CN113215099B - 一种til细胞扩增培养基及其使用方法 - Google Patents
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Abstract
本发明提供了一种TIL细胞扩增培养基及其使用方法，该培养基包括灭活牛血清、白介素‑5、白介素‑9、白介素‑21、单克隆抗体、灵芝多糖、谷胱甘肽、卡拉胶、半乳糖醛酸、乙酰氨基酚和基础培养基；灭活牛血清和基础培养基为细胞扩增提供养分，促进细胞生长；卡拉胶和乙酰氨基酚合用可以让细胞自由生长，促进细胞增殖，在增殖结束后还能保持细胞的杀伤活性；半乳糖醛酸可以减少TIL中Treg数量，而且该培养基中未添加白介素‑2，避免了TIL中Treg数量的大幅度增长；本发明通过白介素‑5、白介素‑9和白介素‑21刺激TIL细胞增殖，灵芝多糖促进TIL细胞的快速增殖，提高细胞扩增倍数；单克隆抗体可以提高细胞杀伤活性。
Description
技术领域
本发明涉及细胞培养领域，特别涉及一种TIL细胞扩增培养基及其使用方法。
背景技术
TIL(Tumor Infiltrating Lymphocytes，肿瘤浸润淋巴细胞)是肿瘤间质中的异质性淋巴细胞，包括T细胞及NK细胞等。这些淋巴细胞中有部分是针对肿瘤特异性突变抗原的T细胞，是深入到敌军内部打击能力最强的免疫细胞，被认为是一种机体对肿瘤细胞特异性免疫反应。
近年来，TILs疗法已被证实能够用于治疗黑色素瘤、宫颈癌、肺癌、肉瘤和卵巢癌，在结直肠癌，乳腺癌等恶性肿瘤中也显示出巨大潜力，充分显示出TIL作为一种细胞免疫治疗手段在肿瘤治疗中的潜力；因此，从肿瘤组织中分离TIL，并在体外进行活化及扩增后，可作为一种实用有效的针对肿瘤患者的特异性免疫治疗手段。
TIL主要通过组织块培养、酶消化、机械解离、细针抽吸等4种方法获得。初次从肿瘤组织分离得到的TIL其免疫功能处于抑制状态，加入IL-2(白介素-2)后其免疫功能得到显著提高,但此时的TIL并不能满足临床有效治疗所需的免疫细胞数量。因此，需要对其进行体外快速扩增。目前，TIL的体外扩增需要高剂量的IL-2，但IL-2用量过高可导致培养的TIL中Treg(调节性T细胞)数量的增加，从而产生免疫抑制。
专利号CN107384867A公开一种肿瘤组织TIL细胞制备方法及专用培养基，公开了采用原代培养基和传代培养基对TIL细胞进行针对性培养，减少IL-2的使用量，降低对IL-2的依赖性，但其传代培养基中IL-2的用量较原代培养基高，培养周期较长。
专利号CN106754703A公开一种TIL细胞体外扩增培养基组合和培养方法，公开了一种使用IL-7(白介素-7)和IL-15(白介素-15)替代IL-2的体外扩增培养基，但该培养基为诱导培养基、增殖培养基和活化适应培养基的组合，使用起来比较麻烦。
因此，需要研究一种不含IL-2，且使用简单、扩增培养时间短的TIL体外扩增培养基。
发明内容
针对上述问题，本发明提供了一种TIL细胞扩增培养基及其使用方法。
本发明的一种TIL细胞扩增培养基，包括以下成分：80～100mL/L灭活牛血清、800～1000IU/mL白介素-5、700～950IU/mL白介素-9、1200～1400IU/mL白介素-21、50～85ng/L单克隆抗体、2～5g/L灵芝多糖、30～60mg/L谷胱甘肽、10～25mg/L卡拉胶、80～120mg/L半乳糖醛酸、0.1～0.5mg/L乙酰氨基酚和余量的基础培养基。
优选的，所述TIL细胞扩增培养基，包括以下成分：85～90mL/L灭活牛血清、900～950IU/mL白介素-5、700～800IU/mL白介素-9、1200～1250IU/mL白介素-21、65～85ng/L单克隆抗体、4g/L灵芝多糖、50～60mg/L谷胱甘肽、10～15mg/L卡拉胶、95～115mg/L半乳糖醛酸、0.1～0.3mg/L乙酰氨基酚和余量的基础培养基。
优选的，所述单克隆抗体为抗CD3单克隆抗体、抗CD56单克隆抗体中的至少一种。
优选的，所述灵芝为白灵芝和赤灵芝中的至少一种。
优选的，所述半乳糖醛酸为D-半乳糖醛酸、鼠李聚糖半乳糖醛酸和寡聚半乳糖醛酸中的至少一种。
优选的，所述基础培养基为DMEM培养基、RPMI-1640培养基和AIM-V培养基中的至少一种。
本发明还提供了一种TIL细胞扩增培养基的使用方法，包括以下步骤：
S1、将TIL细胞密度调整为(1～2)×105个/mL接入扩增培养基中进行扩增培养；
S2、首次更换扩增培养基的时间为扩增培养的第2～3天，而后每隔1～2天更换扩增培养基，扩增培养9～12d后，得TIL细胞。
优选的，所述扩增培养为在温度35±2℃，CO2体积浓度5±0.3％的条件下培养。
优选的，所述首次更换扩增培养基的时间为扩增培养的第2天。
优选的，在更换扩增培养基的同时，将细胞密度调整(1～2)×105个/mL。
本发明与现有技术相比具有如下有益效果：
本发明的TIL细胞扩增培养基的组分中，灭活牛血清和基础培养基为细胞体外扩增提供了所需的养分，可以有效促进细胞的生长；卡拉胶和乙酰氨基酚合用可以让细胞自由生长，促进细胞增殖，在增殖结束后还能保持细胞的杀伤活性；半乳糖醛酸可以减少TIL中Treg数量，而且本发明的扩增培养基中未添加白介素-2，避免了TIL中Treg数量的大幅度增长；本发明通过白介素-5、白介素-9和白介素-21刺激TIL细胞进行增殖，灵芝多糖促进TIL细胞的快速增殖，从而提高细胞扩增倍数；本发明TIL细胞扩增培养基中的各组分相互作用发挥出更好的效果，能够进行有效扩增，经过短时间的培养便可获得大量的TIL细胞，还可提高TIL细胞杀伤活性；单克隆抗体可以提高细胞杀伤活性。
经研究发现，白灵芝多糖和赤灵芝多糖促进TIL细胞增殖效果更好，细胞扩增倍数更高；选用抗CD3单克隆抗体和抗CD56单克隆抗体进行培养，TIL细胞的杀伤活性更高。
附图说明
图1为TIL细胞扩增倍数和杀伤活性测定结果示意图；
具体实施方式
为了更好理解本发明技术内容，下面提供具体实施例，对本发明做进一步的说明。
本发明实施例所使用的TIL细胞均为使用非连续密度梯度离心法从肝癌组织中分离所得的TIL细胞。
本发明的一种TIL细胞扩增培养基，包括以下成分：80～100mL/L灭活牛血清、800～1000IU/mL白介素-5、700～950IU/mL白介素-9、1200～1400IU/mL白介素-21、50～85ng/L单克隆抗体、2～5g/L灵芝多糖、30～60mg/L谷胱甘肽、10～25mg/L卡拉胶、80～120mg/L半乳糖醛酸、0.1～0.5mg/L乙酰氨基酚和余量的基础培养基。
所述单克隆抗体为抗CD3单克隆抗体、抗CD56单克隆抗体中的至少一种。
所述灵芝为白灵芝和赤灵芝中的至少一种。
所述半乳糖醛酸为D-半乳糖醛酸、鼠李聚糖半乳糖醛酸和寡聚半乳糖醛酸中的至少一种。
所述基础培养基为DMEM培养基、RPMI-1640培养基和AIM-V培养基中的至少一种。
实施例与对比例的TIL细胞扩增培养基如下表所示：
实施例5
一种TIL细胞扩增培养基，包括以下成分：85mL/L灭活牛血清、900IU/mL白介素-5、800IU/mL白介素-9、1250IU/mL白介素-21、30ng/L抗CD3单克隆抗体、30ng/L抗CD56单克隆抗体、2g/L紫灵芝多糖、2g/L无柄灵芝多糖、50mg/L谷胱甘肽、10mg/L卡拉胶、35mg/L D-半乳糖醛酸、40mg/L鼠李聚糖半乳糖醛酸、40mg/L寡聚半乳糖醛酸、0.1～0.5mg/L乙酰氨基酚、RPMI-1640培养基补足。
实施例6
一种TIL细胞扩增培养基，包括以下成分：85mL/L灭活牛血清、900IU/mL白介素-5、800IU/mL白介素-9、1250IU/mL白介素-21、30ng/L抗CD3单克隆抗体、30ng/L抗CD56单克隆抗体、2g/L白灵芝多糖、2g/L赤灵芝多糖、50mg/L谷胱甘肽、10mg/L卡拉胶、35mg/L D-半乳糖醛酸、40mg/L阿拉伯半乳糖醛酸、40mg/L多聚半乳糖醛酸、0.1～0.5mg/L乙酰氨基酚、RPMI-1640培养基补足。
实施例7
一种TIL细胞扩增培养基，包括以下成分：85mL/L灭活牛血清、900IU/mL白介素-5、800IU/mL白介素-9、1250IU/mL白介素-21、30ng/L抗CD3单克隆抗体、30ng/L抗CD28单克隆抗体、2g/L白灵芝多糖、2g/L赤灵芝多糖、50mg/L谷胱甘肽、10mg/L卡拉胶、35mg/L D-半乳糖醛酸、40mg/L鼠李聚糖半乳糖醛酸、40mg/L寡聚半乳糖醛酸、0.1～0.5mg/L乙酰氨基酚、RPMI-1640培养基补足。
上述扩增培养基的使用方法，包括以下步骤：
S1、将TIL细胞密度调整为1×105个/mL接入扩增培养基中，在温度35±2℃，CO2体积浓度5±0.3％的条件下进行扩增培养；
S2、首次更换扩增培养基的时间为扩增培养的第2天，在更换扩增培养基的同时将细胞密度调整为1×105个/mL，而后每隔1天更换扩增培养基，扩增培养9d后，得TIL细胞。
使用效果实施例
采用细胞计数法检测扩增倍数；采用流式细胞术检测扩增后TIL中Treg细胞的比例；
细胞杀伤活性检测采用MTT法，以K562细胞为靶细胞，使用含20％NCS的RPMI-1640培养液调整细胞密度至1×106个/ml，按效应细胞：靶细胞＝20:1的比例混合后，分别吸取100μl放入96孔板，另设单独靶细胞和效应细胞组，每个试验做三个复孔；孵育20h后，每孔加入10μl MTT溶液，继续培养孵育4h，吸弃上清，每孔加入二甲亚砜溶液150μl，混匀，用酶标仪(λ＝570nm)测定光密度值(OD值)，结果用均值表示，按照公式计算杀伤活性，结果见表1；
杀伤活性(％)＝[靶细胞OD值+(效应细胞OD值-实验组OD值)]/靶细胞OD值×100％
表1TIL细胞扩增培养基的使用效果
| 扩增倍数 | 杀伤活性(％) | Treg比例(％) | |
| 实施例1 | 383 | 82.5 | 2.6 |
| 实施例5 | 346 | 74.8 | 4.3 |
| 实施例6 | 371 | 72.5 | 6.9 |
| 实施例7 | 374 | 71.9 | 3.4 |
| 对比例1 | 226 | 62.4 | 9.5 |
| 对比例2 | 254 | 65.7 | 9.8 |
| 对比例3 | 319 | 53.5 | 12.5 |
| 对比例4 | 104 | 33.1 | 10.8 |
由上述实验结果可知，采用本发明扩增培养基对TIL细胞进行扩增培养的扩增倍数高，扩增后的TIL细胞对K562细胞具有较高的杀伤活性，TIL中的Treg比例小；与实施例1相比，实施例5使用的灵芝多糖与本发明不同，TIL细胞的扩增倍数较低；实施例6使用的半乳糖醛酸与本发明不同，扩增后TIL中的Treg比例较高；实施例7使用的单克隆抗体与本发明不同，TIL细胞的杀伤活性较低；对比例1未使用灵芝多糖，TIL细胞的扩增倍数低；对比例2未使用卡拉胶和乙酰氨基酚，TIL细胞的扩增倍数和杀伤活性都较低；对比例3未使用半乳糖醛酸，扩增后TIL中的Treg比例高；对比例4未使用白介素-21，白介素-5和白介素-9的用量高，TIL细胞的扩增倍数低，说明本发明白介素-5、白介素-9和白介素-21的组合及用量能够有效刺激TIL细胞增殖。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (2)
1.一种TIL细胞扩增培养基，其特征在于，包括以下成分：85mL/L灭活牛血清、900 IU/mL白介素-5、800 IU/mL白介素-9、1250 IU/mL白介素-21、30 ng/L抗CD3单克隆抗体、35ng/L抗CD56单克隆抗体、2 g/L白灵芝多糖、2 g/L赤灵芝多糖、50 mg/L谷胱甘肽、10 mg/L卡拉胶、35 mg/LD-半乳糖醛酸、40 mg/L鼠李聚糖半乳糖醛酸、40 mg/L寡聚半乳糖醛酸、0.1mg/L乙酰氨基酚和余量的RPMI-1640培养基。
2.如权利要求1所述的一种TIL细胞扩增培养基的使用方法，其特征在于，包括以下步骤：
S1、将TIL细胞密度调整为1×105个/mL接入扩增培养基中，在温度35±2℃，CO2体积浓度5±0.3％的条件下进行扩增培养；
S2、首次更换扩增培养基的时间为扩增培养的第2天，在更换扩增培养基的同时，将细胞密度调整1×105个/mL，而后每隔1天更换扩增培养基，扩增培养9d后，得TIL细胞。
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