CN116875542A - 一种间充质干细胞培养增强剂及其应用 - Google Patents
一种间充质干细胞培养增强剂及其应用 Download PDFInfo
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Abstract
本发明公开了一种间充质干细胞培养增强剂及其应用,该增强剂由维生素E 0.5mg/mL、聚乙烯醇2.5mg/mL、右旋糖酐40 0.5g/mL、α‑酮戊二酸5mg/mL、脂多糖1µg/mL、β‑巯基乙醇150mg/mL、硫辛酸20mg/mL、还原性谷胱甘肽30mg/mL、腐胺1g/mL、孕酮0.6mg/mL、rhNRG1 10µg/mL、rhBMP‑4 50µg/mL组成,间充质干细胞增生无血清完全培养基与其的体积比为99.9:0.1~97.5:2.5时,可有效调高脐带间充质干细胞的原代分离效率,细胞收获量提高20%以上;并保持原代细胞的标志物特性不改变。
Description
技术领域
本发明涉及细胞培养基领域,具体为一种间充质干细胞培养增强剂及其应用。
背景技术
间充质干细胞(mesenchymal stem cells,MSCs)是成体干细胞的一种,属于中胚层的多能干细胞,具有非常强的分化能力,在特定的条件培养下,具有分化为脂肪细胞、骨细胞、软骨细胞、神经细胞、肌细胞等多种类型细胞的潜能。间充质干细胞主要存在于结缔组织和器官间质中,广泛存在于脐带组织、脐带血、胎盘、羊膜、羊水、脂肪组织、骨髓组织、骨骼肌组织、骨外膜组织、肺、肝、胰腺等的组织中,来源十分广泛,在合适的条件下可以进行长期传代培养,这些特性使得间充质干细胞适用于干细胞制剂的规模化培养与应用。
间充质干细胞已经被广泛应用于临床医疗研究、美容等领域。随着对干细胞的认识越来越深入,形成一种观点认为“靠近移植”更有优势,以靠近病患部位组织来源的干细胞,进行细胞移植及组织修复效果更佳。而脂肪作为人体分布最广泛地组织,其中含有的间充质干细胞极为丰富,是唯一一种能够满足“靠近移植”的组织。但是如何获得高产量、高活性、高一致性、更安全的脂肪间充质干细胞,用于进一步的应用研究,仍然是间充质干细胞研究应用的重要课题。而骨髓间充质干细胞,组织来源极为有限,更迫切需要能够培养获得高产量、高一致性、安全、稳定、成分明确的培养基。
传统的间充质干细胞培养通常采用基础培养基如DMEM/F12、MEM-alpha,添加胎牛血清后进行细胞培养。如商业化含血清间充质干细胞培养基(STEMCELL,MesenCult™ 扩增试剂盒(人),#05411),再如中国专利申请CN103146647A公开了一种间充质干细胞培养基,以MEM-alpha为基础培养基添加了10%胎牛血清,这类存在含血清培养基有诸多缺点:成分不确定,限制应用;含有异源蛋白,扩增的间充质干细胞产率较低、传代次数有限,细胞通过胞吞摄取牛血清蛋白而携带牛血清蛋白,能够引起受体免疫反应,同时有引入异源血清携带的细菌、病毒的风险,极大限制了间充质干细胞的应用。
目前也有市售的间充质干细胞无血清培养基,包括含血小板裂解物(hPL)的无血清培养基和成分明确的无血清培养基。含血小板裂解物的培养基(如Helios)添加至MEM-a或DMEM/F12培养基使用,细胞生长迅速,但是这种培养基成分不明确,临床应用安全性及批间一致性差,下游干细胞制剂等应用需要更严苛的质控放行要求。成分明确的无血清培养基(如:STEMCELL,MesenCult™-ACF Plus 培养试剂盒,货号:05448;Gibco,StemPro™ 间充质干细胞无血清无异源培养基,货号:A1067501;ExcellBIOS,MSC增生无血清培养基,货号:ME000-N023),以及中国专利申请CN110331130A公开了的一种间充质干细胞无血清培养基及其用途,这些培养基均为成分明确的培养基,可用于间充质干细胞的培养,但是这些培养基细胞培养效果,特别是在原代种子细胞分离方面,与含血小板裂解物的培养基仍存在较大差距。
发明内容
针对上述问题,本发明开发了一种间充质干细胞培养增强剂及其应用,以提高无血清、成分明确的培养体系下细胞的生长效率,特别是提高脐带间充质干细胞植块法分离效率,提高种子细胞收获量。
本发明的目的在于提供一种间充质干细胞培养增强剂及其应用,以解决上述背景技术中提出的问题。
为了解决上述技术问题,本发明提供如下技术方案:
一种间充质干细胞培养增强剂,由以下成分组成,具体为:维生素E 0.5mg/mL、聚乙烯醇2.5mg/mL、右旋糖酐40 0.5g/mL、α-酮戊二酸5mg/mL、脂多糖1µg/mL、β-巯基乙醇150mg/mL、硫辛酸20mg/mL、还原性谷胱甘肽30mg/mL、腐胺1g/mL、孕酮0.6mg/mL、rhNRG1 10µg/mL、rhBMP-4 50µg/mL。
优选的,所述的间充质干细胞培养增强剂与间充质干细胞增生无血清完全培养基的体积比为0.1:99.9~2.5:97.5。
优选的,所述的间充质干细胞培养增强剂与间充质干细胞增生无血清完全培养基的体积比为1:99。
前述任一所述的间充质干细胞培养增强剂用于间充质干细胞的培养。
优选的,所述的间充质干细胞为人脐带间充质干细胞。
与现有技术相比,本发明所达到的有益效果是:
提高了成分明确培养基体系条件下,脐带组织来源间充质干细胞的原代分离效率,可获得更多的种子细胞。
本发明的细胞培养增强剂,不含有异源体成分,成分明确,可以保持原培养体系仍为成分明确体系,保证下游细胞培养使用(如干细胞制剂使用)的安全性及批间一致性,不增加干细胞制剂等应用的质控放行要求。与血小板裂解物等体系相比也不需要增加艾滋病病毒(HIV-1/2)抗体、乙肝表面抗原(HBsAg)抗体和丙肝病毒(HCV)检测等质控检查。
本发明添加聚乙烯醇、右旋糖酐40、α-酮戊二酸、脂多糖,增加了培养基粘稠度,同时能够产生与人血白蛋白接近的效能,减少了组织细胞的机械损伤,同时具有调控基因合成的作用。
本发明添加孕酮,作为类固醇激素,可参与干细胞的功能调节,可增加免疫调节蛋白的分泌,如HLA-G、PIBF等。
本发明添加腐胺,有助于维持细胞干性及自我更新能力。
本发明添加rhNRG1、rhBMP-4等因子,这些因子与bFGF等因子相互作用,进一步提高细胞生长效率。
本发明添加还原性谷胱甘肽、β-巯基乙醇等成分,减少原代培养过程中因换液周期长等积累的氧化自由基成分,有利于保持原代细胞活力。
本发明的间充质干细胞培养增强剂,经过多重材料配比及剂量配比设计,在优选的添加配比条件下,可有效调高脐带间充质干细胞的原代分离效率,细胞收获量提高20%以上;并保持原代细胞的标志物特性不改变。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。在附图中:
图1为脐带间充质干细胞原代分离显微观察示意图(40倍显微成像);
图2为脐带间充质干细胞原代分离显微观察示意图(100倍显微成像);
图3间充质干细胞原代细胞表面标志物CD90及其阴性对照检测结果;
图4间充质干细胞原代细胞表面标志物CD19及其阴性对照检测结果;
图5间充质干细胞原代细胞表面标志物CD45及其阴性对照检测结果;
图6间充质干细胞原代细胞表面标志物CD34及其阴性对照检测结果;
图7间充质干细胞原代细胞表面标志物CD105及其阴性对照检测结果;
图8间充质干细胞原代细胞表面标志物HLA-DR及其阴性对照检测结果;
图9间充质干细胞原代细胞表面标志物CD73及其阴性对照检测结果;
图10间充质干细胞原代细胞表面标志物CD14及其阴性对照检测结果。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
以下实施例中所述的间充质干细胞培养增强剂所用各种组分均采购自西格玛(Sigma)或阿拉丁;间充质干细胞增生无血清完全培养基(依科赛ExcellBio,货号:ME000-N023,该培养基对应申请专利公开号CN110938590A);人脐带间充质干细胞,该细胞来源于太仓第一人民医院合作研究项目,所使用的组织样本为志愿者捐献,同时经过太仓市第一人民医院伦理委员会批准;康宁细胞结合表面处理培养板(厂家:康宁Corning,货号:3335)。
实施例1:间充质干细胞培养增强剂由以下成分组成,维生素E 0.5mg/mL、聚乙烯醇2.5mg/mL、右旋糖酐40 0.5g/mL、α-酮戊二酸5mg/mL、脂多糖1µg/mL、β-巯基乙醇150mg/mL、硫辛酸20mg/mL、还原性谷胱甘肽30mg/mL、腐胺1g/mL、孕酮0.6mg/mL、rhNRG1 10µg/mL、rhBMP-4 50µg/mL。
本例中的组成成分需经过以0.22微米滤膜过滤除菌。
间充质干细胞培养增强剂的使用方案为:向间充质干细胞增生无血清完全培养基添加一定比例的间充质干细胞培养增强剂,例如取间充质干细胞增生无血清完全培养基99mL,添加如上所描述的增强剂1mL,即使用剂量配比为99:1(体积比)。
实施例2:本实施例提供实施例1的间充质干细胞细胞培养增强剂的效果验证。
本实施例的实验中采用人脐带间充质干细胞,该细胞来源于太仓第一人民医院合作研究项目,所使用的组织样本为志愿者捐献,同时经过太仓市第一人民医院伦理委员会批准,此脐带组织用于下述分离实验。
一、实验方法:
植块法脐带原代间充质干细胞的分离:
取样:使用新鲜脐带组织,样品置于含1-2倍PS抗生素的DPBS内低温运输;
清洗:DPBS清洗,洗去血液等;整根脐带浸入75%无水乙醇内30秒,立即取出用DPBS洗净;
分离:去除两端少量易被污染的组织,剪成2-3cm小段,去除静脉、动脉组织,取华通氏胶组织;
剪碎、清洗:剪碎组织,加入DPBS清洗,转移组织至50mL离心管内;
收集:1000RMP,5分钟离心,弃上清,保留组织;
接种:加入适量培养基,用剪口的枪头吸吹,使组织松散、均匀,吸取1~2mL组织接种至1个T75培养瓶或100mm培养皿中,补加培养基至15mL,摇匀后,移入培养箱培养;
换液:静置培养,第5到6天(D5~6)进行第一次换液,继续静置培养,第8到10天(D8~10)第二次换液,第12到16天(D12~16)收细胞;
传代:分离后第12天开始,每天观察细胞生长状态,较多组织周围细胞生长密集时,进行细胞传代。
实验所用细胞培养皿皆为康宁细胞结合表面处理培养板。
采用本实施例1的增强剂的使用方案,取间充质干细胞增生无血清完全培养基99mL,添加如上所描述的增强剂1mL,即使用剂量配比为99:1,在细胞分离第14天拍摄电镜图如图1~图2所示,图1中40倍电镜下可见组织球贴壁,间充质干细胞致密生长,局部放大图1,如图2所示,100倍镜下可见间充质干细胞贴壁生长良好。
在细胞分离第16天收集并记录原代细胞数,并进行标志物鉴定检测。
二、细胞鉴定:
脐带间充质干细胞的细胞表面标志物检测:
以常规方法进行细胞免疫染色,通过细胞流式术进行标志物分析。阳性标志物:CD90、CD105、CD73阳性率需达到95%以上;阴性标志物:CD19、CD14、CD34、CD45、HLA-DR阳性率需在2%以下;同时分别以对应的同型对照作为参照。
使用BD FACSCanto II进行细胞流式术分析。
检测结果参见图3~10所示,结果显示,采用本实施例1的间充质干细胞培养增强剂获得的原代细胞,可保持间充质干细胞特性不改变,CD90、CD105、CD73阳性率均在95%以上,CD19、CD14、CD34、CD45、HLA-DR阳性率均在2%以下。
实施例3:本实施例提供多种间充质干细胞培养增强剂添加配比的效果验证。
一、实验方法:
培养基的配制:
表1
编号 | 完全培养基与间充质干细胞培养增强剂配比 |
M1 | 100:0 |
M2 | 99.9:0.1 |
M3 | 99.5:0.5 |
M4 | 99:1 |
M5 | 97.5:2.5 |
M6 | 95:5 |
M7 | 90:10 |
根据实施例1的技术方法配制以上培养基,具体配比见上表1。
二、实验方法和结果:
1、植块法脐带原代间充质干细胞的分离,参见实施例2:
实验设置6个实验组(M2~M7)进行对比测试,M1为对照组,每组设置2个重复组。在分离第16天收获原代细胞,进行细胞收获量计数分析。
2、检测结果:
表2
编号 | 活细总数 |
M1 | 6.02E+05 |
M2 | 6.65E+05 |
M3 | 7.25E+05 |
M4 | 9.99E+05 |
M5 | 7.17E+05 |
M6 | 4.80E+05 |
M7 | 1.27E+05 |
按上述实施例检测,如表2脐带原代分离细胞数计数结果所示:在商业化无血清培养基基础上添加本发明所述的间充质干细胞培养增强剂可有效提高脐带原代分离效率,特别是在添加量为1%时,效果最佳,细胞收获量提高50%以上;使用剂量在0.5%~2.5%之间,均显示出促进原代分离的效果,细胞收获量提高19%以上;添加剂量超过5%时,产生毒性作用,减弱了分离效果。
需要说明的是,在本文中,诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来,而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (5)
1.一种间充质干细胞培养增强剂,其特征在于,由以下成分组成,具体为:维生素E0.5mg/mL、聚乙烯醇2.5mg/mL、右旋糖酐40 0.5g/mL、α-酮戊二酸5mg/mL、脂多糖1µg/mL、β-巯基乙醇150mg/mL、硫辛酸20mg/mL、还原性谷胱甘肽30mg/mL、腐胺1g/mL、孕酮0.6mg/mL、rhNRG1 10µg/mL、rhBMP-4 50µg/mL。
2.根据权利要求1所述的一种间充质干细胞培养增强剂,其特征在于,所述的间充质干细胞培养增强剂与间充质干细胞增生无血清完全培养基的体积比为0.1:99.9~2.5:97.5。
3.根据权利要求2所述的一种间充质干细胞培养增强剂,其特征在于,所述的间充质干细胞培养增强剂与间充质干细胞增生无血清完全培养基的体积比为1:99。
4.根据权利要求1~3中任一所述的一种间充质干细胞培养增强剂的应用,其特征在于,该间充质干细胞培养增强剂用于间充质干细胞的培养。
5.根据权利要求4所述的一种间充质干细胞培养增强剂的应用,其特征在于,所述的间充质干细胞为人脐带间充质干细胞。
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