技术领域technical field
本发明属于生物技术领域，涉及一种用于细胞培养肉的纤维支架及其制备方法和应用。The invention belongs to the field of biotechnology, and relates to a fiber support for cell cultured meat, a preparation method and application thereof.
背景技术Background technique
目前，肉类食品作为人们日常饮食中最重要的蛋白质来源，其主要由传统畜牧行业生产，随着人口数量的增加，人们生活水平的提高以及对环境友好产业的关注，开发新型的替代蛋白质越来越受到重视。细胞培养肉是一种由体外培养细胞生产肉类食品的新兴技术，相比传统畜牧业更加环境资源友好。At present, meat food, as the most important source of protein in people's daily diet, is mainly produced by the traditional animal husbandry industry. With the increase in population, the improvement of people's living standards and the concern for environmentally friendly industries, the development of new alternative proteins is becoming more and more important. are getting more and more attention. Cell-cultured meat is an emerging technology for the production of meat food from in vitro cultured cells, which is more environmentally friendly and resource-friendly than traditional animal husbandry.
在细胞培养肉的培养过程中，支架材料作为细胞的黏附载体扮演着重要的角色，其为细胞的黏附、增殖、分化提供场所，并且提供一定的食用口感。目前用于细胞培养肉的支架大致可以分为水凝胶支架、多孔支架以及纤维支架。其中水凝胶支架最为常见，并且可以配合3D生物打印技术实现各种形状的支架制备，多孔支架多由冷冻干燥方法制备，其具有多孔的海绵状结构，但是这两种支架都不具备取向结构，无法诱导分化后的肌肉纤维定向排列。而纤维支架由细长的纤维组成，在与细胞大小相匹配的尺度下可以为细胞的生长提供地形引导，从而使肌肉纤维取向排列，更加有助于肌肉纤维的成熟，可以从外观和口感方面提高细胞培养肉产品与真正肉类的相似度。In the cultivation process of cell culture meat, the scaffold material plays an important role as the adhesion carrier of cells, which provides a place for cell adhesion, proliferation and differentiation, and provides a certain edible taste. The scaffolds currently used for cell culture meat can be roughly divided into hydrogel scaffolds, porous scaffolds, and fiber scaffolds. Among them, hydrogel scaffolds are the most common, and can be combined with 3D bioprinting technology to realize the preparation of scaffolds of various shapes. Porous scaffolds are mostly prepared by freeze-drying methods, which have a porous sponge-like structure, but these two scaffolds do not have an orientation structure. , unable to induce the directional alignment of differentiated muscle fibers. The fiber scaffold is composed of elongated fibers, which can provide topographic guidance for the growth of cells at a scale that matches the cell size, so that the orientation of muscle fibers is arranged, which is more conducive to the maturation of muscle fibers. Improve the resemblance of cell-cultured meat products to real meat.
纤维支架的制备方法包括各种纺丝技术，如干法纺丝、湿法纺丝、静电纺丝等。海藻酸钠是一种来自于海洋的天然多糖，其溶液可以与二价金属阳离子发生耦合形成“蛋盒”结构，快速固化形成水凝胶。目前海藻酸钠基支架材料的制备，多是利用其可与二价金属阳离子快速耦合固化形成水凝胶的特性来加工，出于生物安全考虑，金属离子多选用钙离子，如CN114622296A公开了一种连续化制备细胞培养肉可食用纤维支架的方法。然而钙离子交联的海藻酸水凝胶如果暴露于含有钠钾阳离子的生理盐水或培养基中，其“蛋盒”结构中的钙离子易与钠钾离子发生置换，从而导致“蛋盒”结构瓦解，致使水凝胶在细胞培养的过程中快速降解，不适宜进行长时间的细胞培养。此外，由于海藻酸钠的聚合物分子链上含有大量羧基，这使海藻酸盐基的材料带有负电荷，不利于细胞在材料上的黏附。The preparation methods of fibrous scaffolds include various spinning techniques, such as dry spinning, wet spinning, electrospinning, etc. Sodium alginate is a natural polysaccharide from the ocean. Its solution can be coupled with divalent metal cations to form an "egg box" structure, which can quickly solidify to form a hydrogel. At present, the preparation of sodium alginate-based scaffold materials is mostly based on the characteristics that it can be quickly coupled and solidified with divalent metal cations to form a hydrogel. For biological safety considerations, calcium ions are mostly used as metal ions. For example, CN114622296A discloses a A method for continuously preparing edible fiber scaffolds for cell cultured meat. However, if the calcium ion-crosslinked alginate hydrogel is exposed to physiological saline or medium containing sodium and potassium cations, the calcium ions in its "egg box" structure are easily replaced with sodium and potassium ions, resulting in "egg box" The structure disintegrates, causing the hydrogel to degrade rapidly during the cell culture process, which is not suitable for long-term cell culture. In addition, because the polymer molecular chain of sodium alginate contains a large number of carboxyl groups, this makes the alginate-based material negatively charged, which is not conducive to the adhesion of cells on the material.
综上所述，开发新型用于细胞培养肉的纤维支架，具备高稳定性及粘附性，对于细胞培养肉领域具有重要意义。In summary, the development of a new type of fiber scaffold for cell cultured meat with high stability and adhesion is of great significance to the field of cell cultured meat.
发明内容Contents of the invention
针对现有技术的不足和实际需求，本发明提供一种用于细胞培养肉的纤维支架及其制备方法和应用，开发新型具备高稳定性及粘附性的用于细胞培养肉的纤维支架。Aiming at the deficiencies of the prior art and actual needs, the present invention provides a fiber scaffold for cell cultured meat and its preparation method and application, and develops a new type of fiber scaffold for cell cultured meat with high stability and adhesion.
为达上述目的，本发明采用以下技术方案：For reaching above-mentioned purpose, the present invention adopts following technical scheme:
第一方面，本发明提供一种用于细胞培养肉的纤维支架的制备方法，所述制备方法包括：In a first aspect, the present invention provides a method for preparing a fiber scaffold for cell cultured meat, the preparation method comprising:
采用湿法纺丝制备所述用于细胞培养肉的纤维支架；Prepare the fiber scaffold for cell culture meat by wet spinning;
所述湿法纺丝的纺丝溶液包括具有醛基及甲基丙烯酰胺修饰的海藻酸钠。The spinning solution for wet spinning includes sodium alginate modified with aldehyde groups and methacrylamide.
本发明中，采用具有醛基修饰及甲基丙烯酰胺修饰的海藻酸钠进行湿法纺丝，可以增强纤维支架的黏附性能；同时使纤维支架具有光固化特性，碳碳双键之间形成稳定的共价连接，从而延长纤维支架的降解时间，实现可以使用单一一种主原料来制备纤维支架，同时在培基中实现较长时间的结构稳定性和更高的细胞粘附性。In the present invention, sodium alginate modified with aldehyde groups and methacrylamide is used for wet spinning, which can enhance the adhesion performance of the fiber scaffold; at the same time, the fiber scaffold has photocuring properties, and the formation of carbon-carbon double bonds is stable. The covalent connection of the fibrous scaffold prolongs the degradation time of the fibrous scaffold, and realizes that a single main raw material can be used to prepare the fibrous scaffold, while achieving longer structural stability and higher cell adhesion in the culture medium.
优选地，所述纺丝溶液还包括溶剂和光引发剂。Preferably, the spinning solution further includes a solvent and a photoinitiator.
优选地，所述溶剂包括水。Preferably, the solvent comprises water.
优选地，所述光引发剂包括2,4,6-三甲基苯甲酰基苯基膦酸乙酯(TPO)、2-羟基-4′-(2-羟乙氧基)-2-甲基苯丙酮(I2959)或苯基(2,4,6-三甲基苯甲酰基)磷酸锂(LAP)中任意一种或至少两种的组合。Preferably, the photoinitiator includes ethyl 2,4,6-trimethylbenzoylphenyl phosphonate (TPO), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methyl Any one or a combination of at least two of phenylpropiophenone (I2959) or lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP).
优选地，所述纺丝溶液中具有醛基及甲基丙烯酰胺修饰的海藻酸钠的浓度为50～100mg/mL，包括但不限于51mg/mL、52mg/mL、55mg/mL、60mg/mL、80mg/mL、90mg/mL、91mg/mL、95mg/mL、96mg/mL、97mg/mL、98mg/mL或99mg/mL。Preferably, the concentration of sodium alginate with aldehyde groups and methacrylamide modification in the spinning solution is 50-100 mg/mL, including but not limited to 51 mg/mL, 52 mg/mL, 55 mg/mL, 60 mg/mL , 80mg/mL, 90mg/mL, 91mg/mL, 95mg/mL, 96mg/mL, 97mg/mL, 98mg/mL or 99mg/mL.
优选地，所述纺丝溶液中光引发剂的浓度为1～10mg/mL，包括但不限于2mg/mL、3mg/mL、4mg/mL、5mg/mL、6mg/mL、7mg/mL、8mg/mL或9mg/mL。Preferably, the concentration of the photoinitiator in the spinning solution is 1-10 mg/mL, including but not limited to 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg /mL or 9mg/mL.
优选地，所述湿法纺丝的凝固液包括氯化钙溶液。Preferably, the coagulation solution for wet spinning includes calcium chloride solution.
优选地，所述湿法纺丝具体包括：Preferably, the wet spinning specifically includes:
利用所述纺丝溶液在凝固液中进行纺丝得到初生丝，对所述初生丝进行拉伸及紫外光照射，进行后处理，得到所述用于细胞培养肉的纤维支架。Spinning the spinning solution in a coagulation solution to obtain spun silk, stretching the spun silk, irradiating with ultraviolet light, and performing post-treatment to obtain the fiber scaffold for cell cultured meat.
可以理解，本领域常用的湿法纺丝方法及设备均适用于本发明，湿法纺丝设备可以是任何能够给聚合物溶液提供一定挤出速度的设备，能够提供一定卷绕拉伸功能的设备，能够为挤出的聚合物溶液提供凝固浴场所的设备的组合。It can be understood that the wet spinning methods and equipment commonly used in the art are applicable to the present invention, and the wet spinning equipment can be any equipment that can provide a certain extrusion speed for the polymer solution, and can provide a certain winding stretching function. Equipment, a combination of equipment capable of providing a coagulation bath for the extruded polymer solution.
本发明一具体实施例中，湿法纺丝设备为自制，其组成包括微量灌注泵、注射器、悬臂式机械搅拌器、搅拌桨、聚四氟乙烯接收管、接受浴、紫外光灯。通过微量灌注泵控制，将注射器中的纺丝溶液按一定速度从针头挤出至接受浴中迅速固化形成较粗的纤维丝，再由悬臂式机械搅拌器带动固定在搅拌桨上的接收管旋转牵引拉伸固化的纤维，拉伸过程中使用紫外光灯照射纤维丝以实现二次交联。所选用注射器内径为0.33～1.4cm，微量灌注泵设置推进速度为50～200μL/min，所选用针头规格为20～30G，搅拌器旋转速度为50～100rpm，接收辊外径为2～3cm，紫外光灯波长选用365～405nm。纺丝结束后将纤维束从接受辊上取下流水冲洗5～20min。In a specific embodiment of the present invention, the wet spinning equipment is self-made, and its composition includes a micro-perfusion pump, a syringe, a cantilever mechanical stirrer, a stirring paddle, a polytetrafluoroethylene receiving tube, a receiving bath, and an ultraviolet lamp. Controlled by the micro-perfusion pump, the spinning solution in the syringe is extruded from the needle at a certain speed to the receiving bath to quickly solidify to form thicker fiber filaments, and then the receiving tube fixed on the stirring paddle is driven by the cantilever mechanical stirrer to rotate The cured fiber is pulled and stretched, and the fiber filament is irradiated with ultraviolet light to achieve secondary crosslinking during the stretching process. The inner diameter of the selected syringe is 0.33-1.4 cm, the propulsion speed of the micro-perfusion pump is set at 50-200 μL/min, the selected needle size is 20-30 G, the rotation speed of the stirrer is 50-100 rpm, and the outer diameter of the receiving roller is 2-3 cm. The wavelength of the ultraviolet lamp is selected from 365 to 405nm. After the spinning is finished, the fiber bundle is removed from the receiving roll and rinsed with running water for 5-20 minutes.
优选地，所述氯化钙溶液中钙离子浓度为0.2M～0.6M，包括但不限于0.3M、0.4M或0.5M。Preferably, the calcium ion concentration in the calcium chloride solution is 0.2M-0.6M, including but not limited to 0.3M, 0.4M or 0.5M.
优选地，所述紫外光波长为365～405nm，包括但不限于370nm、380nm、385nm、390nm、395nm、399nm、400nm、402nm、403nm或404nm。Preferably, the wavelength of the ultraviolet light is 365-405nm, including but not limited to 370nm, 380nm, 385nm, 390nm, 395nm, 399nm, 400nm, 402nm, 403nm or 404nm.
优选地，所述纺丝溶液以50～200μL/min的速度进入到凝固液中，包括但不限于52μL/min、55μL/min、56μL/min、60μL/min、80μL/min、90μL/min、120μL/min、150μL/min、180μL/min、190μL/min、195μL/min、196μL/min、198μL/min或199μL/min。Preferably, the spinning solution enters the coagulation solution at a rate of 50-200 μL/min, including but not limited to 52 μL/min, 55 μL/min, 56 μL/min, 60 μL/min, 80 μL/min, 90 μL/min, 120 μL/min, 150 μL/min, 180 μL/min, 190 μL/min, 195 μL/min, 196 μL/min, 198 μL/min, or 199 μL/min.
优选地，所述后处理包括清洗和干燥。Preferably, said post-treatment includes washing and drying.
优选地，所述干燥的方法为风干或冷冻干燥。Preferably, the drying method is air drying or freeze drying.
可以理解，本发明利用经化学改造后的海藻酸钠包括醛基的修饰和甲基丙烯酰胺的修饰，结合湿法纺丝，显著改善了纤维支架的性能，本领域常用的醛基的修饰和甲基丙烯酰胺的修饰方法均适用于本发明，例如向海藻酸钠大分子链修饰醛基的方法可以是高碘酸钠氧化，也可以是经氨基修饰后再接枝二醛类化合物等向海藻酸钠大分子链修饰醛基的任何方法；甲基丙烯酰胺的修饰可以是2-氨基乙基甲基丙烯酸酯的接枝，也可以是甲基丙烯酸缩水甘油酯、甲基丙烯酸酐等任何可以向海藻酸钠大分子链上修饰共轭双键基团的方法。It can be understood that the present invention uses the chemically modified sodium alginate to include modification of aldehyde groups and modification of methacrylamide, combined with wet spinning, to significantly improve the performance of the fiber scaffold. Modification of aldehyde groups commonly used in the field and The modification methods of methacrylamide are applicable to the present invention, for example, the method of modifying the aldehyde group to the macromolecular chain of sodium alginate can be sodium periodate oxidation, or it can be modified by amino groups and then grafted with dialdehyde compounds, etc. Any method for modifying aldehyde groups with sodium alginate macromolecular chains; the modification of methacrylamide can be the grafting of 2-aminoethyl methacrylate, or any method such as glycidyl methacrylate, methacrylic anhydride, etc. A method for modifying conjugated double bond groups on the macromolecular chain of sodium alginate.
优选地，所述具有醛基及甲基丙烯酰胺修饰的海藻酸钠的制备方法包括：Preferably, the preparation method of sodium alginate modified with aldehyde groups and methacrylamide comprises:
将海藻酸钠的溶液与高碘酸钠混合，进行反应，终止反应后进行后处理得到醛基修饰的海藻酸钠；将所述醛基修饰的海藻酸钠的溶液与N-羟基琥珀酰亚胺和1-乙基-3-(3-二甲氨基丙基)-碳化二亚胺盐酸盐混合，再与2-氨基乙基甲基丙烯酸酯盐酸盐混合，进行反应，进行后处理得到具有醛基及甲基丙烯酰胺修饰的海藻酸钠。Mix the solution of sodium alginate with sodium periodate to react, and after the reaction is terminated, perform post-treatment to obtain aldehyde-modified sodium alginate; mix the solution of aldehyde-modified sodium alginate with N-hydroxysuccinyl Amine mixed with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, mixed with 2-aminoethyl methacrylate hydrochloride, reacted, and post-treated Sodium alginate modified with aldehyde group and methacrylamide was obtained.
优选地，所述海藻酸钠的重均分子量为2×105～2×106。Preferably, the weight average molecular weight of the sodium alginate is 2×105 -2×106 .
优选地，所述海藻酸钠的溶液的浓度为5～10mg/mL，包括但不限于6mg/mL、7mg/mL、8mg/mL或9mg/mL。Preferably, the concentration of the sodium alginate solution is 5-10 mg/mL, including but not limited to 6 mg/mL, 7 mg/mL, 8 mg/mL or 9 mg/mL.
优选地，所述高碘酸钠的工作浓度为0.2～1mg/mL，包括但不限于0.3mg/mL、0.4mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL或0.9mg/mL。Preferably, the working concentration of sodium periodate is 0.2-1 mg/mL, including but not limited to 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg /mL or 0.9mg/mL.
优选地，所述N-羟基琥珀酰亚胺的工作浓度为0.5～2mg/mL，包括但不限于0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL、1mg/mL、1.5mg/mL、1.6、1.8mg/mL或1.9mg/mL。Preferably, the working concentration of N-hydroxysuccinimide is 0.5-2 mg/mL, including but not limited to 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1 mg/mL, 1.5mg/mL, 1.6, 1.8mg/mL or 1.9mg/mL.
优选地，所述1-乙基-3-(3-二甲氨基丙基)-碳化二亚胺盐酸盐的工作浓度为2～5mg/mL。Preferably, the working concentration of the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride is 2-5 mg/mL.
优选地，所述2-氨基乙基甲基丙烯酸酯盐酸盐的工作浓度为0.5～2mg/mL，包括但不限于0.6mg/mL、0.7mg/mL、0.8mg/mL、1mg/mL、1.5mg/mL、1.6mg/mL、1.8mg/mL或1.9mg/mL。Preferably, the working concentration of 2-aminoethyl methacrylate hydrochloride is 0.5-2 mg/mL, including but not limited to 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 1 mg/mL, 1.5mg/mL, 1.6mg/mL, 1.8mg/mL or 1.9mg/mL.
本发明一具体实施例中，所述醛基修饰的海藻酸钠的溶液可以为溶解有醛基修饰的海藻酸钠的MES缓冲溶液，所述MES缓冲溶液中2-吗啉乙磺酸(MES)浓度为5～20mg/mL，氯化钠浓度为20～50mg/mL。In a specific embodiment of the present invention, the solution of the sodium alginate modified by the aldehyde group can be an MES buffer solution dissolved with the sodium alginate modified by the aldehyde group, and in the MES buffer solution, 2-morpholineethanesulfonic acid (MES ) concentration is 5-20mg/mL, and the concentration of sodium chloride is 20-50mg/mL.
优选地，所述高碘酸钠的添加量为海藻酸钠单体摩尔量的5％～10％。Preferably, the added amount of the sodium periodate is 5%-10% of the molar amount of the sodium alginate monomer.
优选地，所述后处理包括透析和冷冻干燥。Preferably, said post-treatment includes dialysis and freeze-drying.
本发明一具体实施例中，使用透析袋透析，透析袋截留分子量为3500Da，透析过程换水4～8次，透析温度为20～30℃，冷冻干燥时间为12～36h，温度为-80℃～-50℃。In a specific embodiment of the present invention, a dialysis bag is used for dialysis, the molecular weight cut-off of the dialysis bag is 3500 Da, the water is changed 4-8 times during the dialysis process, the dialysis temperature is 20-30°C, the freeze-drying time is 12-36h, and the temperature is -80°C ~-50°C.
优选地，所述用于细胞培养肉的纤维支架的制备方法包括以下步骤：Preferably, the preparation method of the fiber scaffold for cell culture meat comprises the following steps:
(1)将海藻酸钠的溶液与高碘酸钠混合，进行反应，终止反应后进行后处理得到醛基修饰的海藻酸钠；(1) The solution of sodium alginate is mixed with sodium periodate, reacted, after terminating the reaction, post-treatment is carried out to obtain aldehyde-modified sodium alginate;
将所述醛基修饰的海藻酸钠的溶液与N-羟基琥珀酰亚胺和1-乙基-3-(3-二甲氨基丙基)-碳化二亚胺盐酸盐混合，再与2-氨基乙基甲基丙烯酸酯盐酸盐混合，进行反应，进行后处理得到具有醛基及甲基丙烯酰胺修饰的海藻酸钠；The solution of sodium alginate modified by the aldehyde group is mixed with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, and then mixed with 2 -Aminoethyl methacrylate hydrochloride is mixed, reacted, and post-treated to obtain sodium alginate modified with aldehyde groups and methacrylamide;
(2)利用所述具有醛基及甲基丙烯酰胺修饰的海藻酸钠配制纺丝溶液；(2) Utilizing the sodium alginate modified with aldehyde groups and methacrylamide to prepare a spinning solution;
(3)利用所述纺丝溶液在凝固液中进行纺丝得到初生丝，对所述初生丝进行拉伸及紫外光照射，进行后处理，得到所述用于细胞培养肉的纤维支架。(3) Spinning the spinning solution in a coagulation solution to obtain as-spun silk, stretching and irradiating the as-spun silk with ultraviolet light, and performing post-treatment to obtain the fiber scaffold for cell cultured meat.
第二方面，本发明提供一种用于细胞培养肉的纤维支架，所述用于细胞培养肉的纤维支架由第一方面所述的制备方法制备得到。In a second aspect, the present invention provides a fiber scaffold for cell cultured meat, which is prepared by the preparation method described in the first aspect.
第三方面，本发明提供第二方面所述的用于细胞培养肉的纤维支架在制备细胞培养肉中的应用。In a third aspect, the present invention provides the application of the fiber scaffold for cell-cultured meat described in the second aspect in preparing cell-cultured meat.
第三方面，本发明提供一种制备细胞培养肉的方法，所述方法包括利用第二方面所述的用于细胞培养肉的纤维支架培养细胞，得到所述细胞培养肉。In a third aspect, the present invention provides a method for preparing cell cultured meat, the method comprising using the fiber scaffold for cell cultured meat described in the second aspect to cultivate cells to obtain the cell cultured meat.
优选地，所述细胞为动物细胞，进一步优选地，所述细胞为哺乳动物细胞。Preferably, the cells are animal cells, further preferably, the cells are mammalian cells.
与现有技术相比，本发明具备以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
本发明采用具有醛基修饰及甲基丙烯酰胺修饰的海藻酸钠进行湿法纺丝，可以增强纤维支架的黏附性能；同时使纤维支架具有光固化特性，碳碳双键之间形成稳定的共价连接，从而延长纤维支架的降解时间，实现可以使用单一一种主原料来制备纤维支架，同时在培基中实现较长时间的结构稳定性和更高的细胞粘附性，提高培养细胞的数量和伸展情况。The present invention uses sodium alginate modified with aldehyde group and methacrylamide to carry out wet spinning, which can enhance the adhesion performance of the fiber support; at the same time, the fiber support has photocuring characteristics, and a stable co-cohesive bond is formed between carbon-carbon double bonds. valence linkage, thereby prolonging the degradation time of the fibrous scaffold, realizing that a single main raw material can be used to prepare the fibrous scaffold, and at the same time achieving a longer period of structural stability and higher cell adhesion in the culture medium, improving the quality of cultured cells. quantity and extension.
附图说明Description of drawings
图1为实施例1制备的湿纺纤维以及经冷冻干燥之后的纤维支架图和通过显微镜观察的湿纺纤维图；Fig. 1 is the wet-spun fiber prepared by embodiment 1 and the fiber scaffold figure after freeze-drying and the wet-spun fiber figure observed by microscope;
图2为海藻酸钠原料、修饰有醛基的海藻酸钠、修饰有醛基和甲基丙烯酰胺的海藻酸钠以及最终制得的纤维支架的红外光谱图；Fig. 2 is the infrared spectrogram of the sodium alginate raw material, the sodium alginate modified with aldehyde group, the sodium alginate modified with aldehyde group and methacrylamide and the final fiber support;
图3为将C2C12细胞培养在仅修饰有甲基丙烯酰胺的海藻酸钠纤维(MA-Alg)支架上2天后的活死染色图；Fig. 3 is the life-death staining figure after C2C12 cells are cultivated on the sodium alginate fiber (MA-Alg) support that only has been modified with methacrylamide for 2 days;
图4为将C2C12细胞培养在修饰有醛基和甲基丙烯酰胺的海藻酸钠纤维支架(CHO+MA-Alg)上2天后的活死染色图；Fig. 4 is the life-death staining picture of C2C12 cells cultured on the sodium alginate fiber support (CHO+MA-Alg) modified with aldehyde group and methacrylamide after 2 days;
图5为图3、图4中活死染色活细胞绿色荧光面积统计结果图；Fig. 5 is a graph of the statistical results of the green fluorescent area of live cells stained in Fig. 3 and Fig. 4;
图6为纺丝过程中未经紫外灯照射和经紫外灯照射所制备的两种纤维支架在细胞培养基中浸泡不同时间后的显微镜观察图；Fig. 6 is the microscopic observation diagram of two kinds of fiber scaffolds prepared without ultraviolet light irradiation and ultraviolet light irradiation in the cell culture medium for different time in the spinning process;
图7为经过紫外灯照射的纤维支架在浸泡第21天显微镜观察图。Fig. 7 is a microscope observation picture of the fiber scaffold irradiated by ultraviolet light on the 21st day of immersion.
具体实施方式Detailed ways
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。In order to further illustrate the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with the embodiments and accompanying drawings. It should be understood that the specific implementation manners described here are only used to explain the present invention, rather than to limit the present invention.
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道购买获得的常规产品。If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field, or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased through regular channels.
实施例1Example 1
本实施例制备用于细胞培养肉的纤维支架，其制备过程包括海藻酸钠的化学改性、纺丝溶液的制备以及湿法纺丝纤维的制备。In this example, a fiber scaffold for cell cultured meat was prepared, and the preparation process included chemical modification of sodium alginate, preparation of spinning solution, and preparation of wet-spun fibers.
首先向海藻酸钠的分子链上修饰醛基，向7mg/mL海藻酸钠(购自麦克林生化科技有限公司，货号S875337)的溶液中加入0.6mg/mL高碘酸钠，在25℃下避光反应9h。反应结束后加入0.01mL/mL乙二醇继续搅拌2h以终止反应，后经透析冷冻干燥后得到醛基修饰的海藻酸钠。然后使用透析袋透析以去除小分子杂质，透析袋截留分子量为3500Da，透析过程换水4-8次，透析温度为25℃。冷冻干燥时间为24h，温度为-60℃。First, modify the aldehyde group on the molecular chain of sodium alginate, add 0.6 mg/mL sodium periodate to the solution of 7 mg/mL sodium alginate (purchased from McLean Biochemical Technology Co., Ltd., product number S875337), and add 0.6 mg/mL sodium periodate at 25 ° C. Protect from light for 9 hours. After the reaction was completed, 0.01 mL/mL ethylene glycol was added to continue stirring for 2 h to terminate the reaction, and then dialysis and freeze-drying were performed to obtain aldehyde-modified sodium alginate. Then use the dialysis bag for dialysis to remove small molecular impurities. The molecular weight cut-off of the dialysis bag is 3500Da. During the dialysis process, the water is changed 4-8 times, and the dialysis temperature is 25°C. The freeze-drying time was 24 hours, and the temperature was -60°C.
向修饰有醛基的海藻酸钠分子链上接枝甲基丙烯酰胺，向溶解有含醛基的海藻酸钠的MES缓冲溶液中加入N-羟基琥珀酰亚胺(NHS)和1-乙基-3-(3-二甲氨基丙基)-碳化二亚胺盐酸盐(EDC)以活化海藻酸钠的羧基，然后加入2-氨基乙基甲基丙烯酸酯盐酸盐(AEMA)，25℃下避光反应24h，后经透析冷冻干燥得到双键接枝的含醛基修饰的海藻酸钠。所述MES缓冲溶液中2-吗啉乙磺酸(MES)浓度为17mg/mL，氯化钠浓度为35mg/mL；含醛基的海藻酸钠浓度为7mg/mL；NHS加入后的浓度为1.7mg/mL，EDC加入后的浓度为3mg/mL；AEMA加入后的浓度为1.6mg/mL；透析同上步骤，透析袋截留分子量为3500Da，透析过程换水6次，透析温度为25℃。冷冻干燥时间为30h，温度为-65℃。Graft methacrylamide on the molecular chain of sodium alginate modified with aldehyde group, add N-hydroxysuccinimide (NHS) and 1-ethyl -3-(3-Dimethylaminopropyl)-carbodiimide hydrochloride (EDC) to activate the carboxyl group of sodium alginate, then add 2-aminoethyl methacrylate hydrochloride (AEMA), 25 The reaction was carried out at ℃ for 24 hours in the dark, and then dialysis and freeze-drying were performed to obtain aldehyde-modified sodium alginate grafted with double bonds. In the MES buffer solution, the concentration of 2-morpholineethanesulfonic acid (MES) is 17 mg/mL, and the concentration of sodium chloride is 35 mg/mL; the concentration of sodium alginate containing aldehyde groups is 7 mg/mL; the concentration after NHS is added is 1.7mg/mL, the concentration after adding EDC is 3mg/mL; the concentration after adding AEMA is 1.6mg/mL; the dialysis is the same as the above step, the molecular weight cut-off of the dialysis bag is 3500Da, the water is changed 6 times during the dialysis process, and the dialysis temperature is 25°C. The freeze-drying time was 30 hours, and the temperature was -65°C.
配制纺丝溶液，组成包括去离子水(溶剂)、2,4,6-三甲基苯甲酰基苯基膦酸乙酯(TPO)和具有醛基及甲基丙烯酰胺修饰的海藻酸钠。光引发剂的浓度为6mg/mL，具有醛基及甲基丙烯酰胺修饰的海藻酸钠浓度为75mg/mL。The spinning solution was prepared, and the composition included deionized water (solvent), ethyl 2,4,6-trimethylbenzoylphenyl phosphonate (TPO) and sodium alginate modified with aldehyde groups and methacrylamide. The concentration of the photoinitiator was 6 mg/mL, and the concentration of sodium alginate modified with aldehyde groups and methacrylamide was 75 mg/mL.
湿法纺丝纤维的制备包括纺丝凝固液的配置以及湿法纺丝设备的工作参数配置。纺丝凝固液为氯化钙溶液，钙离子浓度为0.4M。湿法纺丝设备为自制，其组成包括微量灌注泵、注射器、悬臂式机械搅拌器、搅拌桨、聚四氟乙烯接收管、接受浴、紫外光灯。通过微量灌注泵控制，将注射器中的纺丝溶液按一定速度从针头挤出至接受浴中迅速固化形成较粗的纤维丝，再由悬臂式机械搅拌器带动固定在搅拌桨上的接收管旋转牵引拉伸固化的纤维，拉伸过程中使用紫外光灯照射纤维丝以实现二次交联。所选用注射器内径为0.8cm，微量灌注泵设置推进速度为130μL/min，所选用针头规格为25G，搅拌器旋转速度为80rpm，接收辊外径为2.5cm，紫外光灯波长选用400nm。纺丝结束后将纤维束从接受辊上取下流水冲洗15min，后经干燥即得用于细胞培养肉的纤维支架。The preparation of wet spinning fibers includes the configuration of spinning coagulation liquid and the configuration of working parameters of wet spinning equipment. The spinning coagulation solution is a calcium chloride solution with a calcium ion concentration of 0.4M. The wet spinning equipment is self-made, and its composition includes a micro-perfusion pump, a syringe, a cantilever mechanical stirrer, a stirring paddle, a polytetrafluoroethylene receiving tube, a receiving bath, and an ultraviolet lamp. Controlled by the micro-perfusion pump, the spinning solution in the syringe is extruded from the needle at a certain speed to the receiving bath to quickly solidify to form thicker fiber filaments, and then the receiving tube fixed on the stirring paddle is driven by the cantilever mechanical stirrer to rotate The cured fiber is pulled and stretched, and the fiber filament is irradiated with ultraviolet light to achieve secondary crosslinking during the stretching process. The inner diameter of the selected syringe is 0.8cm, the propulsion speed of the microperfusion pump is set to 130μL/min, the selected needle size is 25G, the rotation speed of the stirrer is 80rpm, the outer diameter of the receiving roller is 2.5cm, and the wavelength of the ultraviolet light is 400nm. After spinning, the fiber bundle is taken off from the receiving roll, rinsed with running water for 15 minutes, and then dried to obtain a fiber support for cell cultured meat.
制备的湿纺纤维以及经冷冻干燥之后的纤维支架的数码照片和通过显微镜观察的湿纺纤维的照片如图1所示，海藻酸钠原料、修饰有醛基的海藻酸钠、修饰有醛基和甲基丙烯酰胺的海藻酸钠以及最终制得的纤维支架的红外光谱图如图2所示，其中修饰有醛基的海藻酸钠、修饰有醛基和甲基丙烯酰胺的海藻酸钠以及最终制得得纤维支架均在1750cm-1处出现了新的醛基对应的特征吸收峰，证明了醛基的成功修饰。The prepared wet-spun fiber and the digital photo of the fiber support after freeze-drying and the photo of the wet-spun fiber observed by microscope are shown in Figure 1, sodium alginate raw material, sodium alginate modified with aldehyde group, modified with aldehyde group and the sodium alginate of methacrylamide and the infrared spectrogram of the fibrous scaffold that finally makes as shown in Figure 2, wherein the sodium alginate that is modified with aldehyde group, the sodium alginate that is modified with aldehyde group and methacrylamide and The new characteristic absorption peak corresponding to the aldehyde group appeared at 1750 cm-1 in the finally prepared fiber scaffolds, which proved the successful modification of the aldehyde group.
实施例2Example 2
本实施例制备用于细胞培养肉的纤维支架，其制备过程包括海藻酸钠的化学改性、纺丝溶液的制备以及湿法纺丝纤维的制备。In this example, a fiber scaffold for cell cultured meat was prepared, and the preparation process included chemical modification of sodium alginate, preparation of spinning solution, and preparation of wet-spun fibers.
首先向海藻酸钠的分子链上修饰醛基，向海藻酸钠溶液中加入高碘酸钠，在25℃下避光反应6h。反应结束后加入乙二醇继续搅拌2h以终止反应，后经透析冷冻干燥后得到醛基修饰的海藻酸钠。所述海藻酸钠溶液浓度为5mg/mL；高碘酸钠加入后的浓度为1mg/mL；乙二醇加入后的浓度为0.02mL/mL。然后使用透析袋透析以去除小分子杂质，透析袋截留分子量为3500Da，透析过程换水4次，透析温度为25℃。冷冻干燥时间为12h，温度为-50℃。First, modify the aldehyde group on the molecular chain of sodium alginate, add sodium periodate to the sodium alginate solution, and react at 25° C. in the dark for 6 hours. After the reaction was completed, ethylene glycol was added and the mixture was stirred for 2 hours to terminate the reaction, and aldehyde-modified sodium alginate was obtained after dialysis and freeze-drying. The concentration of the sodium alginate solution is 5 mg/mL; the concentration after adding sodium periodate is 1 mg/mL; the concentration after adding ethylene glycol is 0.02 mL/mL. Then, the dialysis bag was used for dialysis to remove small molecular impurities. The molecular weight cut-off of the dialysis bag was 3500 Da. During the dialysis process, the water was changed 4 times, and the dialysis temperature was 25°C. The freeze-drying time was 12 hours, and the temperature was -50°C.
然后向修饰有醛基的海藻酸钠分子链上接枝甲基丙烯酰胺，向溶解有含醛基的海藻酸钠的MES缓冲溶液中加入N-羟基琥珀酰亚胺(NHS)和1-乙基-3-(3-二甲氨基丙基)-碳化二亚胺盐酸盐(EDC)以活化海藻酸钠的羧基，然后加入2-氨基乙基甲基丙烯酸酯盐酸盐(AEMA)，25℃下避光反应24h，后经透析冷冻干燥得到双键接枝的含醛基修饰的海藻酸钠。所述MES缓冲溶液中2-吗啉乙磺酸(MES)浓度为20mg/mL，氯化钠浓度为50mg/mL；含醛基的海藻酸钠浓度为10mg/mL；NHS加入后的浓度为2mg/mL，EDC加入后的浓度为2mg/mL；AEMA加入后的浓度为0.5mg/mL。透析同上步骤，透析袋截留分子量为3500Da，透析过程换水8次，透析温度为25℃。冷冻干燥时间为36h，温度为-80℃。Then, methacrylamide is grafted onto the molecular chain of sodium alginate modified with aldehyde group, and N-hydroxysuccinimide (NHS) and 1-ethanediene are added to the MES buffer solution of sodium alginate containing aldehyde group Base-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) to activate the carboxyl group of sodium alginate, and then add 2-aminoethyl methacrylate hydrochloride (AEMA), React in the dark at 25°C for 24 hours, and then freeze-dry by dialysis to obtain double bond-grafted sodium alginate modified with aldehyde group. In the MES buffer solution, the concentration of 2-morpholineethanesulfonic acid (MES) is 20mg/mL, and the concentration of sodium chloride is 50mg/mL; the concentration of sodium alginate containing aldehyde groups is 10mg/mL; the concentration after NHS is added is 2mg/mL, the concentration after adding EDC is 2mg/mL; the concentration after adding AEMA is 0.5mg/mL. The dialysis is the same as the above steps, the molecular weight cut-off of the dialysis bag is 3500Da, the water is changed 8 times during the dialysis process, and the dialysis temperature is 25°C. The freeze-drying time was 36 hours, and the temperature was -80°C.
配制纺丝溶液，组成包括去离子水(溶剂)、2-羟基-4′-(2-羟乙氧基)-2-甲基苯丙酮(I2959)和具有醛基及甲基丙烯酰胺修饰的海藻酸钠。光引发剂的浓度为1mg/mL，具有醛基及甲基丙烯酰胺修饰的海藻酸钠浓度为50mg/mL。Prepare the spinning solution, the composition includes deionized water (solvent), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959) and aldehyde group and methacrylamide modified sodium alginate. The concentration of the photoinitiator was 1 mg/mL, and the concentration of sodium alginate modified with aldehyde groups and methacrylamide was 50 mg/mL.
湿法纺丝纤维的制备包括纺丝凝固液的配置以及湿法纺丝设备的工作参数配置。纺丝凝固液为氯化钙溶液，钙离子浓度为0.2M。湿法纺丝设备为自制，其组成包括微量灌注泵、注射器、悬臂式机械搅拌器、搅拌桨、聚四氟乙烯接收管、接受浴、紫外光灯。通过微量灌注泵控制，将注射器中的纺丝溶液按一定速度从针头挤出至接受浴中迅速固化形成较粗的纤维丝，再由悬臂式机械搅拌器带动固定在搅拌桨上的接收管旋转牵引拉伸固化的纤维，拉伸过程中使用紫外光灯照射纤维丝以实现二次交联。所选用注射器内径为1.4cm，微量灌注泵设置推进速度为200μL/min，所选用针头规格为30G，搅拌器旋转速度为100rpm，接收辊外径为3cm，紫外光灯波长选用405nm。纺丝结束后将纤维束从接受辊上取下流水冲洗20min，后经干燥即得用于细胞培养肉的纤维支架。The preparation of wet spinning fibers includes the configuration of spinning coagulation liquid and the configuration of working parameters of wet spinning equipment. The spinning coagulation liquid is a calcium chloride solution with a calcium ion concentration of 0.2M. The wet spinning equipment is self-made, and its composition includes a micro-perfusion pump, a syringe, a cantilever mechanical stirrer, a stirring paddle, a polytetrafluoroethylene receiving tube, a receiving bath, and an ultraviolet lamp. Controlled by the micro-perfusion pump, the spinning solution in the syringe is extruded from the needle at a certain speed to the receiving bath to quickly solidify to form thicker fiber filaments, and then the receiving tube fixed on the stirring paddle is driven by the cantilever mechanical stirrer to rotate The cured fiber is pulled and stretched, and the fiber filament is irradiated with ultraviolet light to achieve secondary crosslinking during the stretching process. The inner diameter of the selected syringe is 1.4cm, the propulsion speed of the microperfusion pump is set to 200μL/min, the selected needle size is 30G, the rotation speed of the stirrer is 100rpm, the outer diameter of the receiving roller is 3cm, and the wavelength of the ultraviolet lamp is 405nm. After spinning, the fiber bundle is taken off from the receiving roll, rinsed with running water for 20 minutes, and then dried to obtain a fiber support for cell cultured meat.
实施例3Example 3
本实施例制备用于细胞培养肉的纤维支架，其制备过程包括海藻酸钠的化学改性、纺丝溶液的制备以及湿法纺丝纤维的制备。In this example, a fiber scaffold for cell cultured meat was prepared, and the preparation process included chemical modification of sodium alginate, preparation of spinning solution, and preparation of wet-spun fibers.
首先向海藻酸钠的分子链上修饰醛基，向海藻酸钠溶液中加入高碘酸钠，在25℃下避光反应12h。反应结束后加入乙二醇继续搅拌1h以终止反应，后经透析冷冻干燥后得到醛基修饰的海藻酸钠。所述海藻酸钠溶液浓度为10mg/mL；高碘酸钠加入后的浓度为0.2mg/mL；乙二醇加入后的浓度为0.005mL/mL。然后使用透析袋透析以去除小分子杂质，透析袋截留分子量为3500Da，透析过程换水8次，透析温度为25℃。冷冻干燥时间为36h，温度为-80℃。First, modify the aldehyde group on the molecular chain of sodium alginate, add sodium periodate to the sodium alginate solution, and react at 25° C. in the dark for 12 hours. After the reaction was completed, ethylene glycol was added to continue stirring for 1 h to terminate the reaction, and then dialysis and freeze-drying were performed to obtain aldehyde-modified sodium alginate. The concentration of the sodium alginate solution is 10 mg/mL; the concentration after adding sodium periodate is 0.2 mg/mL; the concentration after adding ethylene glycol is 0.005 mL/mL. Then, the dialysis bag was used for dialysis to remove small molecular impurities. The molecular weight cut-off of the dialysis bag was 3500 Da. During the dialysis process, the water was changed 8 times, and the dialysis temperature was 25°C. The freeze-drying time was 36 hours, and the temperature was -80°C.
然后向修饰有醛基的海藻酸钠分子链上接枝甲基丙烯酰胺，向溶解有含醛基的海藻酸钠的MES缓冲溶液中加入N-羟基琥珀酰亚胺(NHS)和1-乙基-3-(3-二甲氨基丙基)-碳化二亚胺盐酸盐(EDC)以活化海藻酸钠的羧基，然后加入2-氨基乙基甲基丙烯酸酯盐酸盐(AEMA)，25℃下避光反应24h，后经透析冷冻干燥得到双键接枝的含醛基修饰的海藻酸钠。所述MES缓冲溶液中2-吗啉乙磺酸(MES)浓度为5mg/mL，氯化钠浓度为20mg/mL；含醛基的海藻酸钠浓度为5mg/mL；NHS加入后的浓度为0.5mg/mL，EDC加入后的浓度为5mg/mL；AEMA加入后的浓度为2mg/mL。透析同上步骤，透析袋截留分子量为3500Da，透析过程换水8次，透析温度为25℃。冷冻干燥时间为12h，温度为-80℃。Then, methacrylamide is grafted onto the molecular chain of sodium alginate modified with aldehyde group, and N-hydroxysuccinimide (NHS) and 1-ethanediene are added to the MES buffer solution of sodium alginate containing aldehyde group Base-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) to activate the carboxyl group of sodium alginate, and then add 2-aminoethyl methacrylate hydrochloride (AEMA), React in the dark at 25°C for 24 hours, and then freeze-dry by dialysis to obtain double bond-grafted sodium alginate modified with aldehyde group. In the MES buffer solution, the concentration of 2-morpholineethanesulfonic acid (MES) is 5 mg/mL, and the concentration of sodium chloride is 20 mg/mL; the concentration of sodium alginate containing aldehyde groups is 5 mg/mL; the concentration after NHS is added is 0.5mg/mL, the concentration after adding EDC is 5mg/mL; the concentration after adding AEMA is 2mg/mL. The dialysis is the same as the above steps, the molecular weight cut-off of the dialysis bag is 3500Da, the water is changed 8 times during the dialysis process, and the dialysis temperature is 25°C. The freeze-drying time was 12 hours, and the temperature was -80°C.
配制纺丝溶液，组成包括去离子水(溶剂)、苯基(2,4,6-三甲基苯甲酰基)磷酸锂(LAP)和具有醛基及甲基丙烯酰胺修饰的海藻酸钠。光引发剂的浓度为10mg/mL，具有醛基及甲基丙烯酰胺修饰的海藻酸钠浓度为100mg/mL。A spinning solution was prepared, which consisted of deionized water (solvent), lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) and sodium alginate modified with aldehyde groups and methacrylamide. The concentration of the photoinitiator was 10 mg/mL, and the concentration of sodium alginate modified with aldehyde groups and methacrylamide was 100 mg/mL.
湿法纺丝纤维的制备包括纺丝凝固液的配置以及湿法纺丝设备的工作参数配置。纺丝凝固液为氯化钙溶液，钙离子浓度为0.6M。湿法纺丝设备为自制，其组成包括微量灌注泵、注射器、悬臂式机械搅拌器、搅拌桨、聚四氟乙烯接收管、接受浴、紫外光灯。通过微量灌注泵控制，将注射器中的纺丝溶液按一定速度从针头挤出至接受浴中迅速固化形成较粗的纤维丝，再由悬臂式机械搅拌器带动固定在搅拌桨上的接收管旋转牵引拉伸固化的纤维，拉伸过程中使用紫外光灯照射纤维丝以实现二次交联。所选用注射器内径为0.33cm，微量灌注泵设置推进速度为50μL/min，所选用针头规格为20G，搅拌器旋转速度为50rpm，接收辊外径为2cm，紫外光灯波长选用365nm。纺丝结束后将纤维束从接受辊上取下流水冲洗5min，后经干燥即得用于细胞培养肉的纤维支架。The preparation of wet spinning fibers includes the configuration of spinning coagulation liquid and the configuration of working parameters of wet spinning equipment. The spinning coagulation solution is a calcium chloride solution with a calcium ion concentration of 0.6M. The wet spinning equipment is self-made, and its composition includes a micro-perfusion pump, a syringe, a cantilever mechanical stirrer, a stirring paddle, a polytetrafluoroethylene receiving tube, a receiving bath, and an ultraviolet lamp. Controlled by the micro-perfusion pump, the spinning solution in the syringe is extruded from the needle at a certain speed to the receiving bath to quickly solidify to form thicker fiber filaments, and then the receiving tube fixed on the stirring paddle is driven by the cantilever mechanical stirrer to rotate The cured fiber is pulled and stretched, and the fiber filament is irradiated with ultraviolet light to achieve secondary crosslinking during the stretching process. The inner diameter of the selected syringe is 0.33cm, the propulsion speed of the microperfusion pump is set to 50μL/min, the selected needle size is 20G, the rotation speed of the stirrer is 50rpm, the outer diameter of the receiving roller is 2cm, and the wavelength of the ultraviolet light is 365nm. After spinning, the fiber bundle is removed from the receiving roll, rinsed with running water for 5 minutes, and then dried to obtain a fiber support for cell cultured meat.
对比例1Comparative example 1
本对比例与实施例1相比区别仅在于：不对海藻酸钠的分子修饰醛基，而直接修饰甲基丙烯酰胺，进行湿法纺丝制备纤维支架。其余相应方法参照实施例1。The difference between this comparative example and Example 1 is that the aldehyde group is not modified on the molecule of sodium alginate, but the methacrylamide is directly modified, and the fiber scaffold is prepared by wet spinning. Refer to Example 1 for the remaining corresponding methods.
对比例2Comparative example 2
本对比例与实施例1相比区别仅在于：在湿法纺丝过程中，不对纺丝进行紫外灯照射。The only difference between this comparative example and Example 1 is that during the wet spinning process, the spinning is not irradiated with ultraviolet lamps.
测试例1test case 1
本测试例利用实施例1和对比例1制备的纤维支架培养小鼠成肌细胞(C2C12细胞)，具体培养方法包括：在体积约为0.1cm3的纤维支架上，接种数量为1.5×104的小鼠成肌细胞，在二氧化碳培养箱中37℃恒温静态培养2天，培养基配方为：89％DMEM高糖培养基+10％胎牛血清+1％双抗；In this test example, the fiber scaffolds prepared in Example 1 and Comparative Example 1 were used to culture mouse myoblasts (C2C12 cells). The specific culture method includes: on a fiber scaffold with a volume of about 0.1 cm3 , the inoculated number is 1.5×104 The mouse myoblasts were cultured in a carbon dioxide incubator at 37°C for 2 days, and the medium formula was: 89% DMEM high-glucose medium + 10% fetal bovine serum + 1% double antibody;
图3为将C2C12细胞培养在仅修饰有甲基丙烯酰胺的海藻酸钠纤维(MA-Alg)支架上2天后的活死染色图片，图4为将C2C12细胞培养在修饰有醛基和甲基丙烯酰胺的海藻酸钠纤维支架(CHO+MA-Alg)上2天后的活死染色图片，对绿色荧光面积的统计结果如图5所示，在接种了相同细胞密度的条件下，修饰有醛基的纤维支架上细胞的数量和伸展情况都相比未修饰醛基的支架有一定提升，表明醛基的修饰增强了细胞对纤维支架的黏附性。Figure 3 is a live-dead staining picture of C2C12 cells cultured on a sodium alginate fiber (MA-Alg) scaffold modified only with methacrylamide for 2 days, and Figure 4 is a picture of C2C12 cells cultured on a scaffold modified with aldehyde and methyl The live-dead staining pictures of acrylamide sodium alginate fiber scaffolds (CHO+MA-Alg) after 2 days, and the statistical results of the green fluorescent area are shown in Figure 5. Under the condition of seeding the same cell density, the modified aldehyde Compared with unmodified aldehyde-based scaffolds, the number and extension of cells on the fibrous scaffolds with aldehyde groups were improved to a certain extent, indicating that the modification of aldehyde groups enhanced the adhesion of cells to the fibrous scaffolds.
测试例2test case 2
本测试例对实施例1和对比例2制备的纤维支架在使用过程中进行显微镜观察，培养细胞过程参照测试例1。In this test example, the fiber scaffolds prepared in Example 1 and Comparative Example 2 were observed under a microscope during use, and the process of culturing cells was referred to in Test Example 1.
图6为纺丝过程中未经紫外灯照射和经紫外灯照射所制备的两种纤维支架在细胞培养基中浸泡不同时间后的显微镜观察图。未经紫外灯照射的纤维支架在浸泡第3天(3d)时已经发生了较大程度的溶胀和一定程度的降解，并且在浸泡第7天(7d)时已经发生了大部分的降解；而经过紫外灯照射的纤维支架在浸泡的第7天仍保持着稳定的结构形态，且在浸泡的第21天仍保持着稳定的纤维外貌(图7)，表明本发明中甲基丙烯酰胺的修饰可以为聚合物提供光交联位点，在光引发剂的作用下可以在紫外灯照射下快速交联，显著提高纤维支架稳定性。Fig. 6 is a microscope observation diagram of two kinds of fiber scaffolds prepared without ultraviolet light irradiation and ultraviolet light irradiation in the spinning process after soaking in cell culture medium for different time. The fiber scaffolds without ultraviolet light had a large degree of swelling and a certain degree of degradation on the 3rd day (3d) of immersion, and most of the degradation had occurred on the 7th day (7d) of immersion; while The fiber scaffold irradiated by ultraviolet light still maintained a stable structural form on the 7th day of immersion, and still maintained a stable fiber appearance (Fig. 7) on the 21st day of immersion, indicating that the modification of methacrylamide in the present invention It can provide photocrosslinking sites for polymers, and under the action of photoinitiators, it can be quickly crosslinked under the irradiation of ultraviolet lamps, which significantly improves the stability of fiber scaffolds.
综上所述，本发明采用具有醛基修饰及甲基丙烯酰胺修饰的海藻酸钠进行湿法纺丝，可以增强纤维支架的黏附性能；同时使纤维支架具有光固化特性，碳碳双键之间形成稳定的共价连接，从而延长纤维支架的降解时间，实现可以使用单一一种主原料来制备纤维支架，同时在培基中实现较长时间的结构稳定性和更高的细胞粘附性。In summary, the present invention uses sodium alginate modified with aldehyde groups and methacrylamide for wet spinning, which can enhance the adhesion performance of the fiber scaffold; at the same time, the fiber scaffold has photocuring properties, and the carbon-carbon double bond Form a stable covalent link between them, thereby prolonging the degradation time of the fibrous scaffold, so that a single main raw material can be used to prepare the fibrous scaffold, and at the same time, a longer period of structural stability and higher cell adhesion can be achieved in the culture medium sex.
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。The applicant declares that the present invention illustrates the detailed methods of the present invention through the above-mentioned examples, but the present invention is not limited to the above-mentioned detailed methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed methods to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117344456A (en)* | 2023-10-26 | 2024-01-05 | 青岛大学 | Method for preparing sodium alginate electrospun nanofiber by adopting photo-crosslinking |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103174017A (en)* | 2013-04-15 | 2013-06-26 | 青岛明月生物医用材料有限公司 | Sodium alginate oxide modified chitosan fiber and preparation method and application thereof |
| WO2017136837A1 (en)* | 2016-02-06 | 2017-08-10 | President And Fellows Of Harvard College | Recapitulating the hematopoietic niche to reconstitute immunity |
| CN109395150A (en)* | 2018-10-25 | 2019-03-01 | 恒天嘉华非织造有限公司 | A kind of degradability medical haemostatic material and preparation method thereof |
| CN111662464A (en)* | 2020-07-23 | 2020-09-15 | 南京工业大学 | Preparation method of chitosan/sodium alginate double-network hydrogel |
| CN113518635A (en)* | 2018-12-17 | 2021-10-19 | 哈佛学院院长等 | Bioengineered scaffolds for modulating immune system and uses thereof |
| CN114622296A (en)* | 2022-02-28 | 2022-06-14 | 上海食未生物科技有限公司 | Method for continuously preparing edible fiber scaffold for cell culture meat |
| CN114892311A (en)* | 2022-04-25 | 2022-08-12 | 中国科学院苏州纳米技术与纳米仿生研究所 | A kind of alginate fiber and its preparation method and application |
| KR20230022392A (en)* | 2021-08-06 | 2023-02-15 | 한국재료연구원 | Composition for praparing organic-inorganic complex hydrogel and kit for preparing organic-inorganic complex hydrogel comprising the same |
| CN116121174A (en)* | 2022-09-30 | 2023-05-16 | 山东农业大学 | Method for three-dimensional culture and separation of chicken embryo fibroblasts in vitro |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103174017A (en)* | 2013-04-15 | 2013-06-26 | 青岛明月生物医用材料有限公司 | Sodium alginate oxide modified chitosan fiber and preparation method and application thereof |
| WO2017136837A1 (en)* | 2016-02-06 | 2017-08-10 | President And Fellows Of Harvard College | Recapitulating the hematopoietic niche to reconstitute immunity |
| CN109395150A (en)* | 2018-10-25 | 2019-03-01 | 恒天嘉华非织造有限公司 | A kind of degradability medical haemostatic material and preparation method thereof |
| CN113518635A (en)* | 2018-12-17 | 2021-10-19 | 哈佛学院院长等 | Bioengineered scaffolds for modulating immune system and uses thereof |
| CN111662464A (en)* | 2020-07-23 | 2020-09-15 | 南京工业大学 | Preparation method of chitosan/sodium alginate double-network hydrogel |
| KR20230022392A (en)* | 2021-08-06 | 2023-02-15 | 한국재료연구원 | Composition for praparing organic-inorganic complex hydrogel and kit for preparing organic-inorganic complex hydrogel comprising the same |
| CN114622296A (en)* | 2022-02-28 | 2022-06-14 | 上海食未生物科技有限公司 | Method for continuously preparing edible fiber scaffold for cell culture meat |
| CN114892311A (en)* | 2022-04-25 | 2022-08-12 | 中国科学院苏州纳米技术与纳米仿生研究所 | A kind of alginate fiber and its preparation method and application |
| CN116121174A (en)* | 2022-09-30 | 2023-05-16 | 山东农业大学 | Method for three-dimensional culture and separation of chicken embryo fibroblasts in vitro |
| Title |
|---|
| KWANG-HWAN CHOI ET AL.: ""Muscle stem cell isolation and in vitro culture for meat production: A methodological review"", 《COMPR REV FOOD SCI FOOD SAF》, vol. 20, no. 1, 31 January 2021 (2021-01-31), pages 429 - 457, XP055904480, DOI: 10.1111/1541-4337.12661* |
| 付小兵: "《生物3D打印与再生医学》", vol. 1, 29 February 2020, 华中科技大学出版社, pages: 225* |
| 朱美芳: "中国战略性新兴产业.新材料.高性能纤维》", vol. 2017, 30 November 2017, 中国铁道出版社, pages: 26* |
| 窦雪宇 等: ""海藻酸钠基静电纺丝纳米纤维膜的制备"", 《山东工业技术》, 31 October 2018 (2018-10-31), pages 35* |
| 马小军 等: "《海藻酸基海洋生物医用材料》", vol. 1, 31 January 2020, 上海科学技术出版社, pages: 328* |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117344456A (en)* | 2023-10-26 | 2024-01-05 | 青岛大学 | Method for preparing sodium alginate electrospun nanofiber by adopting photo-crosslinking |
| Publication | Publication Date | Title |
|---|---|---|
| Unal et al. | Glioblastoma cell adhesion properties through bacterial cellulose nanocrystals in polycaprolactone/gelatin electrospun nanofibers | |
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