CN107354132A - 一种nk免疫细胞培养基及其制备方法 - Google Patents

一种nk免疫细胞培养基及其制备方法 Download PDF

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CN107354132A
CN107354132A CN201710739751.9A CN201710739751A CN107354132A CN 107354132 A CN107354132 A CN 107354132A CN 201710739751 A CN201710739751 A CN 201710739751A CN 107354132 A CN107354132 A CN 107354132A
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朱学义
徐峰波
王秋文
闫爽
程志远
李雯雯
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Abstract

本发明公开了一种NK免疫细胞培养基，其由以下组分组成：基础培养基，壳聚糖，γ‑干扰素，胰岛素，过氧化氢酶，二巯基乙醇，亚油酸，氨基酸，维生素。各组分的含量为：基础培养基5‑20g/L，壳聚糖0.5‑2g/L，γ‑干扰素10‑20μg/L，胰岛素10‑20μg/L，过氧化氢酶3‑9mg/L，二巯基乙醇5‑10mg/L，亚油酸1‑3mg/L，氨基酸20‑50mg/L，维生素20‑50mg/L。本发明的NK免疫细胞培养基，能够提供NK免疫细胞生长增殖所需的充足营养与良好环境，能够显著提高NK免疫细胞的扩增速度，大大缩短NK免疫细胞培养的时间。

Description

一种NK免疫细胞培养基及其制备方法
技术领域
本发明属于生物技术领域，特别涉及一种NK免疫细胞培养基及其制备方法。
背景技术
NK细胞(natural killer cell)，又叫自然杀伤细胞，是与T、B细胞并列的第三类群淋巴细胞。NK细胞数量较少，在外周血中约占淋巴细胞总数的15％，在脾内约有3％～4％，也可出现在肺脏、肝脏和肠粘膜，但在胸腺、淋巴结和胸导管中罕见。
NK细胞较大，含有胞浆颗粒，故称大颗粒淋巴细胞。NK细胞可非特异直接杀伤靶细胞，这种天然杀伤活性既不需要预先由抗原致敏，也不需要抗体参与，且无MHC限制。NK细胞杀伤的靶细胞主要是肿瘤细胞、病毒感染细胞、较大的病原体(如真菌和寄生虫)、同种异体移植的器官、组织等。
NK细胞表面受体(NKR)可以识别被病毒感染的细胞表面表达的多糖分子。NK细胞的杀伤效应是由其活化后释放出的毒性分子介导，如穿孔素、颗粒酶和TNFα(肿瘤坏死因子)等。
发明内容
本发明的目的是提供一种NK免疫细胞培养基及其制备方法，用于NK免疫细胞的培养。
为实现上述目的，本发明采用的技术方案是：
一种NK免疫细胞培养基，其由以下组分组成：基础培养基，壳聚糖，γ-干扰素，胰岛素，过氧化氢酶，二巯基乙醇，亚油酸，氨基酸，维生素。
各组分的含量为：
基础培养基5-20g/L，壳聚糖0.5-2g/L，γ-干扰素10-20μg/L，胰岛素10-20μg/L，过氧化氢酶3-9mg/L，二巯基乙醇5-10mg/L，亚油酸1-3mg/L，氨基酸20-50mg/L，维生素20-50mg/L。
所述基础培养基为IMDM培养基。
所述维生素为水溶性维生素。
所述氨基酸为多种氨基酸复合物。
一种NK免疫细胞培养基的制备方法，包括以下步骤：
(1)向基础培养基中加入γ-干扰素，胰岛素，二巯基乙醇，亚油酸，氨基酸，维生素，搅拌15-30min，然后加入壳聚糖、过氧化氢酶，继续搅拌15-30min，得到混合物；
(2)将步骤(1)得到的混合物的pH调节至7.2-7.4，然后除菌，得到所述NK免疫细胞培养基。
步骤(2)中，除菌的方式为机械过滤除颗粒杂质、紫外线杀菌、高分子滤膜除菌三种方法的一种或多种组合。
有益效果：本发明的NK免疫细胞培养基，能够提供NK免疫细胞生长增殖所需的充足营养与良好环境，能够显著提高NK免疫细胞的扩增速度，大大缩短NK免疫细胞培养的时间。
具体实施方式
根据下述实施例，可以更好的理解本发明。然而，本领域的技术人员容易理解，实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1
一种NK免疫细胞培养基，各组分的含量为：
基础培养基5g/L，壳聚糖2g/L，γ-干扰素15μg/L，胰岛素15μg/L，过氧化氢酶5mg/L，二巯基乙醇8mg/L，亚油酸3mg/L，氨基酸50mg/L，维生素30mg/L。
基础培养基为IMDM培养基，维生素为水溶性维生素，氨基酸为多种氨基酸复合物。
其制备方法为：
(1)向基础培养基中加入γ-干扰素，胰岛素，二巯基乙醇，亚油酸，氨基酸，维生素，搅拌30min，然后加入壳聚糖、过氧化氢酶，继续搅拌30min，得到混合物；
(2)将步骤(1)得到的混合物的pH调节至7.2-7.4，然后除菌，得到所述NK免疫细胞培养基。
其中，除菌的方式为机械过滤除颗粒杂质、紫外线杀菌、高分子滤膜除菌三种方法的组合。
实施例2
一种NK免疫细胞培养基，各组分的含量为：
基础培养基20g/L，壳聚糖0.5g/L，γ-干扰素10μg/L，胰岛素20μg/L，过氧化氢酶9mg/L，二巯基乙醇8mg/L，亚油酸2mg/L，氨基酸20mg/L，维生素50mg/L。
基础培养基为IMDM培养基，维生素为水溶性维生素，氨基酸为多种氨基酸复合物。
其制备方法为：
(1)向基础培养基中加入γ-干扰素，胰岛素，二巯基乙醇，亚油酸，氨基酸，维生素，搅拌15min，然后加入壳聚糖、过氧化氢酶，继续搅拌15min，得到混合物；
(2)将步骤(1)得到的混合物的pH调节至7.2-7.4，然后除菌，得到所述NK免疫细胞培养基。
其中，除菌的方式为高分子滤膜除菌。
实施例3
一种NK免疫细胞培养基，各组分的含量为：
基础培养基20g/L，壳聚糖2g/L，γ-干扰素20μg/L，胰岛素10μg/L，过氧化氢酶3mg/L，二巯基乙醇5mg/L，亚油酸1mg/L，氨基酸30mg/L，维生素20mg/L。
基础培养基为IMDM培养基，维生素为水溶性维生素，氨基酸为多种氨基酸复合物。
其制备方法为：
(1)向基础培养基中加入γ-干扰素，胰岛素，二巯基乙醇，亚油酸，氨基酸，维生素，搅拌20min，然后加入壳聚糖、过氧化氢酶，继续搅拌20min，得到混合物；
(2)将步骤(1)得到的混合物的pH调节至7.2-7.4，然后除菌，得到所述NK免疫细胞培养基。
其中，除菌的方式为紫外线杀菌、高分子滤膜除菌的组合。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (7)

1.一种NK免疫细胞培养基，其特征在于：其由以下组分组成：基础培养基，壳聚糖，γ-干扰素，胰岛素，过氧化氢酶，二巯基乙醇，亚油酸，氨基酸，维生素。
2.根据权利要求1所述的NK免疫细胞培养基，其特征在于：各组分的含量为：
基础培养基5-20g/L，壳聚糖0.5-2g/L，γ-干扰素10-20μg/L，胰岛素10-20μg/L，过氧化氢酶3-9mg/L，二巯基乙醇5-10mg/L，亚油酸1-3mg/L，氨基酸20-50mg/L，维生素20-50mg/L。
3.根据权利要求1所述的NK免疫细胞培养基，其特征在于：所述基础培养基为IMDM培养基。
4.根据权利要求1所述的NK免疫细胞培养基，其特征在于：所述维生素为水溶性维生素。
5.根据权利要求1所述的NK免疫细胞培养基，其特征在于：所述氨基酸为多种氨基酸复合物。
6.一种权利要求1-5任一所述的NK免疫细胞培养基的制备方法，其特征在于：包括以下步骤：
(1)向基础培养基中加入γ-干扰素，胰岛素，二巯基乙醇，亚油酸，氨基酸，维生素，搅拌15-30min，然后加入壳聚糖、过氧化氢酶，继续搅拌15-30min，得到混合物；
(2)将步骤(1)得到的混合物的pH调节至7.2-7.4，然后除菌，得到所述NK免疫细胞培养基。
7.根据权利要求6所述的NK免疫细胞培养基的制备方法，其特征在于：步骤(2)中，除菌的方式为机械过滤除颗粒杂质、紫外线杀菌、高分子滤膜除菌三种方法的一种或多种组合。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110305840A (zh) * 2019-07-12 2019-10-08 赛德特生物科技开发有限公司 改善自身免疫疾病的免疫细胞培养基及其制备方法与应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104818248A (zh) * 2015-03-25 2015-08-05 苏州佰通生物科技有限公司 一种免疫细胞培养基、培养方法及应用
CN106148266A (zh) * 2015-04-20 2016-11-23 烟台赛泽生物技术有限公司 一种免疫细胞用培养基及该培养基的添加剂
CN106834225A (zh) * 2016-12-24 2017-06-13 严志海 一种免疫细胞培养基

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104818248A (zh) * 2015-03-25 2015-08-05 苏州佰通生物科技有限公司 一种免疫细胞培养基、培养方法及应用
CN106148266A (zh) * 2015-04-20 2016-11-23 烟台赛泽生物技术有限公司 一种免疫细胞用培养基及该培养基的添加剂
CN106834225A (zh) * 2016-12-24 2017-06-13 严志海 一种免疫细胞培养基

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110305840A (zh) * 2019-07-12 2019-10-08 赛德特生物科技开发有限公司 改善自身免疫疾病的免疫细胞培养基及其制备方法与应用

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