CN116606809A - 一种用于肿瘤治疗的nk细胞培养方法 - Google Patents
一种用于肿瘤治疗的nk细胞培养方法 Download PDFInfo
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Abstract
本发明公开了一种用于肿瘤治疗的NK细胞培养方法,包括如下步骤:(1)分离单个核细胞,将单个核细胞接种于NK细胞培养基中培养1‑3天,培养温度为35‑38℃,得到一次培养物;(2)向一次培养物中补加OK‑432、IL‑21和去乙酰毛花苷,继续培养1‑3天;(3)向二次培养物中IL‑2、IL‑15、IL‑21、亚叶酸、姜黄素,继续培养1‑3天。本发明创造性将去乙酰毛花苷添加至NK细胞培养过程中,发现其在避免培养过程中使用具有复杂成分动物血清的基础上,一方面降低给细胞培养带来的不安全因素,另一方面能促进NK细胞的活化和增殖。
Description
技术领域
本发明涉及细胞扩增技术领域,尤其涉及一种用于肿瘤治疗的NK细胞培养方法。
背景技术
肿瘤免疫治疗被国内外医学界公认为第四大肿瘤治疗法。近年来自然杀伤细胞(NK)免疫治疗技术在世界范围内正在成为一种可靠的抗癌疗法,NK细胞是除T、B细胞之外的第三类淋巴细胞,具有独特功能的细胞亚群。NK细胞是机体重要的免疫效应细胞,在抵抗肿瘤和病毒感染等方面起着非常重要的作用。近年来关于NK细胞及其抗肿瘤功能的研究已经成为免疫学和肿瘤学研究的热点内容之一。
目前,NK细胞免疫治疗在临床上已成为一种重要的治疗手段。但是其在外周血中的数量仅占淋巴细胞总量的5%左右,如何实现NK细胞体外扩增培养从而应用于治疗是关键的技术问题。NK细胞的培养体系复杂,添加的细胞因子种类繁多,而扩增效果往往不佳。
去乙酰毛花苷是一种有机化合物,抑制心肌细胞膜上的Na-K-ATP酶,减少钠-钾交换,细胞内钠离子增多,钠-钙交换也增多,使细胞内钙离子增多,作用于收缩蛋白,发挥正性肌力作用。目前常用作强心药使用。目前还未发现采用去乙酰毛花苷对NK扩增,并提高扩增效率与纯度的研究。
发明内容
本发明的目的是为了解决现有技术中存在的缺点,而提出的一种用于肿瘤治疗的NK细胞培养方法。
一种用于肿瘤治疗的NK细胞培养方法,包括如下步骤:
(1)分离单个核细胞,将单个核细胞接种于NK细胞培养基中培养1-3天,培养温度为35-38℃,得到一次培养物;
其中,NK细胞培养基中包括基础培养基和添加物;基础培养基由RPMI-1640培养基和GT-T551培养基混合得到;添加物包括:胰岛素、人血清白蛋白、胆固醇、谷氨酰胺、抗CD3抗体、FK506和IL-15;
(2)向一次培养物中补加OK-432、IL-21和去乙酰毛花苷,继续培养1-3天;
(3)向二次培养物中IL-2、IL-15、IL-21、亚叶酸、姜黄素,继续培养1-3天。
优选地,步骤(1)中,细胞密度为5×105-2×106个/mL。
优选地,步骤(1)中,基础培养基由RPMI-1640培养基和GT-T551培养基按体积比为1:1-2混合得到。
优选地,步骤(1)的添加物中,胰岛素的浓度为10-30IU/mL,人血清白蛋白的浓度为120-200μg/mL,胆固醇的浓度为1-5μg/mL,谷氨酰胺的浓度为5-15μg/mL,抗CD3抗体的浓度为5-15μg/mL,FK506的浓度为1-6ng/mL,IL-15的浓度为1-5ng/mL。
优选地,步骤(2)中,OK-432的终浓度为2-10ng/mL,IL-21的终浓度为1-10ng/mL,去乙酰毛花苷的终浓度为1-10ng/mL。
优选地,IL-2的终浓度为25-45ng/mL,IL-15的终浓度为10-20ng/mL,IL-21的终浓度为1-5ng/mL,亚叶酸的终浓度为1-5ng/mL,姜黄素的终浓度为1-5ng/mL。
本发明的技术效果如下所示:
本发明首先将分离的单个核细胞加入NK细胞培养基中,使单个核细胞在培养阶段细胞生物状态好。而申请人通过实验发现向一次培养物中加入去乙酰毛花苷并与OK-432、IL-21配合进而二次培养,可有效对NK细胞激活,并促使其分泌细胞因子进一步加快细胞增殖,但其在促进NK细胞增殖的同时容易因细胞扩增过多引发细胞凋亡。
因此,申请人在三次培养时加入姜黄素并与IL-2、IL-15、IL-21、亚叶酸配合,能起到协同增效作用,既可促进NK细胞的活化和增殖,又能可有效避免因细胞扩增而引起的细胞凋亡。
本发明经扩增后,扩增倍数可达1325倍,且经流式细胞仪检测,NK细胞纯度在92.4%以上,并且本发明对不同的人体血液都有相同的结果,稳定性很好,同时方法简单、高效、有利于规模化生产。
本申请创造性将去乙酰毛花苷添加至NK细胞培养过程中,发现其在避免培养过程中使用具有复杂成分动物血清的基础上,一方面降低给细胞培养带来的不安全因素,另一方面能促进NK细胞的活化和增殖。
附图说明
图1为采用实施例5所得培养基培养NK细胞,台盼蓝染色后采样图。
图2为采用实施例5所得培养基培养NK细胞的细胞直径分布图。
图3为采用实施例5所得培养基培养NK细胞的聚团分布图。
图4为流式细胞术分析采用实施例5所得培养基培养NK细胞后表达CD3-CD56+NK细胞百分数图。
图5为实施例5和对比例1-2的培养方法扩增所得NK细胞的细胞扩增效率与纯度对比图。
图6为实施例5和对比例1-2的培养方法扩增所得NK细胞的细胞杀伤活性对比图。
具体实施方式
下面结合具体实施例对本发明作进一步解说。
实施例1
一种用于肿瘤治疗的NK细胞培养方法,包括如下步骤:
(1)分离单个核细胞,将单个核细胞接种于NK细胞培养基中培养1天,其中细胞密度为5×105个/mL,培养温度为35℃,得到一次培养物;
其中,NK细胞培养基中包括基础培养基和添加物;基础培养基由RPMI-1640培养基和GT-T551培养基按体积比为1:1混合得到;
添加物包括:浓度为10IU/mL的胰岛素,浓度为120μg/mL的人血清白蛋白,浓度为1μg/mL的胆固醇,浓度为5μg/mL的谷氨酰胺,浓度为5μg/mL的抗CD3抗体,浓度为1ng/mL的FK506和浓度为1ng/mL的IL-15;
(2)向一次培养物中补加OK-432、IL-21和去乙酰毛花苷,使OK-432的终浓度为2ng/mL,IL-21的终浓度为1ng/mL,去乙酰毛花苷的终浓度为1ng/mL,继续培养1-3天;
(3)向二次培养物中IL-2、IL-15、IL-21、亚叶酸、姜黄素,使IL-2的终浓度为25ng/mL,IL-15的终浓度为10ng/mL,IL-21的终浓度为1ng/mL,亚叶酸的终浓度为1ng/mL,姜黄素的终浓度为1ng/mL,继续培养1-3天。
实施例2
一种用于肿瘤治疗的NK细胞培养方法,包括如下步骤:
(1)分离单个核细胞,将单个核细胞接种于NK细胞培养基中培养3天,其中细胞密度为2×106个/mL,培养温度为38℃,得到一次培养物;
其中,NK细胞培养基中包括基础培养基和添加物;基础培养基由RPMI-1640培养基和GT-T551培养基按体积比为1:2混合得到;
添加物包括:浓度为30IU/mL的胰岛素,浓度为200μg/mL的人血清白蛋白,浓度为5μg/mL的胆固醇,浓度为15μg/mL的谷氨酰胺,浓度为15μg/mL的抗CD3抗体,浓度为6ng/mL的FK506和浓度为5ng/mL的IL-15;
(2)向一次培养物中补加OK-432、IL-21和去乙酰毛花苷,使OK-432的终浓度为10ng/mL,IL-21的终浓度为10ng/mL,去乙酰毛花苷的终浓度为10ng/mL,继续培养3天;
(3)向二次培养物中IL-2、IL-15、IL-21、亚叶酸、姜黄素,使IL-2的终浓度为45ng/mL,IL-15的终浓度为20ng/mL,IL-21的终浓度为5ng/mL,亚叶酸的终浓度为5ng/mL,姜黄素的终浓度为5ng/mL,继续培养3天。
实施例3
一种用于肿瘤治疗的NK细胞培养方法,包括如下步骤:
(1)分离单个核细胞,将单个核细胞接种于NK细胞培养基中培养60h,其中细胞密度为1×106个/mL,培养温度为37℃,得到一次培养物;
其中,NK细胞培养基中包括基础培养基和添加物;基础培养基由RPMI-1640培养基和GT-T551培养基按体积比为1:1.3混合得到;
添加物包括:浓度为25IU/mL的胰岛素,浓度为140μg/mL的人血清白蛋白,浓度为4μg/mL的胆固醇,浓度为8μg/mL的谷氨酰胺,浓度为12μg/mL的抗CD3抗体,浓度为3ng/mL的FK506和浓度为4ng/mL的IL-15;
(2)向一次培养物中补加OK-432、IL-21和去乙酰毛花苷,使OK-432的终浓度为4ng/mL,IL-21的终浓度为6ng/mL,去乙酰毛花苷的终浓度为4ng/mL,继续培养48h;
(3)向二次培养物中IL-2、IL-15、IL-21、亚叶酸、姜黄素,使IL-2的终浓度为40ng/mL,IL-15的终浓度为13ng/mL,IL-21的终浓度为4ng/mL,亚叶酸的终浓度为2ng/mL,姜黄素的终浓度为4ng/mL,继续培养36h。
实施例4
一种用于肿瘤治疗的NK细胞培养方法,包括如下步骤:
(1)分离单个核细胞,将单个核细胞接种于NK细胞培养基中培养48h,其中细胞密度为1.6×106个/mL,培养温度为36℃,得到一次培养物;
其中,NK细胞培养基中包括基础培养基和添加物;基础培养基由RPMI-1640培养基和GT-T551培养基按体积比为1:1.8混合得到;
添加物包括:浓度为15IU/mL的胰岛素,浓度为180μg/mL的人血清白蛋白,浓度为2μg/mL的胆固醇,浓度为12μg/mL的谷氨酰胺,浓度为8μg/mL的抗CD3抗体,浓度为5ng/mL的FK506和浓度为2ng/mL的IL-15;
(2)向一次培养物中补加OK-432、IL-21和去乙酰毛花苷,使OK-432的终浓度为8ng/mL,IL-21的终浓度为2ng/mL,去乙酰毛花苷的终浓度为8ng/mL,继续培养36h;
(3)向二次培养物中IL-2、IL-15、IL-21、亚叶酸、姜黄素,使IL-2的终浓度为30ng/mL,IL-15的终浓度为17ng/mL,IL-21的终浓度为2ng/mL,亚叶酸的终浓度为4ng/mL,姜黄素的终浓度为2ng/mL,继续培养60h。
实施例5
一种用于肿瘤治疗的NK细胞培养方法,包括如下步骤:
(1)分离单个核细胞,将单个核细胞接种于NK细胞培养基中培养48h,其中细胞密度为1.5×106个/mL,培养温度为36.5℃,得到一次培养物;
其中,NK细胞培养基中包括基础培养基和添加物;基础培养基由RPMI-1640培养基和GT-T551培养基按体积比为1:1.5混合得到;
添加物包括:浓度为20IU/mL的胰岛素,浓度为160μg/mL的人血清白蛋白,浓度为3μg/mL的胆固醇,浓度为10μg/mL的谷氨酰胺,浓度为10μg/mL的抗CD3抗体,浓度为4ng/mL的FK506和浓度为3ng/mL的IL-15;
(2)向一次培养物中补加OK-432、IL-21和去乙酰毛花苷,使OK-432的终浓度为6ng/mL,IL-21的终浓度为4ng/mL,去乙酰毛花苷的终浓度为6ng/mL,继续培养48h;
(3)向二次培养物中IL-2、IL-15、IL-21、亚叶酸、姜黄素,使IL-2的终浓度为35ng/mL,IL-15的终浓度为15ng/mL,IL-21的终浓度为3ng/mL,亚叶酸的终浓度为3ng/mL,姜黄素的终浓度为3ng/mL,继续培养48h。
对比例1
一种用于肿瘤治疗的NK细胞培养方法,包括如下步骤:
(1)分离单个核细胞,将单个核细胞接种于NK细胞培养基中培养48h,其中细胞密度为1.5×106个/mL,培养温度为36.5℃,得到一次培养物;
其中,NK细胞培养基中包括基础培养基和添加物;基础培养基由RPMI-1640培养基和GT-T551培养基按体积比为1:1.5混合得到;
添加物包括:浓度为20IU/mL的胰岛素,浓度为160μg/mL的人血清白蛋白,浓度为3μg/mL的胆固醇,浓度为10μg/mL的谷氨酰胺,浓度为10μg/mL的抗CD3抗体,浓度为4ng/mL的FK506和浓度为3ng/mL的IL-15;
(2)向一次培养物中补加OK-432、IL-21和去乙酰毛花苷,使OK-432的终浓度为6ng/mL,IL-21的终浓度为4ng/mL,去乙酰毛花苷的终浓度为6ng/mL,继续培养48h;
(3)向二次培养物中IL-2、IL-15、IL-21、亚叶酸,使IL-2的终浓度为35ng/mL,IL-15的终浓度为15ng/mL,IL-21的终浓度为3ng/mL,亚叶酸的终浓度为3ng/mL,继续培养48h。
对比例2
一种用于肿瘤治疗的NK细胞培养方法,包括如下步骤:
(1)分离单个核细胞,将单个核细胞接种于NK细胞培养基中培养48h,其中细胞密度为1.5×106个/mL,培养温度为36.5℃,得到一次培养物;
其中,NK细胞培养基中包括基础培养基和添加物;基础培养基由RPMI-1640培养基和GT-T551培养基按体积比为1:1.5混合得到;
添加物包括:浓度为20IU/mL的胰岛素,浓度为160μg/mL的人血清白蛋白,浓度为3μg/mL的胆固醇,浓度为10μg/mL的谷氨酰胺,浓度为10μg/mL的抗CD3抗体,浓度为4ng/mL的FK506和浓度为3ng/mL的IL-15;
(2)向一次培养物中补加OK-432、IL-21,使OK-432的终浓度为6ng/mL,IL-21的终浓度为4ng/mL,继续培养48h;
(3)向二次培养物中IL-2、IL-15、IL-21、亚叶酸、姜黄素,使IL-2的终浓度为35ng/mL,IL-15的终浓度为15ng/mL,IL-21的终浓度为3ng/mL,亚叶酸的终浓度为3ng/mL,姜黄素的终浓度为3ng/mL,继续培养48h。
采用实施例5和对比例1-2的培养方法扩增NK细胞,将扩增后的NK细胞继续扩增培养24h,将两次扩增所得NK细胞分别采用台盼蓝染色,然后稀释相同倍数,采用细胞计数板计数细胞总数量,计算扩增倍数。
如图1所示,实施例5组中的总细胞浓度为4.17×106/mL,活细胞浓度为3.71×106/mL,则细胞活率为89.02%。如图2和图3所示,实施例5组中的细胞平均直径为14.32μm,结团率为19.08%。
采用流式检测仪对实施例5和对比例1-2的培养方法扩增所得NK细胞进行细胞纯度检测。如图4所示,实施例5组中的NK细胞纯度91.64%,而其余对比例组分别为81.25%和74.46%。
将实施例5组和对比例1-2组的扩增倍数和纯度进行对比,如图5所示,实施例5所提供的方法对于NK细胞扩增效率最高,而且纯度也是最高。而对比例1组在正常扩增情况下与实施例5相近,但继续培养24h后,扩增效率急剧下降,这是由于对比例1未采用姜黄素,导致其能对NK细胞进行有效激活,促使其分泌细胞因子进一步加快细胞增殖,但随着细胞高速增殖会引发细胞凋亡,导致扩增效率在24h后会发生急剧下降。对比例2组由于未采用去乙酰毛花苷,导致扩增倍数始终处于较低水平,而且纯度也较低。
采用96孔平底培养板,设实验孔、单纯效应细胞孔、单纯靶细胞孔各3个,并设空白孔。于实验孔及单纯靶细胞孔,加入K562。在实验孔及单纯效应细胞孔中分别加入实施例5和对比例1-2扩增所得NK细胞,效靶比设为1:1、1:10、1:20和1:50四组,培养24h。加入20μLWST,继续培养4h,酶标仪检测OD值。
杀伤活性(%)=[1-(实验组OD值-单纯效应细胞组OD值)×单纯靶细胞OD值]×100%。
如图6所示,采用实施例5方法能有效提高NK细胞的杀伤活性。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (6)
1.一种用于肿瘤治疗的NK细胞培养方法,其特征在于,包括如下步骤:
(1)分离单个核细胞,将单个核细胞接种于NK细胞培养基中培养1-3天,培养温度为35-38℃,得到一次培养物;
其中,NK细胞培养基中包括基础培养基和添加物;基础培养基由RPMI-1640培养基和GT-T551培养基混合得到;添加物包括:胰岛素、人血清白蛋白、胆固醇、谷氨酰胺、抗CD3抗体、FK506和IL-15;
(2)向一次培养物中补加OK-432、IL-21和去乙酰毛花苷,继续培养1-3天;
(3)向二次培养物中IL-2、IL-15、IL-21、亚叶酸、姜黄素,继续培养1-3天。
2.根据权利要求1所述用于肿瘤治疗的NK细胞培养方法,其特征在于,步骤(1)中,细胞密度为5×105-2×106个/mL。
3.根据权利要求1所述用于肿瘤治疗的NK细胞培养方法,其特征在于,步骤(1)中,基础培养基由RPMI-1640培养基和GT-T551培养基按体积比为1:1-2混合得到。
4.根据权利要求1所述用于肿瘤治疗的NK细胞培养方法,其特征在于,步骤(1)的添加物中,胰岛素的浓度为10-30IU/mL,人血清白蛋白的浓度为120-200μg/mL,胆固醇的浓度为1-5μg/mL,谷氨酰胺的浓度为5-15μg/mL,抗CD3抗体的浓度为5-15μg/mL,FK506的浓度为1-6ng/mL,IL-15的浓度为1-5ng/mL。
5.根据权利要求1所述用于肿瘤治疗的NK细胞培养方法,其特征在于,步骤(2)中,OK-432的终浓度为2-10ng/mL,IL-21的终浓度为1-10ng/mL,去乙酰毛花苷的终浓度为1-10ng/mL。
6.根据权利要求1所述用于肿瘤治疗的NK细胞培养方法,其特征在于,IL-2的终浓度为25-45ng/mL,IL-15的终浓度为10-20ng/mL,IL-21的终浓度为1-5ng/mL,亚叶酸的终浓度为1-5ng/mL,姜黄素的终浓度为1-5ng/mL。
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