CN114099781A - 一种人源性生物组织材料及培养细胞刺激方法和装置 - Google Patents
一种人源性生物组织材料及培养细胞刺激方法和装置 Download PDFInfo
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Abstract
本发明提供了一种人源性生物组织材料及动态培养细胞刺激方法和装置。该生物组织材料包含体外培养基体,并含有多个可容纳待培养细胞的可塑形腔。培养过程中将细胞置于可塑形腔分泌细胞外基质(ECM)。动态培养方法包括将细胞置于所述可塑形腔,并在不同的时间点对其施加不同方式的外力以刺激细胞分泌ECM。根据不同的待植入部位,选择不同方向、大小及角度的机械力刺激细胞,使其分泌更多的ECM,并起到塑形作用,形成“生物材料+塑形细胞+ECM”的复合生物材料。通过力学刺激复合生物材料成为一个有机整合体,避免了化学交联剂的使用。复合材料中的细胞成分(免疫成分)可以去除。获得的生物组织材料包括血管、疝气补片、肌腱、软骨、肌肉等。
Description
技术领域
本发明涉及生物组织材料技术领域,更具体地,涉及一种人源性生物组织材料及培养细胞刺激方法和装置。
背景技术
目前临床上广泛使用的生物材料如人工血管、疝气补片主要缺点有:(1)人工顺应性较差、不具备人体动脉的柔韧性与弹性,这个缺陷在与小口径动脉吻合时就表现的非常明显,这也是血栓易在吻合口部位形成的主要原因,导致血栓形成及内膜增生,远期通畅率极低;(2)疝气补片容易发生感染、瘢痕、不适感、局部积液、纤维包膜的形成,若感染需取出;(3)普通的天然材料要化学交联剂把一个一个分子强行连接在一起,否则在体内会被快速降解,而化学交联都有毒性,如戊二醛交联剂,它可以让蛋白质变性,有致癌性;(4)用自体细胞培养人源性生物材料如血管、肌腱等,则周期太长,而异体细胞会产生免疫排斥。
细胞外基质(ECM)处于力学环境中,许多ECM成分都直接或间接地受到机械力地调控,并能将力学信号转化为适当的反应。ECM处在不断代谢更新、降解重塑的动态平衡之中。力学刺激不仅能直接引起ECM成分的表达变化,还可通过调控ECM基因表达而使ECM成分变化。
通过力学性能刺激下动态培养生物材料,产生人源性ECM,能够很好的解决生物材料来源有限、免疫排斥和炎症反应等问题;而通过对高分子材料进行相应的结构设计使其具有抗感染和自洁净的能力,同时又兼顾了移植物特有的力学性能。如经过体外塑形的人源性天然材料与高分子材料交错在一起的生物血管,具有近似人体动脉的顺应性,弹性和力学性能,而缝合时由于血管壁的胶原蛋白纤维可以避免针眼渗血。
公开于该背景技术部分的信息仅仅旨在加深对本发明的总体背景技术的理解,而不应当被视为承认或以任何形式暗示该信息构成已为本领域技术人员所公知的现有技术。
发明内容
本发明的第一目的在于,针对上述缺陷和不足,解决现有技术中生物组织材料顺应性差,不能很好的适应人体活动部位的运动趋势的技术问题,提供一种人源性生物组织材料。
本发明的第二目的在于,针对上述缺陷和不足,解决现有技术中缺乏改善生物组织材料顺应性的有效方法,提供一种塑形方法以提高顺应性、刺激分泌以及提高生物材料的弹性恢复能力。
本发明的第三目的在于,针对上述缺陷和不足,解决现有技术中缺少有效改善生物组织材料顺应性的装置,提供一种机械力学刺激下动态培养人源性生物组织材料的塑形装置。
为了实现上述目的,本发明采用的具体技术方案为:
本发明所述的提高人体植入处的顺应性的一种人源性生物组织材料,包括体外培养基体,所述体外培养基体包括形成于体外培养基体内部的多个可塑形腔,所述可塑形腔能够容纳待培养细胞分泌出的细胞外基质,培养过程中待培养细胞分泌细胞外基质进入可塑形腔,细胞外基质的增加对可塑形腔的形状产生塑形作用。
优选地,所述体外培养基体包括细胞培养层,所述细胞培养层形成能够容纳待培养细胞并用于为待培养细胞培养提供生长空间的第一可塑形腔。
优选地,所述体外培养基体还包括与细胞培养层相互结合的加强层,所述加强层形成小体积且密集排布的第二可塑形腔,所述加强层用于加强体外培养基体的结构强度。
优选地,所述细胞培养层和加强层通过静电纺丝或3D打印或发泡的方式相互结合。
优选地,所述细胞培养层和加强层均有高分子材料构成,所述高分子材料包括聚氨酯、聚四氟乙烯、膨化聚四氟乙烯、丝素蛋白、聚己内酯、聚乳酸、聚对苯二甲酸乙二醇酯、聚羟基乙酸、聚乳酸聚羟基乙酸、羧甲基纤维素、明胶、胶原、透明质酸、聚乙烯醇、聚丙烯酰胺、聚丙烯酸、聚乙烯吡咯烷酮或海洋动植物来源的高分子聚合物中的至少一种。
本发明另一目的提供一种塑形方法,提高顺应性、刺激分泌以及提高生物材料的弹性恢复能力,包括步骤:将待培养细胞置于所述可塑形腔,并对其施加不同方式的外力以刺激细胞分泌细胞外基质,形成“生物材料+塑型细胞+塑型ECM”的复合生物材料,并在不同的时间点赋予不同的力学刺激进行体外塑型;其中,外力施加方式以适于待植入人体部位运动趋势的方式设置。
优选地,所述外力施加方式包括以不同角度、方向和大小的拉伸、沿体外培养基体轴向的旋转、或沿体外培养基体长度方向的伸缩中的至少一种方式,既有针对细胞本身的力也有对ECM和对整个材料的力,力学刺激可以持续施加也可以间断交互收缩舒张。
在本发明的一个优选实施方案中,所述生物材料可以是细胞与生物材料相结合,也可以将生物材料脱细胞处理,去除细胞膜及细胞内成分。
优选地,所述人源性细胞分泌包括多种胶原蛋白、弹力蛋白、氨基聚糖、透明质酸、纤维粘连蛋白和层粘连蛋白等相互连接的细胞外基质;在体外经过力学塑形过的细胞外基质不需要交联剂,避免其对人体的毒性作用。
优选地,所述待培养细胞包括成纤维细胞、间充质干细胞、肝星状细胞、平滑肌细胞、脐带血干细胞,脂肪干细胞和肌细胞中的至少一种,可通过基因工程改造,使之分泌特定细胞外基质,如胶原、弹性蛋白、纤连蛋白、层粘连蛋白等。
本发明的另一目的为提供一种塑形装置,包括架体、用于为待培养细胞提供生长环境培养容器、和用于为体外培养基体提供刺激力的动力单元,所述培养容器和动力单元安装于架体,所述动力单元包括依次连接的动力装置、传动机构和连接结构,所述体外培养基体置于培养容器中,并与连接结构连接,通过连接结构的带动实现体外培养基体的运动。
优选的,连接培养容器(如10cm培养皿)的单个设备放置于培养箱中动态培养人源性生物组织材料,而进行大规模批量化生产;多个设备或者单个设备连接成培养容器组(如直径5m),将其放置于培养室(面积30m2)进行培养,培养箱和培养室都提供适合细胞粘附、生长、增殖和代谢的培养环境(如图8)。
本发明与现有技术相比,具有以下有益效果:
本发明提供了一种人源性生物组织材料,包括体外培养基体,所述体外培养基体包括细胞培养层,所述细胞培养层包括多个可容纳待培养细胞的可塑形腔,培养过程中待培养细胞置于可塑形腔并分泌细胞外基质,细胞外基质的增加对可塑形腔的形状产生塑形作用。相比于现有技术,本发明通过在可塑形腔内培养细胞,利用细胞分泌细胞外基质对可塑形腔进行塑形,可以根据不同的待植入生物组织材料的人体部位运动趋势,对可塑形腔塑形,以提高生物组织材料的顺应性,具有适于人体活动部位的效果。
在此基础上,本发明提供了一种人源性生物组织材料的塑形方法,将待培养细胞置于所述可塑形腔,并对其施加不同方式的外力以刺激细胞分泌细胞外基质;其中,外力施加方式以适于待植入人体部位运动趋势的方式设置。根据不同的待植入部位,选择不同的外力刺激方式对细胞进行力学刺激,在外力刺激下,细胞会相对更多的分泌细胞外基质,起到塑形作用。同时,外力刺激不仅具有促进细胞分泌细胞外基质的作用,而且在单位体积内通过外力刺激使得细胞外基质层更加紧实,使其具有较强的弹性恢复力,这种弹性恢复力尤其体现在,例如透析针头插入血管(穿刺人体血管的过程是穿刺针挤出一条路径而不破坏血管壁的结构)再拔出后,生物材料组织快速恢复,具有快速止血的效果。
此外,本发明提供了一种人源性生物组织材料的塑形装置,包括架体、用于为待培养细胞提供生长环境培养容器、和用于为体外培养基体提供刺激力的动力单元,所述培养容器和动力单元安装于架体,所述动力单元包括依次连接的动力装置、传动机构和连接结构,所述体外培养基体置于培养容器中,并与连接结构连接,通过连接结构的带动实现体外培养基体的运动。通过本发明提供的装置,可以实现拉伸、旋转、伸缩的外力方式对体外培养基体承载的细胞进行刺激,以适用于不同的待植入人体部位。
下面结合附图对本发明作进一步的说明。
附图说明
图1为本发明优选实施方式中复合型生物组织材料结构及显微图。
图2为本发明优选实施方式中细胞培养层充分浸润高糖DMEM培养基后固定于六孔板的图片。
图3为通过静电纺丝获得的致密高分子纳米纤维层(加强层)血管材料。
图4为本发明优选实施方式中未种植细胞并未包含细胞外基质的复合血管的图片。
图5为本发明优选实施方式中不同转速下肝星状细胞分泌ECM的含量变化数据图。
图6为本发明优选实施方式中一种塑形装置结构示意图。
图7为本发明优选实施方式中另一种塑形装置结构示意图。
图8为本发明培养室批量生产人源性生物组织材料示意图。
附图标记说明:
10体外培养基体、11加强层;
20细胞培养层、21可塑形腔、22第一可塑形腔、23第二可塑形腔;
30培养容器;
40动力单元、41动力装置、42传动机构、43连接结构;
50架体。
具体实施方式
下面通过具体实施方式对本发明做进一步的解释及说明,应当理解下面的实施方式的目的是为了使本发明的技术方案更加清楚、易于理解,并不限制权利要求的保护范围。
如图1所示,本发明提供了一种人源性生物组织材料,包括体外培养基体10,所述体外培养基体10包括形成于体外培养基体10内部的多个可塑形腔21,所述可塑形腔21能够容纳待培养细胞分泌出的细胞外基质,培养过程中待培养细胞分泌细胞外基质进入可塑形腔21,细胞外基质的增加对可塑形腔21的形状产生塑形作用。
在优选的实施方式中,所述体外培养基体10包括细胞培养层20,所述细胞培养层20形成能够容纳待培养细胞并用于为待培养细胞培养提供生长空间的第一可塑形腔22。
在优选的实施方式中,所述体外培养基体10还包括与细胞培养层20相互结合的加强层11,所述加强层11形成小体积且密集排布的第二可塑形腔23,所述加强层11用于加强体外培养基体10的结构强度。
在优选的实施方式中,所述细胞培养层20和加强层11通过静电纺丝或3D打印或发泡的方式相互结合。
在优选的实施方式中,所述细胞培养层20和加强层11均有高分子材料构成,所述高分子材料包括聚氨酯、聚四氟乙烯、膨化聚四氟乙烯、丝素蛋白、聚己内酯、聚乳酸、聚对苯二甲酸乙二醇酯、聚羟基乙酸、聚乳酸聚羟基乙酸、羧甲基纤维素、明胶、胶原、透明质酸、聚乙烯醇、聚丙烯酰胺、聚丙烯酸、聚乙烯吡咯烷酮或海洋动植物来源的高分子聚合物中的至少一种。
具体地,所述细胞培养层与加强层11通过静电纺丝或者3D打印相结合。
细胞培养层20与加强层11的结合方法包括以下步骤:
步骤1,多孔支架(即一种细胞培养层20)材质的制备:将高分子聚合原料配制成溶液,经静电纺丝或3D打印成型;
步骤2,致密高分子纳米纤维层(即一种加强层11)的制备:将高分子聚合原料配制成溶液,经静电纺丝或3D打印成型;
步骤3,二者结合:将上述脱细胞血管材料和致密高分子纳米纤维覆结构通过静电纺丝或者3D打印复合在一起。
本发明的一种培养方法,在细胞培养过程中,以不同方向的力刺激细胞分泌细胞外基质,这些力的方向是根据塑形的需要而进行的,以适于人体植入处运动趋势的,提高人体顺应性的方式塑形。这样的好处是:第一,可以根据人体植入处运动趋势对体外培养基体10进行适于运动趋势的塑形;第二,在力的刺激下,单位时间内可以分泌更多的细胞外基质;第三,在可塑形腔21空间范围的限制下,单位体积产生更多的外基质使得外基质更加紧实,其作用和效果是在打针后,例如透析针头插入血管(穿刺人体血管的过程是穿刺针挤出一条路径而不破坏血管壁的结构)再拔出后,外基质由于结构紧实,收缩性强,入针处可以快速恢复状态,有利于止血。
具体地,人源性细胞分泌包括多种胶原蛋白、弹力蛋白、氨基聚糖、透明质酸、纤维粘连蛋白和层粘连蛋白等相互连接的细胞外基质;在体外经过力学塑形过的细胞外基质不需要交联剂,避免其对人体的毒性作用。
具体地,人源性细胞包括成纤维细胞、间充质干细胞、肝星状细胞、平滑肌细胞、脐带血干细胞,脂肪干细胞和肌细胞中的至少一种,可通过基因工程改造,使之分泌特定细胞外基质,如胶原、弹性蛋白、纤连蛋白、层粘连蛋白等。
具体地,力学刺激的方式包括以不同角度的拉伸、沿轴向的旋转、以适于人体弯曲运动的方式弯曲、以及沿长度方向的伸缩。
如图6、7所示,本发明提供了一种人源性生物组织材料的塑形装置,包括架体50、用于为待培养细胞提供生长环境培养容器30、和用于为体外培养基体10提供刺激力的动力单元40,所述培养容器30和动力单元40安装于架体50,所述动力单元40包括依次连接的动力装置41、传动机构42和连接结构43,所述体外培养基体10置于培养容器30中,并与连接结构43连接,通过连接结构43的带动实现体外培养基体10的运动。
如图8所示,连接小型的培养容器(如10cm的培养皿为培养容器)的单个设备放置于培养箱中动态培养人源形生物组织材料,进而实现大规模批量化生产;多个设备或者单个设备连接成大型的培养容器(大型的培养容器为培养容器组,其直接可达5m),将其放置于培养室(培养室的面积为30㎡)进行培养,培养箱和培养室都提供适合细胞粘附、生长、增殖和代谢的培养环境,其中,培养环境的温度为37℃,二氧化碳的含量为5%。
下面通过具体实施例对本发明作进一步的说明。
实施例1
一种力学性能刺激下动态培养高分子材料生物复合食管的方法及设备。所述致密高分子纳米纤维层(即一种加强层11)和所述多孔支架(即一种细胞培养层20)的材质包括聚氨酯、聚四氟乙烯、膨化聚四氟乙烯、蚕丝蛋白(fibroin)、丝素蛋白(silk fibroin)、聚己内酯(PCL)、聚乳酸(PLA)、聚对苯二甲酸乙二醇酯、聚羟基乙酸(PGA)、聚乳酸-聚羟基乙酸(PLGA)、羧甲基淀粉、醋酸淀粉、壳聚糖(Chitosan)、羧甲基壳聚糖、海藻酸/海藻酸盐、羧甲基纤维素、明胶、胶原(Ⅰ、Ⅱ、Ⅲ、Ⅳ)、透明质酸(HA)、聚乙烯醇(PVA)、聚丙烯酰胺(PAM)、聚丙烯酸、聚乙烯吡咯烷酮(PVP)中的至少一种。
优选的:所述致密高分子纳米纤维层的厚度为5-200μm,其孔隙的直径为1-20μm,纤维直径为10-5000nm;所述多孔支架层的厚度为20-500μm;所述致密高分子纳米纤维层由静电纺丝或3D打印制备而成;所述多孔支架由发泡快速冷冻成型或3D打印制备而成。
上述静电纺丝采用配制一定浓度的静电纺丝原液,进行干法纺丝。静电纺丝原液由微量泵挤出,流速0.1-2mL/h,喷口接高压正极,接收装置接地,接收器的转速为10-1000r.p.m,静电纺丝电压10-30kV,喷口距离接收装置5-30cm,喷口直径0.1-1mm,环境温度10-30℃,环境湿度20-80%,收集纳米纤维管;
上述发泡快速冷冻成型具体为:配制一定浓度的发泡液,使用一定模具冻成管状结构,冷冻干燥后获得单独的多孔支架管状物,采用一定方法使之与静电纺丝制备的内膜两者紧密结合;或者将发泡液均匀涂抹在上述致密高分子纳米纤维层上,于一定温度下预冷冻一段时间,持续低温抽真空一段时间,后于一定温度真空干燥一段时间,即成。
所述设备包括培养容器30以及附着的动力单元40,前者包括圆柱形培养瓶,内含固定体外培养基体10的连接结构43;后者控制力学的方向、大小以及不同力学模式,既有针对细胞本身的力,也有对ECM刺激的力和对整个材料的力。
如图6所示,所述人源性细胞包括成纤维细胞、间充质干细胞、肝星状细胞和平滑肌细胞中的至少一种,将细胞培养在上述细胞培养层20内。体外培养基体10根据所需食管的具体情况而确定不同的大小长度和内外径。将人源性细胞种植于含高糖DMEM培养基的体外培养基体10,并将体外培养基体10固定于圆柱形培养器30,在37℃、5%CO2条件下培养,细胞贴壁增殖约3天后,给予围绕圆柱形培养器30轴心旋转的力学刺激,并逐渐加大。3-5天25rpm,5-7天75rpm,7-9天以后加至250rpm,同时让培养器来回运动(约2秒1个来回)。
实施例2
一种机械力学刺激下动态培养细胞外基质高分子材料生物复合补片的方法及设备。
本实施例制备的细胞外基质高分子材料生物复合补片的外观如图1所示,其制备方法具体如下:
(1)将PCL和明胶溶解于六氟异丙醇中,获得静电纺丝原液,其具体参数如下表1所示:
表1静电纺丝溶液配制参数
(2)将上述静电纺丝原液进行静电纺丝,获得如图2所示的致密高分子纳米纤维层;静电纺丝的具体参数如下表2所示:
表2静电纺丝参数控制
(3)在步骤(2)制得的致密高分子纳米纤维层的上表面均匀涂抹制备好的发泡液,于-80℃进行预冷冻处理,再于-4℃进行冷冻真空干燥24h,获得多孔支架;上述发泡液的组成和制备参数如下表3和表4所示:
表3发泡液配制参数
表4发泡液制备参数
所获得的生物材料补片由手术时紧贴加强层11和覆盖在该致密高分子纳米纤维层之上的细胞培养层20组成,其中不含有具有免疫原性的细胞成分,允许上述人源性细胞粘附、生长和繁殖于该细胞培养层20内。在上述细胞培养层20上进行细胞培养,分泌细胞外基质,形成“生物材料+塑型细胞+塑形ECM”的复合生物材料,并在不同的时间点赋予不同的力学刺激进行体外塑形。通过调节力学的方向、大小、角度,以及不同力学模式的互相结合进行动态培养,制备不同外形和结构的生物材料。
所述设备包括细胞培养部分以及附着的机械力学部分,前者包括立方体多层培养皿,内含固定生物材料的装置;后者控制力学的方向、大小以及不同力学模式,既有针对细胞本身的力,也有对ECM刺激的力和对整个材料的力。
(4)如图7所示,将上述细胞培养层20充分浸润高糖DMEM培养基后固定于上述设备的直径10cm圆盘型细胞培养皿(即培养容器30的下位概念)中,植入人源性肝星状细胞,37℃、5%CO2培养,约3天后细胞铺满支架形成单层。从第3天开始,持续对不同单层的细胞施加不同方向的力,其中一层细胞培养层20施加直向力10N,另外一层施加横向力10N,其他层施加不同角度(分别向左45°和向右45°)的斜向力10N,分别对应腹直肌,腹横肌,腹外斜肌和腹内斜肌(本发明中细胞培养层20本身就是可以视为多层结构)。第6天将不同单层结合,置于同一层继续培养3天。
(5)采用酶消化法将细胞消化,PBS清洗,于-80℃预冷冻,于-4℃真空干燥,获得所述细胞外基质高分子材料生物复合补片。
实施例3
一种力学性能刺激下动态培养高分子材料生物复合血管的方法及装置。
(1)将PCL和明胶溶解于六氟异丙醇中,获得静电纺丝原液,其具体参数如下表1所示:
表1静电纺丝溶液配制参数
(2)将上述静电纺丝原液进行静电纺丝,获得致密高分子纳米纤维层,如图4,其中A为未弯曲状态,B为弯曲状态,可见在弯曲状态仍然维持管状结构,
静电纺丝的具体参数如下表3所示:
表3静电纺丝参数控制
(3)在步骤(2)制得的致密高分子纳米纤维层的外表面均匀涂抹制备好的发泡液(聚氨酯、聚四氟乙烯、膨化聚四氟乙烯、蚕丝蛋白(fibroin)、丝素蛋白(silk fibroin)、聚己内酯(PCL)、聚乳酸(PLA)、聚对苯二甲酸乙二醇酯、聚羟基乙酸(PGA)、聚乳酸-聚羟基乙酸(PLGA)、羧甲基淀粉、醋酸淀粉、壳聚糖(Chitosan)、羧甲基壳聚糖、海藻酸/海藻酸盐、羧甲基纤维素、明胶、胶原(Ⅰ、Ⅱ、Ⅲ、Ⅳ)、透明质酸(HA)、聚乙烯醇(PVA)、聚丙烯酰胺(PAM)、聚丙烯酸、聚乙烯吡咯烷酮(PVP)中的至少一种),于液氮进行冷冻定型,再于-4℃进行冷冻真空干燥24h,获得多孔支架(未种植细胞并未包含细胞外基质的复合血管的照片如图3所示);
所述设备包括培养容器30以及附着的动力单元40,前者包括圆柱形培养器30,内含固定生物材料的装置;后者控制力学的方向、大小以及不同力学模式,既有针对细胞本身的力,也有对ECM刺激的力和对整个材料的力。
(4)如图6所示,将肝星状细胞培养在上述细胞培养层20内。体外培养基体10根据所需血管的部位而确定不同的大小长度和内外径。将人源性细胞种植于含高糖DMEM培养基的体外培养基体10,并将体外培养基体10固定于圆柱形培养器30,在37℃、5%CO2条件下培养,细胞贴壁增殖约3天后,给予围绕圆柱轴心旋转的力学刺激,并逐渐加大。4-7天50rpm,7-10天100rpm,10-15天以后加至200rpm。
(5)采用酶消化法将细胞消化,PBS清洗,于-80℃预冷冻,于-4℃真空干燥,获得所述细胞外基质高分子材料生物复合血管。此血管壁内成分有回弹性能,可用于透析,被反复穿刺。穿刺针穿刺人体血管的过程是穿刺针挤出的一条路径,而不是破坏血管壁的结构。血管穿刺针拔出后,穿刺途径的血管壁成分可弹性回缩,轻度压迫止血即可。
本发明是通过实施例来描述的,但并不对本发明构成限制,参照本发明的描述,所公开的实施例的其他变化,如对于本领域的专业人士是容易想到的,这样的变化应该属于本发明权利要求限定的范围之内。
Claims (10)
1.一种人源性生物组织材料,其特征在于:
包括体外培养基体(10),所述体外培养基体(10)包括形成于体外培养基体(10)内部的多个可塑形腔(21),所述可塑形腔(21)能够容纳待培养细胞分泌出的细胞外基质;培养过程中待培养细胞分泌细胞外基质进入可塑形腔(21),细胞外基质的增加对可塑形腔(21)的形状产生塑形作用。
2.根据权利要求1所述的人源性生物组织材料,其特征在于:
所述体外培养基体(10)包括细胞培养层(20),所述细胞培养层(20)形成能够容纳待培养细胞并用于为待培养细胞培养提供生长空间的第一可塑形腔(22)。
3.根据权利要求2所述的人源性生物组织材料,其特征在于:
所述体外培养基体(10)还包括与细胞培养层(20)相互结合的加强层(11),所述加强层(11)形成小体积且密集排布的第二可塑形腔(23),所述加强层(11)用于加强体外培养基体(10)的结构强度。
4.根据权利要求3所述的人源性生物组织材料,其特征在于:
所述细胞培养层(20)和加强层(11)通过静电纺丝或3D打印或发泡的方式相互结合。
5.根据权利要求3所述的人源性生物组织材料,其特征在于:
所述细胞培养层(20)和加强层(11)均有高分子材料构成,所述高分子材料包括聚氨酯、聚四氟乙烯、膨化聚四氟乙烯、丝素蛋白、聚己内酯、聚乳酸、聚对苯二甲酸乙二醇酯、聚羟基乙酸、聚乳酸聚羟基乙酸、羧甲基纤维素、明胶、胶原、透明质酸、聚乙烯醇、聚丙烯酰胺、聚丙烯酸、聚乙烯吡咯烷酮或海洋动植物来源的高分子聚合物中的至少一种。
6.根据权利要求1-5任一项所述的人源性生物组织材料的塑形方法,其特征在于:
将待培养细胞置于所述可塑形腔(21),并对其施加不同方式的外力以刺激细胞分泌细胞外基质;其中,外力施加方式以适于待植入人体部位运动趋势的方式设置。
7.根据权利要求6所述的人源性生物组织材料的塑形方法,其特征在于:
所述外力施加方式包括以不同角度、方向和大小的拉伸、沿体外培养基体轴向的旋转、弯曲或沿体外培养基体长度方向的伸缩中的至少一种方式,既有针对细胞本身的力也有对ECM和对整个材料的力,力学刺激可以持续施加也可以间断交互收缩舒张。
8.根据权利要求6所述的人源性生物组织材料的塑形方法,其特征在于:
所述待培养细胞包括成纤维细胞、间充质干细胞、肝星状细胞、平滑肌细胞、脐带血干细胞,脂肪干细胞和肌细胞中的至少一种,可通过基因工程改造,使之分泌特定细胞外基质,如胶原、弹性蛋白、纤连蛋白、层粘连蛋白等。
9.根据权利要求1-5任一项所述的人源性生物组织材料的塑形装置,其特征在于:
包括架体(50)、用于为待培养细胞提供生长环境培养容器(30)、和用于为体外培养基体(10)提供刺激力的动力单元(40),
所述培养容器(30)和动力单元(40)安装于架体(50),
所述动力单元(40)包括依次连接的动力装置(41)、传动机构(42)和连接结构(43),
所述体外培养基体(10)置于培养容器(30)中,并与连接结构(43)连接,通过连接结构(43)的带动实现体外培养基体(10)的运动。
10.根据权利要求9所述的一种人源性生物组织材料的塑形装置,其特征在于:
连接培养容器的单个设备放置于培养箱中动态培养人源性生物组织材料,而进行批量化生产,多个设备或者单个设备连接成培养容器组,将其放置于培养室进行培养,培养箱和培养室都提供适合细胞粘附、生长、增殖和代谢的培养环境。
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CN115721781A (zh) * | 2022-09-09 | 2023-03-03 | 哈尔滨工业大学(深圳) | 一种兼具细胞密度和机械强度的人工肌腱的制备工艺 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2008072967A (ja) * | 2006-09-21 | 2008-04-03 | Setsunan Univ | 骨格筋の培養用足場、同培養装置及び骨格筋の形成方法 |
CN109758614A (zh) * | 2018-12-17 | 2019-05-17 | 太阳雨林(厦门)生物医药有限公司 | 一种细胞外基质高分子材料生物复合补片 |
CN111603611A (zh) * | 2020-03-06 | 2020-09-01 | 广东省人民医院(广东省医学科学院) | 一种细胞衍生基质管状支架及其制备方法 |
-
2021
- 2021-11-25 CN CN202111411688.9A patent/CN114099781A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2008072967A (ja) * | 2006-09-21 | 2008-04-03 | Setsunan Univ | 骨格筋の培養用足場、同培養装置及び骨格筋の形成方法 |
CN109758614A (zh) * | 2018-12-17 | 2019-05-17 | 太阳雨林(厦门)生物医药有限公司 | 一种细胞外基质高分子材料生物复合补片 |
CN111603611A (zh) * | 2020-03-06 | 2020-09-01 | 广东省人民医院(广东省医学科学院) | 一种细胞衍生基质管状支架及其制备方法 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115721781A (zh) * | 2022-09-09 | 2023-03-03 | 哈尔滨工业大学(深圳) | 一种兼具细胞密度和机械强度的人工肌腱的制备工艺 |
CN115721781B (zh) * | 2022-09-09 | 2023-11-07 | 哈尔滨工业大学(深圳) | 一种兼具细胞密度和机械强度的人工肌腱的制备工艺 |
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