CN115044549B - 用于保持免疫细胞多样性的培养基和细胞组合物的培养方法 - Google Patents
用于保持免疫细胞多样性的培养基和细胞组合物的培养方法 Download PDFInfo
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Abstract
本发明涉及一种用于保持免疫细胞多样性的培养基，所述培养基含有M‑CSF、laminin、collagen I、collagen IV、抗CD3抗体、抗LILRB2抗体、Heparansulphate、IL‑2、IL‑10、IL‑15、人血浆、vitamin D和AS1842856。本发明还提供了一种具有免疫微环境的细胞组合物的培养方法。通过上述技术方案，本发明提供的用于保持混合细胞群体中免疫细胞多样性的培养基，在培养离体的胸水和/或腹水中分离的混合细胞群体时，能够更好地维持免疫细胞的多样性。
Description
技术领域
本发明涉及生物医药技术领域，具体涉及一种用于保持免疫细胞多样性的混合细胞群体培养基和一种具有免疫微环境的细胞组合物的培养方法。
背景技术
恶性肿瘤的临床病理表现之一是患者会出现胸水和/或腹水。
胸水和/或腹水中的主要细胞成分中含有大量的肿瘤细胞和免疫细胞，这些免疫细胞和肿瘤细胞形成的混合细胞群体，保留了肿瘤所面临的免疫微环境。其中包含各种T细胞，B细胞，NK细胞和骨髓来源的细胞。这些细胞不仅包括直接或间接参与抗击肿瘤细胞效应细胞，也包括调节肿瘤免疫效应的调节性细胞。
如果将胸水和/或腹水中的免疫细胞提取并加以扩增，可以扩增某种细胞类型，例如T细胞，NK细胞。但是会丢失很多其他细胞类型。例如，CN111286486A公开了一种培养基，所述培养基用于体外扩增恶性肿瘤腹水来源的免疫细胞，其包括：基础培养基以及促增殖因子，所述基础培养基为RPMI-1640，所述促增殖因子由重组人IL-2以及Dynabeads人T细胞激活因子CD3/CD28组成。
但是，目前的免疫细胞的培养基和培养方法所扩增出来的免疫细胞的种类过于简单，缺乏多样性，无法代表真实的免疫微环境，对于肿瘤免疫评价的价值和对肿瘤免疫药物检测的能力非常有限。如何在能够实现一定时间体外培养的前提下，保持混合细胞群体中免疫细胞的多样性，对发现新的肿瘤免疫机制和评价肿瘤免疫药物具有重要价值。
发明内容
本发明的目的是为了增加培养后的免疫细胞的多样性，进而为肿瘤免疫药物筛选和评价提供了更加接近人体的条件。
为了实现上述目的，本发明提供了一种用于保持免疫细胞多样性的培养基（Immunology Microenvironment sustainable medium, IMS培养基），所述IMS培养基含有0.5-50ng/mL的M-CSF、1-50μg/mL的laminin、0.5-2μg/mL的collagen I、0.5-10μg/mL的collagen IV、0.05-0.5μg/mL的抗CD3抗体、5-50μg/mL的抗LILRB2抗体、0.1-1 μg/mL的Heparansulphate、0.5-40U/mL 的IL-2、0.1-20ng/mL的IL-10、0.1-30ng/mL的IL-15、10-50%(w/w)的人血浆、1 -20ng/ml的vitamin D、1nM-100nM的AS1842856。
本发明还提供了一种具有免疫微环境的细胞组合物的培养方法，该方法包括：从离体的胸水和/或腹水中分离混合细胞群体，然后将所分离的混合细胞群体接种至如上所述的培养基中进行培养。
通过上述技术方案，本发明提供的用于保持混合细胞群体中免疫细胞多样性的IMS培养基，在培养离体的胸水和/或腹水中分离的混合细胞群体时，进行一定时间的培养后，还能够较好地维持免疫细胞的多样性，由此可以用于免疫能力检测和免疫药物药效评价。
本发明的其他特征和优点将在随后的具体实施方式部分予以详细说明。
附图说明
附图是用来提供对本公开的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本公开，但并不构成对本公开的限制。在附图中：
图1是测试实施例1中的细胞活力稳定性折线图。
图2是测试实施例1中培养不同时间点的不同类型细胞占比图。
图3是测试实施例1中培养不同时间点的多样性指数变化图。
图4是测试实施例2中不同培养板下测培养前后的免疫细胞多样性对比结果图。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。
本发明提供了一种用于保持免疫细胞多样性的IMS培养基，所述培养基含有0.5-50ng/mL的M-CSF、1-50μg/mL的laminin、0.5-2μg/mL的collagen I、0.5-10μg/mL的collagen IV、0.05-0.5μg/mL的抗CD3抗体、5-50μg/mL的抗LILRB2抗体、0.1-1 μg/mL的Heparansulphate、0.5-40U/mL 的IL-2、0.1-20ng/mL的IL-10、0.1-30ng/mL的IL-15、10-50%(w/w)的人血浆、1 -20ng/ml的vitamin D、1nM-100nM的AS1842856。
其中，laminin为层粘连蛋白，CAS号为114956-81-9，例如可以为购自Sigma-Aldrich的商品号为L2020的产品。
其中，collagen I为I型胶原蛋白，CAS号为9007-34-5，例如可以为购自Sigma-Aldrich的商品号为C3867的产品。
其中，collagen IV为IV型胶原蛋白，CAS号为9007-34-5，例如可以为购自Sigma-Aldrich的商品号为C6745的产品。
其中，抗CD3抗体可以为单克隆抗体，例如可以为购自Sigma-Aldrich的商品号为SAB4700041的产品。
其中，抗LILRB2抗体可以为单克隆抗体，例如可以为购自Abcam的商品号为ab224701的产品。
其中，Heparansulphate为硫酸乙酰肝素，例如可以为购自Sigma-Aldrich的商品号为H4777的产品。
其中，IL-2为白介素-2，例如可以为购自Sigma-Aldrich的商品号为SRP3085的产品。
其中，IL-10为白介素-10，例如可以为购自Sigma-Aldrich的商品号为SRP3312的产品。
其中，IL-15为白介素-15，例如可以为购自Sigma-Aldrich的商品号为SRP3077的产品。
其中，人血浆可以为自体或异体的人血浆，优选为自体人血浆。
其中，vitamin D为维生素D，例如可以为购自Sigma-Aldrich的商品号为C9756的产品。
其中，AS1842856为Foxo1抑制剂中的一种常用化合物，例如可以为购自Sigma-Aldrich的商品号为344355的产品。
其中，可选地，所述IMS培养基还含有基础培养基，所述基础培养基为RPMI-1640培养基、MEM培养基、DMEM培养基、IMEM培养基和F12培养基中的至少一种。
其中，优选地，所述IMS培养基含有还含有50-200U/mL的Penicillin-Streptomycin，1-3mM的L-Glutamine和0.5-2体积%的NEAA。
其中，Penicillin-Streptomycin为青霉素和链霉素的混合物，L-Glutamine为L-谷氨酰胺；NEAA为非必须氨基酸培养液，例如可以为购自Sigma-Aldrich的商品号为M7145的产品。
其中，所述用于保持肿瘤免疫微环境中的免疫细胞多样性的IMS培养基为用于保持来源于离体的胸水和/或腹水的免疫细胞多样性的培养基。
其中，所述胸水和/或腹水为来源于肿瘤患者的胸水和/或腹水。
其中，所述肿瘤包括肺癌、卵巢癌、胃癌、结肠癌、肝癌、乳腺癌、胰腺癌、胆管癌、食管癌、肾癌、宫颈癌、前列腺癌和黑色素瘤中的至少一种。
本发明还提供了一种具有免疫微环境的细胞组合物的培养方法，该方法包括：从离体的胸水和/或腹水中分离混合细胞群体，然后将所分离的混合细胞群体接种至如上所述的培养基中进行培养，培养的容器为超低吸附培养皿或超低吸附培养孔板。
其中，超低吸附培养皿可以为表面涂覆有超低吸附高分子涂层的培养皿，例如可以为购自Corning的商品号为I0905-071的超低吸附培养皿。
其中，可选地，从离体的胸水和/或腹水中分离包含免疫细胞的混合细胞培养物操作包括：将离体的胸水和/或腹水在200-600×g下离心5-20分钟，收集沉淀；然后将所述沉淀用Accutase蛋白酶或者Dispase II中性蛋白酶进行酶解，得到酶解产物；再将所述酶解产物用孔径为65-75μm的细胞筛过滤，得到滤出的悬液。
可选地，该方法还包括：将所述滤出的悬液进行离心以收集混合细胞群体，并将所收集的混合细胞群体接种到如上所述的IMS培养基中进行培养，培养的容器为超低吸附培养皿或超低吸附培养孔板。
其中，接种的细胞密度为1-20×106细胞/mL；培养的温度为36-38℃；培养的时间为1-10天。
下面通过实施例来进一步说明本发明，但是本发明并不因此而受到任何限制。
本发明实施例中涉及的原料、试剂、仪器和设备，如无特殊说明，均可通过购买获得，此外，在本发明实施例中未注明具体实验温度时，实验温度均为室温（20-25℃）。
实施例1
按照如下组成配制IMS培养基、条件缺失的IMS培养基及对照培养基：IMS培养基含有溶于基础培养基RPMI-1640中的15ng/mL的M-CSF、10μg/mL的laminin、0.8μg/mL的collagen I、3μg/mL的collagen IV、0.3μg/mL的抗CD3抗体、20μg/mL的抗LILRB2抗体、0.8μg/mL的Heparansulphate、10U/mL的IL-2、3ng/mL的IL-10、10ng/mL的IL-15、20%(w/w)的人血浆、10ng/mL的vitamin D、30nM的AS1842856、100U/mL的Penicillin-Streptomycin，2mM的L-Glutamine和1% v/v的NEAA。
对比例1
按照如下组成配制培养基：
对照培养基（Tmedium）含有溶于RPMI-1640中的10体积%的FBS，120 IU/mL的IL-2，100U/mL的Penicillin-Streptomycin，2mM的L-Glutamine和1% v/v的NEAA。
对比例2
按照实施例1的组成配制Laminin缺失的IMS培养基（IMS-Lamd-培养基），所不同的是，培养基中缺失Laminin。
对比例3
按照实施例1的组成配制抗LILRB2抗体缺失的IMS培养基（IMS-aLILd-培养基），所不同的是，培养基中缺失抗LILRB2抗体。
对比例4
按照实施例1的组成配制Heparansulphate缺失的IMS培养基（IMS-HSd-培养基），所不同的是，培养基中缺失Heparansulphate。
对比例5
按照实施例1的组成配制AS1842856缺失的IMS培养基（IMS-ASd-培养基），所不同的是，培养基中缺失AS1842856。
测试实施例1
按照如下步骤进行复杂免疫微环境混合培养物的培养稳定性测试。
腹腔积液来自卵巢癌患者，从离体的癌性腹腔积液中分离混合细胞培养物，将离体的腹水在400×g下离心5分钟，收集沉淀；然后加入红细胞裂解液，室温裂解10分钟，400×g离心5分钟，收集所有沉淀，将所述沉淀用Accutase®酶，37℃进行酶解20分钟，得到酶解产物；再将所述酶解产物用孔径为70μm的细胞筛过滤，得到滤出的悬液，滤后悬液400×g离心5分钟，收集细胞沉淀。
将所述滤出的悬液离心所收集沉淀即为混合细胞群体，将所述混合细胞群体进行计数，按照2×106细胞数/mL铺板，分别种植在表面有高分子涂层的超低吸附培养皿上，并分别添加实施例1和对比例1-5的培养基进行培养。通过细胞计数仪（VICELL-XR）计算不同培养基条件及在初始时间点（Day 1）和第3，5，7天的三个测试时间点中，细胞的活细胞的比例，通过台盼蓝染色，计数从0.1-30μm体积范围内活细胞总量除以总颗粒数，通过统一不同培养条件和活细胞的占比，绘制细胞活力稳定性折线图，结果如图1所示。
对初始时间点（Day 1）和第3，5，7天的不同类型细胞亚群所占比例的检测，是通过使用流式细胞仪进行FACS免疫细胞多样性分析，分析指标包括：标记死活、CD326、CD45、CD11b、CD11c、CD86、CD163、CD3、CD56、CD14、CD19。使用CytExpert软件分析FACS检测结果。其中，活细胞且CD45-CD326+的细胞为上皮细胞；活细胞且CD45+CD11b+的细胞为髓系细胞，活细胞且CD45+CD19+的细胞为B细胞，活细胞且CD45+CD3+代表T细胞；活细胞且CD45+CD3+CD4+代表CD4+T细胞；活细胞且CD45+CD3+CD8+代表CD8+T细胞；活细胞且CD45+CD3-CD56+代表NK细胞；活细胞且CD45+CD3+CD56+代表NKT细胞；活细胞且CD45+CD11b+CD86+CD163+的细胞为M2型髓系细胞，活细胞且CD45+CD11b+ CD86+CD163-的细胞为M1型髓系细胞。分析结果如图2和图3所示。图2表示经过不同培养基培养后细胞类型分布比例。图3中，亚群的多样性采用辛普森多样性指数（simpson index）来统计经过培养的多样性指数D，采用所有亚群（N）占总的活细胞的数量的比值（n/N）的平方和表示，多样性指数越小，指示淋巴细胞多样性越高。可以看到在IMS培养基中各种细胞的比例和原始样品的比例保持很好的一致。但是在对比例1的T细胞培养基中，髓系细胞和NK细胞的比例下降非常明显，T细胞会很快占据主要的细胞群体，丢失肿瘤免疫微环境的多样性。在对比例2-5的4种培养基中，也存在不同程度的髓系细胞和NK细胞比例的降低，特别是IMS-aLILd培养基，髓系细胞丢失严重。因此，在IMS培养基中，Laminin、抗LILRB2抗体、Heparansulphate和AS1842856对于维持细胞亚群的多样性有重要作用。
测试实施例2
按照测试实施例1的方法进行细胞的培养和测试，在IMS培养基条件下，对比在未加处理的常规细胞培养板（购自JET BIOFIL，商品号TCP001024）和表面经过处理的超低吸附板（购自科途医学，商品号K2OS-24-M）中进行腹水免疫细胞的培养，并检测培养前后的免疫细胞多样性，结果如图4所示。图4显示了经过不同培养板条件下，各种类型的细胞的回收率。回收率的计算方法为，用第三天回收的细胞的总数乘以不同细胞亚群的比例然后除以第一天该亚型细胞的总数。可以看出，超低吸附板培养的总CD45+细胞的回收率高出未加处理的常规细胞培养板所培养的细胞约140%，髓系细胞的数量和NK细胞的数量也有非常大的差异，因此在超低吸附板培养的情况下，髓系细胞和NK细胞才得以保持。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。
Claims (9)
1.一种用于保持免疫细胞多样性的培养基，其特征在于，所述培养基含有15ng/mL的M-CSF、10μg/mL的laminin、0.8μg/mL的collagen I、3μg/mL的collagen IV、0.3μg/mL的抗CD3抗体、20μg/mL的抗LILRB2抗体、0.8μg/mL的Heparansulphate、10U/mL的IL-2、3ng/mL的IL-10、10ng/mL的IL-15、20体积%的人血浆、10ng/ml的vitamin D、30nM的AS1842856、50-200U/mL的Penicillin-Streptomycin、1-3mM的L-Glutamine和0.5-2体积%的NEAA。
2.根据权利要求1所述的培养基，其中，所述培养基还含有基础培养基，所述基础培养基为RPMI-1640培养基、MEM培养基、DMEM培养基、IMEM培养基和F12培养基中的至少一种。
3.根据权利要求1所述的培养基，其中，所述用于保持免疫细胞多样性的培养基为用于保持来源于离体的胸水和/或腹水的免疫细胞多样性的培养基。
4.根据权利要求3所述的培养基，其中，所述胸水和/或腹水为来源于肿瘤患者的胸水和/或腹水。
5.根据权利要求4所述的培养基，其中，所述肿瘤包括肺癌、卵巢癌、胃癌、结肠癌、肝癌、乳腺癌、胰腺癌、胆管癌、食管癌、肾癌、宫颈癌、前列腺癌和黑色素瘤中的至少一种。
6.一种具有免疫微环境的细胞组合物的培养方法，其特征在于，该培养方法包括：从离体的胸水和/或腹水中分离混合细胞群体，然后将所分离的混合细胞群体接种至权利要求1-5中任意一项所述的培养基中进行培养，培养的容器为超低吸附培养皿或超低吸附培养孔板。
7.根据权利要求6所述的培养方法，其中，从离体的胸水和/或腹水中分离混合细胞群体的操作包括：将离体的胸水和/或腹水进行红细胞裂解，然后在200-600×g下离心5-20分钟，收集沉淀；然后将所述沉淀用Accutase蛋白酶或者Dispase II中性蛋白酶进行酶解，得到酶解产物；再将所述酶解产物用孔径为65-75μm的细胞筛过滤，得到滤出的悬液。
8.根据权利要求7所述的培养方法，其中，该培养方法还包括：将所述滤出的悬液进行离心以收集包含肿瘤细胞和免疫细胞的混合细胞群体，并将所收集的混合细胞群体接种到所述培养基中进行培养，培养的容器为超低吸附培养皿或超低吸附培养孔板。
9.根据权利要求6或8所述的培养方法，其中，接种的细胞密度为1-20×106细胞/mL；培养的温度为36-38℃；培养的时间为1-10天。
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