WO2023241094A1 - Foie d'ingénierie tissulaire fondé sur un échafaudage décellularisé végétal et son procédé de préparation - Google Patents

Foie d'ingénierie tissulaire fondé sur un échafaudage décellularisé végétal et son procédé de préparation Download PDF

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WO2023241094A1
WO2023241094A1 PCT/CN2023/078369 CN2023078369W WO2023241094A1 WO 2023241094 A1 WO2023241094 A1 WO 2023241094A1 CN 2023078369 W CN2023078369 W CN 2023078369W WO 2023241094 A1 WO2023241094 A1 WO 2023241094A1
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plant
decellularized scaffold
preparation
decellularized
celery
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PCT/CN2023/078369
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Chinese (zh)
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任昊桢
王经琳
赵远锦
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南京鼓楼医院
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    • 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
    • C12N5/067—Hepatocytes
    • C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
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    • 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
    • C12N5/067—Hepatocytes
    • 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
    • A61L27/3637—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 characterised by the origin of the biological material other than human or animal, e.g. plant extracts, algae
    • A—HUMAN NECESSITIES
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    • 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
    • A61L27/3641—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 characterised by the site of application in the body
    • 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
    • A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • A—HUMAN NECESSITIES
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    • 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
    • A61L27/3804—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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
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    • 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
    • A61L27/3839—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 characterised by the site of application in the body
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/28—Materials or treatment for tissue regeneration for liver reconstruction
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
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    • 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 belongs to the field of biomedical materials, and specifically relates to a tissue engineering liver based on a plant decellularized scaffold and a preparation method.
  • liver decellularized scaffolds refer to the removal of immunogenic cellular components while retaining non-immunogenic ECM, retaining the natural 3D structure of the tissue, and providing a stable physical structure for subsequent cell-cell interactions and cell-ECM interactions.
  • Commonly used liver decellularized scaffolds are mainly derived from human and animal livers. Due to species differences, this Unavoidable immune rejection may result, thus limited availability. Therefore, in the biomedical field, there is an urgent need for a biocompatible, non-animal-derived decellularized scaffold that can replace animal tissue.
  • the present invention provides a tissue engineering liver based on a plant decellularized scaffold and a preparation method thereof, which helps the proliferation and adhesion of liver cells with the help of the natural porous microstructure of the decellularized celery scaffold.
  • a method for preparing tissue-engineered liver based on plant decellularized scaffolds including the following steps:
  • the celery is celery with a hollow hole structure, which is similar to the structure of liver lobules.
  • step S2 the concentration of the sodium dodecyl sulfate solution is 10 wt%.
  • step S2 the celery stems were immersed in sodium dodecyl sulfate solution for 5 days, and the solvent was changed every day.
  • step S2 in the trition x-100 solution containing hypochlorous acid, the content of hypochlorous acid is 0.1v/v%, and the content of trition x-100 is 1v/v%.
  • step S2 the celery stems are rinsed with a trition x-100 solution containing hypochlorous acid for two days.
  • the decellularized scaffold obtained in step S2 is washed with PBS solution to provide an environment suitable for cell survival.
  • step S3 mix the hepatocyte mixture and Matrigel with a volume ratio of 1:4 and plant it on the decellularized scaffold. After incubating it in the incubator for 30 minutes, add culture medium to help the hepatocytes grow on the decellularized scaffold. adhesion and proliferation.
  • a tissue-engineered liver based on plant decellularized scaffold prepared by the above method A tissue-engineered liver based on plant decellularized scaffold prepared by the above method.
  • the beneficial effects of the invention are: plants are natural renewable materials with abundant sources and are easy to obtain, providing a low-cost substitute for animal tissues.
  • Celery stem tissue has a natural vascular bundle-like porous structure, is cheap, easy to obtain, non-toxic, safe, and can be eaten in daily life.
  • the present invention obtains a decellularized scaffold by soaking celery stems in sodium dodecyl sulfate (SDS) solution, and then rinsing the celery stems with trition x-100 solution containing hypochlorous acid.
  • SDS sodium dodecyl sulfate
  • the method is simple and the operation is Convenient, low-cost, safe, non-toxic, cheap and easily available compared to other sources of decellularized scaffolds.
  • the prepared decellularized scaffold has high biocompatibility, suitable porosity and hydrophilicity, and excellent mechanical properties, providing a suitable biological microenvironment for the growth and proliferation of cells, and its natural 3D microenvironment cannot easily replicated by current 3D printing and microfluidic technologies.
  • a method for preparing tissue-engineered livers based on plant decellularized scaffolds was developed.
  • the tissue-engineered liver prepared by this method has high biocompatibility and can maintain the morphology and function of liver cells for a period of time. In addition, it can also maintain certain liver functions in vitro.
  • the tissue-engineered liver based on the plant decellularized scaffold provided by the present invention shows broad application prospects in tissue repair and regeneration, disease treatment and other aspects.
  • Figure 1 shows the electron microscopy characterization of the celery scaffold prepared by the present invention before and after decellularization.
  • the ratio bars are 100 ⁇ m (i), 20 ⁇ m (ii), and 5 ⁇ m (iii);
  • Figure 2 is a confocal scanning fluorescence image of the bioengineered liver tissue prepared by the present invention after culture for 14 days.
  • the scale bars are all 100 ⁇ m;
  • Figure 3 shows the bioengineered liver tissue prepared by the present invention, and the cells cultured in 2D conventional plane and matrix gel.
  • Immunofluorescence image of ALB (red), nuclei stained blue, scale bar is 50 ⁇ m;
  • Figure 4 shows the expression of liver function of bioengineered liver tissue prepared by the present invention, and cells cultured in 2D conventional plane culture and matrix gel culture.
  • This embodiment provides a tissue-engineered liver based on a plant decellularized scaffold, which is prepared by the following method. The specific steps are as follows:
  • the prepared tissue-engineered livers were cultured for another 14 days, while hepatocytes suspended in Matrigel were seeded on 24-well plates as a control group.
  • cell number was determined using GFP staining.
  • the number of GFP-positive cells was higher than that on the first day.
  • the number of GFP-positive cells increased with the prolongation of culture time, indicating that the decellularized scaffold promoted cell proliferation, confirming its high biocompatibility.
  • hepatocytes Due to the lack of extracellular matrix components and cell-cell interactions, hepatocytes lose their differentiated state over time when cultured in 2D dishes.
  • the composition of Matrigel is heterogeneous, with over 1500 different proteins in its composition, including the most common proteins such as laminin and type IV collagen.
  • Metabolic activity in the scaffolds albumin secretion, glycogen synthesis and mRNA expression levels of key hepatic transcription factors and functional genes were quantified. As shown in Figure 3, the fluorescence image of albumin confirmed that the number of albumin-positive cells was greater in both the Matrigel and acellular scaffold groups compared with the dish group.
  • Hepatocyte-specific gene expression was performed during culture to determine the extent to which liver-specific functions are maintained over time under different culture conditions.
  • the genes detected in each group included ALB, cytochrome P-4503A4 (CYP3A4), cytochrome P-450 1A2 (CYP1A2), cytokeratin 18 (CK18), and asialoglycoprotein receptor (ASGPR).
  • ALB cytochrome P-4503A4
  • CYP1A2 cytochrome P-450 1A2
  • CK18 cytokeratin 18
  • ASGPR asialoglycoprotein receptor
  • the mRNA levels in the decellularized scaffold group were lower than those in the other two groups, and similar results were also shown in CK18 and ASGPR1. This culture condition improves the functional status of the cells. Therefore, the liver engineering structure constructed on the scaffold has a more stable liver phenotype.

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Abstract

La présente invention porte sur un procédé de préparation d'un foie d'ingénierie tissulaire en se fondant sur un échafaudage décellularisé végétal. Le procédé comprend les étapes suivantes : S1, découpe de la tige du céleri d'eau creux ; S2, immersion de la tige du céleri dans une solution de dodécyl sulfate de sodium, puis rinçage de la tige du céleri à l'aide d'une solution de trition x-100 contenant de l'acide hypochloreux pour obtenir un échafaudage décellularisé ; et S3, implantation d'hépatocytes sur l'échafaudage décellularisé et mise en culture de ceux-ci.
PCT/CN2023/078369 2022-06-14 2023-02-27 Foie d'ingénierie tissulaire fondé sur un échafaudage décellularisé végétal et son procédé de préparation WO2023241094A1 (fr)

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CN114836371B (zh) * 2022-06-14 2023-02-24 南京鼓楼医院 一种基于植物脱细胞支架的组织工程肝脏和制备方法
CN116173300B (zh) * 2022-11-21 2023-08-22 郑州大学第一附属医院 一种植物脱细胞人工血管及其制备方法

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