CN106929470A - 一种用于间充质干细胞体外培养和扩增的无血清培养基 - Google Patents
一种用于间充质干细胞体外培养和扩增的无血清培养基 Download PDFInfo
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Abstract
一种用于间充质干细胞体外培养和扩增的无血清培养基。本发明属于生物技术领域，为一种化学成分明确的，用于间充质干细胞体外培养和扩增的无血清培养基。本发明在基础培养基中添加生长因子、胰岛素、油酸、转铁蛋白(不含铁)、亚硒酸钠、牛血清白蛋白、抗氧化剂(β‑巯基乙醇)地塞米松、庆大霉素，能使间充质干细胞帖附于培养板基质，在无血清条件下实现体外培养和扩增，并维持间充质干细胞的特性—多向分化潜能，在体外诱导培养基处理下能够被诱导成脂肪细胞、成骨细胞和软骨细胞。本发明能够有效地避免因使用含血清培养基培养的细胞产品带来的潜在危险。
Description
技术领域
本发明涉及生物技术领域的一种细胞培养基，是一种用于间充质干细胞体外培养和扩增的无血清培养基。
背景技术
间充质干细胞(mesenchymal stem/stromal cell，MSCs)是起源于中胚层的一类成体干细胞；其同样具有多向分化潜能，在体内外适宜条件下，间充质干细胞可以分化为成骨细胞，软骨细胞、脂肪细胞、成纤维细胞、肌细胞等；由于其高效的分化潜能使其临床应用备受人们关注，现已经被用于多种疾病的治疗研究。MSCs可以通过分泌大量生物活性分子包括多种生长因子、细胞因子(cytokine)和趋化因子(chemokine)等，通过对所处微环境的影响来调控自身的各种基因的表达进而通过旁分泌的方式来影响周围的细胞，发挥调控作用进而刺激受损细胞修复并抑制炎症；且越来越多的研究表明，MSCs通过特定的分化途径取代那些受损的细胞可能仅是MSCs发挥了其治疗作用的一小部分功能而已；在机体受损如发生炎症和/或肿瘤时，自身或外源的间充质干细胞在一系列归巢信号的引导下，可以自发的定向趋化性迁移到达损伤部位并定植存活发挥调节与组织修复作用，向缺血或损伤组织归巢是MSCs重要特征，而不会考虑是否是相关的组织；众多研究均表明，MSCs归巢的数量与疗效紧密相连，但归巢的数量与移植的数量并无相关性，因此，MSC的特异性归巢与植入靶组织的能力和效率，是其对疾病有效性的关键；且大量的文献表明，MSCs可以与各种不同的免疫细胞如T淋巴细胞、自然杀伤性细胞等相互作用，影响细胞表型及调控细胞的增殖分化等达到调节免疫系统的功能；由于其高效的多向分化潜能和免疫抑制功能等优越特性，在过去的几十年中，间充质干细胞治疗作为一种新的可治疗多种疾病的治疗方法，对它的临床应用已经取得长足的进步,目前已经有部分产品得到一些国家食品药品管理局的批准，用于不同疾病的治疗。
由间充质干细胞在正常成体组织或器官包括但不局限于骨髓、脐带、脂肪组织、心脏、肝脏、十字韧带、眼睑、牙髓等部位；研究表明，这些低量的由于仅仅通过自体或异体分离的间充质干细胞无法满足临床或科研需求，需要对间充质干细胞进行体外培养且扩增以满足治疗或科研需要；通常体外培养和扩增间充质干干细胞需要在培养基中加入10％-20％的胎牛血清以满足间充质干细胞生长且保持其生物学特性。使用胎牛血清虽然可以满足间充质干细胞的正常生长增殖还能提供贴壁因子以满足贴壁依赖细胞的贴壁生长等；但使用动物血清弊端随着治疗研究的开展也越渐明显；动物血清存在成分不明确的未知蛋白为科研和治疗带来很大的干扰；且往往批次不同，动物血清之间的成分差异也大，稳定性较差，对研究的稳定性影响较大，动物血清还可能携带未知的感染物和具有免疫原性的异种蛋白将对治疗带来极大的威胁等；综上，为了能更好的在体外培养和扩增间充质干细胞，克服动物血清带来的克重弊端，在细胞培养过程中寻找FBS等的替代物或采用无血清培养条件培养，取消非人产品的使用来降低潜在的风险是有必要的；学者们很早就开始了对无血清的间充质干细胞培养基的研究。
发明内容
本发明克服现有培养技术的不足，避免了含动物血清的培养基对细胞产品带来的潜在风险，为体外培养和扩增间充质干细胞提供了一种化学成分明确的无血清培养基。
根据上述培养基的需求，本发明用于体外培养和扩增间充质干细胞无血清，其特征在于，本发明是一种水溶液，其组分包含如下：
所述的基础培养基为α-MEM、DMEM、IMDM/F12(体积比：IMDM:F12＝1:1)、DMEM/F12(体积比：DMEM:F12＝1:1)的培养基中的一种。
所述的培养基的细胞因子中，碱性成纤维细胞生长因子(bFGF)为培养间充质干细胞所必需成分，成纤维细胞生长因子(EGF)、表皮生长因子(TGFβ1)和血小板衍生因子(PDGF-BB)可任选两种组分组合配置。
所述的胰岛素添加量为1-20ug/mL，可为临床级用药或重组蛋白。
所述的微量元素以亚硒酸钠化合物形式提供，亚硒酸钠添加量为10～50uM。
所述培养基的抗氧化剂为巯基乙醇、过氧化氢酶、超氧化物歧化酶、亚硒酸钠的一种或几种。所述组分中的10～50uM的巯基乙醇和10～50uM的亚硒酸钠可被50-150U/ml的氧化氢酶、或50-150U/ml的超氧化物歧化酶所替代，或多种组合使用。
所述的α-MEM、DMEM、IMDM与Ham’s F12、DMEM与Ham’s F12基础培养基的组分在已有的文献中已有公开报道，这里不再赘述。
其中各组分及其作用如下表：
本发明的有益效果为：体外培养和扩增间充质干细胞培养基既要能满足细胞正常的生长增殖，又要能维持其多向分化潜能。培养基中各营养成分需要合理均衡，以满足细胞生长、代谢和功能维持的需求。碳水化合物、氨基酸、无机盐、激素生长因子、微量元素等众多成分需要按照一定的比例添加，过多过少都会对细胞的生长状态造成不良影响。因此在体外培养过程中，本发明为了维持间充质干细胞的多向分化潜能，在培养基中加入适当比例的碱性成纤维细胞生长因子、表皮生长因子或转化生长因子等，在促进细胞正常生长增殖的同时又维持了间充质干细胞的多向分化潜能特性。利用本发明培养扩增的间充质干细胞在体外诱导培养基处理下能分化为脂肪细胞、成骨细胞和软骨细胞，且能够有效地避免因使用含血清培养基培养的细胞产品带来的潜在危险。
附图说明
图1中1.1－1.4是本发明中无血清培养基培养脂肪间充质干细胞至汇合的实物图(其中，左侧图均为显微镜下4X放大的细胞状态实物图，右侧图均为显微镜下10X放大的细胞状态实物图)；
其中；图1.1脂肪间充质干细胞分离培养第2天时细胞状态实物图；
图1.2脂肪间充质干细胞分离培养第5天时细胞状态实物图；
图1.3脂肪间充质干细胞分离培养第8天时细胞状态实物图；
图1.4脂肪间充质干细胞分离培养第12天时细胞状态实物图；
图2是本发明中无血清培养基和DMEM+10％FBS培养基培养的脂肪间充质干细胞同时培养十天的生长形态学的比较实物图(左、右侧图均为显微镜下4X放大的细胞状态实物图)；
图3是RTCA实时无标记细胞分析仪监测本发明中无血清培养基和DMEM+10％FBS培养基培养的脂肪间充质干细胞的细胞生长速率的示意图；
图4是本发明中无血清培养基培养脐带间充质干细胞至汇合的实物图(三图均为显微镜下10X放大的细胞状态实物图)；
图5是流式细胞仪检测本发明中无血清培养基培养的脂肪间充质干细胞表面标志物示意图；
图6是采用本发明的无血清培养基培养扩增后诱导脂肪、成骨和软骨分化后的染色图(三图均为显微镜下10X放大的细胞状态实物图)。
具体实施方式
下面结合附图说明就具体实施方式，进一步阐明本发明，应理解这些实施方式仅用于说明本发明而不用于限制本的、发明的范围，在阅读了本发明之后，本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所限定的范围。
为了理解本发明，下面对本发明进行进一步的详细说明。
以下实施例给出本发明用于间充质干细胞体外培养和扩增的无血清培养基的具体实施方式，但本发明不限于这些实施例。
实施例1
以DMEM为基础培养基，其组分如下，
将上述组分溶于超纯水或三蒸水中，在室温(18-25℃)情况下搅拌溶解，并用0.5mol/L的盐酸调节pH至7.1±0.1，定容至1L，0.22um滤膜过滤除菌后，即可用于间充质干细胞体外培养和扩增。
以脂肪间充质干细胞为例：
脂肪间充质干细胞(ADMSCs)体外培养：将脂肪组织置于75cm2细胞培养瓶中，加入等体积的内含1％双抗(青霉素和链霉素)和5ug/ml的两性霉素B的PBS(磷酸盐缓冲液)，旋紧瓶盖，剧烈摇晃5-10s后，静置，使脂肪组织与PBS分层，脂肪组织位于上层，随后移去PBS。上述步骤反复3次以上，直至下层PBS清亮无血色时停止清洗。在清洗好的脂肪组织中加入等体积的I型胶原酶，添加DNase I(DNA酶1型)，使得DNase I的终浓度为10ug/mL；在所述三者混合溶液中再添加1％的双抗和5ug/ml的两性霉素B，剧烈摇晃5-10s后，放入37℃水浴锅中消化1-2h,消化过程中每10分钟取出培养瓶剧烈摇晃5-10s，使脂肪组织与I型胶原酶充分接触；直至脂肪组织呈现汤状时停止，加入等体积的上述配制的无血清培养基，终止消化；静置5min，待其分层后，弃掉上层未消化的脂肪组织，将下层的细胞悬液转移到50ml离心管中在400g条件下离心10min，离心结束后去除上清溶液；将留置于离心管中的细胞团用20ml配制好的红细胞裂解液重悬，并于室温(18-25℃)下孵育10min,其中在孵育5min时重新混匀细胞与红细胞裂解液，以使红细胞完全破裂；随后于300g条件下离心10min；将离心后的细胞去除上清，加入5mL的清洗液，吹打重悬后，放置1min，使还未消化的微小组织块沉降，然后将细胞悬液用70um过滤网过滤；随后收集过滤后的滤液，以400g条件下离心10min,弃去上清；加入10ml上述配制的无血清培养基重悬，取出10uL的细胞悬液于细胞计数板中计数，然后调整细胞浓度至1×105个/mL；将调整好的细胞接种到25cm2的细胞培养瓶中，于37℃、5％CO2、100％湿度条件下常规培养，每3天换一次无血清培养基，10天后即可长至90％的汇合度，此时可进行细胞传代培养。
细胞生长速率检测：将RTCA仪器淋洒75％酒精消毒后放入CO2培养箱中；向E-Plate检测板中分别添加DMEM+10％FBS培养基及上述无血清培养基并测定两种培养基的背景阻抗值；收集对数期细胞并计数，调整细胞悬液浓度，分别加入5000个细胞到含有两种培养基(上述配置的无血清培养基培养和DMEM+10％FBS培养基)的E-Plate检测板中，每种培养基设置2个平行，并将检测板放入检测台上，进行实时动态细胞增殖检测；实时监测72小时后，停止监测绘制生长曲线。
上述实施例1的培养过程详见附图1至附图3。
实施例2
以DMEM/F12为基础培养基，其组分如下：
将上述组分溶于超纯水或三蒸水中，在室温(18-25℃)情况下搅拌溶解，，并用0.5mol/L的盐酸调节pH至7.1±0.1，定容至1L，0.22um滤膜过滤除菌后，即可用于间充质干细胞体外培养和扩增。
以脐带间充质干细胞为例：
脐带间充质干细胞(UCMSCs)体外培养：从采集瓶中取出脐带，将脐带放置于预先添加有无菌生理盐水的培养皿中，洗去脐带残留血液；将洗净的脐带用经高压消毒灭菌的剪刀将脐带剪成2-3cm的若干小段，再次漂洗，纵行剖开脐带，剔除1条脐静脉、两条脐动脉，剥离出华通氏胶(Wharton'Jelly)，再次用剪刀将华通胶剪碎成1mm3的若干小组织块，转移至细胞培养瓶中，加入上述配置的间充质干细胞无血清培养基，加入的无血清培养基刚好没过组织块即可；于5％CO2、37℃培养箱中静置培养3天；每3天需更换一次新鲜的无血清培养基，14天后细胞汇合度达到85％时消化收获细胞。
流式细胞仪检测UCMSCs表型：利用上述配制的无血清培养基培养传代到第3代的脐带间充质干细胞进行流式细胞术检测，当第3代的细胞长到85-90％汇合度时，采用常规传代方法收集细胞，用PBS重悬细胞，计数，并将细胞调整到2X106个/mL。分别在5个流式管中添加50uL的细胞；采用单染法，分别添加3种不同荧光标记的5uL的阳性抗体(CD73,CD90和CD105)、20uL的阴性抗(CD11b or CD14,CD34,CD45,CD19or CD79α,HLA-DR)孵育30分钟，加入500ul预冷的PBS洗涤，300g条件下离心8min后，弃上清用100ulPBS重悬，上机检测。
根据ISCT的标准，脂肪间充质干细胞至少需要满足阳性表达CD73,CD90and CD105三种表面标志物，且纯度要求在95％以上，不表达造血系统来源的表面标志物CD11b orCD14,CD34,CD45,CD19or CD79α,HLA-DR，并且表达这些表面标志物的细胞数量应控制在2％以下。
UCMSCs分化能力检测：利用上述配制的无血清培养基培养传代到第3代的细胞进行流式细胞术检测，当第3代的细胞长到85-90％汇合度时，采用常规传代方法收集细胞，用PBS重悬细胞，计数，并将细胞调整到1×105个/mL并分别接种至6孔板上培养。当长至80％汇合度时，将常规的完全培养基替换成分化培养基。继续培养1个月，每3天换液。1个月后，去除培养板中培养基，用PBS清洗，去除PBS后每孔添加1mL的4％多聚甲醛于常温下孵育15-30min。然后去除多聚甲醛，灭菌水清洗。
成脂分化染色：添加1000uL的Oil Red O染色液于细胞培养板中，室温孵育5min。去除染色液，灭菌水至少清洗3次以除去未结合的染料。苏木精染色10min后，去除染液，并用灭菌水清洗。倒置显微镜下观察染色情况，脂滴会被染成红色，核质染成蓝色或紫色。
成骨分化染色：添加1000uL的Alizarin Red S染色液于细胞培养板中，并室温孵育5min。去除染液，灭菌水充分清洗至少3次以除去未结合的染料。倒置显微镜下观察染色情况，分化后的细胞外基质存在大量的钙质，染色后细胞被染成红色。
成软骨分化染色：添加1000uL的Alcian Blue染色液于细胞培养板中，并室温孵育5min。去除染液，灭菌水充分清洗至少3次以除去未结合的染料，倒置显微镜下观察染色情况，分化后的细胞外基质存在大量氨基萄聚糖，被染成蓝色。
上述实施例2的培养过程详见附图4至附图6。
Claims (10)
1.一种用于间充质干细胞体外培养和扩增的无血清培养基，其特征在于，所述的无血清培养基是一种组分明确的水溶液，其组分包含如下：
2.根据权利要求1所述的一种用于间充质干细胞体外培养和扩增的无血清培养基，其特征在于，所述的基础培养基是α-MEM、DMEM、IMDM/F12培养基和DMEM/F12培养基中的任意一种的培养基。
3.根据权利要求1所述的一种用于间充质干细胞体外培养和扩增的无血清培养基，其特征在于，所述的碱性成纤维细胞生长因子为培养和扩增间充质干细胞所必需成分，所述的表皮生长因子、转化生长因子1和血小板衍生因子可任选两种或者两种以上组分组合配置。
4.根据权利要求1所述的一种用于间充质干细胞体外培养和扩增的无血清培养基，其特征在于，所述的胰岛素可为临床级用药或重组蛋白。
5.根据权利要求1所述的一种用于间充质干细胞体外培养和扩增的无血清培养基，其特征在于，所述的β-巯基乙醇、亚硒酸钠均为抗氧化剂；所述的抗氧化剂还包括过氧化氢酶、超氧化物歧化酶；
所述组分中的10～50uM的β-巯基乙醇和10～50uM的亚硒酸钠可被50-150U/ml的氧化氢酶、或50-150U/ml的超氧化物歧化酶所替代使用；
所述的抗氧化剂可以是上述四种化合物中的一种或几种的组合配置。
6.根根据权利要求1所述的一种用于间充质干细胞体外培养和扩增的无血清培养基，其特征在于，所述的亚硒酸钠化合物提供微量元素硒。
7.权利要求1-6任一项所述的一种培养间充质干细胞的培养基在培养间充质干细胞中的应用。
8.根据权利要求7所述的应用，其特征在于：所述的间充质干细胞来源于人体脂肪、脐带或脐带血，也包括来源于人体其它组织或动物组织的间充质干细胞。
9.根据权利要求1所述的一种用于间充质干细胞体外培养和扩增的无血清培养基，其特征在于,以DMEM为基础培养基，其组分如下：
10.根据权利要求1所述的一种用于间充质干细胞体外培养和扩增的无血清培养基，其特征在于,以DMEM/F12为基础培养基，其组分如下：
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| CN109161520A (zh) * | 2018-08-17 | 2019-01-08 | 北昊干细胞与再生医学研究院有限公司 | 间充质干细胞的分离培养方法及相应的保存方法和无血清培养基 |
| CN110331130A (zh) * | 2019-07-03 | 2019-10-15 | 依科赛生物科技（太仓）有限公司 | 一种间充质干细胞无血清培养基及其用途 |
| CN110338187A (zh) * | 2018-04-08 | 2019-10-18 | 生物角（厦门）科技有限公司 | 一种间充质干细胞无血清冻存液组合物 |
| CN112708591A (zh) * | 2020-12-24 | 2021-04-27 | 南京农业大学 | 一种用于肌肉干细胞体外分化的化学成分明确的培养基 |
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| CN110338187A (zh) * | 2018-04-08 | 2019-10-18 | 生物角（厦门）科技有限公司 | 一种间充质干细胞无血清冻存液组合物 |
| CN109161520A (zh) * | 2018-08-17 | 2019-01-08 | 北昊干细胞与再生医学研究院有限公司 | 间充质干细胞的分离培养方法及相应的保存方法和无血清培养基 |
| CN109022356A (zh) * | 2018-08-30 | 2018-12-18 | 丰泽康生物医药（深圳）有限公司 | 一种提高间充质干细胞向软骨细胞分化的无血清培养基 |
| CN110331130A (zh) * | 2019-07-03 | 2019-10-15 | 依科赛生物科技（太仓）有限公司 | 一种间充质干细胞无血清培养基及其用途 |
| CN112852719A (zh) * | 2019-11-12 | 2021-05-28 | 武汉济源高科技有限公司 | 一种临床级干细胞无血清培养基 |
| CN112852719B (zh) * | 2019-11-12 | 2022-07-15 | 武汉济源高科技有限公司 | 一种临床级干细胞无血清培养基 |
| CN112708591A (zh) * | 2020-12-24 | 2021-04-27 | 南京农业大学 | 一种用于肌肉干细胞体外分化的化学成分明确的培养基 |
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