CN114533961B - 一种3d打印负载干细胞外泌体的气管支架的制备方法 - Google Patents

一种3d打印负载干细胞外泌体的气管支架的制备方法 Download PDF

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CN114533961B
CN114533961B CN202210184091.3A CN202210184091A CN114533961B CN 114533961 B CN114533961 B CN 114533961B CN 202210184091 A CN202210184091 A CN 202210184091A CN 114533961 B CN114533961 B CN 114533961B
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史宏灿
沈志明
卢丹
孙飞
单一波
卢毅
袁磊
朱剑炜
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Abstract

本发明涉及一种3D打印负载干细胞外泌体的气管支架的制备方法，包括以下步骤：步骤（1）、骨髓间充质干细胞外泌体提取及鉴定；步骤（2）、3D打印PCL气管支架的制备；步骤（3）、光固化水凝胶负载干细胞外泌体；步骤（4）、负载干细胞外泌体复合气管支架的制备；将步骤（2）打印得到的PCL支架浸泡在负载干细胞外泌体的SilMA溶液Exo‑SilMA中，37℃下浸泡30分钟，之后使用蓝光灯交联5分钟，使Exo‑SilMA均匀包裹PCL支架。通过本发明，PCL支架可以提供良好的力学性能，而SilMA具有良好生物相容性、生物降解性、溶胀作用，能够富集促进组织再生的生物成分，间充质干细胞外泌体作为干细胞衍生物，能够发挥干细胞的修复功能，且避免了移植干细胞的致瘤、排斥等风险。

Description

一种3D打印负载干细胞外泌体的气管支架的制备方法
技术领域
本发明涉及一种3D打印负载干细胞外泌体的气管支架的制备方法，属于气管组织工程支架技术领域。
背景技术
3D生物打印是一种能够生成复杂度类似组织且可精确控制的3D组织构建物的新型技术，它实质上是采用3D打印技术逐层构建材料并生成3D组织构建物的增材制造过程。外泌体是细胞分泌的纳米级脂质体囊泡，其直径在 30‐200nm范围内，其包裹了干细胞的重要功能物质如蛋白质、mRNAs 以及micro RNAs等。移植外泌体既可发挥干细胞的功能又可以避免直接移植干细胞的风险，如致瘤风险、伦理、免疫排异反应、栓塞等，因此，其可能成为组织工程领域中的一项具有前景的生物材料。目前还未有将干细胞外泌体应用于气管支架中的发明，因此，本发明利用3D打印技术，结合光固化水凝胶和聚己内酯（PCL）材料，负载骨髓间充质干细胞来源的外泌体，制备一种新型的复合气管支架，从而构建更加合理有效的气管替代物。
发明内容
本发明的目的针对现有技术中存在的不足，将骨髓间充质干细胞外泌体与气管支架有机结合，构建新型负载干细胞外泌体气管支架，发挥干细胞外泌体促气管组织修复再生的作用，从而提供一种3D打印负载干细胞外泌体的气管支架的制备方法。
本发明是通过以下技术方案实现：一种3D打印负载干细胞外泌体的气管支架的制备方法，其特征在于，包括以下步骤：
步骤（1）、骨髓间充质干细胞外泌体提取及鉴定；
收集骨髓间充质干细胞培养基的上清液，采用超速离心法提取干细胞外泌体，PBS溶解后，使用0.22μm滤膜过滤除菌，于-80℃保存；应用透射电子显微镜，鉴定提取的外泌体，观察干细胞外泌体的形态并采图；
步骤（2）、3D打印PCL气管支架的制备；
将PCL材料加入至3D打印机打印PCL支架；
步骤（3）、光固化水凝胶负载干细胞外泌体；
选择光固化水凝胶作为外泌体的载体，光固化水凝胶为光固化丝素蛋白，即甲基丙烯酰化丝素蛋白SilMA溶液，之后将经步骤（1）提取到的干细胞外泌体加入至SilMA溶液中，避光4℃保存；
步骤（4）、负载干细胞外泌体复合气管支架的制备；
将步骤（2）打印得到的PCL支架浸泡在负载干细胞外泌体的SilMA溶液Exo-SilMA中，37℃下浸泡30分钟，之后使用蓝光灯交联5分钟，使SilMA溶液Exo-SilMA均匀包裹PCL支架。
步骤（3）中，所述光固化水凝胶也可以选择光固化海藻酸钠、光固化硫酸软骨素、光固化明胶或光固化透明质酸。
步骤（3）中，所述SilMA溶液的制备方法为：
将再生丝素蛋白用溴化锂溶解，加入甲基丙烯酸缩水甘油酯反应，经透析后冷冻干燥后获得SilMA；使用PBS配置0.4%W/V光引发剂苯基-2,4,6-三甲基苯甲酰基亚磷酸锂，将冻干后的SilMA溶解于苯基-2,4,6-三甲基苯甲酰基亚磷酸锂溶液中得到SilMA溶液。
本发明结构合理、使用方便，本发明的支架中，3D打印的PCL支架可以提供良好的力学性能，而SilMA具有良好的生物相容性、生物降解性、溶胀作用，能够富集促进组织再生的生物成分，间充质干细胞外泌体作为干细胞衍生物，能够发挥干细胞的修复功能，且避免了移植干细胞的致瘤、排斥等风险，其参与细胞间通讯，能够稳定发挥促修复作用，本发明可以有效应用三者的功能，加速气管的组织再生修复。
附图说明
图1为透射电镜鉴定提取的骨髓间充质干细胞外泌体；
图2为负载骨髓间充质干细胞外泌体的复合气管支架实物；
图3为气管支架的微观结构扫描电镜图。
具体实施方式
下面结合具体实施例对本发明做进一步的说明。
一种3D打印负载干细胞外泌体的气管支架的制备方法，步骤为：
1、骨髓间充质干细胞外泌体提取及鉴定；
收集骨髓间充质干细胞培养基的上清液，采用超速离心法提取干细胞外泌体，PBS溶解后，使用0.22μm滤膜过滤除菌，于-80℃保存。应用透射电子显微镜，鉴定提取的外泌体，观察干细胞外泌体的形态并采图；
2、3D打印PCL气管支架的制备；
将PCL材料加入至3D打印机，调整打印参数，打印喷头沿旋转轴转动打印，依次重复打印两层多孔结构，层厚为0.1mm；
3、光固化水凝胶负载干细胞外泌体；
选择光固化水凝胶作为外泌体的载体，可选择的有光固化明胶、管固化透明质酸、光固化丝素蛋白等，在此我们选择光固化丝素蛋白即甲基丙烯酰化丝素蛋白（SilMA）作为载体；
将再生丝素蛋白用溴化锂溶解，加入甲基丙烯酸缩水甘油酯反应，经透析后冷冻干燥后获得SilMA。使用PBS配置0.4%W/V光引发剂苯基-2,4,6-三甲基苯甲酰基亚磷酸锂，将冻干后的SilMA溶解于苯基-2,4,6-三甲基苯甲酰基亚磷酸锂溶液中，之后将提取到的干细胞外泌体加入至SilMA溶液中，避光4℃保存。
4、负载干细胞外泌体复合气管支架的制备；
将PCL支架浸泡在负载干细胞外泌体的SilMA溶液（Exo-SilMA）中，37℃下浸泡30分钟，之后使用蓝光灯交联5分钟，使Exo-SilMA均匀包裹PCL支架。
进一步，详细具体为：
1.骨髓间充质干细胞外泌体提取与鉴定；
收集50mL骨髓间充质干细胞培养基的上清液，之后于300×g低离心力下离心10分钟，去除多余的细胞碎片，最后在100,000×g高离心力下离心70分钟，弃去上清液，加入1mLPBS缓冲液，继续100,000×g离心70分钟，最终获取骨髓间充质干细胞外泌体，溶于PBS，之后使用0.22μm滤膜过滤除菌，于-80℃保存。
将载样铜网固定在支架上，将 20µL 含外泌体的PBS溶液滴加至铜网上，于室温下静置5分钟后，在铜网的一侧使用滤纸将多余液体吸去，之后滴加3%磷钨酸溶液30µL，对样本进行负染5分钟，滤纸吸去表面负染液，将铜网转移至透射电子显微镜，观察干细胞外泌体的形态并采图。
2、光固化丝素蛋白制备；
将丝素蛋白于70℃条件下溶解于溴化锂溶液中2小时，加入甲基丙烯酸缩水甘油酯，于60℃,300rpm条件下反应3小时，收集混合溶液，于透析袋中进行透析，每4小时更换去水离子，透析4天后置于-80℃冰箱冷冻一天，取出后进行冻干，冷冻干燥后获得甲基丙烯酰化丝素蛋白（SilMA），-20℃保存。
3、负载骨髓间充质干细胞外泌体；
将冻干的SilMA于37℃温度下溶解于PBS中，调整浓度为10%（w/v）,避光条件下，加入0.5%（w/v）光引发剂（LAP），搅拌混匀直至完全溶解，收集混合溶液，加入之前提取到的骨髓间充质干细胞外泌体，调整外泌体浓度10-100ug/mL，获得负载骨髓间充质干细胞外泌体的甲基丙烯酰化丝素蛋白Exo-SilMA。
4、气管支架制备；
将PCL材料装载3D打印机中，打印机喷头调整至旋转轴上方，设置打印温度90℃，旋转角度180。，打印速度2mm/s,喷头沿着旋转轴方向往返移动，打印出首层支架，之后喷头沿相反方向移动，打印出外层支架，构建双层PCL支架。
5、复合气管支架制备；
将负载骨髓间充质干细胞外泌体的SilMA置于37℃水浴锅，搅拌混匀直至完全溶解，抽真空去除气泡，之后将PCL支架浸泡在Exo-SilMA溶液中30分钟，取出支架，进行蓝光（波长405nm）照射交联5分钟，获得负载外泌体复合气管支架。

Claims (2)

1.一种3D打印负载干细胞外泌体的气管支架的制备方法，其特征在于，包括以下步骤：
步骤（1）、骨髓间充质干细胞外泌体提取及鉴定；
收集骨髓间充质干细胞培养基的上清液，采用超速离心法提取干细胞外泌体，PBS溶解后，使用0.22μm滤膜过滤除菌，于-80℃保存；应用透射电子显微镜，鉴定提取的外泌体，观察干细胞外泌体的形态并采图；
步骤（2）、3D打印PCL气管支架的制备；
将PCL材料加入至3D打印机打印PCL支架；
步骤（3）、光固化水凝胶负载干细胞外泌体；
选择光固化水凝胶作为外泌体的载体，光固化水凝胶为光固化丝素蛋白，即甲基丙烯酰化丝素蛋白SilMA溶液，之后将经步骤（1）提取到的干细胞外泌体加入至SilMA溶液中，避光4℃保存；所述SilMA溶液的制备方法为：
将再生丝素蛋白用溴化锂溶解，加入甲基丙烯酸缩水甘油酯反应，经透析后冷冻干燥后获得SilMA；使用PBS配置0.4%W/V光引发剂苯基-2,4,6-三甲基苯甲酰基亚磷酸锂，将冻干后的SilMA溶解于苯基-2,4,6-三甲基苯甲酰基亚磷酸锂溶液中得到SilMA溶液；
步骤（4）、负载干细胞外泌体复合气管支架的制备；
将步骤（2）打印得到的PCL支架浸泡在负载干细胞外泌体的SilMA溶液Exo-SilMA中，37℃下浸泡30分钟，之后使用蓝光灯交联5分钟，使SilMA溶液Exo-SilMA均匀包裹PCL支架。
2.根据权利要求1所述的一种3D打印负载干细胞外泌体的气管支架的制备方法，其特征在于，步骤（3）中，所述光固化水凝胶也可以选择光固化海藻酸钠、光固化硫酸软骨素、光固化明胶或光固化透明质酸。
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