WO2022237002A1 - 一种基于凝胶微球的3d打印生物墨水及其应用 - Google Patents

一种基于凝胶微球的3d打印生物墨水及其应用 Download PDF

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WO2022237002A1
WO2022237002A1 PCT/CN2021/113652 CN2021113652W WO2022237002A1 WO 2022237002 A1 WO2022237002 A1 WO 2022237002A1 CN 2021113652 W CN2021113652 W CN 2021113652W WO 2022237002 A1 WO2022237002 A1 WO 2022237002A1
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gel
printing
cells
cell
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方永聪
熊卓
张婷
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Tsinghua University
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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
    • A61L27/3804—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
    • A61L27/3826—Muscle cells, e.g. smooth muscle 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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/222—Gelatin
    • 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
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/225—Fibrin; Fibrinogen
    • 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
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • 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
    • A61L27/3839—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
    • A61L27/3882—Hollow organs, e.g. bladder, esophagus, urether, uterus
    • 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
    • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—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
    • B33Y10/00—Processes of additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—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
    • B33Y70/00—Materials specially adapted for additive manufacturing
    • B33Y70/10—Composites of different types of material, e.g. mixtures of ceramics and polymers or mixtures of metals and biomaterials
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—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
    • B33Y80/00—Products made by additive manufacturing
    • 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
    • A61L2430/22—Materials or treatment for tissue regeneration for reconstruction of hollow organs, e.g. bladder, esophagus, urether, uterus

Definitions

  • the invention relates to a 3D printing bioink based on gel microspheres and its application, belonging to the technical fields of tissue engineering and biomanufacturing.
  • Bio-3D printing technology forms 3D tissues and organs with complex structures by stacking cell-containing bio-inks according to a predefined path, and has great advantages in the construction of complex tissues and organs.
  • Bio-3D printing technology can generally be divided into three types according to the dimension of the building unit: droplet-based (zero-dimensional) printing method, micro-filament-based (one-dimensional) micro-extrusion printing method, and surface-based (two-dimensional) printing method. photocuring printing method.
  • micro-extrusion bio-3D printing technology has the potential to form and manufacture complex tissue structures, and has become the current mainstream printing process. In this process, viscous bioink is squeezed by mechanical force or air pressure as it passes through tiny nozzles to form cell-laden microfibers.
  • the micro-extrusion 3D printing process requires bio-ink materials to have suitable viscosity and rheological properties, which greatly limits the source and concentration range of materials available for printing.
  • the viscosity range of the bio-ink is required to be in the range of 0.03-5 ⁇ 10 4 Pa ⁇ s. Specifically, if the viscosity of the bio-ink is too high, the greater the shear force the bio-ink bears when extruded through the nozzle, the greater the damage to the cells; if the viscosity is too low, although it can be extruded smoothly, the extrusion Afterwards, it is difficult to maintain the basic morphological structure.
  • the micro-extrusion 3D printing process requires the bio-ink material to have shear thinning properties, that is, when the bio-ink is subjected to a high shear rate when passing through the nozzle, the viscosity of the bio-ink becomes lower, which is conducive to its smooth extrusion; When extruded, the bio-ink returns to its original higher viscosity, which is conducive to maintaining the shape and structure after printing.
  • the bio-ink used in the micro-extrusion 3D printing process is hydrogel, which usually has poor mechanical properties, which limits its application in the construction of complex tissues and organs.
  • the purpose of the present invention is to provide a new type of 3D printing bio-ink based on gel microspheres, which has shear thinning and self-healing properties, can improve the mechanical strength and stability of 3D printing structures, thereby greatly expanding the bio-ink
  • the material source and printing ability overcome the shortcomings of existing 3D printing bio-inks such as material viscosity limitations and poor mechanical properties.
  • the 3D printing bio-ink based on gel microspheres provided by the present invention is formed by cross-linked cell-loaded gel microspheres, or by cross-linked cell-loaded gel microspheres and one or more uncrosslinked gel microspheres.
  • the combined gel material is mixed to obtain;
  • the cell-laden gel microspheres serve as the dispersed phase and the gel material serves as the continuous phase.
  • the gel material may be subjected to secondary cross-linking in the following ways: at least one of light cross-linking, temperature cross-linking, ion cross-linking, enzyme cross-linking and covalent cross-linking.
  • the gel microspheres loaded with cells can be prepared according to the following method:
  • At least one of hanging drop culture, ultra-low attachment culture plate, magnetic suspension culture, dynamic rotation culture and microfluidic technology At least one of hanging drop culture, ultra-low attachment culture plate, magnetic suspension culture, dynamic rotation culture and microfluidic technology.
  • the cell density in the gel microspheres may be 10 6 cells/mL-10 8 cells/mL;
  • the mass-volume concentration of the gel material in the cell-laden gel microspheres may be 10-100 mg/mL, such as 20-50 mg/mL.
  • the diameter of the cell-laden gel microspheres may be 50 ⁇ m-1000 ⁇ m, such as 200 ⁇ m-250 ⁇ m, 250 ⁇ m-300 ⁇ m or 350 ⁇ m-400 ⁇ m;
  • the volume content of the cell-laden gel microspheres in the 3D printing bio-ink may be 40% to 100%, such as 60% to 80%, 60%, 80% or 100%, and when it is 100%, it is forming said 3D printed bioink solely from said cell-laden gel microspheres;
  • the mass-volume concentration of the gel material as the continuous phase may be 1-100 mg/mL, such as 4-25 mg/mL;
  • said gel material as said continuous phase can be loaded with cells
  • the cell density in the gel material may be 10 6 cells/mL to 5 ⁇ 10 7 cells/mL.
  • the gel material used in the cell-laden gel microspheres and the gel material used as the continuous phase are both natural polymer hydrogel and/or synthetic polymer hydrogel glue;
  • the natural polymer hydrogel material can be sodium alginate, gelatin, collagen, Matrigel, chitosan, silk fibroin, hyaluronic acid, fibrinogen, chondroitin sulfate, albumin and their formazan At least one of acryloyl products (such as methacryloyl gelatin (GelMA), methacryloyl sodium alginate (AlgMA), etc.);
  • the synthetic polymer hydrogel material can be polyethylene glycol (PEG), polypropylene alcohol (PVA), polyethylene glycol diacrylate (PEGDA), polyethylene oxide (PEO), polyacrylamide ( PAM), polyacrylic acid (PAA), polyphosphazene (PAMPS), poly-N-isopropylacrylamide hydrogel (PNIPAAm) and their methacrylylated products (such as concave arm polyethylene glycol acrylate (4-arm-PEG-AC), methacrylated polyvinyl alcohol (PVAMA), etc.) at least one.
  • PEG polypropylene alcohol
  • PEGDA polyethylene glycol diacrylate
  • PEO polyethylene oxide
  • PAM polyacrylamide
  • PAA polyacrylic acid
  • PAMPS polyphosphazene
  • PNIPAAm poly-N-isopropylacrylamide hydrogel
  • methacrylylated products such as concave arm polyethylene glycol acrylate (4-arm-PEG-AC), methacrylated polyvinyl
  • the cells in the cell-loaded gel microspheres and the cells in the gel material are at least one of parenchymal cells, tumor cells, stromal cells and endothelial cells;
  • the parenchymal cells are derived from at least one of the following tissues/organs: heart, liver, kidney, pancreas and brain structure.
  • the 3D printing bio-ink of the present invention (when including the continuous phase) can multi-level suspension 3D print tissue/organ models with complex vascular channels and/or heterogeneous cell structures, which can be used for repair of damaged tissues and organs, drug development and screening, and pathological research Wait;
  • the tissue/organ model includes at least one of heart, liver, kidney, pancreas and brain structures;
  • the size of the tissue/organ model is 500 ⁇ m-100 mm.
  • Figure 1 is a schematic diagram of the 3D printing bioink based on gel microspheres of the present invention, in the figure, 1 represents the gel microspheres loaded with cells, 2 represents the continuous phase gel material around the gel microspheres, and 3 represents the continuous phase cells within the gel material.
  • Fig. 2 is the ring structure and microscopic observation result of the microsphere ink printing prepared in Example 1 of the present invention.
  • Fig. 3 is the rheological performance characterization of the 3D printing bio-ink based on gel microspheres prepared in Example 1 of the present invention.
  • Fig. 3a is the variation curve of viscosity with shear rate
  • Fig. 3b is the shear stress, storage
  • Figure 3c is the change curve of storage modulus under alternating high and low strain.
  • Fig. 4 shows the bronchus structure and microscopic observation results printed by the microsphere ink prepared in Example 2 of the present invention.
  • Figure 5 shows the grid and double ring structure and microscopic observation results printed by the microsphere ink prepared in Example 3 of the present invention.
  • Embodiment 1 prepare the GelMA gel microsphere bio-ink loaded with cardiomyocytes
  • Cardiomyocytes were extracted from the heart of newborn neonatal rats, and photocrosslinkable methacrylate gelatin (GelMA) was used as the microsphere carrier material to prepare a GelMA solution with a mass volume fraction of 5.0%, which contained cardiomyocytes at a density of 1 ⁇ 10 7 cells/mL. Pass the GelMA solution loaded with cardiomyocytes into the dispersed phase inlet of the T-shaped microfluidic chip at a flow rate of 0.5mL/h, and pass mineral oil containing 10% Span 80 into the continuous phase inlet at a flow rate of 4.0mL/h , light cross-linking is carried out at the outlet of the chip to obtain cardiomyocyte-loaded gel microspheres with a diameter of 200 ⁇ m to 250 ⁇ m.
  • GelMA photocrosslinkable methacrylate gelatin
  • the gel microspheres loaded with cardiomyocytes were washed and centrifuged to remove the mineral oil in the gel microspheres, and at the same time mixed with the GelMA solution loaded with endothelial cells at a volume ratio of 3:2 to obtain a bioink.
  • the schematic diagram of the structure is shown in Figure 1. Wherein, the volume content of the cell-loaded GelMA gel microspheres in the bioink is 60%, the mass-volume concentration of the GelMA gel material in the GelMA gel microspheres is 50 mg/mL, and the mass of the GelMA gel material as the continuous phase - The volume concentration is 25 mg/mL, and the endothelial cell density is 10 6 cells/mL.
  • the GelMA gel microsphere ink prepared in this example is used to print a ring structure (Fig. 2a) with a diameter of 10 mm and a height of 5 mm at room temperature.
  • the printing speed is 5 mm/s, and the extrusion speed is 0.24 mm 3 /s.
  • UV irradiation is used for crosslinking, the light intensity parameter is 100mW/cm 2 , and the time is 50s.
  • the cross-linked ring structure has good mechanical properties and can be stretched repeatedly (Fig. 2b). Observed under a fluorescence microscope, it can be seen that the continuous phase marked in green and the dispersed phase of gel microspheres marked in red are uniformly mixed (Fig. 2c).
  • the GelMA gel microsphere ink prepared in this example not only exhibits shear thinning (Fig. 3a, Fig. 3b), but also has self-healing properties (Fig. 3c). Among them, the self-healing property is not available in conventional GelMA gel bioinks. It should be noted that the minimum concentration of GelMA printing that can usually be achieved in existing studies is generally 75 mg/mL, and the 3D printing bio-ink based on gel microspheres provided by the present invention can achieve a concentration of 50 mg/mL and lower. Meet the requirements of special cells for ultra-soft matrix environment.
  • the commercially purchased myoblast cell line C2C12 cells were used for in vitro culture, the fibrinogen solution with a mass fraction of 2.0% and the Matrigel solution with a volume fraction of 40% were prepared, and the C2C12 cells were mixed, and the final cell density was 5 ⁇ 10 6 cells/ mL. Pass the fibrinogen/Matrigel solution loaded with C2C12 cells into the dispersed phase inlet of the coaxial focusing microfluidic chip at a flow rate of 0.3 mL/h, and pass mineral oil containing 5% Span 80 into the coaxial focusing microfluidic chip.
  • the continuous phase inlet of the flow control chip has a flow rate of 3.0mL/h, and a ten-meter-long silica gel tube placed in a water bath at a temperature of 37°C flows through the outlet of the chip to make the fibrinogen/Matrigel gel microspheres Temperature crosslinking occurs to obtain fibrinogen/Matrigel gel microspheres with a diameter of 250 ⁇ m to 300 ⁇ m.
  • the mass-volume concentration of fibrinogen in the cell-loaded gel microspheres is 20 mg/mL, and the volume fraction of Matrigel is 40%. .
  • the fibrinogen/Matrigel gel microspheres loaded with C2C12 cells were deoiled by centrifugation (1000 rpm, 5 minutes) at room temperature, and the microspheres were cross-linked again by using 50 U/mL thrombin solution, and then in Wash with PBS buffer solution, mix type I rat tail collagen by volume ratio of 1:1 to obtain fibrinogen/Matrigel/collagen gel microsphere ink, wherein, the volume content of gel microsphere in the gel microsphere ink is 50%, the mass-volume concentration of collagen as the continuous phase was 4 mg/mL.
  • the gel microsphere ink prepared in this example was loaded into the nozzle of the 3D printing equipment, and the bronchial structure was directly printed.
  • the printing temperature was 22°C
  • the printing speed was 2 mm/s
  • the extrusion speed was 0.5 mm 3 /s.
  • After the printing is completed put it into an incubator (37° C. and 5% CO 2 ) and incubate for 30 minutes, so that the entire printed structure undergoes overall temperature cross-linking.
  • Figure 4a-4b it can be seen that both ends of the printed bronchial structure have a uniform circular cross-section, and at the same time have stable mechanical properties.
  • the continuous phase marked in green and the dispersed phase of gel microspheres marked in red can be seen, and the microspheres are in a compacted state while the continuous phase is mixed around them (Fig. 4c).
  • Embodiment 3 prepare the pure GelMA gel microsphere ink of loading myoblast
  • myoblast cell line C2C12 cells were used for in vitro culture, using photo-crosslinkable methacrylate gelatin (GelMA) as the microsphere carrier material, preparing a GelMA solution with a mass volume fraction of 7.5%, and mixing the C2C12 cells, The final cell density was 7.5 ⁇ 10 6 cells/mL. Pass the GelMA solution loaded with C2C12 cells into the dispersed phase inlet of the coaxial focusing microfluidic chip, the flow rate is 1.0mL/h, and pass the mineral oil containing 20% Span 80 into the coaxial focusing microfluidic chip.
  • GelMA photo-crosslinkable methacrylate gelatin
  • the flow rate at the inlet of the continuous phase is 10.0mL/h, and light cross-linking is performed at the outlet of the chip to obtain cell-loaded gel microspheres with a diameter of 350 ⁇ m to 400 ⁇ m.
  • the gel microspheres loaded with cells are washed, centrifuged and other steps in sequence to obtain the bioink.
  • the volume content of the cell-loaded GelMA gel microspheres in the bioink is 100%, that is, it does not contain the gel material as the continuous phase, and the mass-volume concentration of the GelMA gel material in the GelMA gel microspheres is 75mg/mL .
  • the printing temperature is 27°C
  • the printing speed is 4mm/s
  • the extrusion speed is 2.5mm 3 /s.
  • ultraviolet irradiation is used for crosslinking
  • the light intensity parameter is 150mW/cm 2
  • the time is 60s.
  • the 3D printing bio-ink containing cell-loaded gel microspheres or the 3D printing bioink with cell-loaded gel microspheres as the dispersed phase and the gel material as the continuous phase, compared with the existing bio-inks, has the following advantages and outstanding sexual effects are as follows:
  • the new 3D printing bioink based on gel microspheres of the present invention has shear thinning and self-healing properties, and can directly 3D print low-concentration or low-viscosity gels without adding rheology modifiers materials, so as to realize the three-dimensional printing of conventional gel materials at ultra-low concentrations, and endow the difficult-to-print hydrogel materials with excellent printability, which greatly expands the bioink library;
  • gel microspheres can provide a customized microenvironment for cell growth, proliferation and differentiation, and at the same time protect cells from shear stress damage in the 3D printing process, which is helpful for the function of 3D printed tissues and organs Mature, greatly promoting the application of bio-3D printing technology in the construction of complex tissues/organs.

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Abstract

本发明公开了一种基于凝胶微球的3D打印生物墨水及其应用。所述3D打印生物墨水由已交联的载细胞的凝胶微球形成，或由已交联的载细胞的凝胶微球与一种或多种未交联的凝胶材料混合得到；载细胞的凝胶微球在3D打印生物墨水中的体积含量为40％～100％。本发明基于凝胶微球的3D打印生物墨水具有剪切变稀和自愈合特性，可以在不添加流变改性剂的条件下直接3D打印低浓度或低粘度凝胶材料，从而实现常规凝胶材料在超低浓度下的三维打印，并赋予难打印水凝胶材料优异的打印性；凝胶微球作为细胞载体，可以为细胞生长、增殖和分化等活动提供定制微环境，同时可以保护细胞免受3D打印工艺中剪切应力的损伤，极大促进生物3D打印技术在复杂组织/器官构建方面的应用。

Description

一种基于凝胶微球的3D打印生物墨水及其应用 技术领域
本发明涉及一种基于凝胶微球的3D打印生物墨水及其应用，属于组织工程和生物制造技术领域。
背景技术
生物3D打印技术通过将含细胞的生物墨水按照预定义路径，层层堆积形成具有复杂结构的3D组织与器官，在复杂组织和器官的构建方面具有巨大的优势。生物3D打印技术按照构筑单元的维度，通常可以分为三种：基于微滴(零维)的打印方法，基于微丝(一维)的微挤出打印方法，和基于面成形(二维)的光固化打印方法。其中，微挤出式生物3D打印技术具有成形制造复杂组织结构的潜力，成为目前主流的打印工艺。这种工艺将粘性生物墨水通过微型喷嘴时，在机械力或气压的驱动下受到挤压从而形成载细胞的微纤维。
微挤出式3D打印工艺要求生物墨水材料具备合适的粘度和流变性能，因而极大限制了可供打印的材料来源和浓度范围。一方面，生物墨水粘度范围要求在0.03～5×10 4Pa·s区间。具体而言，生物墨水的粘度过高，会使得生物墨水挤出经过喷嘴时承受的剪切力越大，对细胞造成的损伤也越大；粘度过低，虽然可以顺利挤出，然而挤出后难以维持基本的形态结构。另一方面，微挤出式3D打印工艺要求生物墨水材料具备剪切变稀性能，即当生物墨水经过喷嘴时承受高剪切速率时，生物墨水粘度变低，从而有利于其顺利挤出；当挤出后，生物墨水又恢复到原来的较高粘度，有利于维持打印后的形态结构。另外，微挤出式3D打印工艺所使用生物墨水为水凝胶，通常力学性能较差，限制了其在复杂组织器官构建上的应用。
因此，针对微挤出式3D打印工艺中现有生物墨水的材料、粘度限制和力学性能较差等不足，提供一种新型生物墨水开发策略以拓展现有技术不足甚为必要。
发明内容
本发明的目的是提供一种新型基于凝胶微球的3D打印生物墨水，其具有剪切变稀和自愈合特性，能够提高3D打印结构的力学强度和稳定性，从而极大拓展生物墨水的材料来源和打印能力，克服了现有3D打印生物墨水面临材料粘度限制和力学性能较差等不足。
本发明提供的基于凝胶微球的3D打印生物墨水，由已交联的载细胞的凝胶微球形成，或由已交联的载细胞的凝胶微球与一种或多种未交联的凝胶材料混合得到；
当包括两种材料时，所述载细胞的凝胶微球作为分散相，所述凝胶材料作为连续相。
所述生物墨水经3D打印完成后，通过作为所述连续相的凝胶材料的二次交联，以提高打印结构的力学强度和稳定性；
可采用下述方式对所述凝胶材料进行二次交联：光照交联、温度交联、离子交联、酶交联和共价交联方式中至少一种。
其中，所述载细胞的凝胶微球可按照下述方法制备：
悬滴法培养、超低附着性培养板、磁力悬浮培养、动态旋转培养和微流控技术中至少一种。
上述的3D打印生物墨水中，所述凝胶微球内细胞密度可为10 6个/mL～10 8个/mL；
所述载细胞的凝胶微球中凝胶材料的质量-体积浓度可为10～100mg/mL，如20～50mg/mL。
上述的3D打印生物墨水中，所述载细胞的凝胶微球的直径可为50μm～1000μm，如可为200μm～250μm、250μm～300μm或350μm～400μm；
所述载细胞的凝胶微球在所述3D打印生物墨水中的体积含量可为40％～100％，如60％～80％、60％、80％或100％，当为100％时即仅由所述载细胞的凝胶微球形成所述3D打印生物墨水；
上述的3D打印生物墨水中，作为所述连续相的所述凝胶材料的质量-体积浓度可为1～100mg/mL，如4～25mg/mL；
作为所述连续相的所述凝胶材料可载有细胞；
所述凝胶材料内细胞密度可为10 6个/mL～5×10 7个/mL。
上述的3D打印生物墨水中，所述载细胞的凝胶微球采用的凝胶材料与作为所述连续相的所述凝胶材料均为天然高分子水凝胶和/或合成高分子水凝胶；
具体地，所述天然高分子水凝胶材料可为海藻酸钠、明胶、胶原、Matrigel、壳聚糖、丝素蛋白、透明质酸、纤维蛋白原、硫酸软骨素、白蛋白以及它们的甲基丙烯酰化产物(如甲基丙烯酰化明胶(GelMA)、甲基丙烯酰化海藻酸钠(AlgMA)等)中的至少一种；
所述合成高分子水凝胶材料可为聚乙二醇(PEG)、聚丙烯醇(PVA)、聚乙二醇二丙烯酸酯(PEGDA)、聚环氧乙烷(PEO)、聚丙烯酰胺(PAM)、聚丙烯酸(PAA)、聚磷腈(PAMPS)、聚N-异丙基丙烯酰胺类水凝胶(PNIPAAm)以及它们的甲基丙烯酰化产物(如凹臂聚乙二醇丙烯酸酯(4-arm-PEG-AC)、甲基丙烯酰化聚乙烯醇(PVAMA)等)至少一种。
本发明3D打印生物墨水中，所述载细胞的凝胶微球内的细胞、所述凝胶材料内的细胞均为实质细胞、肿瘤细胞、基质细胞和内皮细胞中至少一种；
所述实质细胞来源于如下组织/器官：心脏、肝脏、肾脏、胰腺和脑结构中至少一种。
本发明3D打印生物墨水(包括连续相时)能够多级悬浮3D打印具有复杂血管通道和/或异质细胞结构的组织/器官模型，可用于病损组织器官修复、药物开发与筛选和病理研究等；
所述组织/器官模型包括心脏、肝脏、肾脏、胰腺和脑结构中至少一种；
所述组织/器官模型的尺寸为500μm～100mm。
附图说明
图1为本发明基于凝胶微球的3D打印生物墨水的示意图，图中，1表示载细胞的凝胶微球，2表示凝胶微球周围的连续相凝胶材料，3表示作为连续相的凝胶材料内细胞。
图2为本发明实施例1制备的微球墨水打印的圆环结构和显微观察结 果。
图3为本发明实施例1制备的基于凝胶微球的3D打印生物墨水的流变学性能表征，图中，图3a为粘度随剪切速率的变化曲线，图3b为剪切应力、储存模量和损耗模量随剪切应变的变化曲线，图3c为在交替高低应变下的储能模量变化曲线。
图4为本发明实施例2制备的微球墨水打印的支气管结构和显微观察结果。
图5为本发明实施例3制备的微球墨水打印的网格和双环结构和显微观察结果。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、制备载心肌细胞的GelMA凝胶微球生物墨水
从刚出生乳鼠心脏提取心肌细胞，采用可光交联的甲基丙烯酸酯明胶(GelMA)作为微球载体材料，配制质量体积分数为5.0％的GelMA溶液，其中含有心肌细胞的密度为1×10 7个/mL。分别将载心肌细胞的GelMA溶液通入T型微流控芯片的分散相入口，流速为0.5mL/h，将含10％司盘80的矿物油通入连续相入口，流速为4.0mL/h，在芯片出口处进行光照交联，得到直径为200μm～250μm的载心肌细胞凝胶微球。在室温下将载心肌细胞的凝胶微球依次通过清洗、离心等步骤去除凝胶微球中的矿物油，同时与载内皮细胞的GelMA溶液按按3：2的体积比混合得到生物墨水，结构示意图如图1所示。其中，生物墨水中载细胞GelMA凝胶微球的体积含量为60％，该GelMA凝胶微球内GelMA凝胶材料的质量-体积浓度为50mg/mL，作为连续相的GelMA凝胶材料的质量-体积浓度为25mg/mL，内皮细胞密度为10 6个/mL。
采用本实施例制备的GelMA凝胶微球墨水在室温下打印直径为 10mm、高度为5mm的圆环结构(图2a)，打印速度为5mm/s，挤出速度为0.24mm 3/s，打印后采用紫外照射交联，光强参数为100mW/cm 2，时间为50s。交联后圆环结构具有良好的力学性能，可以进行反复拉伸(图2b)。在荧光显微镜下观察，可以看出绿色标记的连续相和红色标记的凝胶微球分散相，两者均匀混合(图2c)。
进一步，通过流变学测试，可以发现本实施例制备的GelMA凝胶微球墨水除了表现出剪切变稀(图3a，图3b)，还具有自愈合的特性(图3c)。其中，自愈合特性是常规GelMA凝胶生物墨水所不具备的。需要说明的是，已有研究通常能实现的GelMA打印最低浓度一般为75mg/mL，本发明提供的基于凝胶微球的3D打印生物墨水，能够实现浓度在50mg/mL及更低浓度，可以满足特殊细胞对超软基质环境的要求。
实施例2、制备载成肌细胞的纤维蛋白原/Matrigel/胶原凝胶微球墨水
采用商业购买的成肌细胞系C2C12细胞进行体外培养，配制质量分数为2.0％的纤维蛋白原溶液与体积分数为40％的Matrigel溶液，并混合C2C12细胞，最终细胞密度为5×10 6个/mL。将载C2C12细胞的纤维蛋白原/Matrigel溶液通入同轴聚焦型微流控芯片的分散相入口，流速为0.3mL/h，将含5％司盘80的矿物油通入同轴聚焦型微流控芯片的连续相入口，流速为3.0mL/h，在芯片出口处流经长达十米的、放置在温度为37℃的水浴中的硅胶管，使纤维蛋白原/Matrigel凝胶微球发生温度交联，得到直径为250μm～300μm的纤维蛋白原/Matrigel凝胶微球，该载细胞凝胶微球内纤维蛋白原的质量-体积浓度为20mg/mL，Matrigel的体积分数为40％。
在室温下将载C2C12细胞的纤维蛋白原/Matrigel凝胶微球通过离心(1000转每分钟，5分钟)去油，通过用50U/mL的凝血酶溶液对微球进行再次交联，然后在用PBS缓冲溶液进行清洗，按1：1的体积比混合I型鼠尾胶原得到纤维蛋白原/Matrigel/胶原凝胶微球墨水，其中，凝胶微球墨水中凝胶微球的体积含量为50％，作为连续相的胶原的质量-体积浓度为4mg/mL。
将本实施例制备的凝胶微球墨水载入3D打印设备的喷头内，直接进行打印支气管结构，打印温度为22℃，打印速度为2mm/s，挤出速度为0.5mm 3/s。打印完成后放入培养箱(37℃和5％CO 2)中孵育30min，使得整个打印结构进行整体的温度交联。如图4a-图4b所示，可以看出打印支气管结构两端具有均匀的圆形横截面，同时具有稳定的力学性能。在荧光显微镜下观察，可以看出绿色标记的连续相和红色标记的凝胶微球分散相，微球处于压实状态，同时周围混合着连续相(图4c)。
实施例3、制备载成肌细胞的纯GelMA凝胶微球墨水
采用商业购买的成肌细胞系C2C12细胞进行体外培养，采用可光交联的甲基丙烯酸酯明胶(GelMA)作为微球载体材料，配制质量体积分数为7.5％的GelMA溶液，并混合C2C12细胞，最终细胞密度为7.5×10 6个/mL。将载C2C12细胞的GelMA溶液通入同轴聚焦型微流控芯片的分散相入口，流速为1.0mL/h，将含20％司盘80的矿物油通入同轴聚焦型微流控芯片的连续相入口，流速为10.0mL/h，在芯片出口处进行光照交联，得到直径为350μm～400μm的载细胞凝胶微球。在室温下将载细胞的凝胶微球依次通过清洗、离心等步骤得到生物墨水。其中，生物墨水中载细胞GelMA凝胶微球的体积含量为100％，即不含有作为连续相的凝胶材料，该GelMA凝胶微球内GelMA凝胶材料的质量-体积浓度为75mg/mL。
将本实施例制备的凝胶微球墨水载入3D打印设备的喷头内直接进行打印网格结构(图5a)，打印温度为27℃，打印速度为4mm/s，挤出速度为2.5mm 3/s。打印后采用紫外照射交联，光强参数为150mW/cm 2，时间为60s。在光学显微镜下观察，可以看出本实施例生物墨水完全由凝胶微球分散相组成(图5b)。
进一步，打印径为15mm、高度为2mm的双环结构(图5c)，打印速度为2.5mm/s，挤出速度为0.20mm 3/s，打印后采用紫外照射交联，光强参数为100mW/cm 2，时间为30s。在荧光显微镜下观察，可以看出本实施例生物墨水完全由绿色标记的凝胶微球分散相组成，不含有作为连续相的凝胶材料(图5d)。
工业应用
本发明提供的含载细胞凝胶微球或以含载细胞凝胶微球作为分散相和凝胶材料作为连续相的3D打印生物墨水，相比于现有的生物墨水，具有以下优点及突出性效果，体现如下：
(1)本发明新型基于凝胶微球的3D打印生物墨水，具有剪切变稀和自愈合特性，可以在不添加流变改性剂的条件下直接3D打印低浓度或低粘度凝胶材料，从而实现常规凝胶材料在超低浓度下的三维打印，并赋予难打印水凝胶材料优异的打印性，极大地扩充了生物墨水库；
(2)凝胶微球作为细胞载体，可以为细胞生长、增殖和分化等活动提供定制微环境，同时可以保护细胞免受3D打印工艺中剪切应力的损伤，有助3D打印组织器官的功能成熟，极大促进生物3D打印技术在复杂组织/器官构建方面的应用。

Claims (11)

  1. 一种基于凝胶微球的3D打印生物墨水，由已交联的载细胞的凝胶微球形成，或由已交联的载细胞的凝胶微球与一种或多种未交联的凝胶材料混合得到；
    当包括所述载细胞的凝胶微球和所述凝胶材料时，所述载细胞的凝胶微球作为分散相，所述凝胶材料作为连续相。
  2. 根据权利要求1所述的3D打印生物墨水，其特征在于：所述载细胞的凝胶微球按照下述方法制备：
    悬滴法培养、超低附着性培养板、磁力悬浮培养、动态旋转培养和微流控技术中至少一种。
  3. 根据权利要求1或2所述的3D打印生物墨水，其特征在于：所述已交联的载细胞的凝胶微球内细胞密度为10 6个/mL～10 8个/mL；
    所述已交联的载细胞的凝胶微球中凝胶材料的质量-体积浓度为10～100mg/mL。
  4. 根据权利要求1-3中任一项所述的3D打印生物墨水，其特征在于：所述已交联的载细胞的凝胶微球的直径为50μm～1000μm；
    所述已交联的载细胞的凝胶微球在所述3D打印生物墨水中的体积含量为40％～100％。
  5. 根据权利要求1-4中任一项所述的3D打印生物墨水，其特征在于：作为所述连续相的所述凝胶材料的质量-体积浓度为5～100mg/mL；
  6. 根据权利要求1-5中任一项所述的3D打印生物墨水，其特征在于：作为所述连续相的所述凝胶材料载有细胞；
    所述凝胶材料内细胞密度为10 6个/mL～5×10 7个/mL。
  7. 根据权利要求1-6中任一项所述的3D打印生物墨水，其特征在于：所述载细胞的凝胶微球采用的凝胶材料与作为所述连续相的所述凝胶材料均为天然高分子水凝胶和/或合成高分子水凝胶。
  8. 根据权利要求7所述的3D打印生物墨水，其特征在于：所述天然高分子水凝胶材料为海藻酸钠、明胶、胶原、Matrigel、壳聚糖、丝素蛋白、透明质酸、纤维蛋白原、硫酸软骨素、白蛋白以及它们的甲基丙烯酰 化产物中的至少一种；
    所述合成高分子水凝胶材料为聚乙二醇、聚丙烯醇、聚乙二醇二丙烯酸酯、聚环氧乙烷、聚丙烯酰胺、聚丙烯酸、聚磷腈、聚N-异丙基丙烯酰胺类水凝胶以及它们的甲基丙烯酰化产物中的至少一种。
  9. 根据权利要求5-8中任一项所述的3D打印生物墨水，其特征在于：所述载细胞的凝胶微球内的细胞、所述凝胶材料内的细胞均为实质细胞、肿瘤细胞、基质细胞和内皮细胞中至少一种；
    所述实质细胞来源于如下组织/器官：心脏、肝脏、肾脏、胰腺和脑结构中至少一种。
  10. 权利要求1-9中任一项所述3D打印生物墨水在构建具有复杂血管通道和/或异质细胞结构的组织/器官模型中的应用；
    所述组织/器官模型包括心脏、肝脏、肾脏、胰腺和脑结构中至少一种；
    所述组织/器官模型的尺寸为500μm～100mm。
  11. 根据权利要求10所述的应用，其特征在于：所述3D打印生物墨水打印完成后，对作为所述连续相的凝胶材料进行二次交联；
    采用下述方式对所述凝胶材料进行二次交联：光照交联、温度交联、离子交联、酶交联和共价交联方式中至少一种。
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