CN113774019A - 一种脐带血间充质干细胞无血清培养基、培养方法及其应用 - Google Patents
一种脐带血间充质干细胞无血清培养基、培养方法及其应用 Download PDFInfo
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- CN113774019A CN113774019A CN202110919053.3A CN202110919053A CN113774019A CN 113774019 A CN113774019 A CN 113774019A CN 202110919053 A CN202110919053 A CN 202110919053A CN 113774019 A CN113774019 A CN 113774019A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了CDCM培养基及其应用,所述CDCM培养基包括所述CDCM培养基包括α‑MEM培养基、胰岛素、硒元素、L‑抗坏血酸、b‑FGF、EGF、TGF‑β、氢化可的松、IGF、肝素、NaHCO3和转运蛋白。本发明还公开了脐带血间充质干细胞的培养方法。本发明使用了化学成分确定的培养基对细胞进行培养。该化学成份确定的培养基能够稳定的维持干细胞生长,实现脐带血间充质干细胞的大量扩增。本发明优化培养皿包被蛋白基质材料,有效支持了脐带血间充质干细胞在CDCM中的生长。本发明最显著的优点是,解决了常规用一定浓度的胎牛血清培养带来的各种隐患或自体血浆带来的规模化扩增受限等问题,对细胞治疗领域意义重大。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种脐带血间充质干细胞无血清培养基、培养方法及其应用。
背景技术
近年来干细胞治疗取得了猛进的发展,作为一种替代医学方案,效果已经得到了普遍认可。但是,大部分试验表明MSCs归巢能力和分化能力都较弱小,对疾病往往存在效果不明显,个体疗效差异大等问题。造成上述结果的原因主要有:第一,移植的干细胞数量不足;第二,即便获得了足够量的干细胞,但在体外培养扩增过程当中,干细胞负载了基因内突变或基因外修饰状态的改变,可能不能正常行使其功能;第三,受体的体内环境不能支持移植的干细胞归巢定植,妨害干细胞发挥功能,或加速了干细胞的衰老。因此,要达到干细胞移植的理想治疗效果,需要满足两个条件:足够量的功能良好的成体干细胞和足够支持移植干细胞发挥功能的受体体内微环境。在上述两个条件中,大量获取高质量的年轻态成体干细胞是决定性的。
采集成体干细胞应该尽量从最年轻的机体的组织中采取。这是分离获得高质量年轻态干细胞的基本保障。毫无疑问,最年轻的机体是刚刚分娩的胎儿。目前已经有较为成熟的技术从胎儿的组织,比如脐带、脐血、胎盘和羊膜等处中提取间充质干细胞。其中脐血间充质干细胞有特殊的优势。具体表现在:1)与其他来源间充质干细胞相比,脐血间充质干细胞由于其是最年轻的细胞,干性最强,同质性更强,质量可控性更高,更适合做细胞药物。2)脐血间充质干细胞来源于新生儿组织,它和来源于成体组织的其它间充质干细胞相比,免疫原型较弱,适应性更强,干细胞细胞活性和免疫调节能力也更好。3)脐血间充质干细胞可广泛应用于是皮炎、关节炎和细胞因子风暴(如新冠肺炎重症患者引起病情恶化的“炎症风暴”)等免疫过激型疾病和糖尿病足、冻疮等血管坏死型疾病4)脐血间充质干细胞来源于血液组织,它和来源于新生儿的脐带或胎盘组织的间充质干细胞相比,在分化潜能和分泌因子方面有所不同。我们自己的研究团队已经通过小RNA深度测序和细胞外因子分泌谱两个角度进行了较为深入研究。
然而,脐血间充质干细胞的分离和培养过程仍然需要使用胎牛血清(CN201210437524.8一种高效分离和扩增人脐带血间充质干细胞的方法)或自体脐带血富血小板血浆(202011578876.6一种脐带血间充质干细胞培养方法),给脐血间充质干细胞的最终应用带来安全隐患,而且受限于胎牛血清的批次质量不稳定或自体血浆有限的收集体积,难以进行质量可控的规模化制备。
化学成份限定的培养基能够标准化生产制备,如能使用特殊配方的化学成分限定的适用于脐血间充质干细胞的培养基,实现大规模扩增,可以避免传统方法中存在的血清质量批次不稳定,引入外来风险因素等问题,使得我们能够生产出质量可控、均一稳定的临床级脐血间充质干细胞,充分应用脐血间充质干细胞本身的优势,将极大的促进影响人类寿命的最主要因素-难治疾病的治疗,产生巨大的社会和经济价值。
发明内容
发明目的:本发明所要解决的技术问题是提供了一种脐带血间充质干细胞无血清培养基(CDCM)培养基。
本发明还要解决的技术问题是提供了一种脐带血间充质干细胞的培养方法。
本发明最后要解决的技术问题是提供了所述的培养基、所述的方法培养获得的脐带血间充质干细胞在制备降低血糖方面的药物中的应用。
技术方案:本发明提供一种CDCM培养基,所述CDCM培养基包括α-MEM 培养基、以α-MEM培养基为基准计算,还包括15~25mg L-1胰岛素、10~20μg L-1硒元素、55~75mg L-1L-抗坏血酸、5~25μg L-1b-FGF、5~25μg L-1EGF、1~5μg L-1TGF-β、200~500μg L-1氢化可的松、10~30μg L-1IGF、15~30mg/L肝素、 450~500mg L-1NaHCO3和250~400mg L-1转运蛋白。
本发明内容还包括一种脐带血间充质干细胞的培养方法,包括以下步骤:
1)收集脐带血样品;
2)分离脐带血中单核细胞成分:将步骤1)的脐带血样品均经过肝素抗凝,羟甲基纤维素裂解红细胞,密度梯度离心获取单核细胞成分之后接种于包被蛋白基质成分的培养皿;
3)分离间充质干细胞:在步骤2)的培养皿中加入所述的CDCM培养基培养,及时除去未贴壁细胞并更换培养基;
4)培养和扩增间充质干细胞:继续使用所述的CDCM培养基培养,每5-8 天换液一次,直至细胞为80-95%融合;细胞用胰酶进行消化,接种于一般培养板,使用所述的CDCM培养基继续培养,2-4天换液一次,直至融合,并进行下一次传代,实现间充质干细胞的扩增。
其中,所述脐带血样品为医院采集脐带血样品或自脐带血库中获取的人脐带血样品,或在无菌条件下获取正常或早产胎儿的脐带血样品。
其中,所述步骤2)中的蛋白基质成分包括明胶蛋白、层连蛋白、副纤维连接蛋白、纤连蛋白按优选体积比1~5∶1~5∶0.5~2∶0.5~2混合。
其中,所述步骤2)具体包括以下处理步骤:首先使用同体积α-MEM培养基稀释抗凝过的脐带血,与4-6g/L的甲基纤维素混合,静置20-40分钟,沉降红细胞,吸取上清,离心后,用PBS制成单细胞悬液,叠加到相对密度在1-1.1的淋巴细胞分离液上,在2000-3000转/分钟离心15-30分钟,取界面层,加PBS 制成单核细胞悬液,离心洗涤。
本发明内容还包括所述的培养基、所述的方法培养获得的脐带血间充质干细胞在制备降低血糖方面的药物中的应用。
本发明内容还包括所述的培养基、所述的方法培养获得的脐带血间充质干细胞在制备治疗糖尿病药物中的应用。
有益效果:与现有技术相比,本发明具备以下优点:本发明使用了化学成分确定的培养基对细胞进行培养。该化学成份确定的培养能够稳定的维持干细胞生长,实现脐带血间充质干细胞的大量扩增。同时了本发明在以往专利的基础上 (CN201210437524.8一种高效分离和扩增脐带血间充质干细胞的方法)进一步优化了培养皿包被蛋白基质材料,有效支持了脐带血间充质干细胞在CDCM中的生长。本发明最显著的有益效果是,解决了现有技术常规用一定浓度的胎牛血清培养带来的各种隐患或自体血浆带来的规模化扩增受限等问题,对细胞治疗领域意义重大。
附图说明
图1为本发明培养脐带血间充质干细胞20天后的结果;
图2为三种培养体系所获得的细胞形态;
图3为糖尿病小鼠注射脐带血间充质干细胞后的血糖浓度变化。
具体实施方式
本发明的α-MEM培养基为常规商业购买产品。
实施例1培养基CDCM-1和包被的蛋白基质的制备
本发明提出使用化学成份限定的培养基CDCM,以及于此配合的蛋白基质包被的培养板。CDCM培养基成份为:α-MEM培养基以及添加成份,以α-MEM 培养基为基准计算(体积),添加成份的添加量为19.4mg L-1胰岛素、14μg L-1硒元素、64mg L-1L-抗坏血酸、10μg L-1b-FGF、10μg L-1EGF、2μg L-1TGF- β、362μg L-1氢化可的松、20μg L-1IGF、22.5mg/L肝素、543mgL-1NaHCO3和310.7mg L-1Transferrin(美国默克公司,T3309);
包被的蛋白基质为相同浓度的明胶蛋白、层连蛋白、副纤维连接蛋白、纤连蛋白按体积比3∶1∶0.5∶0.5混合。
实施例2培养基CDCM-2的制备及相应包被蛋白基质
本发明提出使用化学成份限定的培养基CDCM,以及于此配合的蛋白基质包被的培养板。在本发明提出的权利要求保护范围内,调整培养基各成份具体浓度,配制得CDCM-2:α-MEM培养基以及添加成份,以α-MEM培养基为基准计算 (体积),添加成份添加量为25mgL-1胰岛素、20μg L-1硒元素、55mg L-1L-抗坏血酸、25ug L-1b-FGF、25μg L-1EGF、5μg L-1TGF-β、200μg L-1氢化可的松、 10ug L-1IGF、30mg L-1肝素、500mg L-1NaHCO3和250L- 1Transferrin;包被的蛋白基质为相同浓度的明胶蛋白、层连蛋白、副纤维连接蛋白、纤连蛋白按优选的体积比1∶5∶2∶0.5混合。
实施例3培养基CDCM-3的制备及相应包被蛋白基质
本发明提出使用化学成份限定的培养基CDCM,以及于此配合的蛋白基质包被的培养板。在本发明提出的权利要求保护范围内,调整培养基各成份具体浓度,配制得CDCM-2:α-MEM培养基以及添加成份,以α-MEM培养基为基准计算(体积),添加成份的添加量为15mgL-1胰岛素、10μg L-1硒元素、75mg L-1L- 抗坏血酸、5ug L-1b-FGF、5μg L-1EGF、1μg L-1TGF-β、500μg L-1氢化可的松、 30ug L-1IGF、15mg L-1肝素、450mg L-1NaHCO3和400mg L- 1Transferrin;包被的蛋白基质为相同浓度的明胶蛋白、层连蛋白、副纤维连接蛋白、纤连蛋白按优选的体积比5∶1∶0.5∶2混合。
实施例4脐带血间充质干细胞的制备
在无菌条件下获取正常或早产胎儿的脐带血,25-200ml,肝素抗凝。脐带血间充质干细胞的分离纯化过程在分娩后24小时内进行。
使用同体积α-MEM稀释抗凝过的脐带血,与5g/L的甲基纤维素按4∶1混合,静置30分钟,沉降红细胞。吸取上清,离心后,用PBS制成单细胞悬液,叠加到相对密度1.077的淋巴细胞分离液上,2500rpm离心20min,取界面层,加PBS制成单细胞悬液,离心洗涤分别在实施例1配置的CDCM-1、实施例2 配置的CDCM-2和实施例3配置的CDCM-3中培养。将细胞加入蛋白基质包被的细胞培养板上。细胞。细胞达到90%融合时,进行传代。传代时,使用普通培养皿,并在上述CDCM培养基中进行细胞的扩增。
包被细胞培养板:将明胶蛋白、层连蛋白、副纤维连接蛋白和纤连蛋白按照说明分别稀释至2mg/ml按照实施例1-3相应的体积比混合后,转移到细胞培养板,室温下放置1小时。在无菌环境下,可在4℃存3个月。
分离和扩增脐带血间充质干细胞:将在无菌条件下采集的脐带血以体积比 1∶1与细胞在α-MEM混合稀释,与5g/L的甲基纤维素按4∶1混合,静置30分钟,沉降红细胞。吸取上清,离心后,用PBS制成单细胞悬液,叠加到相对密度1.077 的淋巴细胞分离液Ficoll-Hypaque(美国Sigma公司)上,2500rpm离心20min,取界面层,加PBS制成单细胞悬液,离心洗涤。
因为Ficoll-Hypaque的比重为1.077g/ml,其比单核细胞重但比红细胞轻,因此可以将单核细胞与残留的红细胞分离。收集界层面即可收集到比较纯的单核细胞。
将获取的单核细胞用PBS稀释,2000rpm,离心10min,去掉上清液,并加入新的PBS打散,稀释。以同样的条件再洗涤一次,可见沉积于离心管底部的细胞。
随后,使用实施例1配置的CDCM-1、实施例2配置的CDCM-2或实施例3 配置的CDCM-3均匀打散收集到的细胞。细胞打散后,接种于包被过的细胞培养板,7天后更换培养基。洗涤培养板,除去未贴壁细胞。继续使用添加10%胎牛血清的脐带血间充质干细胞培养基培养,每7天换液一次,直至细胞接近90%融合。
将90%融合细胞用胰酶进行消化,接种于一般培养板,使用与原带培养时相同的CDCM-1、CDCM-2或CDMC-3培养,3天换液一次。直至融合,并进行下一次传代,直至传至第25代。三种培养体系下,细胞倍增时间比较如图1。如图可知,CDCM-1培养环境下,细胞增殖达到了惊人的40代,才出现了倍增时间的明显延长,而CDCM-2在第30代时倍增时间已经达到了82小时,出现了衰老迹象;CDCM-3培养环境在细胞传至第25代时生长缓慢,并很快衰老。第三代时三种培养体系所获得的细胞形态如图2所示。如图表明,在培养至第三代时,CDMC-1、CDMC-2和CDMC-3体系均能较好的维持细胞的状态。
实施例5培养的间充质干下表的细胞表面抗原表征
为了确定实施例4的上述三种培养体系所获得的具有间充质干细胞表面抗原的特征,使用FACs对细胞表面标记物CD34、CD45、CD3、CD73、CD105、 CD9进行分析。结果显示于表1。
表1
表1证实,对于本发明所分离和培养的干细胞,低表达CD34、CD45及CD3,而高表达CD73、CD105及CD90。此结果表明本发明所分离并扩增的细胞是间充质干细胞。
实施例6
应用20周龄雄性非肥胖糖尿病(NOD)小鼠,体重24~27g。在12小时的光照和12小时的黑暗周期中饲养。实施实验时,NOD小鼠分为两组,每组6-8 只。血糖自发增加至750-810mg/dl后,将小鼠束缚住,并通过脾动脉注射悬浮在0.1ml生理盐水中的由实施例4制备的1×106脐带血来源的间充质干细胞。2 周后进行第二次注射,即脾动脉注射悬浮在0.1ml生理盐水中的1×106脐带血来源的间充质干细胞。对照组进行相同的程序,但仅注射生理盐水(假手术组)。第二次注射后,每三天自眼眶采血一次,使用血糖检测仪记录血糖水平,观察血糖恢复情况。血糖变化情况如图3所示,注射实施例4制备的脐带血来源的间充质干细胞后的第21天,细胞处理组小鼠糖尿病模型的血糖水平已经显著低于仅注射生理盐水的对照组。细胞处理30天后,糖尿病小鼠模型的血糖水平已经从第零天时的791mg/dl降至482mg/dl;而生理盐水注射的对照组从第零天的 809mg/dl升到了880mg/dl,展示了实施例4制备的脐带血来源的间充质干细胞具有成为降低糖尿病血糖水平的细胞类药物的能力,从而证明了本发明。
Claims (8)
1.一种CDCM培养基,其特征在于,所述CDCM培养基包括α-MEM培养基、以α-MEM培养基为基准,还包括15~25mg L−1胰岛素、10~20μg L−1硒元素、55~75 mg L−1L-抗坏血酸、5~25μg L− 1b-FGF、5~25μg L−1EGF、1~5μg L−1TGF-β、200~500μg L−1氢化可的松、10~30μg L−1IGF、15~30mg/L肝素、450~500mg L−1 NaHCO3和250~400mg L−1转运蛋白。
2.权利要求1所述的CDCM培养基在脐带血间充质干细胞的培养中的应用。
3.一种脐带血间充质干细胞的培养方法,其特征在于,包括以下步骤:
1)收集脐带血样品;
2)分离脐带血中单核细胞成分:将步骤1)的脐带血样品均经过肝素抗凝,羟甲基纤维素裂解红细胞,密度梯度离心获取单核细胞成分之后接种于包被蛋白基质成分的培养皿;
3)分离间充质干细胞:在步骤2)的培养皿中加入权利要求1所述的CDCM培养基培养,及时除去未贴壁细胞并更换培养基;
4)培养和扩增间充质干细胞:继续使用权利要求1所述的CDCM培养基培养,每5-8天换液一次,直至细胞为80-95%融合;细胞用胰酶进行消化,接种于一般培养板,使用权利要求1所述的CDCM培养基继续培养,2-4天换液一次,直至融合,并进行下一次传代,实现间充质干细胞的扩增。
4.根据权利要求3所述的脐带血间充质干细胞的培养方法,其特征在于,所述脐带血样品为医院采集脐带血样品或自脐带血库中获取的人脐带血样品,或在无菌条件下获取正常或早产胎儿的脐带血样品。
5.根据权利要求3所述的脐带血间充质干细胞的培养方法,其特征在于,所述步骤2)中的蛋白基质成分包括明胶蛋白、层连蛋白、副纤维连接蛋白、纤连蛋白按体积比1~5:1~5:0.5~2:0.5~2混合。
6.根据权利要求3所述的脐带血间充质干细胞的培养方法,其特征在于,所述步骤2)具体包括以下处理步骤:首先使用同体积α-MEM培养基稀释抗凝过的脐带血,与4-6g/L的甲基纤维素混合,静置20-40分钟,沉降红细胞,吸取上清,离心后,用PBS制成单细胞悬液,叠加到相对密度在1-1.1的淋巴细胞分离液上,在2000-3000转/分钟离心15-30分钟,取界面层,加PBS制成单核细胞悬液,离心洗涤。
7.权利要求1所述的培养基、权利要求3-6任一项所述的方法培养获得的脐带血间充质干细胞在制备降低血糖方面的药物中的应用。
8.权利要求1所述的培养基、权利要求3-6任一项所述的方法培养获得的脐带血间充质干细胞在制备治疗糖尿病药物中的应用。
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