CN114984050A - 一种间充质干细胞外泌体冻干粉制备及使用方法 - Google Patents
一种间充质干细胞外泌体冻干粉制备及使用方法 Download PDFInfo
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Abstract
本发明公开了一种间充质干细胞外泌体冻干粉制备及使用方法,本发明公开了一种脂肪、脐带、胎盘、骨髓等干细胞获取外泌体的方法,应用冻干技术获得外泌体冻干剂及制备的方法,包括:干细胞为间充质干细胞,包括但不限于脂肪、脐带、胎盘、骨髓等组织获得的干细胞。从各组织中提取获得间充质干细胞,应用传统的瓶皿二维培养方法获得一定数量的间充质干细胞,将间充质干细胞转移至三维培养体系中进行培养,包括但不限于:微载体、中空纤维、水凝胶、微流体等进行培养。提取获得培养上清,应用离心法获得外泌体,将获得的外泌体与海藻糖、甘露醇等冻干蛋白保护剂混合均匀后,制成外泌体冻干制剂。
Description
技术领域
本发明涉及间充质干细胞制剂领域,具体为一种间充质干细胞外泌体冻干粉制备及使用方法。
背景技术
间充质干细胞(MSC,mesenchymal stem cells)是干细胞家族的重要成员,来源于发育早期的中胚层,属于多能干细胞,MSC最初在骨髓中发现,因其具有多向分化潜能、造血支持和促进干细胞植入、免疫调控和自我复制等特点而日益受到人们的关注,外泌体是一种直径为30-100nm的纳米级脂质包裹体结构,外泌体由细胞分泌释放出来,在血液等体液内传播,最后又可被其他细胞吞噬,是细胞间通讯的重要介质。
在二维培养法的培养环境下,无法模拟细胞体内生长环境,培养环境的不合理无法充分激发细胞活性,使得细胞与细胞之间没有相互的刺激作用,所得外泌体的种类单一、浓度较低,无法获得更多的外泌体,鉴于此,我们提出一种一种间充质干细胞外泌体冻干粉制备及使用方法。
发明内容
本发明的目的在于提供一种间充质干细胞外泌体冻干粉制备及使用方法,以解决上述背景技术中提出的二维培养法的培养环境下,所得外泌体的种类单一、浓度较低,无法获得更多的外泌体的问题。
为实现上述目的,本发明提供如下技术方案:
一种间充质干细胞提取外泌体的方法及外泌体冻干制剂的制备方法,其特征在于,包括:
步骤一、对脂肪、脐带、胎盘、骨髓等组织进行处理,置于瓶皿进行贴壁培养,加入无血清培养基,经1-2次传代后,收集间充质干细胞及培养上清;
步骤二、将间充质干细胞转移至三维培养体系,包括但不限于:微载体、中空纤维、水凝胶、微流体等进行培养。收集培养上清液。
步骤三、使用离心法去除所述培养上清液中的细胞碎片、死细胞和杂质;高速离心获得培养上清中外泌体。
步骤四、外泌体溶液中加入冻干保护剂,使用真空冷冻干燥机将外泌体溶液中水分排出。
作为优选技术方案:所述步骤一,组织来源为脂肪、脐带、胎盘、骨髓。
作为优选技术方案:所述步骤一,具体包括:对组织块或细胞进行平皿贴壁培养,获得间充质干细胞悬液及培养上清液;包括:每皿细胞生长至融合度80%以上,收集培养上清液。贴壁细胞用PBS缓冲液清洗,0.025%-0.25%胰酶或胰酶替代物消化,去除消化酶,将细胞传至培养瓶中,加入培养基,继续传代培养,细胞传代扩增至2代之后,收集培养液及细胞。
作为优选技术方案:所述步骤二,细胞转移至三维培养体系进行培养,具体包括:将所述间充质干细胞悬液导入三维培养装置中培养,每2-4天收集培养上清液,并更换新鲜的无外泌体培养基;培养至28-32天后终止培养。
作为优选技术方案:所述步骤三,对所述培养上清液进行离心、纯化外泌体;包括:将所述培养上清液在400g~600g离心8min~15min,收集上清液,弃沉淀;收集的上清液在4℃以1800g-2200g离心10min~20min,收集上清液,弃沉淀;收集的上清液在4℃以8000g-12000g离心20min~40min,收集上清液,弃沉淀,上清液使用0.22μm滤器过滤,收集过滤液;收集的上清液在4℃以90000g-110000g离心60min~80min,弃上清,收集沉淀,即得外泌体;收集的外泌体使用生理盐水或PBS溶液重悬,在4℃以90000g-110000g离心60min~80min,弃上清,收集沉淀,即得纯化外泌体;将纯化外泌体使用一定体积的生理盐水或PBS溶液重悬,置于4℃冰箱短期保存或置于-80℃冰箱长期保存。
作为优选技术方案:所述步骤四,对所述外泌体溶液进行冻干;包括:外泌体冻干制剂中含有外泌体、氯化钠、海藻糖、甘露醇。
作为优选技术方案:按外泌体溶液的重量体积百分比计,加入各成分的量,包括:3%-10%海藻糖、3%-10%甘露醇。
作为优选技术方案:外泌体冻干保护剂使用0.22μm过滤除菌后,加入到外泌体溶液中。
作为优选技术方案:外泌体溶液加入冻干保护剂,置于-80℃冰箱冷冻2-12h,置于预冷的真空干燥箱中,真空冷冻干燥,排出水分,获得外泌体冻干制剂。
作为优选技术方案:外泌体冻干制剂可置于室温长期保存。
与现有技术相比,本发明的有益效果是:
本发明的二维培养法结合三维培养方法,即可通过二维培养法快速的获得原代组织中获得一定数量及纯度的干细胞,又能通过三维培养法模拟细胞体内生长环境,提供合适的环境可以充分激发细胞活性,使得细胞与细胞之间相互作用,互相刺激,所得外泌体的种类更多、浓度更高,通过培养方法的合理利用,可以达到获得更多外泌体的目的;
本发明基于外泌体的不稳定性,在外泌体中添加合适计量的海藻糖及甘露醇,海藻糖和甘露醇能与蛋白质功能基团结合,使蛋白质分子处于饱和状态从而避免聚集;另外通过束缚水分子,降低“共晶点”温度,减少冰晶体的形成量,隔离和减缓蛋白质聚集,防止蛋白质凝聚变性,从而保证冻藏过程中的外泌体细胞品质,使用冻干技术可让外泌体在常温长期保存。
具体实施方式
实施例一:
一种间充质干细胞提取外泌体的方法及外泌体冻干制剂的制备方法,其特征在于,包括:
步骤一、对脂肪、脐带、胎盘、骨髓等组织进行处理,置于瓶皿进行贴壁培养,加入无血清培养基,经1-2次传代后,收集间充质干细胞及培养上清;
步骤二、将间充质干细胞转移至三维培养体系,包括但不限于:微载体、中空纤维、水凝胶、微流体等进行培养。收集培养上清液。
步骤三、使用离心法去除所述培养上清液中的细胞碎片、死细胞和杂质;高速离心获得培养上清中外泌体。
步骤四、外泌体溶液中加入冻干保护剂,使用真空冷冻干燥机将外泌体溶液中水分排出。
实施例二:
步骤一中,组织来源为脂肪、脐带、胎盘、骨髓,通过采取不同部位的细胞,提高细胞的丰富程度,可以在模拟人体的环境下相互作用,相互刺激,外泌体的种类更多、浓度更高。
具体的,对组织块或细胞进行平皿或者瓶皿贴壁培养,获得间充质干细胞悬液及培养上清液;包括:每皿细胞生长至融合度80%以上,收集培养上清液。贴壁细胞用PBS缓冲液清洗,0.025%-0.25%胰酶或胰酶替代物消化,去除消化酶,将细胞传至培养瓶中,加入培养基,继续传代培养,细胞传代扩增至2代之后,收集培养液及细胞;细胞转移至三维培养体系进行培养,具体包括:将所述间充质干细胞悬液导入三维培养装置中培养,每2-4天收集培养上清液,并更换新鲜的无外泌体培养基;培养至28-32天后终止培养;即可通过二维培养法快速的获得原代组织中获得一定数量及纯度的干细胞,又能通过三维培养法模拟细胞体内生长环境,提供合适的环境可以充分激发细胞活性。
实施例三:
步骤三中,对所述培养上清液进行离心、纯化外泌体;
具体的步骤为,将所述培养上清液在400g~600g离心8min~15min,收集上清液,弃沉淀;收集的上清液在4℃以1800g-2200g离心10min~20min,收集上清液,弃沉淀;收集的上清液在4℃以8000g-12000g离心20min~40min,收集上清液,弃沉淀,上清液使用0.22μm滤器过滤,收集过滤液;收集的上清液在4℃以90000g-110000g离心60min~80min,弃上清,收集沉淀,即得外泌体;收集的外泌体使用生理盐水或PBS溶液重悬,在4℃以90000g-110000g离心60min~80min,弃上清,收集沉淀,即得纯化外泌体;将纯化外泌体使用一定体积的生理盐水或PBS溶液重悬,置于4℃冰箱短期保存或置于-80℃冰箱长期保存,通过进行四次离心力逐步加大的离心操作,可以高效获得高纯度的纯化外泌体,并在4℃的温度下进行分离,细胞收缩紧致,提高分离速度。
实施例四:
对所述外泌体溶液进行冻干;包括:外泌体冻干制剂中含有外泌体、氯化钠、海藻糖、甘露醇;按外泌体溶液的重量体积百分比计,加入各成分的量,包括:3%-10%海藻糖、3%-10%甘露醇;合适配量的海藻糖和甘露醇能与蛋白质功能基团结合,使蛋白质分子处于饱和状态从而避免聚集;另外通过束缚水分子,降低“共晶点”温度,减少冰晶体的形成量,隔离和减缓蛋白质聚集,防止蛋白质凝聚变性,从而保证冻藏过程中的外泌体细胞品质;外泌体冻干保护剂使用0.22μm过滤除菌后,加入到外泌体溶液中,保证洁净避免细胞污染;外泌体溶液加入冻干保护剂,置于-80℃冰箱冷冻2-12h,置于预冷的真空干燥箱中,真空冷冻干燥,排出水分,获得外泌体冻干制剂;外泌体冻干制剂可置于室温长期保存。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的仅为本发明的优选例,并不用来限制本发明,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (10)
1.一种间充质干细胞外泌体冻干粉制备方法,其特征在于:包括如下步骤:
步骤一、对组织进行处理,置于瓶皿进行贴壁培养,加入无血清培养基,经传代后,收集间充质干细胞及培养上清;
步骤二、将间充质干细胞转移至微载体三维培养体系进行培养,收集培养上清液。
步骤三、使用离心法清洁培养上清液;高速离心获得培养上清液中的外泌体。
步骤四、外泌体溶液中加入冻干保护剂,采用冻干机将外泌体溶液中水分排出。
2.根据权利要求1所述的一种间充质干细胞外泌体冻干粉制备方法,其特征在于:步骤一中,所述组织来源为脂肪、脐带、胎盘、骨髓。
3.根据权利要求2所述的一种间充质干细胞外泌体冻干粉制备方法,其特征在于:步骤一中,对所述组织进行平皿贴壁培养;贴壁细胞清洗消化后,去除消化酶将细胞传至培养瓶中培养,细胞传代扩增至2代之后,收集培养液及细胞。
4.根据权利要求3所述的一种间充质干细胞外泌体冻干粉制备方法,其特征在于:步骤二中,所述间充质干细胞使用到的三维培养体系采用微载体、中空纤维、水凝胶和微流体的任一种,将所述间充质干细胞悬液导入三维培养装置中培养,培养至28-32天后终止培养。
5.根据权利要求1所述的一种间充质干细胞外泌体冻干粉制备方法,其特征在于:步骤三中,所述培养上清液进行四次离心力逐步加大的离心操作,即得外泌体,收集的外泌体进行重悬,离心弃上清液,即得纯化外泌体,将纯化外泌体重悬,置于冰箱保存。
6.根据权利要求5所述的一种间充质干细胞外泌体冻干粉制备方法,其特征在于:步骤四中,所述外泌体溶液中加入冻干保护剂得到外泌体冻干制剂,所述外泌体冻干制剂中含有外泌体、氯化钠、海藻糖、甘露醇。
7.根据权利要求6所述的一种间充质干细胞外泌体冻干粉制备方法,其特征在于:所述外泌体冻干制剂按外泌体溶液的重量百分比计,其中海藻糖为外泌体溶液重量的3%-10%,甘露醇为外泌体溶液重量的3%-10%。
8.根据权利要求6所述的一种间充质干细胞外泌体冻干粉制备方法,其特征在于:所述冻干保护剂使用0.22μm过滤除菌后,加入到外泌体溶液中。
9.根据权利要求8所述的一种间充质干细胞外泌体冻干粉制备方法,其特征在于:所述外泌体溶液加入冻干保护剂,置于-80℃冰箱冷冻2-12h,置于预冷的真空干燥箱中,真空冷冻干燥,排出水分,获得外泌体冻干制剂。
10.根据权利要求9所述的一种间充质干细胞外泌体冻干粉使用方法,其特征在于:所述外泌体冻干制剂能够于室温中稳定保存。
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