem cells, stem cells, embryonic stem cells, dedifferentiated stem cells, induced pluripotent stem cells, tooth-derived stem cells, urine-derived stem cells, amniotic fluid-derived stem cells, placenta-derived stem cells, umbilical cord It may be one or more selected from the group consisting of derived stem cells and cord blood stem cells.
  • the cells
  • the cell portion 23 may be printed with at least two or more types of bioinks including different cells.
  • the bioink is hydrogel, decellularized extracellular matrix (dECM), vascular endothelial growth factor, fibroblast growth factor, platelet-derived growth factor, angiopoietin-1, transformation Transforming growth factor beta (TGF- ⁇ ), erythropoietin (EPO), stem cell factor (SCF), epidermal growth factor (EGF), colony stimulating factor ( colony stimulating facor (CSF), endothelial cell growth supplement (ECGS), matrix metalloproteinase (MMP), tissue inhibitor matrix metalloproteinase (TIMP), alginate ), gelatin, collagen, hyaluronic acid, chitosan, fibrin, agarose, silk protein, heparin, heparan At least one selected from the group consisting of sulfuric acid (heparan sulfate), keratin sulfate, dermatan sulfate, chondroitin sulfate, fatty alcohol, and fatty acid It may further include.
  • the cells in the bioink are 10 4 cells/ml to 10 9 cells/ml, 10 4 cells/ml to 10 8 cells/ml, 10 4 cells/ml to 10 7 cells/ml, 10 5 cells/ml to 10 9 cells/ml, 10 5 cells/ml to 10 8 cells/ml, 10 5 cells/ml to 10 7 cells/ml, 10 6 cells/ml to 10 9 cells/ml, 10 6 cells/ml to 10 8 cells/ml, or 10 6 cells/ml to 10 7 cells/ml.
  • the height of the cell part 23 may have a lower limit of 100um, 200um, 300um, 400um, 500um, 600um, 700um, 800um, 900um, 1000um, 1100um, 1100um, 1200um, 1300um, 1400um, or 1500um, and an upper limit of 2000um, It may be 1900um, 1800um, 1700um, 1600um, 1500um, 1400um, 1300um, 1200um, 1000um, 900um, 800um, 700um, 600um, 500um, 400um, 300um, or 200um, and the height of the cell part is a combination of the lower limit and the upper limit. It can be formed in a set range.
  • the height of the cell portion 23 may be 500 to 2000um, 500 to 1800um, 500 to 1600um, 1000 to 2000um, 1000 to 1800um, 1000 to 1600um, 1200 to 2000um, 1200 to 1800um, or 1200 to 1600um have.
  • the size of the gap 3 formed by the artificial tissue patch 2 that is spaced apart from each other may be reduced, and when the height of the cell portion 23 decreases, the spaced apart from each other The size of the gap 3 formed by the artificial tissue patch 2 may be increased.
  • a tissue or organ analog according to an embodiment of the present invention may include a gap 3 formed by stacking a plurality of artificial tissue patches 2 spaced apart from each other.
  • the gap 3 may perform a function of allowing nutrients and/or oxygen to sufficiently reach the cells contained in the cell portion 23.
  • the gap 3 when a plurality of artificial tissue patches 2 are stacked, the gap 3 may be stacked to be spaced apart from each other, and the height of the membrane 21 and the second coupling portion ( 22) and/or the height of the cell portion 23 may have different sizes, and specifically, the height of the second coupling portion 22 and the height of the membrane 21 and the cell portion 23 Can be set by car.
  • the height of the membrane 21 is set to 200 ⁇ m
  • the height of the cell portion 23 is set to 1,200 to 1,600 ⁇ m
  • the height of the second coupling unit 22 is set to 2,000 ⁇ m, so that the size of the gap 3 is It can be formed in 200 to 600um.
  • the gap 3 is 100 to 800um, 100 to 700um, 100 to 600um, 100 to 500um, 100 to 400um, 100 to 300um, 100 to 200um, 200 to 800um, 200 to 700um, 200 to 600um, 200 to 500um, 200-400um, 200-300um, 300-800um, 300-700um, 300-600um, 300-500um, 300-400um, 400-800um, 400-700um, 400-600um, or 400-500um Can be.
  • tissue or organ analog according to an embodiment of the present invention may be characterized in that, as the gap 3 is formed, the generation of a hypoxic area is suppressed.
  • tissue or organ analog according to an embodiment of the present invention may be characterized in that necrosis caused by hypoxia of cells is prevented.
  • the necrosis rate of cells included in the tissue or organ analog is 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10 % Or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.3% or less, or 0.2% or less.
  • Another example of the present invention is to spray a melt of a thermoplastic resin to prepare a support (1) having a first coupling portion (11); Manufacturing an artificial tissue patch; And forming a gap (3) by combining the artificial tissue patch (2) with the first coupling portion (11) of the support (1) to be spaced apart from each other to form a gap (3). It's about the method.
  • the manufacturing of the artificial tissue patch 2 includes the steps of spraying a melt of a thermoplastic resin to prepare a membrane 21 on which bio-ink can be printed; Spraying a melt of a thermoplastic resin to form a second coupling portion (22) capable of being coupled to the first coupling portion (11) of the support on the membrane (21); And printing bio-ink containing cells on the membrane 21 to prepare the cell portion 23.
  • the manufacturing of the membrane 21 includes at least two or more concentric ring portions 211 having different diameters, and a connection portion 212 for interconnecting the at least two or more concentric ring portions 211 It may be manufactured to include.
  • the connection part 212 may be to connect the at least two or more concentric ring parts 211 in a radial direction.
  • the artificial tissue patches 2 may be stacked apart from each other at intervals of 200 to 600 ⁇ m to form the gap 3.
  • the second coupling part 22 is formed to have a height of 1 to 3mm, 1 to 2.5mm, 1 to 2mm, 1.5 to 3mm, 1.5 to 2.5mm, 1.5 to 2mm, 2 to 3mm, or 2 to 2.5mm
  • the plurality of artificial tissue patches (2) are 100 to 800um, 100 to 700um, 100 to 600um, 100 to 500um, 100 to 400um, 100 to 300um, 100 to 200um, 200 to 800um, 200 to 700um, 200 to 600um, 200-500um, 200-400um, 200-300um, 300-800um, 300-700um, 300-600um, 300-500um, 300-400um, 400-800um, 400-700um, 400-600um, or 400-500um It may be stacked spaced apart to form a gap 3 of the interval.
  • the manufacturing of the cell part 23 may be manufacturing by printing at least two or more types of bioinks containing different cells.
  • the manufacturing of the membrane 21 may be such that the membrane 21 has a porosity of 40% or more, 45% or more, 50% or more, 55% or more, or 60% or more.
  • the membrane 21 may be manufactured to have a void 213 formed by a difference in diameter of the at least two ring portions 211, and the void 213 may be 40% or more, 45% or more of the total area of the membrane, It may be 50% or more, 55% or more, or 60% or more.
  • the melt of the thermoplastic resin is 100 to 500 um, 100 to 450 um, 100 to 400 um, 100 to 350 um, 100 to 300 um, 150 to 500 um, 150 to 450 um, 150 to 400 um, 150 to 350 um, 150 To 300um, 200 to 500um, 200 to 450um, 200 to 400um, 200 to 350um, 200 to 300um, 250 to 500um, 250 to 450um, 250 to 400um, 250 to 350um, 250 to 300um, 300 to 500um, 300 to 450um , 300 to 400um, or may be manufactured by printing with a printing line having a width of 300 to 350um.
  • the membrane is 50 to 500um, 50 to 450um, 50 to 400um, 50 to 350um, 50 to 300um, 50 to 250um, 50 to 200um, 100 to 500um, 100 to 450um, 100 to 400um , 100-350um, 100-300um, 100-250um, 100-200um, 150-500um, 150-450um, 150-400um, 150-350um, 150-300um, 150-250um, 150-200um, 200-500um, 200 To 450 um, 200 to 400 um, 200 to 350 um, 200 to 300 um, or 200 to 250 um, for example, may be manufactured by printing to have a height of 200 um.
  • the artificial tissue patch has a necrosis rate of 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less. , 2% or less, 1% or less, 0.5% or less, 0.3% or less, or may be stacked spaced apart on the support so as to be 0.2% or less.
  • the present invention enables a smooth supply of oxygen or nutrients, so that the cell necrosis rate of tissues or organ analogs can be significantly reduced.
  • the present invention can provide a tissue or organ analogue having a flexible mechanical strength, so that a foreign body sensation caused by transplantation and a negative effect on the tissue surrounding the transplant can be reduced.
  • FIG. 1 is a schematic diagram showing a process of manufacturing a 3D artificial tissue or organ using a spraying type 3D cell printing technology, after melting a synthetic polymer at a high temperature, a support having a bonding portion, and a mesh form (with a connecting portion) One concentric circle) is produced, and then artificial tissue patches are prepared by printing bio-ink containing living cells on the membrane in an appropriate pattern, and then the artificial tissue patches are sequentially stacked on the connection part of the support, and artificial tissue. Since nutrients and oxygen can be supplied through the gaps and voids between the patches, it is possible to manufacture an alternative 3D artificial tissue or organ in which cell necrosis caused by hypoxia is prevented.
  • FIG. 2A is a view showing a manufacturing process of a support 1 according to an embodiment of the present invention.
  • FIG. 2B is a view showing a state in which a support 1 according to an embodiment of the present invention is manufactured.
  • 2C is a diagram showing a schematic view of a support 1 according to an embodiment of the present invention.
  • 2D is a view showing the manufacturing process of the membrane 21 and the second coupling portion 22 according to an embodiment of the present invention.
  • FIG. 2E is a view showing a state of manufacturing a membrane 21 having a second coupling portion 22 formed thereon and a PCL membrane on which cells are printed according to an exemplary embodiment of the present invention.
  • 2F is a view showing a schematic view of the membrane 21 in which the second coupling portion 22 is formed according to an embodiment of the present invention.
  • FIG. 2G is an enlarged view of an artificial tissue patch 2 printed with bio-ink on a membrane according to an exemplary embodiment of the present invention.
  • Figure 2h is a view showing the results of culturing by printing bioinks on a membrane (W/PCL membrane) or a petri dish without a membrane (W/O PCL membrane), respectively.
  • Figure 2i is a diagram showing the cell activity according to the bio-ink printing method.
  • 3A is a view showing a fabrication of a tissue or organ analog according to an embodiment of the present invention.
  • 3B is a view showing a perspective view of a tissue or organ analog according to an embodiment of the present invention.
  • 3C is a view showing a front view of a tissue or organ analog according to an embodiment of the present invention.
  • FIG. 4 is a diagram showing that nutrients and oxygen are supplied to cells in the structure according to an embodiment of the present invention to prevent necrosis
  • FIG. 4A is a comparative example prepared without gaps and voids through which oxygen can be supplied.
  • 1 is a diagram showing the structure of 1
  • FIG. 4B is a view showing that cell necrosis occurred in the structure of Comparative Example 1 prepared without gaps and voids (necrosis: green)
  • FIG. 4C is a diagram according to an example of the present invention.
  • FIG. 4D is a diagram showing a decrease in cell necrosis in a tissue or organ analogue according to an embodiment of the present invention (necrosis: green).
  • 5A is a view showing a state of measuring the elastic force of a tissue or organ analog according to an embodiment of the present invention.
  • 5B is a view showing a result of measuring the elastic force of a tissue or organ analog according to an embodiment of the present invention.
  • 6A is a view showing a state in which an analogue and a control group according to an embodiment of the present invention are transplanted into nude mice, respectively.
  • 6B is a diagram showing a volume retention rate (%) after implantation of an analogue and a control group according to an embodiment of the present invention to a nude mouse, respectively.
  • 6C is a view showing the results of H&E staining obtained 4 weeks after implantation of an analogue and a control group according to an example of the present invention into nude mice, respectively.
  • 6D is a diagram showing a quantification of the ratio of the area occupied by fat cells to the cross-sectional area of each structure after H&E staining by securing an analogue and a control group according to an example of the present invention 4 weeks after transplantation into a nude mouse, respectively.
  • 7A is a view showing a method of printing bioink on an analog according to an example of the present invention.
  • FIG. 7B is a diagram showing a CD31 staining result after implantation of an analog according to an embodiment of the present invention.
  • 7C is a diagram showing the number and diameter of blood vessels after implantation of an analog according to an embodiment of the present invention.
  • 7D is a diagram showing the results of Perilipin and HuNU staining to determine the ability to form fat after transplantation of an analog according to an embodiment of the present invention.
  • FIG. 7E is a diagram showing the quantification of the number of HuNu-stained adipocytes per unit area after transplantation of an analog according to an embodiment of the present invention.
  • 7F is a view showing a result of measuring the size of a lipid droplet after implantation of an analog according to an example of the present invention.
  • 7G is a diagram showing the ratio of HuNu-stained cells to all cells (enclosed human cells + mouse cells) per unit area after implantation of an analog according to an example of the present invention.
  • the support may be manufactured to have a first coupling portion that is bondable to the membrane so that the artificial tissue patch can be properly stacked, and may be manufactured in a shape corresponding to the shape of the artificial tissue patch in order to support the artificial tissue patch.
  • a circular support was manufactured.
  • a synthetic polymer polycaprolactone material was mounted on a SUS syringe coupled with a nozzle having an inner diameter of 400 um, and then set in a 3D printing equipment to melt the material at a temperature of 90 degrees.
  • a ring-shaped support part 12 was manufactured by spraying the material with a pneumatic pressure of 500 kPa to have a thickness of 300 um and a height of 200 um on a circular rod having a diameter of 14 mm rotating at a constant speed, and then stopping the rotation and
  • a first coupling part 11 having a length of about 10 mm was manufactured when the support part 12 was laid on the floor as shown in FIG. 2B.
  • the manufacturing process of the support 1 is shown in FIG. 2A, and the state of the manufactured support 1 is shown in FIG. 2B.
  • the shape of the membrane can be variously modified according to the mechanical strength and fabrication shape suitable for the desired tissue or organ.
  • a second coupling portion 22 capable of being coupled to the first coupling portion was manufactured using a synthetic polymer as shown in FIG. 2E.
  • the synthetic polymer polycaprolactone was mounted on a SUS syringe coupled with a nozzle having an inner diameter of 400 um, and then set in a 3D printing equipment to melt the material at a temperature of 90 degrees. Thereafter, concentric circles with a diameter of 1 mm to 13 mm, a thickness of 300 um, and a height of 200 um in increments of 2 mm were sprayed on the plate with a pneumatic pressure of 500 kPa. Thereafter, a straight line having the same thickness and height was sprayed from the innermost concentric circle to the outermost concentric circle at 30° intervals, thereby fabricating a mesh-shaped membrane as shown in FIG. 2E. Finally, a material was laminated to have a height of 2 mm while a second coupling portion 22 having a diameter of 2 mm in contact with the membrane so as to be coupled to the first coupling portion.
  • the height of the second coupling part that can be combined with the first coupling part may be set so that the artificial tissue patch can be properly laminated, taking into account the height of the bioink sprayed on the membrane and the height of the required gap formed by being stacked apart from each other.
  • the height of the connecting ring portion of the two-joining portion increases, the mutual separation becomes wider when stacked on the support, and as the height of the connecting ring portion of the second connecting portion decreases, the mutual separation becomes narrower.
  • a decellularized extracellular matrix powder derived from the milled human abdominal fat was sufficiently dissolved in a 0.5 M acetic acid aqueous solution containing 2 mg/ml pepsin at a concentration of 2 to 5% by weight for 5 days to prepare a liquid. After that, it was neutralized to pH 7 to 7.3 using 10N sodium hydroxide.
  • a first bioink was prepared by uniformly mixing adipose-derived stem cells in a neutralized decellularized substrate-based solution at a concentration of 5 X 10 6 cells/mL. DiO, a dyeing agent, was added so that it could be distinguished from the second bioink to give it green fluorescence.
  • An artificial tissue patch was prepared by performing bioprinting using the prepared membrane and bioink. Specifically, the bio-ink was sprayed at 10° C. and 150 kPa to print the bio-ink on the membrane to prepare a cell part.
  • the sprayed bioink had a thickness of 1 mm (normal range: 800 um to 1.2 um) and a height of 1.5 mm (normal range: 1.2 to 1.6 mm).
  • bio-inks are printed using 3D printing, it is possible to precisely spray a plurality of materials at a desired location, and the printed cells can be expected to exhibit excellent activity.
  • FIG. 2I blood vessels and target tissue parts may be separated and printed.
  • each cell may be encapsulated in tissue-specific bio-ink and printed. After printing, the cells It can show better activity.
  • pre-adipocytes were subjected to in vitro differentiation treatment, followed by experiments.
  • Fig.2iA (1) when the differentiated adipocyte precursors are encapsulated in adipose-derived decellularized extracellular matrix (AdECM) bioink and printed on the membrane, (2) differentiated adipocyte precursors and human umbilical cord
  • AdECM venous endothelial cells
  • VdECM blood vessel-derived decellularized extracellular matrix
  • Differentiated adipocyte precursors were encapsulated in AdECM bioink, human umbilical vein endothelial cells (HUVECs) were encapsulated in VdECM bioink, and each bio-ink was concentrically separated on a membrane and printed, the activities were compared.
  • CD31 was IF stained to see
  • the formation of blood vessels could not be confirmed in the experimental group (1) in which the adipocyte precursors were encapsulated in an adipose-derived decellularized extracellular matrix (AdECM) bioink and printed.
  • AdECM adipose-derived decellularized extracellular matrix
  • the length of the blood vessels produced was 361.7 ⁇ 110.6 ⁇ m. 1.9 times, the thickness was 19.1 ⁇ 4.1 ⁇ m, about 2.5 times, and the number of connections was 154 per unit square meter, showing an improvement of about 2.9 times.
  • C of FIG. 2i is a result of confirming the secretion amounts of leptin and adiponectin, which are representative cytokines secreted from adipose tissue matured in each group, through ELISA analysis.
  • leptin and adiponectin are representative cytokines secreted from adipose tissue matured in each group.
  • the result of measuring the amount of leptin secretion in each group, on day 7 was 97.3 ⁇ 15.6 pg/ml for (1) and 108.5 ⁇ 10.2 pg/ml for (2) (3). Is 188.9 ⁇ 14.9 pg/ml.
  • Example 2 To the support prepared in Example 1, the artificial tissue patch prepared in Example 2 was separated and laminated to have a spacing of 2 mm to prepare a tissue analogue having a gap of 300 um. The appearance of the prepared tissue analog is shown in Figure 3a.
  • a bio-ink containing cells was injected into a hollow cylindrical mold having a shape similar to that of the tissue analog of Example 3, followed by gelation, to prepare a tissue analog having no gaps and voids.
  • tissue analogs prepared in Example 3 and Comparative Example 1 were maintained at 37° C. and 5% CO2, and cell growth medium (Dulbecco's Modified Eagle's medium with low glucose (DMEM/LG), 10% FBS, 1% Penicilin- Streptomycin) was cultured for 7 days while changing the medium every 2 days, and tissue analogues were washed with phosphate buffered saline (PBS).
  • cell growth medium Dulbecco's Modified Eagle's medium with low glucose (DMEM/LG), 10% FBS, 1% Penicilin- Streptomycin
  • FIG. 4 is a view showing that the tissue analogue prepared according to Example 3 can prevent necrosis by supplying nutrients and oxygen to the cells in the structure through the gap formed by the artificial tissue patch.
  • Fig. 4A a lot of cells necrotized by hypoxia were observed at the cut surface (Fig. 4B, necrosis: green), but the present application manufactured according to Example 3
  • the cells in the three-dimensional artificial tissue/organ of the invention did not cause necrosis (FIG. 4D). Therefore, it was confirmed that the 3D artificial tissue/organ according to an example of the present invention is an effective structure for general volume regeneration by preventing necrosis of cells due to hypoxia.
  • a cyclic compression test was performed to confirm the elasticity of the synthetic polymer skeleton that maintains the shape of the artificial tissue analogue.
  • the strength of the artificial tissue analog skeleton prepared according to Example 3 was measured. Measurement conditions were as follows.
  • the force according to the displacement was measured through three cycles, and the state of the measurement process is shown in FIG. 5A.
  • the elasticity measurement results are shown in Fig. 5b.
  • the artificial tissue analog was restored to its original state even in repeated compression experiments, and almost the same displacement-force result was confirmed in three consecutive cycles.
  • the compression process and the recovery process are almost the same for the same displacement, which means that they have high elasticity. (If it has a low elastic force, the force in the restoration process is lower.)
  • the skeleton of the artificial tissue analog of the present invention has elasticity that can be restored to its original state against deformation, which is It means that it can be applied to the same soft tissue.
  • the prepared artificial tissue analogue had an elastic modulus of 0.683 ⁇ 0.006 N/mm.
  • the tissue analogue prepared in Example 3 was implanted into a nude mouse and the progress was observed. Specifically, as shown in FIG. 6A, a 13 mm diameter and 4 mm height were transplanted (Adipose tissue construct of FIG. 6A), and AdECM bioink containing Pre-adipocytes was encapsulated in the control group and the analog of Example 3 After injection into a hollow cylindrical mold of the same size, a cylindrical structure produced by gelation at 37°C was implanted (adECM only in FIG. 6A). The implanted structures in Fig. 6A are indicated by arrows.
  • the volume retention rate of the tissue analog was 88.0 ⁇ 4.3% after 2 weeks and 81.3 ⁇ 6.3% after 4 weeks, which was more than 80% of the initial volume, but in the case of the cylindrical structure as a control group, after 2 weeks The initial volume was not maintained at 45.7 ⁇ 4.7% and 26.8 ⁇ 4.5% after 4 weeks.
  • the structure when the structure is manufactured only with hydrogel-based bioink, the volume cannot be maintained in the in vivo environment due to the weak physical properties of the bioink, but the polymer skeleton of the artificial tissue analog according to an example of the present invention is sufficient to prevent the structure from collapsing. It means that the mechanical strength was maintained for up to 4 weeks.
  • Fig. 6c shows the results of H&E staining by securing an artificial tissue analogue and a cylindrical structure (Fig. 6a) implanted in a nude mouse after 2 weeks and 4 weeks.
  • Fig. 6c when the analog according to the present invention was implanted, a large number of adipocytes were formed, and in the control group, it was confirmed that fat cells were hardly formed due to severe contraction and most of the cells died. Also, the cells were located only on the outer periphery of the structure. This is because the encapsulated cells in the center of the structure have been killed by hypoxia or have moved to the outside.
  • H&E staining of the artificial tissue analogue according to the present invention cells were evenly distributed throughout the structure. This means that the tissue analogue maintained mechanical strength even in the living body, and oxygen and nutrients were supplied to the inside of the structure due to the voids and gaps.
  • the tissue analog according to an example of the present invention can not only stably maintain a volume in vivo, but also aid in maturation of the encapsulated cells and promote regeneration.
  • FIG. 7B After 4 weeks after transplantation, CD31 staining was performed to observe angiogenesis (FIG. 7B), and the number and diameter of blood vessels per group were compared and shown in FIG.
  • the average diameter of the formed blood vessels was measured, and it was also confirmed that the experimental group (3) had a diameter of about 3.9 times larger than that of (1) and about 2.2 times larger than that of (2).
  • microvessels with red blood cells were clearly shown in the experimental group (3), which means that the implanted artificial tissue structure was successfully anastomized with the vascular system of the existing tissue.
  • Perilipin staining was performed to compare fat formation, and staining was performed using human nuclei antibody to confirm the encapsulated cells, and the results are shown in FIG. 7D.
  • Perilipin is a marker for mature adipocytes.
  • the number of HuNu-stained adipocytes per unit area of the sample obtained at the second week was quantified and shown in FIG. 7E. As shown in Fig. 7e, it was found that when the bio-ink of the artificial tissue was separated and printed, tissue formation by the enclosed cells could be achieved in a faster period.
  • Figure 7f shows the results of measuring the size of lipid droplets for samples obtained at the 2nd and 4th weeks. As shown in FIG. 7f, it was confirmed that when the compartmentalization was performed as in an example of the present invention, the size of the lipid droplet (30 ⁇ 10 ⁇ m) of the actual native adipose became similar.
  • the ratio of HuNu-stained cells to all cells per unit area was confirmed at week 4 and shown in FIG. 7G.
  • the proportion of HuNu-stained cells in the compartmentalization group was rapidly reduced, and this was successfully replaced by autologous cells when fully mature adipocytes were replaced with autologous cells in terms of tissue regeneration. It means that it replaced (*p ⁇ 0.05, **p ⁇ 0.01).
  • support 11 first coupling portion
  • connection part 213 void

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Abstract

La présente invention concerne un tissu artificiel ou un analogue d'organe préparé à l'aide d'une impression tridimensionnelle de cellules et leur procédé de préparation.
PCT/KR2020/014918 2019-10-31 2020-10-29 Tissu artificiel ou substituts organiques préparés à l'aide d'une impression de cellules en trois dimensions et leur procédé de préparation WO2021086058A1 (fr)

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