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WO2019113900A1 - Tissu adipeux imprimé en 3d - Google Patents

Tissu adipeux imprimé en 3d Download PDF

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Publication number
WO2019113900A1
WO2019113900A1 PCT/CN2017/116247 CN2017116247W WO2019113900A1 WO 2019113900 A1 WO2019113900 A1 WO 2019113900A1 CN 2017116247 W CN2017116247 W CN 2017116247W WO 2019113900 A1 WO2019113900 A1 WO 2019113900A1
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WO
WIPO (PCT)
Prior art keywords
adipose tissue
fat
brown
printed
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2017/116247
Other languages
English (en)
Chinese (zh)
Inventor
张键
黄晨
赵华山
汪宝蓓
陈指龙
薛丽
杨雅莉
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Institute of Advanced Technology of CAS
Original Assignee
Shenzhen Institute of Advanced Technology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Institute of Advanced Technology of CAS filed Critical Shenzhen Institute of Advanced Technology of CAS
Priority to PCT/CN2017/116247 priority Critical patent/WO2019113900A1/fr
Publication of WO2019113900A1 publication Critical patent/WO2019113900A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
    • 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
    • B33Y10/00—Processes of 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
    • 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
    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0653—Adipocytes; Adipose tissue
    • 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
    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/74—Alginate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/76—Agarose, agar-agar
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • the invention relates to the field of biotechnology, in particular to adipose tissue prepared by 3D printing technology.
  • Adipose tissue is composed of fat cells and is divided into small leaflets by thin layer of loose connective tissue. Adipose tissue is not only a simple energy storage organ, but its more important function is to participate in the pathological and physiological processes of various diseases through the endocrine system. Adipose tissue is divided into white adipose tissue and brown adipose tissue. White adipose tissue is composed of a large number of single-foamed fat cells, which are mainly distributed in the subcutaneous and mesenteric areas. It is an energy reservoir in the body, which stores fat, maintains body temperature, regulates fat metabolism, and supports protection. Brown fat is characterized by the accumulation of capillaries, in which fat cells contain many fat droplets and are rich in mitochondria.
  • Brown fat is mainly distributed in the interscapular region, the back of the neck and other areas, the main function is to be decomposed and radiate a lot of heat energy under the stimulation of cold.
  • brown fat plays an important role in the treatment of metabolic-related diseases, including obesity, type 2 diabetes, and polycystic ovary syndrome, and induces an increase in brown fat through physiological or drug stimulation. Improve metabolic indicators in these conditions.
  • brown fat can secrete a variety of factors that regulate the endocrine system and body hormone levels.
  • brown adipose tissue in different disease models (eg, type 2 diabetes model and polycystic ovary syndrome model) and found the disease table in these models after transplantation.
  • the types have improved to varying degrees, such as weight loss in the type 2 diabetes model, significant improvement in metabolic indicators, and improved ovarian function in polycystic ovary syndrome.
  • simple brown adipose tissue transplantation due to the limited number of donor cell tissues, the difficulty of transplantation of soft tissue, and the limitations of clinical application, limits the use of adipose tissue for the treatment of related diseases.
  • the bioactive 3D printed adipose tissue not only helps to explore the physiological and pathological mechanisms of related lesions involved in fat endocrine metabolism in vitro or in vivo, but also provides a new therapeutic tool for the study of human fat-related endocrine metabolism.
  • the present invention prints biologically functional 3D adipose tissue in vitro on the basis of fat cell culture, combined with tissue engineering method and current advanced 3D printing technology, and inoculates fat precursor cells in a liquid scaffold.
  • the material was co-cultured and induced by the adipocyte induction solution for about 7 days to form a three-dimensional adipose tissue-like composite structure. This method allows the cells to grow in a three-dimensional structure, better mimics the growth state of the body, and has a cell secretory function. .
  • One aspect of the present invention provides a 3D printed brown adipose tissue made from a biocompatible scaffold material, a fat precursor cell, and a mixed culture solution by 3D bioprinting.
  • a 3D printed brown adipose tissue is prepared by making a biocompatible scaffold material into a biocompatible scaffold by 3D bioprinting, and then mixing and cultured the cells containing brown fat precursor cells.
  • the liquid is injected into the stent; and brown fat tissue is induced by the adipocyte induction solution; or
  • the biocompatible scaffold material is selected from one or a combination of collagen, sodium alginate, gelatin, agarose, matrigel, hyaluronic acid, chitosan, and dextran.
  • the collagen is selected from the group consisting of fibroblast collagen, more preferably one of type I collagen, type II collagen, type III collagen, type XI collagen, type XXIV collagen, and type XXVII collagen. Or any combination of several.
  • gelatin is used as the scaffold material.
  • the mixed culture liquid is a fluid liquid made of a medium and a biologically active substance required for promoting the growth of fat cells;
  • Fat cell induction fluid composition
  • the fat cell is selected from the group consisting of fat. After mechanical separation and digestion, the obtained fat precursor cells are used for the preparation of artificial adipose tissue.
  • the biocompatible stent has pores formed by 3D bioprinting, and the diameter of the voids does not exceed 1 mm.
  • the size of the void has one or more sizes, preferably from 1 to 4 sizes, and more preferably from 2 to 3 sizes.
  • the 3D printing size of the biocompatible stent of the present invention is: pore size (R): 50 ⁇ m to 800 ⁇ m; line stacking angle: 0-90°.
  • the pore size (R) is preferably 100 ⁇ m, 150 ⁇ m, 200 ⁇ m, 250 ⁇ m, 300 ⁇ m, 350 ⁇ m, 400 ⁇ m, 450 ⁇ m, 500 ⁇ m, 550 ⁇ m, 600 ⁇ m, 650 ⁇ m, 700 ⁇ m, 750 ⁇ m;
  • the line stacking angle is preferably 5°, 10°, 15°, 20°, 30°, 40°, 50°, 60°, 70°, 80°, 90°.
  • Another aspect of the present invention provides a method of preparing an artificial adipose tissue according to the present invention, comprising the steps of:
  • the biocompatible scaffold material is made into a biocompatible scaffold by 3D bioprinting, and then the mixed culture medium containing the brown fat precursor cells is injected into the biocompatible scaffold to prepare adipose tissue precursor; or
  • the biocompatible scaffold material is mixed with brown fat cells to prepare a brown adipose tissue precursor by 3D bioprinting;
  • the prepared brown adipose tissue is cultured and stored in a mixed culture solution for use.
  • Another aspect of the present invention provides the use of the artificial brown adipose tissue of the present invention for treating a metabolic disorder disease
  • the metabolic disorder diseases include polycystic ovary syndrome, obesity, central obesity, diabetes (type I diabetes or II) Type 2 diabetes, hypertension, heart qi, ischemic heart disease, hypertension, triglyceride dyslipidemia, HDL dyslipidemia, cholesterol dyslipidemia, elevated fasting blood glucose, abnormal leptin regulation and abnormal fat regulation Things.
  • Another aspect of the present invention provides the use of the artificial brown adipose tissue of the present invention for the preparation of a medical article for treating a metabolic disorder, wherein the metabolic disorder comprises polycystic ovary syndrome, obesity, central obesity, diabetes (type I diabetes) Or type II diabetes), hypertension, qi deficiency, ischemic heart disease, hypertension, triglyceride dyslipidemia, HDL dyslipidemia, cholesterol dyslipidemia, elevated fasting blood glucose, abnormal leptin regulation, and abnormal fat regulation Wait.
  • the metabolic disorder comprises polycystic ovary syndrome, obesity, central obesity, diabetes (type I diabetes) Or type II diabetes), hypertension, qi deficiency, ischemic heart disease, hypertension, triglyceride dyslipidemia, HDL dyslipidemia, cholesterol dyslipidemia, elevated fasting blood glucose, abnormal leptin regulation, and abnormal fat regulation Wait.
  • Another aspect of the present invention provides the use of the artificial brown adipose tissue of the present invention as a physiological process model for studying brown fat; preferably, the physiological process associated with brown fat refers to fat proliferation of brown adipose tissue in vivo , chronic inflammation of adipose tissue, recruitment and differentiation of macrophages, and fat angiogenesis.
  • the fat precursor cells or the tissue or mixed cells containing the fat precursor cells do not comprise germ cells or pluripotent stem cells, or they are all non-human cells, or are used in the present invention.
  • Adipose precursor cells or mixed cells are commercially available.
  • the adipose precursor cells may be derived from autologous cells of the subject.
  • the artificial adipose tissue of the present invention can be used as a research and treatment tool for direct endocrine diseases or indirect related diseases involving fat endocrine metabolism caused by any factor;
  • the artificial adipose tissue of the present invention can be used as a research model for studying physiological problems
  • the artificial adipose tissue of the present invention overcomes the problem of loose structure and unsuitable surgery when the brown fat is transplanted into the body in the prior art, and provides a personalized adipose tissue which can be designed according to the use site and body shape of the subject;
  • the artificial adipose tissue of the present invention can achieve a cell secretory function by activation of an adipocyte-inducing fluid.
  • FIG. 1 3D printed adipose tissue flow diagram: Adipose tissue cells and scaffold materials are prepared by printing first, then by injecting or first mixing and printing to prepare 3D printed adipose tissue.
  • FIG. 2 Adipocytes differentiated into fat cells, and after oil red O staining, a large number of oil droplets secreted by fat cells can be seen.
  • the composition of the mixed culture solution was: DMEM F12 + 10% FBS + 1% streptomycin + 1% penicillin.
  • the mixed culture solution was sterilized by filtration at 0.22 ⁇ m, and then equilibrated in a 37 ° C, 5% CO 2 incubator for 2-4 hours.
  • Fat cell induction solution I MEM/F12 + 10% FBS + streptomycin + penicillin + 5 ug / mL insulin + 1 nM 3, 3 ', 5-triiodo-L-thymidine (T3); 0.22 ⁇ m filtration After sterilization, equilibrate for 2-4 hours in a 37 ° C, 5% CO 2 incubator.
  • Fat cell induction solution II MEM/F12+10%FBS+streptomycin+penicillin+5ug/mL insulin+1nM 3,3',5-T triiodo-L-thymidine (T3)+125uM ⁇ Indomethacin +0.5 mM 3-isobutyl-1-methylxanthine (IBMX) + 0.5 uM rosiglitazone; 0.22 ⁇ m filter sterilized and equilibrated in a 37 ° C, 5% CO 2 incubator 2 4 hours.
  • IBMX 3-isobutyl-1-methylxanthine
  • the collected cells were placed in a 10 cm culture dish, and the mixed precursor culture medium was incubated in a 37 ° C incubator for 1-2 h, and the fat precursor cells were attached. Remove the other impurity cells from the supernatant and add fresh mixed culture solution. After 24 hours, the cells were changed, and the growth of the cultured cells was observed every day, and the cell changes were recorded in detail.
  • the shape of the three-dimensional adipose tissue is designed to resemble the shape of an adipose tissue, and the internal structure is ensured to communicate with a certain pore structure;
  • the prepared biocompatible stent was stored at -80 ° C until use.
  • the cultured progenitor cells of Example 2 were injected into a biocompatible scaffold at a final concentration of 2 x 10 6 cells/mL to obtain a brown adipose tissue precursor.
  • the prepared brown adipose tissue precursor was placed in the adipocyte induction solution I for 2 days, and the adipocyte induction solution II was cultured for 4-5 days to induce brown adipose tissue.
  • the shape of the three-dimensional adipose tissue is designed to resemble the shape of adipose tissue, and the internal structure is ensured to communicate with a certain pore structure;
  • the prepared brown adipose tissue precursor was cultured in adipocyte induction solution I for 2 days, and the adipocyte induction solution II was cultured for 4-5 days to induce brown adipose tissue. After staining with oil red O, a large amount of fat cells were secreted. The oil droplets prove that the active brown adipose tissue has been obtained, and the related oils and active factors can be normally secreted, and the cell secretory function can be achieved.

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  • Manufacturing & Machinery (AREA)
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  • Pharmacology & Pharmacy (AREA)
  • Optics & Photonics (AREA)
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  • Animal Behavior & Ethology (AREA)
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Abstract

L'invention concerne un tissu adipeux imprimé en 3D, et en particulier un tissu adipeux artificiel. Le tissu adipeux imprimé en 3D comprend un échafaudage biocompatible, un tissu adipeux et une solution de culture mixte. L'échafaudage biocompatible et le tissu adipeux permettent de préparer un tissu adipeux artificiel bioactif à l'aide de la bio-impression 3D. La solution de culture mixte est utilisée pour la culture et la conservation du tissu adipeux et des cellules adipeuses. Une endoprothèse biocompatible est préparée au moyen de l'impression 3D. Le tissu adipeux artificiel peut être utilisé comme outil pour la recherche de mécanismes physiopathologiques et comme traitement de maladies endocrines et métaboliques d'origine graisseuse.
PCT/CN2017/116247 2017-12-14 2017-12-14 Tissu adipeux imprimé en 3d Ceased WO2019113900A1 (fr)

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PCT/CN2017/116247 WO2019113900A1 (fr) 2017-12-14 2017-12-14 Tissu adipeux imprimé en 3d

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Application Number Priority Date Filing Date Title
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104769101A (zh) * 2012-09-04 2015-07-08 人类起源公司 组织产生方法
CN104768586A (zh) * 2012-09-04 2015-07-08 人类起源公司 组织生成的方法
WO2016123362A1 (fr) * 2015-01-30 2016-08-04 Northwestern University Ovaire artificiel

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104769101A (zh) * 2012-09-04 2015-07-08 人类起源公司 组织产生方法
CN104768586A (zh) * 2012-09-04 2015-07-08 人类起源公司 组织生成的方法
WO2016123362A1 (fr) * 2015-01-30 2016-08-04 Northwestern University Ovaire artificiel

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG, ZHI ET AL.: "Application of Adipose-Derived Stem Cells Compound with Three-dimensional Printing Scaffold in Tissue Engineering", CHINESE JOURNAL OF REPARATIVE AND RECONSTRUCTIVE SURGERY, vol. 30, no. 3, 31 March 2016 (2016-03-31), pages 320 - 322 *

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