CN116478914A - 无血清细胞培养体系及其细胞消化终止液 - Google Patents
无血清细胞培养体系及其细胞消化终止液 Download PDFInfo
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Abstract
本申请公开一种细胞消化终止液,用于终止胰蛋白酶对体外贴壁培养细胞的消化进程,其包括以下组分:转铁蛋白,终浓度为5‑10mg/mL;胰岛素,终浓度为1‑5mg/mL;维生C,终浓度为5‑10μM;次黄嘌呤钠,终浓度为5‑10mM;胸苷,终浓度为1‑3mM;非必需氨基酸溶液,稀释至每种氨基酸终含量为0.1mM。本申请具备竞争性结合胰蛋白酶作用,使细胞蛋白和胰蛋白酶结合的机会大大减少,从而起着终止胰蛋白酶消化细胞的效果,有效替血清终止消化的作用,有利于在临床上应用,构建无血清细胞培养体系。
Description
技术领域
本申请涉及细胞培养领域,尤其涉及无血清细胞培养体系及其细胞消化终止液。
背景技术
目前,细胞的体外处理,包括收获后的运输和治疗制备,一般是在含有胎牛血清(fatal bovine serum,FBS)的培养基中进行的,它促进细胞的附着、增殖和分化。然而,由于FBS的使用存在安全问题,包括人畜共患病的潜在传播和移植排斥反应,因为FBS中的蛋白质和多肽被认为与培养的细胞结合,然后通过细胞移植过程转移到宿主,导致移植细胞的排斥反应因此临床处理细胞需要异种无血清培养基。
消除培养液中的血清和动物成分是确定细胞培养过程的重要一步。定义介质只包含具有已知化学特性的产品和材料,如小分子、盐和脂肪酸。这些培养基还可能包括动物系统中不产生的重组蛋白。加入无血清或无动物的培养基将在以下几个方面对细胞培养过程有好处:通过减少介质组成的可变性来提高一致性;简化对监管准则的遵从;如果原材料需要改变,简化可比性测试。
细胞脱离是培养贴壁细胞所必需的。胰蛋白酶化是最流行的分离技术,常规方式终止胰蛋白酶的消化作用是加入血清,然而在无血清的培养体系中,为了终止消化而引入血清,不符合临床要求。
发明内容
本申请的目的是,至少部分克服现有技术的不足,提供一种无血清的细胞消化终止液,并且利用该细胞消化终止液构建无血清细胞培养体系。
为达到以上技术目的,本申请采用的技术方案如下:
第一方面,提供一种细胞消化终止液,用于终止胰蛋白酶对体外贴壁培养细胞的消化进程,其包括以下组分:
转铁蛋白,终浓度为5-10mg/mL;
胰岛素,终浓度为1-5mg/mL;
维生素C,终浓度为5-10μM;
次黄嘌呤钠,终浓度为5-10mM;
胸苷,终浓度为1-3mM;
非必需氨基酸溶液,稀释至每种氨基酸终含量为0.1mM。
可选择地,所述细胞消化终止液需加入基础培养基中使用,所述基础培养基选自DMEM/F12细胞培养基、F12细胞培养基和William's E细胞培养基中的一种。
可选择地,所述非必需氨基酸溶液为100倍浓缩液;所述非必需氨基酸溶液包含甘氨酸、L-丙氨酸、L天冬酰胺、L-天冬氨酸、L-谷氨酸、L-脯氨酸和L-丝氨酸。
优选地,所述细胞消化终止液不含血清。
进一步可选择地,所述细胞消化终止液适用于二维培养的细胞和三维培养的细胞。
可选择地,所述细胞消化终止液适用于体外培养的干细胞或肝细胞。
进一步地,所述细胞消化终止液适用于体外培养的人胚胎干细胞或肝癌细胞。
优选地,所述细胞消化终止液的工作温度为4℃。
进一步优选地,所述细胞消化终止液在胰蛋白酶消化细胞60min内加入以终止消化。
第二方面,提供一种无血清细胞培养体系,包括使用无血清细胞培养液和消化终止液,其特征在于,所述消化终止液采用如前所述的细胞消化终止液。
与现有技术相比较,本申请具有如下优势:
(1)本申请的细胞消化终止液,经验证具备竞争性结合胰蛋白酶作用,使细胞蛋白和胰蛋白酶结合的机会大大减少,从而起着终止胰蛋白酶消化细胞的效果,有效替血清终止消化的作用;
(2)本申请的细胞消化终止液与传统的含血清的终止液相比较,无明显的细胞毒性,在消化60min内加入对细胞活性的影响无明显差异;
(3)本申请的细胞消化终止液的组成成分清楚,都是容易获取、价格相对低廉的成分,不会大幅度提高本申请的的制备成本;
(4)本申请的细胞消化终止液,可以配合应用于多种基础培养基,可以适应不同细胞,为各种多类培养应用提供足够条件;不仅可以用于常规二维培养细胞,还可以用于在三维培养所形成的细胞球体,具有临床应用的良好前景。
附图说明
图1示出了本申请的无血清消化终止添加剂与其他配方的添加剂或培养基之间对细胞培养的影响。
图2示出了本申请的无血清消化终止添加剂,与10%胎牛血清作为对照组相比,对不同时间消化的MSC终止消化后,细胞活率柱状图;
图3示出了本申请的无血清消化终止添加剂对进行连续消化的MSC终止消化后,在消化时间60分钟左右,与10%胎牛血清作为对照组相比,细胞死活染色结果(10倍物镜);
图4示出了申请的无血清消化终止添加剂对三维HepG2细胞培养物在消化时间60分钟左右,与10%胎牛血清作为对照组相比,终止消化后,细胞死活染色结果(10倍物镜)。
具体实施方式
以下结合附图和具体实施方式对本申请作进一步详细描述。
本申请提供一种细胞消化终止液,用于终止胰蛋白酶对体外贴壁培养细胞的消化进程,其包括以下组分:转铁蛋白,终浓度为5-10mg/mL;胰岛素,终浓度为1-5mg/mL;维生素C,终浓度为5-10μM;次黄嘌呤钠,终浓度为5-10mM;胸苷,终浓度为1-3mM;非必需氨基酸溶液,稀释至每种氨基酸终含量为0.1mM。
转铁蛋白是一种铁载体,也有助于降低氧自由基和过氧化物的毒性水平。细胞在适当的时间需要适当数量的铁来维持健康并继续生长和分裂。铁太少或太多都会导致生长和生产力低下。在自然界中,铁通过铁结合蛋白转铁蛋白(存在于哺乳动物血液中)输送到细胞中。转铁蛋白是一种万能的铁载体,通过受体介导的铁从转铁蛋白转移到细胞,将适量的铁输送到细胞。表面特定的转铁蛋白受体根据细胞对铁的需求上调或下调。由于细胞具有调节铁吸收的能力,转铁蛋白能够提供细胞所需的确切数量的铁。这很重要,因为它消除了细胞摄入过多铁元素而导致细胞损伤的问题,而其他铁元素来源也会导致细胞损伤。
胰岛素可促进葡萄糖和氨基酸的摄取、脂肪形成、细胞内运输以及蛋白和核酸的合成。胰岛素的作用主要是代谢,在哺乳动物细胞培养中以大约100倍的生理浓度添加重组胰岛素作为生长因子,具有抗凋亡和有丝分裂作用。
维生素C,又称抗坏血酸,是一种辅因子,在培养基中用作抗氧化剂。抗坏血酸是一种天然存在的内酯,由植物和许多动物产生,但不包括人类或其他灵长类动物。它作为电子供体(即还原剂),并显示出抗氧化活性,特别是对活性氧。抗坏血酸保护细胞免受辐射和氧自由基的破坏作用。参与脯氨酸和赖氨酸的羟化离子Na+/Ca2+交换,调节环核苷酸水平抑制培养细胞凋亡等。
非必需氨基酸溶液(non-essential amino acids,NEAA),含7种非必需氨基酸:甘氨酸、L-丙氨酸、L天冬酰胺、L-天冬氨酸、L-谷氨酸、L-脯氨酸和L-丝氨酸,常作为细胞培养补品,商品化的NEAA为100倍浓缩液,使用时需要将该浓缩液稀释100倍。它被用作细胞培养基的补充,以优化细胞生长。大多数细胞可以通过葡萄糖氨解、糖酵解或TCA循环(三羧酸循环)合成非必需氨基酸(NEAA)。当这些氨基酸的浓度较低时,细胞需要消耗培养基中更多的葡萄糖和谷氨酰胺,并产生过量的副产物,这可能会影响细胞的生长。在细胞培养基中添加NEAA可以减轻这些非必需氨基酸自身产生的潜在副作用。
次黄嘌呤和胸苷是一种营养添加剂,适用于动物细胞培养应用。提供预先形成的嘌呤和嘧啶,以克服胞内残留氨基蝶呤的影响。细胞培养基保持正确的pH值,支持多种类型细胞的生长。
本申请的细胞消化终止液,不含血清,在二维和三维条件下,不仅能够通过与胰蛋白酶进行竞争性结合实现终止消化的效果,还能够短暂提供足够的营养,快速降低过度消化对细胞的损害小。本申请可以用于贴壁细胞,也可用于三维细胞球体回收的消化终止。
本申请的用法与血清类似,需要加入基础培养基中使用,所述基础培养基,选自DMEM/F12细胞培养基、高糖培养基和William's E细胞培养基中的一种。DMEM/F-12(Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12)是一种广泛使用的基础培养基,用于支持多种哺乳动物细胞的生长,主要含有3.15g/L葡萄糖、0.365g/L L-谷氨酰胺、酚红、碳酸氢钠以及3.57g/LHEPES。DMEM为广谱型培养基,是Eagle-培养基的改良型,添加更高浓度的氨基酸和维生素等营养物质。广泛应用于细胞培养的各个领域,可用于培养肿瘤细胞系,原代细胞,干细胞等。DMEM培养基有高糖(4.5g/l)、低糖(1g/l)和无糖等类型。William's E培养基是早期培养基(富含氨基酸,葡萄糖含量加倍)的改良产品,William'sE培养基中含有独特的成分,包括锌、铁、锰、非必需氨基酸、还原剂谷胱甘肽和脂质亚油酸甲酯,含酚红,不含L-谷氨酰胺和HEPES。本申请的实施例可以根据目标肝细胞的类型筛选合适的基础培养基,实现对目标肝细胞的充足的营养供给。
为了验证本申请的细胞消化终止液对体外培养细胞的影响,按照下表配制不同的实施例和对比例,并验证各自对细胞的影响。其中,体外培养细胞以肝癌细胞(HepG2)为例。
表1含不同配比的消化终止液的培养基
表2不同实施例的不同孵育时间对培养细胞的影响
注:“0”表示与对照相比较无明显差异;“+”表示与对照相比较有一定的促进作用,“++”表示与对照相比较有明显的促进作用;“-”表示与对照相比较有一定的抑制作用,“--”表示与对照相比较有明显的抑制作用。
表3实施例10和不同对比例对细胞培养的影响
注:“0”表示与对照相比较无明显差异;“+”表示与对照相比较有一定的促进作用,“++”表示与对照相比较有明显的促进作用;“-”表示与对照相比较有一定的抑制作用,“--”表示与对照相比较有明显的抑制作用。
以上结果表明,本申请的细胞消化终止液可提高细胞活率,快速促贴壁且保持结构完整。
如图1所示,对进行连续消化的HepG2终止消化后,重新接种至96孔板(104个细胞每孔)观察细胞贴壁和形态情况。在消化时间60min以内,与各个对比例相比,从实施例10结果表明,本申请的细胞消化终止液对于促细胞贴壁和结构完整情况最佳;结合对比例1和对比例2的结果表明本申请的细胞消化终止液成分各分开单独添加,细胞的贴壁量减少,细胞结构达不到本申请的完全组合效果;结合对比例3~对比例5的结果,单独使用基础培养基虽然可以终止消化,但无法让细胞快速恢复到较佳的生理状态。
下一步验证本申请的细胞消化终止液对细胞的影响。本实施例中,采用所述细胞消化终止液对人胎盘干细胞(mesenchymal stem cell,MSC)和肝癌细胞(HepG2)进行消化终止。为了进行安全、有效和可复制的细胞功能性实验和移植治疗,控制细胞的数量,质量和活力是非常重要的。由于MSC和HepG2是黏附的,通常需要酶消化制备供移植用的细胞悬液,不适当的细胞分离程序可能不仅会极大地破坏细胞的活力,还会破坏细胞的功能。在此,将本申请的细胞消化终止液与常规的血清终止液进行比较,确认本申请的效果。
具体地,在二维条件下,采用所述细胞消化终止液,对MSC进行预设时间的消化终止。在培养箱中,培养条件为5%CO2,37℃。具体的操作步骤参考如下:将约4.8x106个MSC细胞接种至6孔整板中,放置于CO2细胞培养箱中进行静置培养3天后。获取贴壁培养的MSC,舍弃上清液,使用磷酸缓冲溶液洗涤两次;使用胰蛋白酶溶液进行消化;用所述前述的实施例3(本申请的细胞消化终止液搭配DMEM/F12培养基)作为实验组,10%胎牛血清搭配DMEM/F12培养基作为对照组,重悬细胞观察死活情况并再次接种24小时后检测活率。
在三维条件中,采用所述细胞消化终止液,对MSC进行预设时间的消化终止:1.从培养液中取出尽可能多的细胞培养基,注意不要干扰细胞;2.添加预冷消化液,加入消化液体积≥细胞球体体积的2倍;3.移液器上下轻轻地使用宽孔尖端小心地打碎破坏3D细胞球体结构;4.用消化液在4℃下培养约30分钟和1小时;5.在显微镜下观察培养物,是否完全解聚和细胞浮动自由活动后,用所述前述的实施例3(本申请的细胞消化终止液搭配DMEM/F12培养基)作为实验组,10%胎牛血清搭配对应DMEM/F12培养基作为对照组进行终止消化。在另一个实施例中,用HepG2细胞替换MSC细胞进行实验。
经过0~60min的连续消化,对细胞的细胞形态进行观察,进行消化后活率检测,结果如下:
(1)如图2所示,对进行连续消化的MSC终止消化后,重新接种至96孔板(104个细胞每孔)用CC8试剂检测细胞活率。在消化时间60min以内,细胞活率与对照组相比无显著差异。
(2)如图3所示,为验证细胞活率结果,对进行连续消化的MSC终止消化后,在消化时间60min以内,细胞死活成像与对照组相比无显著差异。荧光红色指示死细胞,荧光绿色指示活细胞(10倍物镜)。
(3)如图4所示,为验证细胞活率结果,针对三维HepG2细胞培养物在消化时间60min,达到解聚和细胞浮动自由活动后,细胞死活成像与对照组相比无显著差异。荧光红色指示死细胞,荧光绿色指示活细胞(10倍物镜)。
以上结果表明,本申请的细胞消化终止液基本可以替代血清实现细胞消化的终止。这种效果可以用于构建完整的无血清细胞培养体系,包括使用无血清细胞培养液和本申请的消化终止液。
综上所述,本申请提供一种细胞消化终止液,用于终止胰蛋白酶对体外贴壁培养细胞的消化进程,其包括以下组分:转铁蛋白,终浓度为5-10mg/mL;胰岛素,终浓度为1-5mg/mL;维生素C,终浓度为5-10μM;次黄嘌呤钠,终浓度为5-10mM;胸苷,终浓度为1-3mM;非必需氨基酸溶液,稀释至每种氨基酸终含量为0.1mM。本申请具备竞争性结合胰蛋白酶作用,使细胞蛋白和胰蛋白酶结合的机会大大减少,从而起着终止胰蛋白酶消化细胞的效果,有效替血清终止消化的作用,有利于在临床上应用,构建无血清细胞培养体系。
上述实施例为本申请较佳的实施方式,但并不仅仅受上述实施例的限制,其他的任何未背离本申请的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,均包含在本申请的保护范围之内。
Claims (10)
1.一种细胞消化终止液,用于终止胰蛋白酶对体外贴壁培养细胞的消化进程,其特征在于,其包括以下组分:
转铁蛋白,终浓度为5-10mg/mL;
胰岛素,终浓度为1-5mg/mL;
维生素C,终浓度为5-10μM;
次黄嘌呤钠,终浓度为5-10mM;
胸苷,终浓度为1-3mM;
非必需氨基酸溶液,稀释至每种氨基酸终含量为0.1mM。
2.如权利要求1所述的细胞消化终止液,其特征在于,所述细胞消化终止液需加入基础培养基中使用,所述基础培养基选自DMEM/F12细胞培养基、F12细胞培养基和William's E细胞培养基中的一种。
3.如权利要求1所述的细胞消化终止液,其特征在于,所述非必需氨基酸溶液为100倍浓缩液;所述非必需氨基酸溶液包含甘氨酸、L-丙氨酸、L天冬酰胺、L-天冬氨酸、L-谷氨酸、L-脯氨酸和L-丝氨酸。
4.如权利要求1所述的细胞消化终止液,其特征在于,所述细胞消化终止液不含血清。
5.如权利要求1所述的细胞消化终止液,其特征在于,所述细胞消化终止液适用于二维培养的细胞和三维培养的细胞。
6.如权利要求1所述的细胞消化终止液,其特征在于,所述细胞消化终止液适用于体外培养的干细胞或肝细胞。
7.如权利要求6所述的细胞消化终止液,其特征在于,所述细胞消化终止液适用于体外培养的人胚胎干细胞或肝癌细胞。
8.如权利要求1所述的细胞消化终止液,其特征在于,所述细胞消化终止液的工作温度为4℃。
9.如权利要求1所述的细胞消化终止液,其特征在于,所述细胞消化终止液在胰蛋白酶消化细胞60min内加入以终止消化。
10.一种无血清细胞培养体系,包括使用无血清细胞培养液和消化终止液,其特征在于,所述消化终止液采用如权利要求1~9任意一项所述的细胞消化终止液。
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