WO2024055893A1 - Procédé de préparation pour une fibre bionique à composants multiples portant des cellules d'ensemencement, et son application - Google Patents

Procédé de préparation pour une fibre bionique à composants multiples portant des cellules d'ensemencement, et son application Download PDF

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Publication number
WO2024055893A1
WO2024055893A1 PCT/CN2023/117476 CN2023117476W WO2024055893A1 WO 2024055893 A1 WO2024055893 A1 WO 2024055893A1 CN 2023117476 W CN2023117476 W CN 2023117476W WO 2024055893 A1 WO2024055893 A1 WO 2024055893A1
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WIPO (PCT)
Prior art keywords
tube
solution
iii
phase solution
deionized water
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Application number
PCT/CN2023/117476
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English (en)
Chinese (zh)
Inventor
赵远锦
王经琳
任昊桢
陈国璞
商逸璇
Original Assignee
南京鼓楼医院
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Publication of WO2024055893A1 publication Critical patent/WO2024055893A1/fr

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Classifications

    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F1/00—General methods for the manufacture of artificial filaments or the like
    • D01F1/02—Addition of substances to the spinning solution or to the melt
    • D01F1/08—Addition of substances to the spinning solution or to the melt for forming hollow filaments
    • 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
    • A61L27/02—Inorganic materials
    • A61L27/025—Other specific inorganic materials not covered by A61L27/04 - A61L27/12
    • 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
    • A61L27/16—Macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • 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
    • A61L27/20—Polysaccharides
    • 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
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
    • D01D5/00—Formation of filaments, threads, or the like
    • D01D5/24—Formation of filaments, threads, or the like with a hollow structure; Spinnerette packs therefor
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F1/00—General methods for the manufacture of artificial filaments or the like
    • D01F1/02—Addition of substances to the spinning solution or to the melt
    • D01F1/10—Other agents for modifying properties
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F8/00—Conjugated, i.e. bi- or multicomponent, artificial filaments or the like; Manufacture thereof
    • D01F8/04—Conjugated, i.e. bi- or multicomponent, artificial filaments or the like; Manufacture thereof from synthetic polymers
    • D01F8/10—Conjugated, i.e. bi- or multicomponent, artificial filaments or the like; Manufacture thereof from synthetic polymers with at least one other macromolecular compound obtained by reactions only involving carbon-to-carbon unsaturated bonds as constituent
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F8/00—Conjugated, i.e. bi- or multicomponent, artificial filaments or the like; Manufacture thereof
    • D01F8/18—Conjugated, i.e. bi- or multicomponent, artificial filaments or the like; Manufacture thereof from other substances
    • 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

Definitions

  • the invention relates to the field of biomedical materials, and in particular to a preparation method and application of a bionic seed cell-carrying multi-component fiber.
  • Liver transplantation is the most effective method to treat severe liver failure, but the problem of insufficient liver donors has seriously affected the widespread implementation of liver transplantation. Finding a safe and effective treatment method for liver failure is a current medical research hotspot and a major issue that needs to be solved urgently.
  • bioartificial liver combines biology and engineering. Its principle is to build a three-dimensional space complex of cells and biomaterials as a bioreactor to simulate normal liver function and perform blood circulation outside the body to treat patients with liver failure. Purpose of treatment. This therapy is expected to become an alternative treatment for acute and chronic liver failure, end-stage liver disease and metabolic disorders after liver transplantation, and has broad clinical application prospects.
  • Microfluidic technology creates an exciting route towards the preparation of functional materials.
  • Microfluidic technology is a technology that confines minute amounts of liquid in micro-sized microchannels and precisely controls and operates them. Therefore, microfluidic technology has high hopes in the preparation of functional microfibers.
  • the present invention provides a method for preparing biomimetic seed cell-laden multi-component fibers based on microfluidic technology.
  • the purpose of the present invention is to provide a method for preparing bionic seed cell-laden multi-component fibers to promote the proliferation and adhesion of seed cells.
  • a method for preparing biomimetic seed cell-laden multi-component fibers including the following steps:
  • the IV tube is used as the internal phase solution channel
  • the I tube is the intermediate phase solution channel
  • the gap between the II tube and the III tube is the external phase solution channel
  • the PVA (polyvinyl alcohol) solution is used as the internal phase solution
  • the seed cells are loaded Sodium alginate solution is the intermediate phase solution
  • calcium chloride aqueous solution is the external phase solution
  • step S1 after the preparation of tubes I and II, they are placed in ethanol for ultrasonic cleaning for 5 minutes, and then dried with nitrogen.
  • step S1 the prepared II tube is immersed in an acetone solution containing 5% octadecyltrimethoxysilane. Hydrophobic treatment in liquid.
  • step S2 the needle is connected to the I tube, III tube, and IV tube through the PE tube.
  • step S3 the concentration of the PVA solution is 10 wt%, the concentration of the sodium alginate solution is 2 wt%, and the concentration of the calcium chloride aqueous solution is 1.5 wt%.
  • step S3 the flow rates of the internal phase solution, the intermediate phase solution, and the external phase solution are 0.5 mL/h, 3 mL/h, and 10 mL/h respectively.
  • step S3 the multi-component hollow fiber device is removed by: first stopping the flow of calcium chloride aqueous solution, then feeding deionized water into the external phase solution channel, and then stopping the flow of sodium alginate solution. After the sodium alginate solution no longer flows out of the tube outlet, stop passing deionized water. Finally, remove the PE tube and needle, and use deionized water to clean each solution channel.
  • step S3 the PVA solution, calcium chloride aqueous solution carrying seed cells, and sodium alginate solution were injected into the internal phase solution channel, the external phase solution channel, and the intermediate phase using 1mL, 10mL, and 25mL SGE precision syringes to remove bubbles respectively. solution channel.
  • the invention also provides a biomimetic seed cell-laden multicomponent fiber obtained by any of the above preparation methods and its application in preparing a bioartificial liver.
  • the present invention builds a multi-component hollow fiber device with multi-phase solution channels that is easy to accurately control and operate by drawing and assembling several glass capillary tubes and installing needles through PE tubes; PVA solution, sodium alginate solution, and calcium chloride aqueous solution carrying seed cells are introduced into the channel and the external phase solution channel, relying on the reaction of sodium alginate and calcium chloride solution that can quickly form ultrafine fiber gel to promote seed cells For proliferation and adhesion, biomimetic seed cell-laden multi-component hollow fibers were prepared by adjusting the flow rates of the three solutions.
  • the prepared bionic seed cell-laden multi-component (hollow) fiber has high biocompatibility and can maintain the morphology and function of liver cells for a period of time; it also has a similar structure to the natural liver lobule and is multi-component hollow.
  • the fiber has a bionic bile canalicular structure, which is conducive to material transport and function.
  • Figure 1 is a schematic structural diagram of a multi-component hollow fiber device
  • Figure 2 is a physical diagram of the multi-component hollow fiber device
  • Figure 3 shows the fluorescence microscope images of the cross-section and longitudinal section of the prepared multi-component fiber (non-hollow), (a) is the cross-section, (b) is the longitudinal section;
  • Figure 4 is a cross-sectional fluorescence microscope image of the prepared multi-component hollow fiber with bionic seed cells for bioartificial liver;
  • Figure 5 is a scanning electron microscope image of the prepared multi-component hollow fiber with bionic seed cells for bioartificial liver
  • Figure 6 is a fluorescent image of live and dead staining of the prepared bioartificial liver after culture with multi-component hollow fibers of bionic seed cells;
  • Figure 7 is a schematic diagram of albumin and urea secretion after co-culture of bionic seed cell-laden multi-component fibers and bionic seed cell-laden multi-component hollow fibers.
  • Multi-component hollow fibers are prepared by the following method, specifically including the following steps:
  • tube II Draw a glass capillary tube with an inner diameter larger than the tip end of tube I, and gently polish any port on sandpaper until the port is flat and smooth to obtain tube II; tube I and tube II are placed in ethanol after preparation Ultrasonically clean for 5 minutes, blow dry with nitrogen, and immerse the prepared II tube in an acetone solution containing 5% octadecyltrimethoxysilane for hydrophobic treatment;
  • tube IV as the internal phase solution channel
  • tube I as the intermediate phase solution channel
  • the gap between tube II and tube III as the external phase solution channel
  • 10wt% PVA solution as the internal phase solution
  • 2wt% seed cells The sodium alginate solution is the intermediate phase solution
  • the 1.5wt% calcium chloride aqueous solution is the external phase solution;
  • the diameter of the jet gradually increases; after the jet stabilizes, the seeded cells begin to flow at a flow rate of 3mL/h. of sodium alginate solution. After a few minutes, gradually introduce the calcium chloride aqueous solution into the external phase solution channel. At the same time, reduce the flow rate of the deionized water to ensure that the linear jet flow flows out stably until the deionized water is no longer introduced and the chlorine The flow rate of the calcium chloride aqueous solution is 10ml/L. At this time, the linear jet flow is completely transformed into a gel-like calcium alginate fiber, which flows along the outlet channel driven by the calcium chloride aqueous solution; it is collected and prepared using the calcium chloride solution. of calcium alginate fiber;
  • Multi-component hollow fibers loaded with seed cells were then prepared according to the method of Example 1, and multi-component hollow fibers loaded with seed cells (non-hollow) were prepared according to a method similar to Example 1 (the only difference being that the PVA solution was not passed through).
  • the sodium alginate solution loaded with seed cells that flows into the I tube is a sodium alginate solution loaded with seed cells mixed with red and green fluorescent nanoparticles, that is, each of the six parallel tube openings is cross-introduced with red and green fluorescent nanoparticles. Fluorescent nanoparticles were loaded onto the seeded cells in an alginate solution to characterize the different components.
  • Figure 3 shows the cross-section and longitudinal sections of the prepared multi-component fiber. Light microscope image, Figure 4 is a cross-sectional fluorescence microscope image of the prepared multi-component hollow fiber, and Figure 5 is a scanning electron microscope image of the multi-component hollow fiber.
  • the albumin secretion level and urea synthesis value of HepG2 showed an increasing trend.
  • the introduction of PVA solution improves the biological activity of calcium alginate fiber, provides a better environment for cell growth, and thereby promotes functional expression.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Textile Engineering (AREA)
  • Veterinary Medicine (AREA)
  • Dermatology (AREA)
  • Transplantation (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Manufacturing & Machinery (AREA)
  • Biomedical Technology (AREA)
  • Inorganic Chemistry (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • Botany (AREA)
  • Toxicology (AREA)
  • Mechanical Engineering (AREA)
  • Separation Using Semi-Permeable Membranes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un procédé de préparation pour une fibre bionique à composants multiples portant des cellules d'ensemencement, et son application. Le procédé de préparation comprend les étapes suivantes consistant à : S1, 1) étirer et assembler des tubes I à extrémités pointues ; 2) étirer un tube II ayant une extrémité pointue ; et 3) étirer un tube III ayant un diamètre interne supérieur à ceux du tube I et du tube II ; et insérer le tube I et le tube II dans le tube III à travers deux ouvertures du tube III, respectivement, insérer les extrémités pointues des tubes I dans une extrémité non pointue du tube II, ajuster les axes des tubes I, du tube II et du tube III pour les faire coïncider, et fixer les tubes I, le tube II et le tube III en place ; S2, étirer un tube IV ayant une extrémité pointue, emboîter de manière coaxiale les extrémités pointues du tube IV et des tubes I, et installer des aiguilles à l'extérieur des tubes I, du tube III et du tube IV ; et S3, 1) définir chaque canal de solution et préparer des solutions ; introduire de l'eau désionisée dans un canal de solution externe, puis introduire séquentiellement et de manière correspondante une solution de PVA, une solution d'alginate de sodium et une solution aqueuse de chlorure de calcium, et réduire progressivement le débit de l'eau désionisée jusqu'à ce que l'eau désionisée soit complètement coupée, de façon à obtenir une fibre d'alginate de calcium ; et utiliser une solution de chlorure de calcium pour collecter les fibres d'alginate de calcium.
PCT/CN2023/117476 2022-09-13 2023-09-07 Procédé de préparation pour une fibre bionique à composants multiples portant des cellules d'ensemencement, et son application WO2024055893A1 (fr)

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CN202211023430.6A CN115262028A (zh) 2022-09-13 2022-09-13 一种仿生载种子细胞多组分纤维的制备方法及应用
CN202211023430.6 2022-09-13

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115262028A (zh) * 2022-09-13 2022-11-01 南京鼓楼医院 一种仿生载种子细胞多组分纤维的制备方法及应用

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CN114854677A (zh) * 2022-07-04 2022-08-05 南京农业大学 一种用于细胞培养肉生产的微流控仿生纤维及其制备方法和应用
CN115262028A (zh) * 2022-09-13 2022-11-01 南京鼓楼医院 一种仿生载种子细胞多组分纤维的制备方法及应用

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