CN113502267A - 一种用于外周血中nk细胞扩增的培养基及方法 - Google Patents
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Abstract
本发明公开了一种用于外周血中NK细胞扩增的培养基及方法,包括以NKG2D与CD2为主的包被液及以1640基础培养基为主体、自体灭火血浆、L‑谷氨酰胺、2‑巯基乙醇、葡萄糖、酚红、IL‑2、IL‑15为添加剂的营养液两部分,本发明通过提供的NK细胞培养基及培养方法极大的降低了NK细胞的生产成本,且整个过程操作简单、扩增效率高、纯度良好。利用本发明,10天即可获得大量的高纯度的NK细胞,有利于NK细胞的大规模生产和应用。
Description
技术领域
本发明涉及细胞制备技术领域,具体涉及一种用于外周血中NK细胞扩增的培养基及方法。
背景技术
NK细胞疗法,NK细胞疗法,是指通过向肿瘤患者回输经体外诱导培养的NK细胞,发挥直接或间接杀伤肿瘤细胞的作用,从而达到治疗肿瘤的目的。被广泛用于各种癌症的治疗,包括:血液系统肿瘤、神经母细胞瘤、卵巢肿瘤、横纹肌肉瘤、乳腺癌、胃癌等等,NK细胞疗法在这些病症中均取得了不错的临床治疗效果。大量研究数据表明,NK细胞疗法在提高恶性肿瘤患者免疫功能和生存质量方面都有极为不错的表现,可以与手术、放疗和化疗等传统治疗方案互补,在未来可能成为治疗癌症患者的新手段。
以NK细胞为基础的过继性免疫治疗需要大量的细胞,其应用范围为 患者每公斤体重需1×10 6 —8×10 7 CD3 - CD56 + NK细胞 (即一个80kg的成人需要至少8×10 7 NK细胞,并需多次回输)。此外,NK细胞在外周血中所占比例很低(5-15%),因此在用于过继性免疫治疗之前,有必要对其进行扩增。
目前NK细胞临床试验大多数还处于比较早期的阶段,未来需要在现有技术的基础上,不断提高NK细胞治疗的有效性和安全性,优化生产(降低成本,缩短生产周期,优化工艺),扩大治疗范围。因此,进一步研究一种耗时短、易制备、低成本的体外NK细胞扩增的培养基和培养方法十分必要。
发明内容
发明目的:为降低NK生产成本,缩短NK细胞生产周期,优化NK细胞生产工艺,以NKG2D、CD2为激活剂,以1640基础培养基作为基础培养基;添加浓度为为1200U/mL的IL-2;浓度为50ng/mL的IL-15,浓度为20mM的L-谷氨酰胺,浓度为50uM的2-巯基乙醇的终,浓度为2000mg/L的葡萄糖,终浓度为6mg/L的酚红,体积分数为10%的自体灭活血浆形成本发明的培养基。
本发明的有益效果如下:
以本发明提供的NK细胞培养基及培养方法,操作简单、扩增效率高、纯度良好。利用本发明,10天即可获得大量的高纯度的NK细胞。
附图说明
图1是流式检测实施例及其对比组的CD3-CD56+阳性率的结果图;
图2是流式检测阴性组的CD3-CD56+阳性率的结果图;
图3是实施例及其对比组扩增曲线;
图4 实施例及其对比组扩增倍数柱状图。
具体实施方式
为了使本技术领域的人员更好的理解本发明方案,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整的描述。
实施例
本实施例提供NK细胞扩增的培养基及方法。
本实施例采用的培养基包被液、刺激培养基和扩增培养基:
包被液中,NKG2D、CD2终浓度比为1∶1,具体配方为:DPBS磷酸缓冲液中加入终浓度为5ug/mL的NKG2D、终浓度为5ug/mL的CD2。
营养液中基础培养基为1640基础培养基,IL-2、IL-15、L-谷氨酰胺、2-巯基乙醇、葡萄糖、酚红的终浓度比为8∶1∶6∶0.2∶1000∶0.05,其中IL-2的终浓度为1200U/mL、IL-15的终浓度为50ng/mL、L-谷氨酰胺的终浓度为20mM、2-巯基乙醇的终浓度为50uM、葡萄糖的终浓度为2000mg/L、酚红的终浓度为6mg/L、自体灭活血浆的体积分数为10%。
本实施例采用NK细胞扩增的方法包括:
(1)使用包被液包被T25NK扩增瓶在4℃包被1天;
(2)接种培养基与扩增培养基都用本发明中的营养液代替,单个核细胞接种量为1.a5×106/mL,接种10mL;
(3)接种当天记为第1天,在37℃、5%CO2培养箱中培养4天,第5天将细胞取出静置5min,吸弃之前的营养液,用新的营养液重悬,接种于新的T25培养板中,从第5天开始,隔1天对细胞进行计数并补加培养基使细胞浓度维持在1.5×106/mL。
为了做比较,本实施例还设置了对比组,对比组与本实施例的区别仅在于,刺激培养基中的刺激因子浓度减半:1640基础培养基中加入IL-2的终浓度为600U/mL、IL-15的终浓度为25ng/mL。
实施例的结果测试
CD3-CD56+阳性率的检测:取实施例及其对比组最终所得细胞,收集细胞到1.5mL离心管中1000r/min离心5min,弃上清后用1mL含2%(v/v)血清的PBS重悬,加入抗体CD3-FITC、CD56-APC,4℃孵育30min后用含2%血清的PBS洗三遍。然后以5×105 /ml的密度重悬细胞,使用Agilent NovoCyte 流式仪细胞仪检测并分析其扩增效率。在检测过程中,以并未用流式抗体染色的细胞作为阴性对照。
从第5天开始对NK细胞的数量进行记录至第14天,对扩增的NK数量数据进行折线图展示。
结果实施例1及其对比组测试结果如图1所示,流式细胞分析发现实施例1的CD3-CD56+阳性率上升到70.37% (其对比组为41.76% );数量扩增到322倍(其对比组为176倍)见图4。阴性对比组见图3。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
显然,所描述的实施例仅仅本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都应当属于本发明保护的范围。
Claims (10)
1.一种用于外周血中NK细胞扩增的培养基,其特征在于,包括包被液及营养液两部分构成;所述包被液包括抗体NKG2D、CD2;所述营养液包括基础培养基、自体灭活血浆、一些化合物和细胞因子。
2.根据权利要求1所述的一种用于外周血中NK细胞扩增的培养基,其特征在于,所述营养液具体包括1640基础培养基、自体灭火血浆、L-谷氨酰胺、2-巯基乙醇、葡萄糖、酚红、IL-2、IL-15。
3.根据权利要求1所述的一种用于外周血中NK细胞扩增的培养基,其特征在于,所述的包被液中NKG2D、CD2的终浓度为1-10ug/mL;所述的营养液中的IL-2的终浓度为100-2000U/mL、IL-15的终浓度为5-100ng/mL、L-谷氨酰胺的终浓度为5-40mM、2-巯基乙醇的终浓度为5-100uM、葡萄糖的终浓度为1000-2500mg/L、酚红的终浓度为2-8mg/L、自体灭活血浆的体积分数为10%。
4.根据权利要求2所述的一种用于外周血中NK细胞扩增的培养基,其特征在于,所述包被液中,所述的NKG2D、CD2的终浓度比为1∶1;所述营养液中,所述的IL-2、IL-15、L-谷氨酰胺、2-巯基乙醇、葡萄糖、酚红的终浓度比为8∶1∶6∶0.2∶1000∶0.05。
5.根据权利要求1所述的一种用于外周血中NK细胞扩增的培养基,其特征在于,所述包被液中NKG2D的终浓度为5ug/mL最优、CD2的终浓度为5ug/mL最优;所述的营养液中IL-2的终浓度为1200U/mL最优、IL-15的终浓度为50ng/mL最优、L-谷氨酰胺的终浓度为20mM最优、2-巯基乙醇的终浓度为50uM最优、葡萄糖的终浓度为2000mg/L、最优、酚红的终浓度为6mg/L最优。
6.一种用于外周血中NK细胞扩增的方法,其特征在于,包括:如下步骤:
S1:包被:包被液包被需要使用的NK扩增培养瓶;
S2:培养:营养液扩增NK细胞。
7.根据权利要求6所述的一种用于外周血中NK细胞扩增的方法,其特征在于,提前一天使用包被液包被NK扩增瓶在4℃包被1天。
8.根据权利要求6所述的一种用于外周血中NK细胞扩增的方法,其特征在于,所述包被阶段的包被液包被的的第0天至第1天,不适用任何培养基;所述培养阶段使用营养液激活扩增NK细胞,从第1天至第14天,从第1天至第5天期间不换液;从第6天至第14天期间,隔1天对细胞进行计数并补加培养基。
9.根据权利要求6所述的一种用于外周血中NK细胞扩增的方法,其特征在于,单个核细胞接种量为1×106-2×106/ml,起始接种10ml。
10.根据权利要求6所述的一种用于外周血中NK细胞扩增的方法,其特征在于,所述外周血也包括脐带血。
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