CN114381428A - 作用于注射皮下或损伤组织部位的细胞培养基 - Google Patents
作用于注射皮下或损伤组织部位的细胞培养基 Download PDFInfo
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Abstract
本发明属于细胞生物学领域，公开了一种作用于注射皮下或损伤组织部位的细胞培养基，由基础培养基和添加组分组成，其中，所述基础培养基为RPMI‑1640培养基，所述添加组分为：平衡盐水、血蛋白、葡萄糖、细胞生长因子、水解胶原蛋白液。本发明所提供的一种作用于注射皮下或损伤组织部位的细胞培养基，无血清、不含异源动物成分；所有成分都是明确的，且同种来源(人)，包括蛋白质，且不含抗生素；能够培养人淋巴细胞、CIK、DC、NK等各类免疫细胞；具有很高的成活率和扩增倍数；能够维持高密度培养；具有很高的杀伤活性；成分简单、制作简便、成本低。
Description
技术领域
本发明属于细胞生物学领域，尤其涉及一种作用于注射皮下或损伤组织部位的细胞培养基。
背景技术
细胞免疫治疗技术是一种疗效显著的全新的抗肿瘤治疗的方法。通过采集人体免疫细胞，经过体外富集、活化，并扩增，使其靶向性杀伤力增强，再输回到人体，达到治疗多种免疫疾病的目的。然而体外培养扩增，需要适合免疫细胞生长扩增的培养基，包括适宜的渗透压，为细胞提供生长所必需的营养物质，同时也要保护细胞免受代谢过程中产生的毒性物质的损害。
传统的细胞培养主要是在基础培养基中添加一定浓度的人或动物血清。但由于血清成分复杂，虽含有许多对细胞有利成分，也含有对细胞有害的成分，并且本身有携带细菌、病毒、蛋白传染性疾病的风险。人血清昂贵，不适宜大规模扩增。由于血清供体的不同，导致批次间存在很大差异；动物源无血清培养基中含有从牛或其他动物血清或组织中分离纯化的物质，可能含有动物病原体，如牛血清中含有牛病毒，这就造成这些动物病毒引起疾病的危险。因此，血清在细胞临床大规模培养中有很多不利因素。
虽然目前已存在较多公司开发无血清培养基产品，但是存在培养基成分复杂、制作麻烦、成本高、推广性不高的问题。
发明内容
本发明的目的是，提供一种作用于注射皮下或损伤组织部位的细胞培养基，其成分简单、制作方便、成本较低，易推广。
本发明采用的技术方案如下：
一种作用于注射皮下或损伤组织部位的细胞培养基，起到细胞再生作用，由基础培养基和添加组分组成，其中，所述基础培养基为RPMI-1640 培养基，所述添加组分为：生理盐水、血蛋白、葡萄糖、细胞生长因子、水解胶原蛋白液。
进一步地，每一升RPMI-1640培养基中，生理盐水100ml～200ml、血蛋白2.2～2.6g、葡萄糖2～2.6g、细胞生长因子3～6mg、水解胶原蛋白液10～50 ml。
进一步地，所述细胞培养基的pH值为6.8-7.6，渗透压为280-360 mOsm/kg.H2O。
进一步地，所述生理盐水为Hanks平衡盐溶液或Earle平衡盐溶液。
进一步地，所述细胞生长因子为成纤维细胞生长因子。
与现有技术相比，本发明所提供的一种作用于注射皮下或损伤组织部位的细胞培养基，具有如下技术效果：
1、无血清、不含异源动物成分；所有成分都是明确的，且同种来源(人)，包括蛋白质，且不含抗生素；
2、能够培养人淋巴细胞、CIK、DC、NK等各类免疫细胞；
3、具有很高的成活率和扩增倍数；
4、能够维持高密度培养；
5、具有很高的杀伤活性；
6、成分简单、制作简便、成本低。
具体实施方式
以下实施例仅用于更加清楚地说明本发明的技术方案，而不能以此来限制本发明的保护范围。如在说明书及权利要求当中使用了某些词汇来指称特定部件。本领域技术人员应可理解，硬件或软件制造商可能会用不同名词来称呼同一个部件。本说明书及权利要求并不以名称的差异来作为区分部件的方式，而是以部件在功能上的差异来作为区分的准则。说明书后续描述为实施本发明的较佳实施方式，然所述描述乃以说明本发明的一般原则为目的，并非用以限定本发明的范围。本发明的保护范围当视所附权利要求所界定者为准。
下面结合具体实施例对本发明做进一步详细说明。
本发明实施例所提供的免疫细胞无血清基础培养基是无血清、不含异源动物成分(Xeno-free)的人免疫细胞培养基，可用于人淋巴细胞、CIK、DC、 NK等各类免疫细胞的培养，具有很高的成活率、杀伤活性和扩增倍数。其由各种免疫细胞的基础营养成份，可在无血清条件下添加组分在体外环境长时间维持细胞的生长。
无血清培养基，就是在细胞培养中不需要添加血清，但是在某些应用中要添加生长因子或细胞因子。无血清培养基中添加了血清的主要成分:粘附因子、生长因子、必需的营养物质和激素等，能减少血清带来的不利因素，使细胞培养的条件更稳定。经历了天然培养基、合成培养基后，无血清培养基和无血清培养成为当今细胞培养领域的一大趋势。采用无血清培养可简化纯化和鉴定各种细胞产物的程序，避免病毒污染造成的危害。
需要说明的是，本发明实施例中的细胞培养基仅用于细胞增殖培养，不具备对细胞的选择、诱导、分化功能，培养后的细胞用于体外诊断。本发明实施例公开了一种作用于注射皮下或损伤组织部位的细胞培养基，由基础培养基和添加组分组成，其中，所述的基础培养基为RPMI-1640培养基，所述添加组分为：平衡盐水、血蛋白、葡萄糖、细胞生长因子、水解胶原蛋白液。
本发明适用于高密度细胞培养体系的免疫细胞的无血清培养基，采用 RPMI-1640作为基础培养基以及特定的添加组分的组合，尤其是血蛋白、葡萄糖、细胞生长因子、水解胶原蛋白液，四者同时满足特定的浓度范围时，能够使得免疫细胞快速、大量的增殖，且细胞存活率更高，特别适合免疫细胞的高密度细胞培养体系。
下面来对各物质组分及其含量进行具体说明。
RPMI是Roswell Park Memorial Institute的缩写，代指洛斯维·帕克纪念研究所。RPMI是该研究所研发的一类细胞培养基，1640是培养基代号。其中含有10％胎牛血清。制作时，在无菌条件下将合成的RPMI1640干粉在电子天平上称取适量称重后置于消毒容器内，加9L纯化水稀释后搅拌均匀获得基础培养基。然后再添加组分，搅拌均和均匀，之后再过滤灭菌，调节PH值，保持至7.2±0.4，当细胞培养基配置完成后，过滤灭菌，分装封口，最后冷藏备用。
平衡盐水(BSS):Hanks液和Earle液是常用的BSS基础溶液。PH调整液:3.7％、5.6％、7.4％的NaHCO3溶液、HEPES溶液(二羟乙基哌嗪乙烷磺酸)。本实施例中，每一升RPMI-1640培养基中，平衡盐水100ml～200ml，比如可以设置为120ml或160ml。
人血白蛋白(HSA)是细胞培养基中的重要组分之一，在细胞生理活动中起到重要作用。本实施例中，每一升RPMI-1640培养基中，血蛋白 2.2～2.6g，优选为2.4g。
氨基酸是细胞合成蛋白质的基本成分，氨基酸中有12种是细胞本身不合成的，必须由培养液提供。激素有胰岛素、生长激素和多种生长因子如表皮生长因子、成纤维细胞生长因子、增殖刺激因子(MSA)、类胰岛素生长因子(IGFl、IGF2)等。本实施例中，每一升RPMI-1640培养基中，成纤维细胞生长因子含量为3～6mg，优选为4mg。
碳水化合物是细胞生长主要能量来源，其中有的是合成蛋白质和核酸的成分。主要有葡萄糖、核糖、脱氧核糖、丙酮酸钠和醋酸等。本实施例中，每一升RPMI-1640培养基中，葡萄糖含量为2～2.6g，优选为2.2g。
水解胶原蛋白是在较高温度下用蛋白酶水解胶原或明胶得到的，受温度和酶的双重作用，使水解胶原蛋白的相对分子质量比明胶更小，是相对分子质量从几千到几万的蛋白多肽的混合物。胶原蛋白是生物高分子，动物结缔组织中的主要成分，也是哺乳动物体含量最多、分布最广的功能性蛋白，占蛋白质总量的25％～30％。胶原蛋白的水解产物含有多种氨基酸，其中以甘氨酸最为丰富，其次为丙氨酸、谷氨酸和精氨酸，半胱氨酸、色氨酸、酪氨酸以及蛋氨酸等必需氨基酸含量低，因此，胶原蛋白属不完全蛋白质，胶原是从动物真皮中提取的，具改善细胞表面特性促使其附着生长的作用。添加水解胶原蛋白制作的培养基，能够有效存进菌丝的生长，在古氨量和水溶性方，具有独特优势，同时菌丝生长快、菌丝粗壮等特点，对大罐的发酵具有明显的提高。本实施例中，水解胶原蛋白液10～50ml，优选为30ml。
本发明制作的作用于注射皮下或损伤组织部位的细胞培养基可制作成液体型或干粉型，其中液体型的型号有2ml、3ml、4ml、5ml、6ml、7ml、 8ml、10ml、15ml、20ml、30ml、50ml、100ml、150ml、200ml、300ml、 500ml、1000ml。干粉型的型号有2ml、3ml、4ml、5ml、6ml、7ml、8ml、10ml、15ml、20ml、30ml、50ml、100ml、150ml、200ml、300ml、500ml、 1000ml。
制作成的细胞培养基的产品性能指标说明如下：
1.外观
细胞培养基外观为透明澄清的液体，淡黄色或粉红色。
2.pH值
细胞培养基pH值：6.8-7.6
3.渗透压
细胞培养基渗透压：280-360mOsm/kgH2O
4.细菌内毒素
细胞培养基细菌内毒素：<5EU/ml
5.微生物
细菌：阴性
霉菌：阴性
6.细胞生长试验
VERO细胞(或其他细胞)接种5×104个/mL,培养48～72小时的细胞形态为成纤维样，贴壁生长，细胞数量应达到1×105个/mL。换新鲜培养基继续培养48小时的细胞为成纤维样，贴壁生长，细胞计数应不小于1×105个/mL。
制作成的细胞培养基产品需在2℃～8℃冷藏，并保持干燥、密封。检验方法为：吸取一定量的细胞悬液，加到细胞培养瓶内，向细胞培养瓶内加至10mL的细胞培养液，使细胞接种密度为5×104个/mL。并将细胞培养瓶送入培养箱，在37±1℃温度条件及5±0.1％二氧化碳体积分数条件下进行培养。反复使用同一瓶培养基长时间培养过程中如发现细胞生长速度逐渐减慢，可向该瓶培养基中另行加入一定量的葡萄糖。如培养过程中发现培养液浑浊、沉淀等现象，需关注操作过程及贮藏过程中是否有环节对培养基造成污染；如发现细胞死亡，但并无污染现象，则需要检查培养细胞液浓度是否过大，并可适当减小。
值得注意的是，以上所述仅为本发明的较佳实施例，并非因此限定本发明的专利保护范围，本发明还可以对上述各种零部件的构造进行材料和结构的改进，或者是采用技术等同物进行替换。故凡运用本发明的说明书内容所作的等效结构变化，或直接或间接运用于其他相关技术领域均同理皆包含于本发明所涵盖的范围内。
Claims (7)
1.一种作用于注射皮下或损伤组织部位的细胞培养基，由基础培养基和添加组分组成，其特征在于，所述基础培养基为RPMI-1640培养基，所述添加组分为：平衡盐水、血蛋白、葡萄糖、细胞生长因子、水解胶原蛋白液。
2.如权利要求1所述的作用于注射皮下或损伤组织部位的细胞培养基，其特征在于，每一升RPMI-1640培养基中，平衡盐水100ml～200ml、血蛋白2.2～2.6g、葡萄糖2～2.6g、细胞生长因子3～6mg、水解胶原蛋白液10～50ml。
3.如权利要求1所述的作用于注射皮下或损伤组织部位的细胞培养基，其特征在于，所述平衡盐水为Hanks平衡盐溶液或Earle平衡盐溶液。
4.如权利要求2所述的作用于注射皮下或损伤组织部位的细胞培养基，其特征在于，所述细胞生长因子为成纤维细胞生长因子。
5.如权利要求2所述的作用于注射皮下或损伤组织部位的细胞培养基，其特征在于，所述细胞培养基的pH值为6.8-7.6，渗透压为280-360mOsm/kg.H2O。
6.如权利要求1所述的作用于注射皮下或损伤组织部位的细胞培养基，其特征在于，所述免疫细胞为CIK细胞。
7.如权利要求1所述的作用于注射皮下或损伤组织部位的细胞培养基，其特征在于，制作成的所述细胞培养基为干粉型或液体型。
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