CN115029295B - 一种新型干细胞3d分化方法 - Google Patents
一种新型干细胞3d分化方法 Download PDFInfo
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- CN115029295B CN115029295B CN202210513858.2A CN202210513858A CN115029295B CN 115029295 B CN115029295 B CN 115029295B CN 202210513858 A CN202210513858 A CN 202210513858A CN 115029295 B CN115029295 B CN 115029295B
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Abstract
本发明涉及一种新型干细胞3D分化方法,通过完成定型内胚层诱导阶段或肝祖细胞阶段,将细胞酶解后的细胞悬液与海藻酸钠和间充质干细胞相关制品等溶液混合制备成细胞海藻酸钠混合液,再与氯化钙反应液反应形成细胞水凝胶,并继续完成肝样细胞的定向诱导分化。本发明采用以藻酸盐水凝胶制备的3D微球将人胚胎干细胞体外定向诱导分化为成熟的肝样细胞,大大降低分化成本及简化生产步骤,实现易富集、分化和装载在同一体系中实现,便于下游应用。本发明可获得大量、高质量的分化成熟的细胞,并且该技术因其材料的特殊性,可保护微球内的细胞免受病毒感染,移植后细胞受病毒感染的风险低,可为病毒性患者的细胞移植治疗的提供理想的细胞源。
Description
技术领域
本发明属于生物技术领域,具体涉及一种新型干细胞3D分化方法。
背景技术
肝细胞移植作为临床治疗终末期肝病、急性肝衰竭一种有效的治疗技术,在肝脏再生领域被广泛研究。由于缺乏稳定的肝细胞来源,限制了基于肝细胞治疗的临床应用。干细胞具有高度增殖性,可再生性和多能性,体外可定向诱导分化为多种类型的细胞。因此干细胞技术是解决细胞来源短缺问题的具有潜力的解决方案。研究人员发展了有效地将干细胞分化为多种成熟细胞谱系的2D体外定向诱导分化技术,通过在分化过程中使用细胞因子或小分子来干预激活细胞分化所必需的特定信号通路,达到定向分化目的。但是现有2D技术的实用性受到以下因素的限制:(1)分化完全的细胞产量低;(2)难以规模化培养;(3)诱导分化后的细胞难富集和应用到下游。这些因素均限制了干细胞分化来源的细胞在临床和科研中的应用。因此,亟需开发新的分化体系解决以上问题。
发明内容
本发明的目的在于提供一种新型的干细胞分化方法,降低分化成本及简化生产步骤,分化和装载在同一体系中实现,以实现高产、规模化培养、易富集、成本低、易用于细胞移植、药物筛选和药物毒性测试等下游应用,以解决目前干细胞分化来源的细胞在临床和科研中难以应用等问题。
一种新型干细胞3D分化方法,通过结合并优化干细胞向肝样细胞的分化和细胞水凝胶培养技术,得到了在干细胞定向分化中间阶段(比如干细胞肝向分化的定型内胚层阶段或肝祖细胞阶段)制备细胞水凝胶进行3D分化的方法。具体而言,通过完成一定分化阶段后(如肝向分化为定型内胚层阶段或肝祖细胞阶段),将细胞酶解后的细胞悬液与海藻酸钠混合溶液混合制备成细胞海藻酸钠混合液,再与氯化钙反应液反应形成细胞水凝胶,并继续完成肝样细胞的定向诱导分化。主要包括以下步骤:
(1)在第一阶段培养基中定向诱导分化为定型内胚层细胞或者进一步在第二阶段培养基中分化为肝祖细胞;
(2)将生理盐水、银耳多糖、间充质干细胞相关制品的冻干粉混匀后,再称取海藻酸钠配制成4%海藻酸钠溶液;
(3)用Dispase酶或Accutase酶消化贴壁的定型内胚层细胞或肝祖细胞,将消化下来的定型内胚层细胞用第二阶段培养基重悬,或将消化下来的肝祖细胞用第三阶段培养基重悬,再将所得细胞悬液与步骤(2)的海藻酸钠混合溶液混匀,形成细胞-海藻酸钠混悬液,通过注射器滴落到氯化钙反应液中形成细胞水凝胶,或使用微囊发生器制备更小粒径(50-300μm)的细胞水凝胶;
将步骤(3)处理过的定型内胚层细胞在第二阶段培养基中完成诱导肝祖细胞阶段,继续添加肝细胞生长因子、抑瘤素M诱导分化,在第三阶段培养基中完成诱导肝样细胞阶段,获得肝样细胞;或者将步骤(3)处理过的肝祖细胞添加肝细胞生长因子、抑瘤素M诱导分化,在第三阶段培养基中完成诱导肝样细胞阶段。
本发明在干细胞在单层贴壁培养分化至第一阶段结束时将定型内胚层细胞(或分化至肝祖细胞阶段)经酶解消化后与海藻酸钠混合溶液混合制成得细胞海藻酸钠混合液,进一步制备成细胞水凝胶,使细胞在水凝胶间相互聚集,在3D条件下,细胞相互聚集更有利于加强细胞间的相互作用,从而进一步增强肝样细胞的功能。此外,这种分化方法相比于2D分化,可以大量节省培养空间,这可为以后肝样细胞的规模分化培养奠定一定的基础。
优选的,间充质干细胞相关制品包括间充质干细胞或间充质干细胞质膜囊泡或间充质干细胞外泌体。
优选的,步骤(1)所述培养定型内胚层细胞的方法为:干细胞在基质胶包被的孔板中,隔天添加IDE1使用浓度为0.1~10μM、CHIR99021使用浓度为0.1~10μM和Torin2使用浓度为0.1~1000nM进行诱导分化,在第一阶段培养基中培养;然后在所述第一培养基中添加IDE1使用浓度为0.1~10μM和LDN193189/DM3189使用浓度为0.1~1000nM诱导分化,在第一阶段培养基中培养定型内胚层细胞。本发明均采用小分子化合物进行诱导分化,大大降低分化成本,可为干细胞来源的定型内胚层的大规模生产提供一定的参考依据。
优选的,步骤(2)中银耳多糖的浓度为0.01~2g/ml;被包裹的定型内胚层细胞或被包裹的肝祖细胞与间充质干细胞相关制品比例为1:(0.01-1);和/或间充质干细胞冻干粉(1~5,000,000细胞量/ml聚合物)
优选的,步骤(3)中的氯化钙反应液由CaCl2、D-fructose和HEPES组成,pH为7.4。
优选的,所述第二阶段培养基为由双抗、Glutamax、DMSO、NeAA、2-Mercaptoethanol、Knockout-SR组成的Knockout DMEM培养基,所述第三阶段培养基为含10μM hydrocortisone-21-hemisuccinate和100U/mL青链霉素的HepatoZYME-SFM HZM培养基。
本发明还提供一种海藻酸钠溶液,由生理盐水,银耳多糖,间充质干细胞相关制品的冻干粉和海藻酸钠配置而成。
优选的,银耳多糖的浓度为0.01~2g/ml;和/或所述间充质干细胞相关制品冻干粉(浓度为1~5,000,000细胞量/ml聚合物),和/或所述海藻酸钠溶液为1~6%,优选4%,和/或所述生理盐水的浓度为0.9%;和/或被包裹的定型内胚层细胞或被包裹的肝祖细胞与间充质干细胞相关制品比例为1:(0.01-1)。
上述海藻酸钠溶液的应用,主要用于细胞3D分化。
银耳多糖具有良好的免疫调节和抗炎症功能,无毒副作用,在作为免疫调节及抗炎方面具有巨大的潜力;间充质干细胞分泌多种生长因子(如HGF)可促进肝细胞分化成熟,且其亦具有免疫调节功能,可通过细胞间的相互作用及产生细胞因子抑制T细胞的增殖及其免疫反应,从而发挥免疫重建的功能。水凝胶材料除海藻酸钠外加上银耳多糖、间充质干细胞冻干粉,有助于后续水凝胶类肝细胞移植提供一定的免疫调节和抗炎作用,且生物相容性好,可生物降解。
与现有技术相比,本发明采用以藻酸盐水凝胶制备的3D微球将人胚胎干细胞体外定向诱导分化为肝样细胞,大大降低分化成本及简化生产步骤,实现易富集、分化和装载在同一体系中实现。可获得大量、高质量的分化成熟的细胞,并且该技术因其材料的特殊性,还可为病毒性患者的细胞移植治疗的提供理想的细胞源(移植的细胞受病毒感染的风险低)。本发明制备的3D微球用于细胞的分化,该方法生产成本低,产量高、易富集,并且该方法生产的微球内的细胞可免受病毒感染。
本发明建立了一种新型的3D微球原位分化体系,该体系为今后获得可用于肝细胞移植、代谢性肝病治疗和肝衰竭氨昏迷用的肝细胞源打下坚实的基础。具体表现为其技术仅涉及简单材料(藻酸盐相关、氯化钙、注射器或微囊发生器等),海藻酸盐作为新型组织支架材料因材料本身特有的生物安全性、细胞亲和性、可降解性逐渐被应用到医疗领域。海藻酸盐对宿主细胞无排异现象,不会引起机体过敏、炎症等不良反应,细胞可在其表面维持正常活性,且海藻酸盐可在体内被吸收降解随代谢产物排出,为细胞增殖、迁移、运输营养和代谢活动提供良好场所,且此分化体系分化获得的肝样细胞比常规2D分化获得的肝样细胞特异阶段基因表达更高,白蛋白分泌更高。同时具有肝细胞代谢转化功能和氨清除功能,且可保护微球内的细胞免受病毒感染,这项优点是现有其他3D分化培养技术暂不具备的。这种分化方法获得的肝样细胞可为病毒性患者的细胞移植治疗、代谢性肝病治疗和肝衰竭氨昏迷用提供理想的细胞源。
附图说明
图1是实施例1的新型干细胞3D分化方法的步骤示意图;
图2是实施例2的新型干细胞3D分化方法的步骤示意图;
图3是实施例1的海藻酸钠3D微球明场图;
图4是对比例1的2D分化获得的细胞与实施例1的3D微球分化获得的细胞阶段特异性基因及药物代谢酶及核受体基因mRNA表达水平比较;
图5是荧光显微镜下观察pHCMV-E1E2病毒感染对比例1的2D及实施例1的3D微球内的Huh7和hESC-Heps 48h后的感染情况;
图6是用共聚焦显微镜观察实施例1的包裹细胞在各个分化阶段的细胞活性图;
图7是对比例1的2D分化获得的细胞与实施例1的3D微球分化获得的细胞的蛋白水平表达对比图;
图8是显微镜拍摄实施例1的肝样细胞的ICG释放图;
图9是实施例1的肝样细胞的氨清除功能图;
图10是不同时间点,实施例1在内胚层细胞阶段包埋与实施例2在肝祖细胞阶段包埋分化获得肝样细胞的特异基因表达情况;
图11是实施例1与实施例3不同酶前期处理获得肝样细胞的特异基因表达情况。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明作进一步地详细描述。
实施例1
以诱导分化肝样细胞为例,一种新型3D定向诱导分化方法(在定型内胚层细胞阶段进行细胞水凝胶制备),如图1所示包括如下步骤:
(1)干细胞在37℃、5%CO2培养箱中培养。在含有100U/mL青霉素(Gibco)、100U/mL链霉素(Gibco)的mTesR培养基(Stem Cell Technologies)中保持未分化状态,每天换新鲜的培养基。
(2)干细胞在干细胞基质胶(Corning,BD,354277)包被的24孔板中以1.5*105铺板,隔天添加I(IDE1)、C(CHIR99021)、T(Torin 2)开始进行诱导分化,在第一阶段培养基中培养1天,I的使用浓度为100nM,C为3μM,T为15nM。隔天在所述第一阶段培养基中添加I和D(LDN193189/DM3189)继续诱导分化2天,I的使用浓度为100nM,D为250nM,共3天完成定型内胚层细胞阶段。
(3)完成定型内胚层细胞阶段后,吸弃第一阶段培养基后,用Dispase酶孵育后消化贴壁的细胞(3x 106/ml),将聚集的细胞转移到第二阶段培养基中,与等量的4%(w/v)的海藻酸钠溶液混合,形成2%的海藻酸钠细胞悬液,再将含有分化细胞的海藻酸钠细胞悬液通过27G注射器快速挤出,液滴落入凝胶浴(100mM CaCl2+20mM D-fructose+1x HEPES,pH7.4),将产生的液滴在凝胶浴中沉淀10分钟,以确保凝胶形成,然后用生理盐水洗涤两次,进入诱导肝祖细胞阶段,添加二甲基亚砜(DMSO)诱导分化,在第二阶段培养基中完成诱导肝祖细胞阶段,DMSO使用浓度为1%,共培养5天。
(4)完成诱导肝祖细胞阶段后,进入诱导肝样细胞阶段,用含10μMhydrocortisone-21-hemisuccinate(Sigma-Aldrich)和100U/mL青链霉素的HepatoZYME-SFM(HZM)培养基(Gibco),同时加10ng/ml HGF(PeproTech)、20ng/ml OSM(PeproTech)再处理分化后的细胞10天。
(5)完成诱导肝样细胞阶段,获得肝样细胞。
4%(w/v)海藻酸钠溶液配制方法为:0.9%生理盐水,0.01-2g/ml银耳多糖,间充质干细胞冻干粉(1~5,000,000细胞量/ml聚合物)(被包裹的定型内胚层细胞和间充质干细胞比例为1:1~1:0.01)混匀后再称取相应海藻酸钠配制成4%海藻酸钠溶液,边摇晃边缓慢多次加入,上下颠倒混匀,4℃保存。间充质干细胞可以用间充质干细胞质膜囊泡或间充质干细胞外泌体来代替。
氯化钙反应工作液为100mM CaCl2+20mM D-fructose+1x HEPES,pH7.4。
本实施例中第一阶段培养基段为由双抗以及B-27组成的RPMI1640培养基,第二阶段培养基为由双抗、Glutamax、DMSO、NeAA、2-Mercaptoethanol、Knockout-SR组成的Knockout DMEM培养基。
采用本实施例的制备方法探索不同酶前期处理获得肝样细胞的特异基因表达。
实施例2
以诱导分化肝样细胞为例,一种新型3D定向诱导分化方法(在肝祖细胞阶段进行细胞水凝胶制备),如图2所示包括如下步骤:
(1)干细胞在37℃、5%CO2培养箱中培养。在含有100U/mL青霉素(Gibco)、100U/mL链霉素(Gibco)的mTesR培养基(Stem Cell Technologies)中保持未分化状态,每天换新鲜的培养基。
(2)干细胞在干细胞基质胶(Corning,BD,354277)包被的24孔板中以1.5*105铺板,隔天添加I(IDE1)、C(CHIR99021)、T(Torin 2)开始进行诱导分化,在第一阶段培养基中培养1天,I的使用浓度为100nM,C为3μM,T为15nM。隔天在所述第一阶段培养基中添加I和D(LDN193189/DM3189)继续诱导分化2天,I的使用浓度为100nM,D为250nM,共3天完成定型内胚层细胞阶段。
(3)完成定型内胚层细胞阶段后,进入诱导肝祖细胞阶段,添加浓度为1%的二甲基亚砜(DMSO)诱导分化,在第二阶段培养基中完成诱导肝祖细胞阶段,共培养5天。
(4)完成诱导肝祖细胞阶段后,进入诱导肝样细胞阶段,吸弃第二阶段培养基后,用Dispase酶孵育后消化贴壁的细胞(3x 106/ml),将聚集的细胞转移到第三阶段培养基中,与等量的4%(w/v)的海藻酸钠溶液混合,形成2%的海藻酸钠细胞悬液,再将含有分化细胞的海藻酸钠细胞悬液通过27G注射器快速挤出,液滴落入凝胶浴(100mM CaCl2+20mMD-Fructose+1x HEPES,pH 7.4),将产生的液滴在凝胶浴中沉淀10分钟,以确保凝胶形成,或使用微囊发生器制备更小颗粒大小的微球,然后用NACl洗涤两次,进入诱导肝样细胞阶段,用含10μM hydrocortisone-21-hemisuccinate(Sigma-Aldrich)和100U/mL青链霉素的HepatoZYME-SFM(HZM)培养基(Gibco),同时加10ng/ml HGF(PeproTech)、20ng/ml OSM(PeproTech)再处理分化后的细胞10天。
(5)完成诱导肝样细胞阶段,获得肝样细胞。
4%(w/v)海藻酸钠溶液配制方法为:0.9%生理盐水,0.01~2g/ml银耳多糖,间充质干细胞冻干粉(1~5,000,000细胞量/ml聚合物)(被包裹的定型内胚层细胞和间充质干细胞比例为1:1)混匀后再称取相应海藻酸钠配制成4%海藻酸钠溶液,边摇晃边缓慢多次加入,上下颠倒混匀,4℃保存。间充质干细胞可以用间充质干细胞质膜囊泡或间充质干细胞外泌体来代替。
氯化钙反应工作液为100mM CaCl2+20mM D-fructose+1x HEPES,pH7.4。
本实施例中第一阶段培养基段为由双抗以及B-27组成的RPMI1640培养基,第二阶段培养基为由双抗、Glutamax、DMSO、NeAA、2-Mercaptoethanol、Knockout-SR组成的Knockout DMEM培养基。
实施例3
一种新型3D定向诱导分化方法,步骤(3)中采用Accutase酶(Stem CellTechnologies)孵育5min后分离细胞,得到单细胞悬液,其它的均与实施例1相同。
对比例1
采用现有2D方法诱导分化干细胞。步骤(3)中完成定型内胚层细胞阶段后,吸弃第一阶段培养基后,进入诱导肝祖细胞阶段,添加二甲基亚砜(DMSO)诱导分化,在第二阶段培养基中完成诱导肝祖细胞阶段,DMSO使用浓度为1%。其它与实施例1相似。
对比例2
将实施例1中的4%海藻酸钠溶液中银耳多糖或间充质干细胞冻干粉去掉,其它与实施例1相似。实验发现,与含间充质干细胞冻干粉的海藻酸钠溶液制备的细胞水凝胶相比,未加入间充质干细胞冻干粉的海藻酸钠溶液制备的细胞水凝胶最终分化得到的肝样细胞特异基因表达有所下降。
对比例3
分化方法是模拟人胚胎发育过程的阶段和模式,在细胞发育的各个关键节点通过外在的信号调控细胞向特定谱系分化,期间需要添加各个不同小分子,为了确保小分子能够及时进入微球且细胞的分泌物能够排出,采用标记了特定分子大小的异硫氰酸荧光素的葡聚糖进行检测,携带与小分子同等大小的异硫氰酸荧光素的标记液能够快速进入海藻酸钠微球,表示海藻酸钠微球具有良好的物质通透性,肝细胞分化所需的中小分子能够及时进入微球及合成的白蛋白、尿素以及其他细胞因子等都能够很好的透过微球分泌到周围环境中。
实验发现,若在Day0,即干细胞阶段直接进行细胞水凝胶制备后分化,在Day 0时进行包埋,可见微球随着分化时间的增加逐渐膨胀,且Day 0包埋后微球内细胞活性不高。
实验还发现,若分化至Day18,即肝样细胞后再进行细胞水凝胶制备,用酶消化后的肝样细胞形态会发生明显变化,胞质明显变大,消化后的肝样细胞白蛋白表达明显降低,特异基因表达也会下降,表明在分化至肝样细胞后再消化下来进行细胞水凝胶制备,可能会导致细胞的去分化。
对比例4
实现发现,4%海藻酸钠溶液配制时需边摇晃边缓慢多次加入,不可一次性加入足量的海藻酸钠,会难以溶解。需保证充分溶解,溶液浓度及密度均匀,过程需严格无菌操作,配制后尽快使用,置于4℃保存。
实验还发现,若造成污染或浓度过低,如1%海藻酸钠溶液会使细胞水凝胶在分化过程中逐渐溶解,浓度过高如8%的浓度,难以形成细胞水凝胶。
实验结果
倒置相差显微镜下观察实施例1制备所得海藻酸钠微球基质半透明,如图3所示。
将实施例1和对比例1所得细胞阶段特异性基因及药物代谢酶及核受体基因mRNA表达水平比较如图4所示,可以看到,这种3D分化方法得到的肝样细胞(hESC-Heps-3D)比常规2D分化得到的肝样细胞(hESC-Heps-2D)更高表达其阶段特异性基因及药物代谢酶和核受体基因。
图5荧光显微镜下观察pHCMV-E1E2病毒感染对比例1的2D及实施例1的3D微球内的Huh7和hESC-Heps 48h后的感染情况。(EGFP为病毒表达报告基因)。结果显示pHCMV-E1E2病毒均能感染2D的Huh7和肝样细胞,表达绿色荧光,而未能感染3D微球内的Huh7和肝样细胞。由此可见,本发明采用的3D分化方法还可有效阻断微球外的病毒进入微球感染细胞。可为获得用于肝细胞移植(尤其是病毒性肝炎患者的细胞移植治疗)的新的肝细胞源提供重要的参考依据。
为了观察细胞活力,在分化后的第3、8、14、18天,收集包埋的细胞,并按照制造商的说明进行Calcein/PI细胞活性与毒性检测(Beyotime,C2015M)。图6结果显示均只表达钙黄绿素荧光产物,而碘化丙啶不能穿透活细胞的细胞膜,只能染色细胞膜完整性被破坏的死细胞中,表明在Day 3包埋后分化的各个阶段细胞均保持较高活性。
从图7可以看出,实施例1的肝样细胞的亦更高表达蛋白水平。
功能性肝细胞能够摄取培养基中吲哚菁绿,图8,用含ICG的培养基孵育hESC-Heps-2D培养3h后,细胞能成功摄取其中的ICG染成绿色,而hESC-Heps-3D孵育24h后亦可摄取ICG染成绿色,并摄取后换成不含ICG的培养基,微球内的细胞能在48h后释放,表明hESC-Heps-3D具有代谢ICG的能力。检查分化后细胞的氨清除能力,结果表明该分化方法得到的类肝细胞具有一定清除氨的能力,在肝衰竭的氨昏迷治疗中可能具有一定应用潜力。此外,这种分化方法还可有效阻断微球外的病毒进入微球感染细胞。这种分化方法获得的肝样细胞可为病毒性患者的细胞移植治疗、代谢性肝病治疗和肝衰竭氨昏迷用提供理想的细胞源。
本发明实施例1的3D分化方法获得的肝样细胞亦具有代谢转化功能和氨清除功能,如图9所示。
在Day3(内胚层细胞)、Day8(肝祖细胞)不同时间点制备细胞水凝胶进行分化,最终得到的肝样细胞的特异性阶段基因表达情况如图10所示。
不同前期处理获得肝样细胞的特异基因表达影响很大,如图11所示。前期采用不同类型的酶消化贴壁的定性内胚层细胞阶段,按实施例1的方法获得的肝样细胞其特异基因表达有一定差异,但采用Dispase酶消化方式最终得到的肝样细胞,其表达的较具代表性特异基因(NTCP、ALB)表达更高。
Claims (6)
1.一种新型干细胞3D分化方法,其特征在于,包括以下步骤:
(1)在第一阶段培养基中定向诱导分化为定型内胚层细胞或者进一步在第二阶段培养基中分化为肝祖细胞;
(2)将生理盐水、银耳多糖、间充质干细胞冻干粉混匀后,再称取海藻酸钠配制成4%海藻酸钠溶液;
(3)用Dispase酶或Accutase酶消化贴壁的定型内胚层细胞或肝祖细胞,将消化下来的定型内胚层细胞用第二阶段培养基重悬,或将消化下来的肝祖细胞用第三阶段培养基重悬,再将所得细胞悬液与步骤(2)的海藻酸钠混合溶液混匀,形成细胞-海藻酸钠混悬液,通过注射器滴落到氯化钙反应液中形成细胞水凝胶,或使用微囊发生器制备细胞水凝胶;
(4)将步骤(3)处理过的定型内胚层细胞在第二阶段培养基中完成诱导肝祖细胞阶段,继续添加肝细胞生长因子、抑瘤素M诱导分化,在第三阶段培养基中完成诱导肝样细胞阶段,获得肝样细胞;或者将步骤(3)处理过的肝祖细胞添加肝细胞生长因子、抑瘤素M诱导分化,在第三阶段培养基中完成诱导肝样细胞阶段;
其中步骤(2)中银耳多糖的浓度为0.01-2g/ml;被包裹的定型内胚层细胞或被包裹的肝祖细胞与间充质干细胞比例为1:(0.01-1);间充质干细胞冻干粉中含有1-5,000,000细胞量/ml聚合物;生理盐水的浓度为0.9%;
步骤(3)中的氯化钙反应液由CaCl2、D-Fructose和HEPES组成,pH为7.4。
2.根据权利要求1所述新型干细胞3D分化方法,其特征在于,步骤(1)所述培养定型内胚层细胞的方法为:干细胞在Matrigel hESC-qualified包被的孔板中,隔天添加IDE1、CHIR99021和Torin2进行诱导分化,在第一阶段培养基中培养;然后在所述第一培养基中添加IDE1和LDN193189/DM3189诱导分化,在第一阶段培养基中培养定型内胚层细胞。
3.根据权利要求1所述新型干细胞3D分化方法,其特征在于,所述第二阶段培养基为由双抗、Glutamax、DMSO、NeAA、2-Mercaptoethanol、Knockout-SR组成的Knockout DMEM培养基,所述第三阶段培养基为含10μMhydrocortisone-21-hemisuccinate和100U/mL青链霉素的HepatoZYME-SFM HZM培养基。
4.根据权利要求1所述新型干细胞3D分化方法,其特征在于,步骤(3)中细胞悬液与海藻酸钠溶液的体积比为1:1。
5.一种海藻酸钠溶液,其特征在于,由生理盐水,银耳多糖,间充质干细胞冻干粉和海藻酸钠配置而成;所述银耳多糖的浓度为0.01-2g/ml;所述间充质干细胞冻干粉中含1~5,000,000细胞量/ml聚合物;所述海藻酸钠溶液为1~6%;所述生理盐水的浓度为0.9%。
6.根据权利要求5所述海藻酸钠溶液的应用,其特征在于,用于细胞3D分化。
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