CN113957050A - 一种nkt细胞的培养方法 - Google Patents
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Abstract
本发明公开了一种NKT细胞的培养方法,包括以下步骤:(1)自外周血中分离出外周血单个核细胞,采用无血清培养基重悬;(2)将步骤(1)重悬后的细胞接种在CD3抗体和缬草油包被的培养容器中,培养12小时后加入IL‑2、IFN‑γ、异丙肌苷、钛酸钙、抗坏血酸,继续培养过程中及时补充添加IL‑2、IFN‑γ、异丙肌苷、钛酸钙、抗坏血酸的无血清培养基;(3)培养3‑4天后补充的无血清培养基中添加以下成分:羟基芫花素、2‑巯基乙醇、维生素B3、IL‑2、IL‑21,每隔2‑3天补液一次,培养至第14天收获NKT细胞。采用上述培养过程得到的NKT细胞数量多,增殖效率有显著提高,而且得到的NKT细胞活性高,临床应用效果更好。
Description
技术领域
本发明涉及细胞培养领域,尤其涉及一种NKT细胞的培养方法。
背景技术
NKT细胞(Natural killer T cell)是一种细胞表面既有T细胞受体TCR,又有NK细胞受体的特殊T细胞亚群。NKT细胞能大量产生细胞因子,且可以发挥与NK细胞相似的细胞毒作用。。NKT细胞主要分布于肝、骨髓、胸腺、脾及外周血中。
NKT细胞作为一类新的免疫调节细胞在抗肿瘤免疫,克服移植排斥,以及抑制自身免疫性疾病的发生中发挥着至关重要的作用。NKT细胞的功能异常与多种临床疾病,如恶性肿瘤、自身免疫性疾病等。研究还显示,调控NKT细胞的功能可达到对肿瘤以及多种人类疾病的治疗效果。
为了满足NKT细胞在临床的应用需求,体外培养NKT细胞成为获得足够数量具有活性的NKT细胞有效途径。常见的NKT细胞体外培养方法是在添加诸多细胞因子的培养基中培养外周血单个核细胞,在细胞因子的刺激下诱导外周血单个核细胞成为NKT细胞。在常见的培养过程中,需要添加各类因子,但是常规培养方法得到的NKT细胞增殖率较低,获得的细胞数量有限,并且细胞活性差,限制了NKT细胞在临床的应用。因此,有必要提供一种NKT细胞的培养方法,提高NKT细胞的体外增殖效率。
发明内容
为了克服现有技术的不足,本发明的目的在于提供一种NKT细胞的培养方法,具有增殖效率高,细胞活性好的特点。
本发明的目的采用如下技术方案实现:
一种NKT细胞的培养方法,包括以下步骤:
(1)自外周血中分离出外周血单个核细胞,采用无血清培养基重悬;
(2)将步骤(1)重悬后的细胞接种在CD3抗体和缬草油包被的培养容器中,培养12小时后加入IL-2、IFN-γ、异丙肌苷、钛酸钙、抗坏血酸,继续培养过程中及时补充添加IL-2、IFN-γ、异丙肌苷、钛酸钙、抗坏血酸的无血清培养基;
(3)培养3-4天后补充的无血清培养基中添加以下成分:羟基芫花素、2-巯基乙醇、维生素B3、IL-2、IL-21,每隔2-3天补液一次,培养至第14天收获NKT细胞。
优先地,步骤(2)中添加的IL-2、IFN-γ、异丙肌苷、钛酸钙、抗坏血酸在培养基中的终浓度为:IL-2 1000-1500U/mL、IFN-γ500-1000 U/mL、异丙肌苷10-15μg/mL、钛酸钙3-8μg/mL、抗坏血酸6-10μg/mL。
优先地,步骤(2)中添加的IL-2、IFN-γ、异丙肌苷、钛酸钙、抗坏血酸在培养基中的终浓度为:IL-2 1300U/mL、IFN-γ800U/mL、异丙肌苷12μg/mL、钛酸钙6μg/mL、抗坏血酸8μg/mL。
优先地,步骤(3)中添加的羟基芫花素、2-巯基乙醇、维生素B3、IL-2、IL-21在培养基中的终浓度为:羟基芫花素20-30ng/mL、2-巯基乙醇10-20 ng/mL、维生素B3 1-5μg/mL、IL-21000-1500U/mL、IL-21 800-1200 U/mL。
优先地,步骤(3)中添加的羟基芫花素、2-巯基乙醇、维生素B3、IL-2、IL-21在培养基中的终浓度为:羟基芫花素25ng/mL、2-巯基乙醇15ng/mL、维生素B3 3μg/mL、IL-21200U/mL、IL-21 1000U/mL。
优先地,无血清培养基为AIM-V培养基。
优先地,步骤(2)中培养容器的包被过程如下:向PBS中加入CD3抗体和缬草油,CD3抗体和缬草油在PBS中的浓度为:CD3抗体4-8μg/mL、缬草油1-5ng/mL,在4℃下包被过夜。
优先地,步骤(1)中细胞密度为1-3×106个/mL。
相比现有技术,本发明的有益效果在于:本发明提供一种NKT细胞的培养方法,将外周血单个核细胞接种在CD3抗体和缬草油包被的容器中,有助于细胞保持稳定的状态,然后在培养基中添加IL-2、IFN-γ、异丙肌苷、钛酸钙、抗坏血酸,有助于激活更多的NKT细胞。培养3-4天后在培养基中添加羟基芫花素、2-巯基乙醇、维生素B3、IL-2、IL-21继续扩增培养NKT细胞,采用上述培养过程得到的NKT细胞数量多,增殖效率有显著提高,而且得到的NKT细胞活性高,临床应用效果更好。
具体实施方式
下面,结合具体实施方式,对本发明做进一步描述,需要说明的是,在不相冲突的前提下,以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。
实施例1
一种NKT细胞的培养方法,包括以下步骤:
(1)自外周血中分离出外周血单个核细胞,采用无血清AIM-V培养基重悬,细胞密度为1×106个/mL;
(2)取T75培养瓶采用包被液在4℃下包被过夜,包被液的制备过程如下:向PBS中加入CD3抗体和缬草油,CD3抗体和缬草油在PBS中的浓度为:CD3抗体6μg/mL、缬草油3ng/mL,取步骤(1)重悬后的细胞15mL接种在包被后T75培养瓶中,培养12小时后加入IL-2、IFN-γ、异丙肌苷、钛酸钙、抗坏血酸,继续培养过程中及时补充添加IL-2、IFN-γ、异丙肌苷、钛酸钙、抗坏血酸的AIM-V培养基,使细胞密度保持在1×106个/mL;IL-2、IFN-γ、异丙肌苷、钛酸钙、抗坏血酸在培养基中的终浓度为:IL-2 1300U/mL、IFN-γ800U/mL、异丙肌苷12μg/mL、钛酸钙6μg/mL、抗坏血酸8μg/mL。
(3)培养3天后补充的AIM-V培养基中添加以下成分:羟基芫花素、2-巯基乙醇、维生素B3、IL-2、IL-21,羟基芫花素、2-巯基乙醇、维生素B3、IL-2、IL-21在培养基中的终浓度为:羟基芫花素25ng/mL、2-巯基乙醇15ng/mL、维生素B3 3μg/mL、IL-2 1200U/mL、IL-211000U/mL,每隔2天补液一次,培养至第14天收获NKT细胞。
实施例2
一种NKT细胞的培养方法,包括以下步骤:
(1)自外周血中分离出外周血单个核细胞,采用无血清AIM-V培养基重悬,细胞密度为2×106个/mL;
(2)取T75培养瓶采用包被液在4℃下包被过夜,包被液的制备过程如下:向PBS中加入CD3抗体和缬草油,CD3抗体和缬草油在PBS中的浓度为:CD3抗体4μg/mL、缬草油1ng/mL,取步骤(1)重悬后的细胞15mL接种在包被后T75培养瓶中,培养12小时后加入IL-2、IFN-γ、异丙肌苷、钛酸钙、抗坏血酸,继续培养过程中及时补充添加IL-2、IFN-γ、异丙肌苷、钛酸钙、抗坏血酸的AIM-V培养基,使细胞密度保持在2×106个/mL;IL-2、IFN-γ、异丙肌苷、钛酸钙、抗坏血酸在培养基中的终浓度为:IL-2 1000U/mL、IFN-γ500U/mL、异丙肌苷10μg/mL、钛酸钙3μg/mL、抗坏血酸6μg/mL。
(3)培养3天后补充的AIM-V培养基中添加以下成分:羟基芫花素、2-巯基乙醇、维生素B3、IL-2、IL-21,羟基芫花素、2-巯基乙醇、维生素B3、IL-2、IL-21在培养基中的终浓度为:羟基芫花素20ng/mL、2-巯基乙醇10 ng/mL、维生素B3 1μg/mL、IL-21000U/mL、IL-21800 U/mL,每隔2天补液一次,培养至第14天收获NKT细胞。
实施例3
一种NKT细胞的培养方法,包括以下步骤:
(1)自外周血中分离出外周血单个核细胞,采用无血清AIM-V培养基重悬,细胞密度为3×106个/mL;
(2)取T75培养瓶采用包被液在4℃下包被过夜,包被液的制备过程如下:向PBS中加入CD3抗体和缬草油,CD3抗体和缬草油在PBS中的浓度为:CD3抗体8μg/mL、缬草油5ng/mL,取步骤(1)重悬后的细胞15mL接种在包被后T75培养瓶中,培养12小时后加入IL-2、IFN-γ、异丙肌苷、钛酸钙、抗坏血酸,继续培养过程中及时补充添加IL-2、IFN-γ、异丙肌苷、钛酸钙、抗坏血酸的AIM-V培养基,使细胞密度保持在3×106个/mL;IL-2、IFN-γ、异丙肌苷、钛酸钙、抗坏血酸在培养基中的终浓度为:IL-2 1500U/mL、IFN-γ1000 U/mL、异丙肌苷15μg/mL、钛酸钙8μg/mL、抗坏血酸10μg/mL。
(3)培养3-4天后补充的AIM-V培养基中添加以下成分:羟基芫花素、2-巯基乙醇、维生素B3、IL-2、IL-21,羟基芫花素、2-巯基乙醇、维生素B3、IL-2、IL-21在培养基中的终浓度为:羟基芫花素30ng/mL、2-巯基乙醇20 ng/mL、维生素B3 5μg/mL、IL-2 1500U/mL、IL-211200 U/mL,每隔3天补液一次,培养至第14天收获NKT细胞。
对比例1
对比例1提供一种NKT细胞的培养方法,和实施例1的区别在于:省去包被液中的缬草油,其余均和实施例1相同。
对比例2
对比例2提供一种NKT细胞的培养方法,和实施例1的区别在于:省去异丙肌苷,其余均和实施例1相同。
对比例3
对比例3提供一种NKT细胞的培养方法,和实施例1的区别在于:省去钛酸钙,其余均和实施例1相同。
对比例4
对比例4提供一种NKT细胞的培养方法,和实施例1的区别在于:省去羟基芫花素,其余均和实施例1相同。
对比例5
对比例5提供一种NKT细胞的培养方法,和实施例1的区别在于:省去羟基芫花素,将2-巯基乙醇的用量调整为40ng/mL,其余均和实施例1相同。
对比例6
对比例6提供一种NKT细胞的培养方法,和实施例1的区别在于:省去2-巯基乙醇,将羟基芫花素的用量调整为40ng/mL,其余均和实施例1相同。
分别统计实施例1至3,对比例1至6中细胞在经14天的培养后的扩增倍数及培养14天后的细胞活率。细胞数量用台盼蓝染色后采用Countstar细胞计数仪进行计数,用流式细胞仪检测培养14天得到的NKT细胞中表达CD3+CD56+细胞占比,结果如表1所示。
表1
组别 | 扩增倍数 | 细胞活率(%) | CD3<sup>+</sup>CD56<sup>+</sup>(%) |
实施例1 | 425 | 97.68 | 85.46 |
实施例2 | 421 | 97.22 | 84.77 |
实施例3 | 424 | 97.95 | 85.06 |
对比例1 | 379 | 91.74 | 61.93 |
对比例2 | 321 | 83.62 | 67.81 |
对比例3 | 356 | 89.48 | 72.83 |
对比例4 | 325 | 82.29 | 79.49 |
对比例5 | 317 | 85.33 | 81.25 |
对比例6 | 346 | 88.78 | 80.66 |
由表1可以看出,实施例1至3中细胞的扩增倍数、活率以及CD3+CD56+细胞所占比例均高于对比例1至6。对比例1中省去了包被液中的缬草油,对比例2和对比例3中分别省去了异丙肌苷、钛酸钙,细胞增殖活性降低,这是因为采用CD3抗体和缬草油包被培养瓶有助于细胞保持稳定的状态,添加的异丙肌苷、钛酸钙和培养基中的其他成分一起有助于激活更多的NKT细胞。对比例4中省去了羟基芫花素,对比例5和对比例6中分别在省去羟基芫花素和2-巯基乙醇中的一种后增加剩余组分的用量,细胞的增殖活性也有不同程度的下降,说明羟基芫花素、2-巯基乙醇两种成分协同作用,提高了细胞的增殖活性。
以HepG2细胞为靶细胞,采用MTT法分别检测效靶比10:1,20:1,40:1 时NKT细胞的杀伤活性,结果如表2所示。
表2
组别 | 10:1 | 20:1 | 40:1 |
实施例1 | 51.49 | 74.93 | 91.26 |
对比例1 | 23.59 | 48.97 | 65.88 |
对比例2 | 31.97 | 52.64 | 70.21 |
对比例3 | 38.28 | 57.99 | 74.08 |
对比例4 | 42.93 | 63.22 | 81.94 |
对比例5 | 44.37 | 65.51 | 86.72 |
对比例6 | 43.05 | 63.76 | 84.27 |
由表2可以看出实施例1中的NKT细胞的杀伤活性较高,在效靶比40:1时,杀伤活性达到91.26%。在对比例1至6中收获的NIK细胞杀伤活性均不及实施例1。说明采用本发明的培养方法有助于NKT细胞保持较好的杀瘤活性。
上述实施方式仅为本发明的优选实施方式,不能以此来限定本发明保护的范围,本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
Claims (8)
1.一种NKT细胞的培养方法,其特征在于,包括以下步骤:
(1)自外周血中分离出外周血单个核细胞,采用无血清培养基重悬;
(2)将步骤(1)重悬后的细胞接种在CD3抗体和缬草油包被的培养容器中,培养12小时后加入IL-2、IFN-γ、异丙肌苷、钛酸钙、抗坏血酸,继续培养过程中及时补充添加IL-2、IFN-γ、异丙肌苷、钛酸钙、抗坏血酸的无血清培养基;
(3)培养3-4天后补充的无血清培养基中添加以下成分:羟基芫花素、2-巯基乙醇、维生素B3、IL-2、IL-21,每隔2-3天补液一次,培养至第14天收获NKT细胞。
2. 根据权利要求1所述NKT细胞的培养方法,其特征在于,步骤(2)中添加的IL-2、IFN-γ、异丙肌苷、钛酸钙、抗坏血酸在培养基中的终浓度为:IL-2 1000-1500U/mL、IFN-γ500-1000 U/mL、异丙肌苷10-15μg/mL、钛酸钙3-8μg/mL、抗坏血酸6-10μg/mL。
3.根据权利要求1所述NKT细胞的培养方法,其特征在于,步骤(2)中添加的IL-2、IFN-γ、异丙肌苷、钛酸钙、抗坏血酸在培养基中的终浓度为:IL-2 1300U/mL、IFN-γ800U/mL、异丙肌苷12μg/mL、钛酸钙6μg/mL、抗坏血酸8μg/mL。
4. 根据权利要求1所述NKT细胞的培养方法,其特征在于,步骤(3)中添加的羟基芫花素、2-巯基乙醇、维生素B3、IL-2、IL-21在培养基中的终浓度为:羟基芫花素20-30ng/mL、2-巯基乙醇10-20 ng/mL、维生素B3 1-5μg/mL、IL-21000-1500U/mL、IL-21 800-1200 U/mL。
5.根据权利要求1所述NKT细胞的培养方法,其特征在于,步骤(3)中添加的羟基芫花素、2-巯基乙醇、维生素B3、IL-2、IL-21在培养基中的终浓度为:羟基芫花素25ng/mL、2-巯基乙醇15ng/mL、维生素B3 3μg/mL、IL-2 1200U/mL、IL-21 1000U/mL。
6.根据权利要求1所述NKT细胞的培养方法,其特征在于,无血清培养基为AIM-V培养基。
7.根据权利要求1所述NKT细胞的培养方法,其特征在于,步骤(2)中培养容器的包被过程如下:向PBS中加入CD3抗体和缬草油,CD3抗体和缬草油在PBS中的浓度为:CD3抗体4-8μg/mL、缬草油1-5ng/mL,在4℃下包被过夜。
8.根据权利要求1所述NKT细胞的培养方法,其特征在于,步骤(1)中细胞密度为1-3×106个/mL。
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