WO2017080117A1 - 三维细胞培养支架及其制备方法 - Google Patents

三维细胞培养支架及其制备方法 Download PDF

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WO2017080117A1
WO2017080117A1 PCT/CN2016/072756 CN2016072756W WO2017080117A1 WO 2017080117 A1 WO2017080117 A1 WO 2017080117A1 CN 2016072756 W CN2016072756 W CN 2016072756W WO 2017080117 A1 WO2017080117 A1 WO 2017080117A1
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cell culture
dimensional
preparation
fiber
dimensional cell
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PCT/CN2016/072756
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French (fr)
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袁建华
苏嘉良
陈勇
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广州洁特生物过滤股份有限公司
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Priority to US15/775,678 priority Critical patent/US11168294B2/en
Publication of WO2017080117A1 publication Critical patent/WO2017080117A1/zh

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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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • 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
    • B33Y40/00—Auxiliary operations or equipment, e.g. for material handling
    • B33Y40/20—Post-treatment, e.g. curing, coating or polishing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y70/00—Materials specially adapted for additive manufacturing
    • 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
    • C12N5/0062—General methods for three-dimensional 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
    • C12N2533/30—Synthetic polymers
    • C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers

Definitions

  • the invention relates to the technical field of cell culture, in particular to a three-dimensional cell culture scaffold and a preparation method thereof.
  • the pharmaceutical industry, life sciences, medicine, clinical quarantine testing, cell therapy and tissue engineering industries have developed rapidly.
  • the large-scale in vitro culture of animal cells to express specific proteins, monoclonal antibodies, interferons and viruses, vaccine preparations, and cell therapy products has become a focus area of current research and application.
  • the commonly used large-scale cell culture devices mainly include cell culture dishes, cultured multi-well plates, culture flasks, culture spinner flasks, bioreactors, and cell factories.
  • the cells are mostly grown in a two-dimensional plane in a culture environment. , amplification.
  • Three-dimensional cell culture refers to inoculation of cells onto a culture vector having a three-dimensional network spatial structure, and then allowing cells to adhere, migrate, and differentiate on the three-dimensional vector.
  • Three-dimensional cell culture overcomes many of the shortcomings of two-dimensional planar culture.
  • the three-dimensional network space structure can provide a larger surface area for cell adhesion, increase cell yield while reducing or even avoiding cell contact inhibition, providing cell growth similar to that in humans.
  • the three-dimensional micro-environment facilitates the interaction between cells and between cells and extracellular matrix and the excretion of metabolites, ensuring that cells maintain their original differentiation and functional expression.
  • the three-dimensional cell culture systems used are collagen, peptide, gelatin, fibrin, A natural polymer such as agarose syrup or alginate is used as a scaffold material for cell adhesion.
  • the specific step is to first make a natural polymer material into a hydrogel, and then mix the cells with nutrients such as hydrogel and culture solution. Together, a three-dimensional cell culture system in a hydrogel state is formed.
  • this kind of three-dimensional culture system has obvious improvement compared with two-dimensional culture, there are still many defects. Since the entire culture system is a relatively high viscosity gel state, it is not conducive to the transport of nutrients to cells located deep in the scaffold, and is also detrimental to the excretion of cellular metabolites.
  • this type of culture system is also not conducive to the separation of cells from the attached stent, resulting in difficult to harvest cells, and the scaffold material is also difficult to recycle. It is also difficult for such a gelled three-dimensional culture system to dynamically observe the cultured cells at any time. Therefore, there is an urgent need for a new three-dimensional cell culture system capable of overcoming the above various drawbacks.
  • the polystyrene fiber porous scaffold prepared by Baker et al. using electrospinning technology can be placed in a multi-well cell culture plate as a three-dimensional cell culture insert, but the size of the fiber in the three-dimensional fiber cell culture insert is difficult to control, and the three-dimensional fiber inter-frame
  • the aperture size and shape are uncertain, uneven, and the average aperture is small (about 15 microns), and the three-dimensional fiber holder is soft in the natural state, and cannot maintain the overall shape of the three-dimensional insert, so that the three-dimensional cell culture plug-in still exists. Many defects.
  • Liu Qing discloses a method for manufacturing a three-dimensional fiber cell culture insert by first producing a single-layer porous sheet support by injection molding, and then performing layer-by-layer assembly by means of pillars or bonding.
  • the preparation method is the final three-dimensional method.
  • the fiber cell culture insert overcomes many of the defects of other three-dimensional cell culture systems, but has a complicated preparation process, high energy consumption, high production cost, and is difficult to prepare on a large scale.
  • a method for preparing a three-dimensional cell culture scaffold comprises the following steps:
  • the surface of the fiber of the three-dimensional cell culture scaffold manufactured by 3d printing is surface-treated with a treating agent.
  • the raw material used in the 3d printer is a non-biodegradable polymer or a biodegradable polymer selected from the group consisting of polystyrene, polypropylene, polyethylene, and polycarbonate.
  • a biodegradable polymer selected from the group consisting of polystyrene, polypropylene, polyethylene, and polycarbonate.
  • the biodegradable polymer is selected from one or more of polylactic acid, polylactic acid-hydroxyacetate, polycaprolactone, and a copolymer thereof .
  • the 3d printer employs a composite of transparent polystyrene and a transparent styrene-butadiene random copolymer in a mass ratio of 0-90:100-10.
  • the mass ratio of the transparent polystyrene to the transparent styrene-butadiene random copolymer in the blend is from 90 to 60: 10-40; the transparent styrene-butadiene The ratio of the amount of styrene to butadiene in the random copolymer is from 90 to 50:10 to 50.
  • the treatment agent is selected from the group consisting of: a plasma hydrophilic treatment agent or a temperature sensitive treatment agent.
  • the plasma hydrophilic treatment agent is selected from the group consisting of oxygen, nitrogen, carbon dioxide, carboxylic acids or derivatives thereof (eg, saturated or unsaturated small molecule aliphatic carboxylic acids or aromatic carboxylic acids having a molecular weight of less than 500) An amino acid having a molecular weight of less than 500 or a derivative thereof.
  • the temperature sensitive treatment agent is N-isopropylacrylamide or a derivative thereof.
  • the cell culture device is selected from the group consisting of a cell culture dish, a cell culture flask, a cell culture multiwell plate, a cell culture roller bottle, a cell culture tube, or a bioreactor.
  • the method for assembling the three-dimensional fiber cell culture scaffold into a cell culture device is to directly place a three-dimensional fibroblast culture scaffold inside a cell culture device for a non-closed cell culture device such as a culture dish, a cultured multiwell plate, or the like, and then perform The cells were cultured inoculation
  • a closed cell culture device such as a culture flask, a culture spinner, a bioreactor, etc.
  • a three-dimensional fiber cell culture scaffold is placed inside an open cell culture device, and then ultrasonic welding, heat welding, laser sintering, etc. are employed. The process is to seal the missing surface of the cell culture device body and finally perform cell seed culture.
  • Another object of the present invention is to provide a three-dimensional cell culture scaffold.
  • the three-dimensional cell culture scaffold prepared by the above preparation method prepared by the above preparation method.
  • the three-dimensional fiber network structure parameters of the three-dimensional cell culture scaffold are as follows: the fiber diameter is 30-1000 micrometers, the spacing of adjacent fibers is 50-1500 micrometers, and the number of parallel layers is 1-100000 layers, the spacing of adjacent planar layers is 30-1000 microns.
  • the inside of the three-dimensional fiber cell culture scaffold may have a cylindrical shape, or a rectangular parallelepiped shape, or a prismatic hollow passage of various shapes, and the hollow passage circumscribed cylinder has a diameter of 300 to 5000 ⁇ m.
  • 3D printing technology has been developed since the end of the 19th century, but until the recent advancement of information technology, the continuous updating of mechanical automation and the promotion of the Internet have made the golden age of 3D printing technology formally come.
  • 3D printing technology is called the “third industrial revolution” and belongs to the field of additive manufacturing. The manufacturing process is to first design and process the 3D graphics of the product, then convert it into a data format recognizable by the 3D printer, and then import it into the 3D printing system for 3D printing. For products with complex structures such as cavities or moving parts, there is no need to design, manufacture and modify the mold, and it can be quickly printed at one time.
  • the three-dimensional fiber cell culture scaffold using the 3D printing technology to manufacture a three-dimensional fiber network structure can simplify the manufacturing process, save raw materials and manufacturing time, reduce production cost, greatly improve production efficiency, reduce energy consumption, and avoid traditional manufacturing techniques. Defects, easy to make large-scale intelligent manufacturing.
  • a transparent polystyrene and a transparent styrene-butadiene random copolymer are preferably used as a material of a three-dimensional cell culture scaffold in a mass ratio of 0-90:100-10 (the transparent polystyrene is selected). From Total's GPPS-1050, GPPS-1240 or GPPS-535N, or Yangzi Petrochemical-BASF's PS One or more of 143E, 158K or 165H.
  • the transparent styrene-butadiene random copolymer is selected from Chevron Phillips KR03, KR03NW or BK10, or one of Maomingzhong and SL-801G, SL-803G, SL-805F or SL-838F, or Several.
  • the material has the advantages of high strength, high flexibility, free bending, high transparency, good fluidity, and no clogging of the printer nozzle, and is particularly suitable for biological fields requiring high transparency.
  • a model of a three-dimensional fiber cell culture insert with a fiber diameter of 300 ⁇ m, a fiber spacing of 300 ⁇ m in the same plane layer, a layer spacing of 300 ⁇ m and a layer of 12 layers was designed according to the size of the 3.5 mm culture dish.
  • the model 3D data is converted into a 3D print code and then entered into a 3D printer.
  • the 3D fiber cell culture insert layer designed by the FDM principle 3D printer is printed at a print head temperature of 210 ° C and a print line speed of 30 mm/min. come out.
  • the surface of the fiber in the three-dimensional fiber cell culture insert was subjected to acrylic graft polymerization surface treatment by low-temperature plasma technique, followed by radiation sterilization by gamma ray, and then the three-dimensional fiber cell culture insert was placed on a biological test bench at a diameter of 3.5 mm. In the culture dish, the cells were subsequently seeded and cultured for 48 hours, and finally the cells were harvested by trypsinization.
  • a model of a three-dimensional fiber cell culture insert with a fiber diameter of 500 ⁇ m, a fiber spacing of 1000 ⁇ m, a layer spacing of 500 ⁇ m, and a layer of 150 layers was designed according to the size of a 50 ml culture bottle.
  • the model 3D data is converted into a 3D print code and then entered into a 3D printer.
  • PS polystyrene
  • the surface of the fiber in the three-dimensional fiber cell culture insert was subjected to a graft-polymerization temperature-sensitive surface treatment using N-isopropylacrylamide, and then the treated cell culture insert was placed in a 50 ml-liter flask having an open side, and then subjected to ultrasonication. Welding The culture insert was mounted and fixed in a culture flask, followed by radiation sterilization using gamma rays, followed by cell seeding and incubation for 48 hours, and finally the system temperature was lowered to 20 ° C for temperature-sensitive treatment to harvest the cells.
  • the AutoCAD software was used to design a three-dimensional fiber with a fiber diameter of 1000 ⁇ m, a fiber spacing of 2000 ⁇ m in the same plane layer, a layer spacing of 1000 ⁇ m and a layer number of 1500 layers.
  • the cell culture plug-in model is then converted into a 3D print code and then imported into a 3D printer.
  • the 3D fiber cell culture insert layer designed by the FDM principle 3D printer is used at a print head temperature of 190 ° C and a print line speed of 80 mm/min. print it out.
  • PE polyethylene
  • the surface of the fiber in the three-dimensional fiber cell culture insert was subjected to a graft-polymerization temperature-sensitive surface treatment using N-isopropylacrylamide, and then the treated cell culture insert was placed in a 850 ml culture flask having an open side, and then subjected to ultrasonication.
  • Welding The culture insert was mounted and fixed in a culture flask, followed by radiation sterilization using gamma rays, followed by cell seeding and incubation for 48 hours, and finally the system temperature was lowered to 20 ° C for temperature-sensitive treatment to harvest the cells.
  • a model of a three-dimensional fiber cell culture insert with a fiber diameter of 600 ⁇ m, a fiber spacing of 1000 ⁇ m, a layer spacing of 600 ⁇ m, and a layer of 4000 layers was designed according to the size of the 2000 ml culture roller bottle.
  • the model 3D data is converted into a 3D print code and then entered into a 3D printer.
  • PS polystyrene
  • the surface of the fiber in the three-dimensional fiber cell culture insert was subjected to a graft-polymerization temperature-sensitive surface treatment using N-isopropylacrylamide, and then the treated cell culture insert was placed in a 50 ml-liter flask having an open side, and then subjected to ultrasonication. Welding The culture insert was mounted and fixed in a culture flask, followed by radiation sterilization using gamma rays, followed by cell seeding and incubation for 48 hours, and finally the system temperature was lowered to 20 ° C for temperature-sensitive treatment to harvest the cells.
  • a model of a three-dimensional fiber cell culture insert with a fiber diameter of 600 ⁇ m, a fiber spacing of 1000 ⁇ m, a layer spacing of 600 ⁇ m, and a layer of 4000 layers was designed according to the size of the 2000 ml culture roller bottle.
  • the model 3D data is converted into a 3D print code and then entered into a 3D printer.
  • the 1.75mm diameter transparent polystyrene linear material prepared by the above method is used as a raw material, and the three-dimensional fiber cell designed by the FDM principle 3D printer is used at a print head temperature of 240 ° C and a printing linear velocity of 50 mm/min.
  • the development plugin layer is printed out.
  • the surface of the fiber in the three-dimensional fiber cell culture insert was subjected to a graft-polymerization temperature-sensitive surface treatment using N-isopropylacrylamide, and then the treated cell culture insert was placed in a perfusion bioreactor, followed by sterilization, followed by cell seeding and The cells were perfused for 48 h, and finally the system temperature was lowered to 20 ° C for temperature-sensitive treatment to harvest the cells.
  • the three-dimensional insert in the bioreactor is then inoculated, and the cells are harvested in a cycle, and the cells are harvested without damage.

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Abstract

一种三维细胞培养支架及其制备方法，制备方法包括如下步骤：根据细胞培养装置的形状设计三维细胞培养支架的形状；采用3D打印机一次打印制造成型；将3D打印制造的三维细胞培养支架的纤维表面利用处理剂进行表面处理，即得。采用3D打印技术来制造具有三维纤维网络结构的三维纤维细胞培养支架可以节约原料和制造时间、降低生产成本、提高生产效率、降低能耗、规避传统制造技术的缺陷、便于大规模智能制造。

Description

三维细胞培养支架及其制备方法 技术领域
本发明涉及细胞培养技术领域，特别是涉及一种三维细胞培养支架及其制备方法。
背景技术
近年来，制药行业、生命科学、医学、临床学检疫检验、细胞治疗和组织工程行业技术飞速发展。通过动物细胞在体外大规模培养来表达特定的蛋白、单克隆抗体、干扰素及病毒、疫苗制品、细胞治疗产品已成为目前研究与应用的重点领域。目前常用的细胞大规模培养的装置主要有细胞培养皿、培养多孔板、培养瓶、培养转瓶、生物反应器、和细胞工厂等，细胞在培养环境中大都以二维平面的方式贴壁生长、扩增。
二维平面细胞培养虽然在一定程度上满足了细胞培养产业的发展需求，但是存在众多缺陷，比如细胞在这种环境下生长很容易发生拥挤现象而导致细胞间发生接触抑制，同时细胞暴露于培养液的表面积减少，并且细胞贴附表面积有限。二维细胞培养缺乏像在体内三维环境一样的细胞与细胞之间、细胞与细胞外基质之间的交互作用，从而使细胞的增生受到一定的限制，不利于细胞的分化和原有功能的表达。因此，三维细胞培养应运而生。三维细胞培养是指将细胞先接种到具有三维网络空间结构的培养载体上，然后使细胞在这种三维载体上粘附、移行和分化扩增。三维细胞培养克服了二维平面培养的众多缺陷，三维网络空间结构可以为细胞粘附提供更大的表面积，提高细胞产量的同时减少或甚至避免细胞接触抑制，提供了近似于人体内的细胞生长三维空间微环境，便于细胞之间和细胞与细胞外基质之间的交互作用以及代谢产物的排出，保障了细胞保持原有的分化和功能表达。
目前大多数所采用的三维细胞培养系统是以胶原、多肽、明胶、纤维蛋白、 琼脂糖浆、藻酸盐类等天然高分子做为细胞粘附的支架材料，其具体步骤是先将天然高分子材料做成水凝胶，然后将细胞与水凝胶、培养液等营养物质混合到一起形成水凝胶态的三维细胞培养系统。这类三维培养系统虽然较二维培养有了比较明显的改善，但是仍然存在着很多缺陷。由于整个培养系统是粘度比较大的凝胶态，首先不利于营养物质向位于支架深处的细胞的传输，同时也不利于细胞代谢产物的排出。此外，该类培养系统也不利于细胞与贴附支架的分离，导致细胞难以收获，而且支架材料也难以回收再利用。这种凝胶态的三维培养系统大多也难于对所培养的细胞进行随时的动态观察。因此，迫切需要一种新的能够克服上述种种缺陷的三维细胞培养系统。
Baker等人采用静电纺丝技术制备的聚苯乙烯纤维多孔支架可以放在多孔培养板中做为三维细胞培养的插件，但是该三维纤维细胞培养插件中的纤维的尺寸大小难以控制，三维纤维间的孔径尺寸和形状不确定、不均匀，平均孔径较小(约15微米)，而且三维纤维支架在自然状态下是软的，不能维持三维插件的整体形状，因此做为三维细胞培养插件还存在很多缺陷。刘青公开了一种采用注塑等工艺先生产单层多孔片层支架，然后通过支柱或粘结等方式进行层层组装的方法来制造三维纤维细胞培养插件的方法，该制备方法虽然最终的三维纤维细胞培养插件克服了已有的其它三维细胞培养系统的众多缺陷，但是制备工艺复杂、能耗高、生产成本高、难于大规模制备。
发明内容
基于此，本发明的目的是提供一种新的三维细胞培养支架的制备方法。
具体的技术方案如下：
一种三维细胞培养支架的制备方法，包括如下步骤：
根据细胞培养装置的形状设计三维细胞培养支架的形状；
采用3d打印机一次打印制造成型；
将3d打印制造的三维细胞培养支架的纤维表面利用处理剂进行表面处理，即得。
在其中一些实施例中，3d打印机采用的原料为非生物降解类聚合物或可生物降解类聚合物，所述非生物降解类聚合物选自：聚苯乙烯、聚丙烯、聚乙烯、聚碳酸酯及其共聚物中的一种或几种；所述可生物降解类聚合物选自：聚乳酸、聚乳酸-羟基乙酸酯、聚己内酯及其共聚物中的一种或几种。
在其中一些实施例中，3d打印机采用的原料为透明聚苯乙烯与透明苯乙烯-丁二烯无规共聚物以质量比为0-90：100-10的复合物。
在其中一些实施例中，所述共混物中透明聚苯乙烯与透明苯乙烯-丁二烯无规共聚物的质量比为90-60：10-40；所述透明苯乙烯-丁二烯无规共聚物中苯乙烯与丁二烯的物质的量的比例为90-50：10-50。
在其中一些实施例中，所述处理剂选自：等离子亲水处理剂或温敏处理剂。
在其中一些实施例中，所述等离子亲水处理剂选自：氧气、氮气、二氧化碳、羧酸或其衍生物(如分子量小于500的饱和或不饱和小分子脂肪族羧酸或芳香族羧酸)、分子量小于500的氨基酸或其衍生物。
在其中一些实施例中，所述温敏处理剂为N-异丙基丙烯酰胺或其衍生物。
在其中一些实施例中，所述细胞培养装置选自细胞培养皿、细胞培养瓶、细胞培养多孔板、细胞培养转瓶、细胞培养管或生物反应器。
将该三维纤维细胞培养支架组装到细胞培养装置中的工艺技术方法为对于非封闭型的细胞培养装置如培养皿、培养多孔板等直接将三维纤维细胞培养支架放置在细胞培养装置内部，然后进行细胞接种培养。对于封闭型的细胞培养装置如培养瓶、培养转瓶、生物反应器等先将三维纤维细胞培养支架放置在某一个面敞开的细胞培养装置内部，然后采用超声焊接、热焊接、激光烧结焊接等工艺将细胞培养装置体的缺失面焊接封闭成型，最后进行细胞接种培养。
本发明的另一目的是提供一种三维细胞培养支架。
具体的技术方案如下：
上述制备方法制备得到的三维细胞培养支架。
在其中一些实施例中，该三维细胞培养支架的三维纤维网络结构参数如下：纤维直径为30-1000微米，相邻纤维的间距为50-1500微米，平行面层数为 1-100000层，相邻平面层的间距为30-1000微米。
该三维纤维细胞培养支架的内部可以具有圆柱体形、或长方体形、或各种形状的棱柱状体型的中空通道，中空通道外接圆柱体的直径为300-5000微米。
本发明的原理及优点如下：
3D打印技术自19世纪末以来就开始发展，但直到最近几年由于信息技术的不断进步，机械自动化的不断更新和互联网的推动才使得3D打印技术应用的黄金时代正式到来。3D打印技术被称为“第三次工业革命”，属于增材智能制造领域。其制造过程为首先对产品进行3D图形设计与处理，然后将其转化为3D打印机可识别的数据格式，接着将其导入到3D打印系统中进行3D打印。对于内部有空腔或活动部件等结构复杂的产品无需设计、制造和修改模具，可以一次性快速打印成型。因此，本发明采用3D打印技术来制造三维纤维网络结构的三维纤维细胞培养支架可以简化制造工艺流程、节约原料和制造时间、降低生产成本、极大地提高生产效率、降低能耗、规避传统制造技术的缺陷、便于大规模智能制造。
本发明中优选采用透明聚苯乙烯与透明苯乙烯-丁二烯无规共聚物以质量比为0-90：100-10的复合物作为三维细胞培养支架的材料(所述透明聚苯乙烯选自道达尔的GPPS-1050、GPPS-1240或GPPS-535N，或者扬子石化-巴斯夫的PS
Figure PCTCN2016072756-appb-000001
143E、158K或165H，中的一种或几种。所述透明苯乙烯-丁二烯无规共聚物选自雪佛龙菲利普KR03、KR03NW或BK10，或茂名众和SL-801G、SL-803G、SL-805F或SL-838F，中的一种或几种。)，该材料具有高强度、高柔韧性、可随意弯折、且透明度高、流动性好、不会堵塞打印机喷嘴等优点，特别适用于对透明性要求高的生物领域。
具体实施方式
下面结合具体实施例对本发明进行详细的描述。所列举的实施例仅是本发明实施例的一部分，而非全部实施例，仅用于说明本发明，而非用于限制本发明的范围。本领域的技术人员在没有做出创造性劳动的前提下所获得的其它实施 例均属于本发明的保护范围。
实施例1
先采用AutoCAD软件根据3.5mm培养皿的尺寸设计出纤维直径为300微米，同平面层纤维间距为300微米，层间距为300微米，层数为12层的三维纤维细胞培养插件的模型，接着将该模型3D数据转化成3D打印代码，然后输入到3D打印机中。采用3mm直径的聚丙烯(PP)线性材料为原料，在打印头温度为210℃，打印线速度为30mm/min的条件下，用FDM原理的3D打印机将设计的三维纤维细胞培养插件层层打印出来。采用低温等离子技术对三维纤维细胞培养插件中纤维表面进行丙烯酸接枝聚合表面处理，接着采用伽玛射线进行辐射消毒，然后在生物实验台上将该三维纤维细胞培养插件放置在直径为3.5mm的培养皿中，随后进行细胞接种并培养48h，最后采用胰酶消化处理收获细胞。
实施例2
先采用AutoCAD软件根据50ml培养瓶的尺寸设计出纤维直径为500微米，同平面层纤维间距为1000微米，层间距为500微米，层数为150层的三维纤维细胞培养插件的模型，接着将该模型3D数据转化成3D打印代码，然后输入到3D打印机中。采用1.75mm直径的聚苯乙烯(PS)线性材料为原料，在打印头温度为240℃，打印线速度为50mm/min的条件下，用FDM原理的3D打印机将设计的三维纤维细胞培养插件层层打印出来。采用N-异丙基丙烯酰胺对三维纤维细胞培养插件中纤维表面进行接枝聚合温敏表面处理，随后将处理过的细胞培养插件放置在侧面敞开的体积为50ml的培养瓶中，然后进行超声焊接将培养插件安装固定在培养瓶中，接着采用伽玛射线进行辐射消毒，随后进行细胞接种并培养48h，最后将系统温度降到20℃进行温敏处理收获细胞。
实施例3
先采用AutoCAD软件根据850ml培养瓶的尺寸设计出纤维直径为1000微米，同平面层纤维间距为2000微米，层间距为1000微米，层数为1500层的三维纤维 细胞培养插件模型，接着将该模型3D数据转化成3D打印代码，然后输入到3D打印机中。采用1.75mm直径的聚乙烯(PE)线性材料为原料，在打印头温度为190℃，打印线速度为80mm/min的条件下，采用FDM原理的3D打印机将设计的三维纤维细胞培养插件层层打印出来。采用N-异丙基丙烯酰胺对三维纤维细胞培养插件中纤维表面进行接枝聚合温敏表面处理，随后将处理过的细胞培养插件放置在侧面敞开的体积为850ml的培养瓶中，然后进行超声焊接将培养插件安装固定在培养瓶中，接着采用伽玛射线进行辐射消毒，随后进行细胞接种并培养48h，最后将系统温度降到20℃进行温敏处理收获细胞。
实施例4
先采用AutoCAD软件根据2000ml培养转瓶的尺寸设计出纤维直径为600微米，同平面层纤维间距为1000微米，层间距为600微米，层数为4000层的三维纤维细胞培养插件的模型，接着将该模型3D数据转化成3D打印代码，然后输入到3D打印机中。采用1.75mm直径的聚苯乙烯(PS)线性材料为原料，在打印头温度为240℃，打印线速度为50mm/min的条件下，用FDM原理的3D打印机将设计的三维纤维细胞培养插件层层打印出来。采用N-异丙基丙烯酰胺对三维纤维细胞培养插件中纤维表面进行接枝聚合温敏表面处理，随后将处理过的细胞培养插件放置在侧面敞开的体积为50ml的培养瓶中，然后进行超声焊接将培养插件安装固定在培养瓶中，接着采用伽玛射线进行辐射消毒，随后进行细胞接种并培养48h，最后将系统温度降到20℃进行温敏处理收获细胞。
实施例5
利用天平秤准确称量道达尔聚苯乙烯GPPS-124010Kg、茂名众和K胶(透明的苯乙烯-丁二烯无规共聚物，其中丁二烯的摩尔百分含量为30％)SL-803G2.5Kg，先在80℃烘干后进行预分散共混，然后在双螺杆共混挤出设备中于230℃进行熔融共混6分钟，接着在直径为1.75mm的圆形模口中挤出，挤出的线材经过冷却水槽冷却定型、两轮牵引机牵引、以30m/min的速度在双工收卷机上高 速收卷，最后烘干、即得透明聚苯乙烯线性材料。
先采用AutoCAD软件根据2000ml培养转瓶的尺寸设计出纤维直径为600微米，同平面层纤维间距为1000微米，层间距为600微米，层数为4000层的三维纤维细胞培养插件的模型，接着将该模型3D数据转化成3D打印代码，然后输入到3D打印机中。采用上述方法制备得到的1.75mm直径的透明聚苯乙烯线性材料为原料，在打印头温度为240℃，打印线速度为50mm/min的条件下，用FDM原理的3D打印机将设计的三维纤维细胞培养插件层层打印出来。采用N-异丙基丙烯酰胺对三维纤维细胞培养插件中纤维表面进行接枝聚合温敏表面处理，随后将处理过的细胞培养插件放置在灌流生物反应器中，消毒后，随后进行细胞接种并灌流培养48h，最后将系统温度降到20℃进行温敏处理收获细胞。然后对生物反应器中的三维插件进行传代接种，循环培养、循环无伤收获细胞。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种三维细胞培养支架的制备方法，其特征在于，包括如下步骤：
    根据细胞培养装置的形状设计三维细胞培养支架的形状；
    采用3d打印机一次打印制造成型；
    将3d打印制造的三维细胞培养支架的纤维表面利用处理剂进行表面处理，即得。
  2. 根据权利要求1所述的制备方法，其特征在于，3d打印机采用的原料为非生物降解类聚合物或可生物降解类聚合物，所述非生物降解类聚合物选自：聚苯乙烯、聚丙烯、聚乙烯、聚碳酸酯及其共聚物中的一种或几种；所述可生物降解类聚合物选自：聚乳酸、聚乳酸-羟基乙酸酯、聚己内酯及其共聚物中的一种或几种。
  3. 根据权利要求1所述的制备方法，其特征在于，3d打印机采用的原料为透明聚苯乙烯与透明苯乙烯-丁二烯无规共聚物以质量比为0-90：100-10的复合物。
  4. 根据权利要求3所述的制备方法，其特征在于，所述共混物中透明聚苯乙烯与透明苯乙烯-丁二烯无规共聚物的质量比为90-60：10-40；所述透明苯乙烯-丁二烯无规共聚物中苯乙烯与丁二烯的物质的量的比例为90-50：10-50。
  5. 根据权利要求1所述的制备方法，其特征在于，所述处理剂选自：等离子亲水处理剂或温敏处理剂。
  6. 根据权利要求5所述的制备方法，其特征在于，所述等离子亲水处理剂选自：氧气、氮气、二氧化碳、羧酸或其衍生物、氨基酸或其衍生物。
  7. 根据权利要求5所述的制备方法，其特征在于，所述温敏处理剂为N-异丙基丙烯酰胺或其衍生物。
  8. 根据权利要求1-7任一项所述的制备方法，其特征在于，所述细胞培养装置选自细胞培养皿、细胞培养瓶、细胞培养多孔板、细胞培养转瓶、细胞培养管或生物反应器。
  9. 权利要求1-8任一项所述的制备方法制备得到的三维细胞培养支架。
  10. 根据权利要求9所述的三维细胞培养支架，其特征在于，该三维细胞培养支架的三维纤维网络结构参数如下：纤维直径为30-1000微米，相邻纤维的间距为50-1500微米，平行面层数为1-100000层，相邻平面层的间距为30-1000微米。
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