CN115125203A - 一种Th2细胞体外培养方法 - Google Patents
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Abstract
本发明提供了一种体外培养Th2细胞的培养方法，属于细胞培养技术领域。获取外周血PBMC，使用含自体血浆的ALyS505N‑0培养基调整细胞密度，DAY0加入CD3、CD28、BCL2L2蛋白、神经肽、吗啡、多西环素、IL‑13、IL‑16、IL‑11、IL‑4、IL‑2，DAY3、DAY5添加含IL‑13、IL‑16、IL‑11、IL‑4、IL‑2的ALyS505N‑0培养基，DAY7、DAY9、DAY11分别添加IL‑11、IL‑4、IL‑2的ALyS505N‑0培养基，培养至14天收获细胞。本发明显著提高了TH2细胞的纯度和增殖倍数，纯度在70％以上，增殖倍数在300倍以上。
Description
技术领域
本发明属于免疫细胞治疗技术领域，尤其涉及一种Th2细胞体外培养方法。
背景技术
CD4+辅助性T细胞(Th细胞)辅对机体的特异性和非特异性免疫均具有重要调节作用，能协助B细胞产生抗体，也能促进其他T细胞的分化成熟，是机体内一类重要的免疫调节细胞。根据其分化的细胞因子类型的不同，可将Th细胞分成三个亚型:Th1细胞、Th2细胞和Th17细胞。
Th2细胞主要功能为刺激B细胞增殖并产生免疫球蛋白G1和免疫球蛋白E抗体，与体液免疫有关。Th2细胞与细胞外免疫有关,其主要功能为清除机体寄生虫感染,同时与哮喘及其他过敏性疾病的诱发和持续有关,主要产生IL-4,IL-5,IL-9,IL-10,IL-13,IL-25和双调蛋白等重要细胞因子，这些细胞因子负责促进抗体合成、活化嗜酸性粒细胞和抑制多种巨噬细胞功能，从而提供独立于吞噬细胞的保护反应。这些细胞因子也会抵消Th1反应，使Th2接受IL-4的刺激活化。在功能上，由于细胞因子受体在多种细胞类型中广泛表达，因此Th2细胞因子对体内的许多细胞类型都有影响。Th2细胞刺激并募集免疫细胞的特定亚群，例如嗜酸性粒细胞和嗜碱性粒细胞，使其到达感染部位或对导致组织嗜酸性粒细胞增多和肥大细胞增生的过敏原或毒素作出反应。Th2细胞介导的免疫应答是机体清除寄生虫感染和变态反应性疾病发生的主要机制。
有研究表明，Th2细胞的分化受多种因素的影响，未致敏(naive)CD4+T细胞经抗原刺激后分化为Th0细胞，作为Th1和Th2的前体细胞，Th0细胞可分泌Th1样细胞因子和Th2样细胞因子。
从前体Th0细胞分化为Th2细胞需抗原的反复刺激，其分化受到微环境和抗原呈递细胞(APC)等因素的影响。Th0细胞向Th2细胞的极化可受到抗原类型和浓度、共刺激分子、细胞因子浓度、免疫活性激素、转录因子、抗原呈递细胞的类型等多种因素的影响。其中细胞因子起着重要的调节作用：细胞因子IL-4、IL-13主要调节Th2细胞分化此外，MHC-抗原肽-TCR亲和力也是影响Th1/Th2细胞分化的重要因素之一。抗原肽与MHC之间亲和力越低越有利于Th2细胞分化；抗原肽-MHC复合体与TCR之间亲和力越低越有利于Th2细胞分化。
目前，一般通过以下两种不同方式体外获得Th2细胞。一是通过磁珠分离或流式细胞术(FACS)分选出部分T细胞(例如，Naive CD4+T细胞)，然后细胞进行体外活化并分化成特定的Th2亚群，该方法虽然能得到较高纯度的Th2细胞，但成本较高，且由于使用磁珠或流式分选，对于后续用于临床治疗存在风险。二是直接从外周血分离PBMC，加入抗CD3和抗CD28抗体、IL-4诱导Th2细胞，该种方法操作简便，但Th2细胞纯度较低，降低了Th2细胞的应用价值。
发明内容
有鉴于此，本发明的目的在于提供一种Th2细胞体外培养方法；以便提高所获得的Th2细胞的纯度、数量和安全性。
为了实现上述发明目的，本发明提供了以下技术方案：
本发明提供了一种Th2细胞体外培养方法，包括以下步骤：
1)培养瓶包被，在T75cm2培养瓶中，加入9ml DPBS和单抗CD31-10ug/ml，单抗CD281-10ug/ml，轻轻摇晃，使溶液均匀的铺满瓶底，在室温下，静置40～60分钟，移除包被液；用10ml DPBS清洗培养瓶一次，
2)制备外周血PBMC，使用含BCL2L2蛋白20-100ng/ml、神经肽1-5ng/ml、吗啡20-50ng/ml、多西环素0.5-2ug/ml、IL-13 2-10ng/ml、IL-165-20ng/ml、IL-11 20-50ng/ml、IL-4 8-20ng/ml、IL-2 2-10ng/ml,无血清培养基培养基制备成细胞液,转入至步骤1)包被好的培养瓶中进行培养。
3)第0天接种细胞后，DAY3、DAY5天添加含IL-13 2-10ng/ml、IL-165-20ng/ml、IL-11 20-50ng/ml、IL-4 8-20ng/ml、IL-2 2-10ng/ml的无血清培养基进行培养，DAY7、DAY9、DAY11天添加含IL-11 20-50ng/ml、IL-48-20ng/ml、IL-2 2-10ngIU/m的无血清培养基培养基进行培养，至第14天收获细胞。
进一步地，所述步骤(2)采用PBMC分离方法采用密度梯度法分离得到。
进一步地，所述无血清培养基为ALyS505N-0。
进一步地，所述步骤(2)中PBMC细胞悬液中细胞密度为1.0-1.3×106个/ml。
进一步地，所述步骤(2)和步骤(3)中细胞培养条件为37℃，5％CO2条件下进行培养。
进一步地，所述步骤(3)中添加新鲜培养基后，细胞悬液的密度在1.0×106个/ml以上。
相比现有技术，本发明的有益效果在于：本发明提供一种Th2细胞体外培养的方法，使用密度梯度法分离得到PBMC，使用含BCL2L2蛋白、神经肽、吗啡、多西环素、IL-13、IL-16、IL-11、IL-4、IL-2的AlyS505N-0培养基重悬PBMC后转入使用单抗CD3、单抗CD28包被的培养瓶中，诱导Th2细胞活化与增殖。在培养过程中不断添加含IL-13、IL-16、IL-11、IL-4、IL-2的AlyS505N-0无血清培养基，进一步促进Th2细胞的生长和分化，提高Th2细胞的数量。同时，使用该方法扩增得到了高纯度的Th2细胞，提高了Th2细胞的应用价值。
附图说明
图1为实施例1体外Th2细胞培养第14天CD4+IL-4+表型检测流式图；
图2为实施例2体外Th2细胞培养第14天CD4+IL-4+表表型检测流式图；
图3为实施例3体外Th2细胞培养第14天CD4+IL-4+表表型检测流式图；
图4为对比例1体外Th2细胞培养第14天CD4+IL-4+表表型检测流式图；
图5为对比例2体外Th2细胞培养第14天CD4+IL-4+表表型检测流式图；
图6为对比例3体外Th2细胞培养第14天CD4+IL-4+表表型检测流式图；
图7为对比例4体外Th2细胞培养第14天CD4+IL-4+表表型检测流式图；
图8为对比例5体外Th2细胞培养第14天CD4+IL-4+表表型检测流式图；
图9为对比例6体外Th2细胞培养第14天CD4+IL-4+表表型检测流式图；
图10为对比例7体外Th2细胞培养第14天CD4+IL-4+表表型检测流式图；
图11为对比例8体外Th2细胞培养第14天CD4+IL-4+表表型检测流式图。
【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。
本发明需要提示说明的实验环境、实验材料和仪器设备如下：
1、实验环境：GMP环境下实验室内的超净工作台中操作。
2、试剂：磷酸盐缓冲液DPBS(珠海贝索生物技术有限公司)、ALyS505N-0(株式会社细胞科学研究所CSTI)、人淋巴细胞分离液、肝素钠采血管、CD3单抗、CD28单抗、BCL2L2蛋白、IL-13、IL-16、IL-11、IL-4、IL-2。
3、仪器与设备：离心机(Thermo，美国)、T75悬浮培养瓶、T175悬浮培养瓶、NIPRO细胞培养袋(日本NIPRO株式会社)、CO2培养箱(三洋，中国)、超净工作台(苏州净化)。
实施例1
一种体外培养Th2细胞的方法，包括以下步骤：
(1)培养瓶包被，在T75cm2培养瓶中，加入9ml DPBS和单抗CD3 10ug/ml，单抗CD2810ug/ml，轻轻摇晃，使溶液均匀的铺满瓶底，在室温下，静置40分钟，移除包被液；用10mlDPBS清洗培养瓶一次。
(2)采用密度梯度离心法得到PBMC，使用含BCL2L2蛋白100ng/ml、神经肽5ng/ml、吗啡50ng/ml、多西环素2ug/ml、IL-13 10ng/ml、IL-16 20ng/ml、IL-11 50ng/ml、IL-420ng/ml、IL-2 10ng/ml无血清培养基培养基制备成细胞液20ml，细胞悬液的密度为1.0×106个/ml，转入至步骤(1)包被好的培养瓶中进行培养。
(3)第0天接种细胞后，DAY3、DAY5天补充添加含IL-13 10ng/ml、IL-16 20ng/ml、IL-11 50ng/ml、IL-4 20ng/ml、IL-2 10ng/ml的无血清培养基培养基进行培养，DAY7、DAY9、DAY11天分别补充添加含IL-11 50ng/ml、IL-4 20ng/ml、IL-2 10ng/ml的无血清培养基培养基进行培养，每次补液后的细胞密度为1.0×106个/ml。
(4)培养至第14天收获成熟Th2细胞，取样检测细胞数，算出增殖倍数，流式检测Th2细胞的纯度，结果见表1。
表1实验结果
实施例2
一种体外培养Th2细胞的方法，包括以下步骤：
(1)培养瓶包被，在T75cm2培养瓶中，加入9ml DPBS和单抗CD3 1ug/ml，单抗CD281ug/ml，轻轻摇晃，使溶液均匀的铺满瓶底，在室温下，静置60分钟，移除包被液；用10mlDPBS清洗培养瓶一次。
(2)采用密度梯度离心法得到PBMC，使用含BCL2L2蛋白20ng/ml、神经肽1ng/ml、吗啡20ng/ml、多西环素0.5ug/ml、IL-13 2ng/ml、IL-16 5ng/ml、IL-11 20ng/ml、IL-4 8ng/ml、IL-2 2ng/ml，无血清培养基培养基制备成细胞悬液20ml，细胞悬液的密度为1.3×106个/ml，转入至步骤(1)包被好的培养瓶中进行培养。
(3)第0天接种细胞后，DAY3、DAY5天补充添加含、IL-13 2ng/ml、IL-16 5ng/ml、IL-11 20ng/ml、IL-4 8ng/ml、IL-2 2ng/ml的无血清培养基培养基进行培养，DAY7、DAY9、DAY11天分别补充添加含IL-11 20ng/ml、IL-4 8ng/ml、IL-2 2ng/ml的无血清培养基培养基进行培养，每次补液后将细胞密度维持在1.3×106个/ml。
(4)培养至第14天收获成熟Th2细胞，取样检测细胞数，算出增殖倍数，流式检测Th2细胞的纯度，结果见表2。
表2实验结果
实施例3
一种体外培养Th2细胞的方法，包括以下步骤：
(1)培养瓶包被，在T75cm2培养瓶中，加入9ml DPBS和单抗CD3 5ug/ml，单抗CD285ug/ml，轻轻摇晃，使溶液均匀的铺满瓶底，在室温下，静置50分钟，移除包被液；用10mlDPBS清洗培养瓶一次。
(2)采用密度梯度离心法得到PBMC，使用含BCL2L2蛋白60ng/ml、神经肽3ng/ml、吗啡40ng/ml、多西环素1.2ug/ml、IL-13 6ng/ml、IL-16 14ng/ml、IL-11 38ng/ml、IL-414ng/ml、IL-2 8ng/ml的无血清培养基培养基制备成细胞悬液20ml，细胞密度为1.1×106个/ml，转入至步骤(1)包被好的培养瓶中进行培养。
(3)第0天接种细胞后，DAY3、DAY5天分别补充添加含IL-13 6ng/ml、IL-16 14ng/ml、IL-11 38ng/ml、IL-4 14ng/ml、IL-2 8ng/ml的无血清培养基培养基进行培养，DAY7、DAY9、DAY14天分别补充添加含IL-11 38ng/ml、IL-4 14ng/ml、IL-2 8ng/mll的无血清培养基培养基进行培养，每次补液后细胞密度维持在1.1×106个/ml。
(4)培养至第14天收获成熟Th2细胞，取样检测细胞数，算出增殖倍数，流式检测Th2细胞的纯度，结果见表3。
表3实验结果
对比例1
对比例1提供一种体外培养Th2细胞的方法，和实施例1的区别为：DAY0天使用单抗CD10 2ug/ml，单抗CD28 10ug/ml包被培养瓶，然后使用IL-4 20ng/ml、IL-2 10ng/ml的AlyS505N-0培养基进行培养，DAY3、DAY5、DAY7、DAY9、DAY11天添加含IL-4 15ng/ml、IL-25ng/ml的AlyS505N-0培养基，其余均与实施例1相同。
对比例2
对比例2提供一种体外培养Th2细胞的方法，和实施例1的区别为：省去步骤(2)中的BCL2L2蛋白，其余均与实施例1相同。
对比例3
对比例3提供一种体外培养Th2细胞的方法，和实施例1的区别为：省去步骤(2)中神经肽，其余均与实施例1相同。
对比例4
对比例4提供一种体外培养Th2细胞的方法，和实施例1的区别为：省去步骤(2)中吗啡，其余均与实施例1相同。
对比例5
对比例5提供一种体外培养Th2细胞的方法，和实施例1的区别为：省去步骤(2)中多西环素，其余均与实施例1相同。
对比例6
对比例6提供一种体外培养Th2细胞的方法，和实施例1的区别为：省去步骤(2)和(3)中IL-13，其余均与实施例1相同。
对比例7
对比例7提供一种体外培养Th2细胞的方法，和实施例1的区别为：省去步骤(2)和(3)中IL-16，其余均与实施例1相同。
对比例8
对比例7提供一种体外培养Th2细胞的方法，和实施例1的区别为：省去步骤(2)和(3)中IL-11，其余均与实施例1相同。
试验例1
(1)采用台盼蓝染色法统计实施例1、对比例1至8中细胞在培养第0天和第14天的数量，计算各组的增殖倍数。
(2)流式表型检测：取实施例1、对比例1至8中培养至第14天的细胞以流式抗体染色，测定Th2细胞CD4+IL-4+双阳性细胞的百分比。
结果如表4所示：
表4实验结果
由表4可知，实施例1的培养方法细胞增殖倍数最高，在第14天时收获的细胞数最多。对比例1-8中使用常规方法制备体外制备Th2细胞或者省去本发明中的某些成分后Th2细胞的细胞数、细胞纯度均有所下降。由各对照组1-8可以看出，常规方法制备体外制备Th2细胞或省去本发明中的某些成分后，其Th2细胞培养效果均不及本发明的实施例，进一步说明上述成分协同作用，提高免疫细胞的数量、纯度。
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 (6)
1.一种Th2细胞体外培养方法，其特征在于，具体步骤如下：
1)培养瓶包被，在T75cm2培养瓶中，加入9ml DPBS和单抗CD31-10ug/ml，单抗CD28 1-10ug/ml，轻轻摇晃，使溶液均匀的铺满瓶底，在室温下，静置40～60分钟，移除包被液；用10ml DPBS清洗培养瓶一次，
2)制备外周血PBMC，使用含BCL2L2蛋白20-100ng/ml、神经肽1-5ng/ml、吗啡20-50ng/ml、多西环素0.5-2ug/ml、IL-13 2-10ng/ml、IL-165-20ng/ml、IL-11 20-50ng/ml、IL-4 8-20ng/ml、IL-2 2-10ng/ml无血清培养基培养基制备成细胞液,转入至步骤1)包被好的培养瓶中进行培养。
3)第0天接种细胞后，DAY3、DAY5天补加含IL-13 2-10ng/ml、IL-165-20ng/ml、IL-1120-50ng/ml、IL-4 8-20ng/ml、IL-2 2-10ng/ml的无血清培养基进行培养；DAY7、DAY9、DAY11天补加含IL-11 20-50ng/ml、IL-48-20ng/ml、IL-2 2-10ng/m的无血清培养基培养基进行培养，至第14天收获细胞。
2.根据权利要求1所述体外培养Th2细胞的方法，其特征在于，所述步骤2)采用密度梯度法分离得到PBMC。
3.根据权利要求1所述体外培养Th2细胞的方法，其特征在于，所述无血清培养基为ALyS505N-0。
4.根据权利要求1所述体外培养Th2细胞的方法，其特征在于，所述步骤2)中PBMC细胞悬液中细胞密度为1.0-1.3×106个/ml。
5.根据权利要求1所述体外培养Th2细胞的方法，其特征在于，所述步骤2)和步骤3)中在37℃，5％CO2条件下进行细胞培养。
6.根据权利要求1所述体外培养Th2细胞的方法，其特征在于，所述步骤3)中补加新鲜培养基后，细胞悬液的密度在1.0-1.5×106个/ml以上。
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