CN115262028A - 一种仿生载种子细胞多组分纤维的制备方法及应用 - Google Patents
一种仿生载种子细胞多组分纤维的制备方法及应用 Download PDFInfo
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Abstract
本发明提供一种仿生载种子细胞多组分纤维的制备方法，包括以下步骤：S1、1)拉制并组装具有尖口端的I管；2)拉制一根具有尖口端的Ⅱ管；3)拉制内径大于I管和Ⅱ管的Ⅲ管；将I管和Ⅱ管分别从Ⅲ管的两个开口插入，I管的尖口端插入Ⅱ管的非尖口端，调节I管、Ⅱ管、Ⅲ管的轴心重合并固定住位置；S2、拉制具有尖口端的Ⅳ管；将Ⅳ管的尖口端与I管的尖口端同轴嵌套；在I管、Ⅲ管、Ⅳ管外分别安装针头；S3、1)定义各溶液通道并配置溶液；向外向溶液通道中通入去离子水，然后依次对应通入PVA溶液、海藻酸钠溶液、氯化钙水溶液，逐渐减小去离子水的流速直至完全关闭，得到海藻酸钙纤维；使用氯化钙溶液收集海藻酸钙纤维。
Description
技术领域
本发明涉及生物医学材料领域，具体涉及一种仿生载种子细胞多组分纤维的制备方法及应用。
背景技术
我国为肝病大国，各种原因引起的肝功能衰竭死亡率高达80％，是目前临床一大难题。肝移植是治疗重症肝功能衰竭的最为有效的手段，但供肝不足的问题已严重影响了肝移植的广泛开展。寻求一种安全、有效的肝功能衰竭的治疗方法，是目前医学的研究热点和亟待解决的重大问题。
近年来，生物人工肝的出现为解决这一难题带来了希望。生物人工肝结合了生物学和工程学，其原理是通过建立一个细胞与生物材料的三维空间复合体作为生物反应器，模拟正常的肝脏功能，在体外进行血液循环，起到对肝功能衰竭患者进行治疗的目的。该疗法有望成为继肝移植后，治疗急、慢性肝功能衰竭、终末期肝病和代谢障碍性疾病的替代治疗手段，具有广阔的临床应用前景。
众所周知，体外培养的肝细胞之所以很难存活，很大程度上是由于脱离了体内的微环境。因此，要想维持细胞的活性和功能，非常重要的一点就是要尽可能地模拟细胞在体内的微环境，这也是目前构建组织工程肝脏的一个共识和努力方向。如何模拟肝脏天然的肝小叶结构来实现完美的物质输送和功能发挥是一个亟待解决的问题，这就提示我们要从肝小叶管道结构方面来模拟肝脏的微环境。微流控技术创造了一条通向制备功能性材料的令人兴奋的途径。微流控技术是一门将微量液体约束在微尺寸的微通道中并对其进行精确控制及操作的技术。因此，在功能性微纤维制备方面，微流控技术被寄子厚望。
因此，本发明提供一种基于微流控技术的仿生载种子细胞多组分纤维的制备方法。
发明内容
本发明的目的在于提供一种仿生载种子细胞多组分纤维制备方法，以促进种子细胞的增殖和黏附。
为实现上述目的，本发明采用以下技术方案：
一种仿生载种子细胞多组分纤维的制备方法，包括以下步骤：
S1、搭建多组分纤维中空微流控装置
1)拉制6根一端具有尖口端的玻璃毛细管，将尖口端在砂纸上轻轻打磨，直至尖口端平整光滑，将其尖口端用速干胶环绕同一轴心同向固定在一起，得到具有尖口端的I管；
2)拉制一根内径大于I管尖口端端口的玻璃毛细管，将其任一端口在砂纸上轻轻打磨直至端口平整光滑，得到Ⅱ管；
3)将一根尺寸大于I管和Ⅱ管的玻璃毛细管固定在玻璃衬底上，得到Ⅲ管；将I管和Ⅱ管分别从Ⅲ管的两个开口插入，且I管的尖口端部分插入Ⅱ管被砂纸打磨过的一端中，调节I管、Ⅱ管、Ⅲ管的轴心重合后，使用速干胶固定住I管、Ⅱ管、Ⅲ管的位置；
S2、搭建多组分中空纤维装置
拉制一根具有尖口端的玻璃毛细管，得到Ⅳ管；将Ⅳ管的尖口端与I管的尖口端同轴嵌套后固定；在I管、Ⅲ管、Ⅳ管外分别安装针头并用速干胶固定，得到多组分中空纤维装置；
S3、制备纤维
1)以Ⅳ管为内向溶液通道、I管为中间相溶液通道、Ⅱ管和Ⅲ管之间的间隙为外向溶液通道，以PVA溶液为内向溶液、搭载种子细胞的海藻酸钠溶液为中间相溶液、氯化钙水溶液为外向溶液；
2)向外向溶液通道中通入去离子水，待去离子水流相稳定后，依次向内向溶液通道、中间相溶液通道、外向溶液通道中分别通入对应的PVA溶液、搭载种子细胞的海藻酸钠溶液、氯化钙水溶液，并逐渐减小去离子水的流速直至完全关闭，制备得到海藻酸钙纤维；使用氯化钙溶液收集制备得到的海藻酸钙纤维后，撤除多组分中空纤维装置。
当装置内出现流体紊乱或堵塞现象时，迅速停止通入氯化钙，通入并增大去离子水流流速至10mL/h以上。
为优化上述技术方案，采取的具体措施还包括：
进一步地，步骤S1中，I管、Ⅱ管制备完成后置于乙醇中超声清洗5分钟，氮气吹干。
进一步地，步骤S1中，制备得到的Ⅱ管浸没于含有5％十八烷基三甲氧基硅烷的丙酮溶液中进行疏水处理。
进一步地，步骤S2中，针头通过PE管和I管、Ⅲ管、Ⅳ管连通。
进一步地，步骤S3中，PVA溶液的浓度为10wt％、海藻酸钠溶液的浓度为2wt％、氯化钙水溶液的浓度为1.5wt％。
进一步地，步骤S3中，通入I管的6根玻璃毛细管中的海藻酸钠溶液中交替掺有红色和绿色的荧光纳米离子。
进一步地，步骤S3中，内向溶液、中间相溶液、外向溶液的流速分别为0.5mL/h、3mL/h、10mL/h。
进一步地，步骤S3中，多组分中空纤维装置撤除的方式为：先停止通入氯化钙水溶液，然后向外向溶液通道中通入去离子水，之后停止通入海藻酸钠溶液，在I管出口不再有海藻酸钠溶液流出后，停止通入去离子水，最后拆除PE管和针头，使用去离子水清洗各溶液通道。
进一步地，步骤S3中，PVA溶液、搭载种子细胞的氯化钙水溶液、海藻酸钠溶液分别用1mL、10mL和25mL的SGE精密注射器去除气泡后注射进内向溶液通道、外向溶液通道、中间相溶液通道。
通过上述任一项制备方法得到的仿生载种子细胞多组分纤维在制备生物人工肝中的应用。
本发明的有益效果是：
本发明通过拉制、组装若干玻璃毛细管并通过PE管安装针头，搭建了一种便于精确控制和操作的具有多相溶液通道的多组分中空纤维装置；分别向内向溶液通道、中间相溶液通道、外向溶液通道中通入PVA溶液、搭载种子细胞的海藻酸钠溶液、氯化钙水溶液，依赖于能够快速形成超细纤维凝胶的海藻酸钠以及氯化钙溶液的反应，促进种子细胞的增殖和黏附，通过调节三者溶液的流速制备了仿生载种子细胞多组分纤维和仿生载种子细胞多组分中空纤维；制备得到的仿生载种子细胞多组分(中空)纤维具有较高的生物相容性，能在一段时间内维持肝细胞的形态和功能；还与天然肝小叶结构相似，且多组分中空纤维具有仿生胆小管结构，有利于实现的物质输送和功能发挥。以上优点证明了本发明制备的纤维在制备生物人工肝中具有潜在的应用价值。
附图说明
图1为多组分中空纤维装置结构示意图；
图2为多组分中空纤维装置的实物图。
图3为制备的多组分纤维的横截面和纵截面荧光显微镜图像，(a)为横截面，(b)为纵截面；
图4为制备的多组分中空纤维的横截面荧光显微镜图像；
图5为制备德生物人工肝用仿生载种子细胞多组分纤维的扫描电镜图像；
图6为制备的生物人工肝用仿生载种子细胞多组分纤维培养后的活死染色的荧光图像；
图7为仿生载种子细胞多组分纤维与仿生载种子细胞多组分中空纤维共培养后白蛋白与尿素分泌示意图。
具体实施方式
实施例1制备多组分中空纤维
多组分中空纤维通过以下方法制备，具体包括以下步骤：
S1、搭建多组分纤维中空微流控装置
1)如图1所示，拉制6根一端具有尖口端的玻璃毛细管，将尖口端在砂纸上轻轻打磨，直至尖口端平整光滑，将其尖口端用速干胶环绕同一轴心同向固定在一起，得到具有尖口端的I管；
2)拉制一根内径大于I管尖口端端口的玻璃毛细管，将其任一端口在砂纸上轻轻打磨直至端口平整光滑，得到Ⅱ管；I管、Ⅱ管制备完成后置于乙醇中超声清洗5分钟，氮气吹干，制备得到的Ⅱ管浸没于含有5％十八烷基三甲氧基硅烷的丙酮溶液中进行疏水处理；
3)将一根尺寸大于I管和Ⅱ管的玻璃毛细管固定在玻璃衬底上，得到Ⅲ管；将I管和Ⅱ管分别从Ⅲ管的两个开口插入，且I管的尖口端部分插入Ⅱ管被砂纸打磨过的一端中，调节I管、Ⅱ管、Ⅲ管的轴心重合后，使用速干胶固定住I管、Ⅱ管、Ⅲ管的位置；
S2、搭建多组分中空纤维装置
拉制一根具有尖口端的玻璃毛细管，得到Ⅳ管；将Ⅳ管的尖口端与I管的尖口端同轴嵌套；如图2所示，在I管、Ⅲ管、Ⅳ管外分别通过PE管安装针头并用速干胶固定，得到多组分中空纤维装置；
S3、制备纤维
1)以Ⅳ管为内向溶液通道、I管为中间相溶液通道、Ⅱ管和Ⅲ管之间的间隙为外向溶液通道，以10wt％的PVA溶液为内向溶液、2wt％的交替掺有红色和绿色的荧光纳米离子的搭载种子细胞的海藻酸钠溶液为中间相溶液、1.5wt％的氯化钙水溶液为外向溶液；
2)分别用1mL、10mL和25mL的SGE精密注射器吸取上一步制备的PVA溶液、氯化钙水溶液、搭载种子细胞的海藻酸钠溶液，然后将注射器放置于蠕动泵的卡槽内，设置好型号、量程、流速等参数。首先向外向溶液通道中通入流速为10mL/h的去离子水，待去离子水流体稳定后，以0.5mL/h的流速开始通入海藻酸钠溶液，半分钟后，内向溶液通道的出口处出现细长且连续的线状喷射流，随着喷射流逐渐远离内向溶液通道的出口，喷射流的口径逐渐增大；待喷射流稳定后，以3mL/h的流速开始通入氯化钙水溶液，数分钟后逐渐增大氯化钙水溶液的流速，同时减小去离子水的流速，保证线状喷射流一直稳定地流出，直至去离子水不再通入，此时线状喷射流完全转化为凝胶状的海藻酸钙纤维，在氯化钙水溶液的驱动下沿着出口通道流动；使用氯化钙溶液收集制备得到的海藻酸钙纤维；
3)制备结束后，先停止通入氯化钙水溶液，然后向外向溶液通道中通入去离子水，之后停止通入海藻酸钠溶液，在I管出口不再有海藻酸钠溶液流出后，停止通入去离子水，最后拆除PE管和针头，使用去离子水清洗各溶液通道。
实施例2形态和相容性测试
称取海藻酸钠粉末置于超净台中的紫外灭菌灯下过夜照射后用灭菌水溶解配置，用胰蛋白酶将其从培养瓶壁消化下来，转移至离心管中离心，去除胰蛋白酶及旧的培养液加入到无菌海藻酸钠溶液，用移液枪轻轻吹打细胞直至完全悬浮且均匀分散在海藻酸钠溶液中，然后按照实施例1的方法制备负载种子细胞的多组分纤维和多组分中空纤维(区别在于是否通入PVA溶液)。
海藻酸钠溶液中交替掺有红色和绿色的荧光纳米离子，用来表征不同的组分。图3为制备的多组分纤维的横截面和纵截面荧光显微镜图像，图4为制备的多组分中空纤维的横截面荧光显微镜图，图5为多组分(中空)纤维的扫描电镜图像。
如图6所示，为了检测细胞在纤维中的生命活性，用Calcein AM和碘化丙啶(PI)对活细胞和死细胞同时进行染色。纤维被染色后大多数细胞呈现绿色，没有细胞被染成红色，说明细胞维持良好的活性，微流控制备的纤维对细胞的损害很小。
实施例3仿生载种子细胞多组分纤维在制备生物人工肝中的应用
按照实施例1提供的方法制备多组分纤维、多组分中空纤维，在相同的实验条件下，保证上述纤维内包裹的细胞量一致，分别用白蛋白试剂盒和尿素试剂盒测量纤维中HepG2白蛋白分泌水平和尿素合成值这两个重要指标，定量地考察HepG2细胞在具有不同细胞分布结构的三维共培养环境下功能的表达情况。
如图7所示，HepG2的白蛋白分泌水平和尿素合成值都呈增长的趋势。PVA溶液的引入，提高了海藻酸钙纤维的生物活性，为细胞生长提供了更好的环境，进而促进了功能的表达。
以上仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，应视为本发明的保护范围。
Claims (10)
1.一种仿生载种子细胞多组分纤维的制备方法，其特征在于，包括以下步骤：
S1、搭建多组分纤维中空微流控装置
1)拉制6根一端具有尖口端的玻璃毛细管，将其尖口端环绕同一轴心同向固定在一起，得到具有尖口端的I管；
2)拉制一根内径大于I管尖口端端口的玻璃毛细管，得到Ⅱ管；
3)将一根尺寸大于I管和Ⅱ管的玻璃毛细管固定在玻璃衬底上，得到Ⅲ管；将I管和Ⅱ管分别从Ⅲ管的两个开口插入，且I管的尖口端部分插入Ⅱ管中，调节I管、Ⅱ管、Ⅲ管的轴心重合后，固定住I管、Ⅱ管、Ⅲ管的位置；
S2、搭建多组分中空纤维装置
拉制一根具有尖口端的玻璃毛细管，得到Ⅳ管；将Ⅳ管的尖口端与I管的尖口端同轴嵌套后固定；在I管、Ⅲ管、Ⅳ管外分别安装针头，得到多组分中空纤维装置；
S3、制备纤维
1)以Ⅳ管为内向溶液通道、I管为中间相溶液通道、Ⅱ管和Ⅲ管之间的间隙为外向溶液通道，以PVA溶液为内向溶液、搭载种子细胞的海藻酸钠溶液为中间相溶液、氯化钙水溶液为外向溶液；
2)向外向溶液通道中通入去离子水，待去离子水流相稳定后，依次向内向溶液通道、中间相溶液通道、外向溶液通道中分别通入对应的PVA溶液、搭载种子细胞的海藻酸钠溶液、氯化钙水溶液，并逐渐减小去离子水的流速直至完全关闭，制备得到海藻酸钙纤维；使用氯化钙溶液收集制备得到的海藻酸钙纤维后，撤除多组分中空纤维装置。
2.根据权利要求1所述的一种仿生载种子细胞多组分纤维的制备方法，其特征在于，
步骤S1中，I管、Ⅱ管制备完成后置于乙醇中超声清洗5分钟，氮气吹干。
3.根据权利要求1所述的一种仿生载种子细胞多组分纤维的制备方法，其特征在于，
步骤S1中，制备得到的Ⅱ管浸没于含有5％十八烷基三甲氧基硅烷的丙酮溶液中进行疏水处理。
4.根据权利要求1所述的一种仿生载种子细胞多组分纤维的制备方法，其特征在于，
步骤S2中，针头通过PE管和I管、Ⅲ管、Ⅳ管连通。
5.根据权利要求1所述的一种仿生载种子细胞多组分纤维的制备方法，其特征在于，
步骤S3中，PVA溶液的浓度为10wt％、海藻酸钠溶液的浓度为2wt％、氯化钙水溶液的浓度为1.5wt％。
6.根据权利要求1所述的一种仿生载种子细胞多组分纤维的制备方法，其特征在于，
步骤S3中，通入I管的6根玻璃毛细管中的海藻酸钠溶液中交替掺有红色和绿色的荧光纳米离子。
7.根据权利要求1所述的一种仿生载种子细胞多组分纤维的制备方法，其特征在于，
步骤S3中，内向溶液、中间相溶液、外向溶液的流速分别为0.5mL/h、3mL/h、10mL/h。
8.根据权利要求4所述的一种仿生载种子细胞多组分纤维的制备方法，其特征在于，
步骤S3中，多组分中空纤维装置撤除的方式为：先停止通入氯化钙水溶液，然后向外向溶液通道中通入去离子水，之后停止通入海藻酸钠溶液，在I管出口不再有海藻酸钠溶液流出后，停止通入去离子水，最后拆除PE管和针头，使用去离子水清洗各溶液通道。
9.根据权利要求1所述的一种仿生载种子细胞多组分纤维的制备方法，其特征在于，
步骤S3中，PVA溶液、氯化钙水溶液、搭载种子细胞的海藻酸钠溶液分别用1mL、10mL和25mL的SGE精密注射器去除气泡后注射进内向溶液通道、外向溶液通道、中间相溶液通道。
10.如权利要求1～9任一项制备方法得到的仿生载种子细胞多组分纤维在制备生物人工肝中的应用。
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| JP6332782B2 (ja) * | 2013-06-07 | 2018-05-30 | 国立大学法人 鹿児島大学 | 細胞凝集塊作製法 |
| CN103820880B (zh) * | 2014-01-21 | 2016-04-20 | 东南大学 | 一种海藻酸钙纤维及其制备方法 |
| JP6439918B2 (ja) * | 2014-10-17 | 2018-12-19 | 国立大学法人 東京大学 | ３次元細胞構造体の製造方法 |
| CN106215987B (zh) * | 2016-08-12 | 2018-09-28 | 四川大学 | 多通道并流微流体芯片及基于该芯片的线性多相异质结构纤维的可控纺丝方法 |
| CN108360088B (zh) * | 2018-02-28 | 2020-03-03 | 清华大学深圳研究生院 | 制备海藻酸钙纤维的方法和装置 |
| EP3835409A4 (en) * | 2018-08-10 | 2022-05-04 | Mochida Pharmaceutical Co., Ltd. | HOLLOW ALGINATE MICROFIBER |
| CN112921436B (zh) * | 2021-03-08 | 2023-04-07 | 南京鼓楼医院 | 一种包裹钙钛矿量子点的纤维、制备方法及装置 |
| CN114457442B (zh) * | 2022-01-19 | 2022-12-06 | 西南交通大学 | 具有集水特性的仿蛛丝中空纺锤节微纤维装置及制备方法 |
| CN114790441A (zh) * | 2022-05-07 | 2022-07-26 | 上海大学 | 一种基于中空微纤维的器官芯片的制备方法及器官芯片 |
| CN114854677B (zh) * | 2022-07-04 | 2022-11-04 | 南京农业大学 | 一种用于细胞培养肉生产的微流控仿生纤维及其制备方法和应用 |
| CN115262028A (zh) * | 2022-09-13 | 2022-11-01 | 南京鼓楼医院 | 一种仿生载种子细胞多组分纤维的制备方法及应用 |
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2022
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- 2023-09-07 LU LU506337A patent/LU506337B1/en active IP Right Grant
- 2023-09-07 WO PCT/CN2023/117476 patent/WO2024055893A1/zh unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024055893A1 (zh) * | 2022-09-13 | 2024-03-21 | 南京鼓楼医院 | 一种仿生载种子细胞多组分纤维的制备方法及应用 |
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| WO2024055893A1 (zh) | 2024-03-21 |
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