CN112300990A - 一种用于体外扩增暨活化γδ-T细胞的培养基 - Google Patents
一种用于体外扩增暨活化γδ-T细胞的培养基 Download PDFInfo
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Abstract
本发明关于一种体外扩增暨活化γδ‑T细胞的培养基，该培养基包含自体树突细胞外泌体，可有效率地提升细胞扩增倍数，并产生对癌细胞具有高度毒杀活性的γδ‑T细胞。
Description
技术领域
本发明关于一种体外扩增暨活化γδ-T细胞的培养基。
背景技术
免疫细胞治疗是将病人本身的免疫细胞在体外增殖和活化后，再回输至体内的一种治疗方式。目前免疫细胞治疗已逐渐被应用于癌症治疗上，成功的关键在于了解各类免疫细胞的特性和功能，并根据癌症患者的状况及基因特性来选择最合适的免疫细胞类型，例如NK细胞、T细胞等。
人体免疫***中的T细胞大致上分为二大类:Alpha-Beta T细胞与Gamma-Delta T(γδ-T)细胞。γδ-T细胞存在于***血液中，其数量只占***血液中所有T细胞的1％～5％。它是藉由辨识细胞表面的异戊烯基焦磷酸(isopentenyl pyrophosphate,IPP)分子来区分正常与异常细胞。IPP是细胞代谢的一个中间产物，它在癌细胞内的产量会增加，尤其是当癌细胞的p53基因突变时。识别IPP的γδ-T细胞会增殖和活化并会增强攻击肿瘤细胞的力量，此为γδ-T细胞的特殊功能，如果其他免疫细胞没有侦测到癌细胞标志的话，γδ-T细胞还是可以发现癌细胞，并加以攻击。以一般情况来说，异常的细胞会拥有和其他正常细胞不同的分子，γδ-T细胞将这些作为癌细胞标志来识别并进行攻击。除了IPP分子之外，γδ-T细胞也能透过MIC A/b、HMB-PP、细胞间粘附分子-1，CD166等标记物来进行辨识。因此，γδ-T细胞能辨识与杀死多种癌细胞。
γδ-T细胞有一个很重要的特征，即在辨识异常细胞时不需要HLA，因此与个体的HLA型别无关。这个特征使得γδ-T细胞可使用在任何人身上而不会产生移植物抗宿主病(graft-versus-host disease,GVHD)。目前在美国、欧洲与日本已有许多细胞疗法的人体临床应用是使用γδ-T细胞来进行的，这些临床应用皆证实γδ-T细胞的使用是安全无虞的。
由于γδ-T细胞在血液中数量稀少且其增生能力因人而异，因此以γδ-T细胞进行细胞疗法的挑战在于是否能够于体外大量且快速的扩增并活化γδ-T细胞。近几年发现，使用唑来膦酸(Zoledronic acid)能使γδ-T细胞大量增殖，其技术也已经确立。唑来膦酸除了使用在培养γδ-T细胞之外，将唑来膦酸注射到癌症患者体内可以发现癌细胞更大量表现IPP，藉此可以提高γδ-T细胞对癌细胞的灵敏度。然而，唑来膦酸是一种治疗骨质疏松症的药物，用于培养细胞或注射至病患体内仍存在一定疑虑，因此仍极需要开发出一种方便、有效、安全的体外扩增暨活化γδ-T细胞的培养基。
发明内容
本发明提供一种用于体外扩增暨活化γδ-T细胞的培养基，其包含浓度范围介于5μg/ml至50μg/ml的自体树突细胞外泌体。于一较佳实施例中，该自体树突细胞外泌体的浓度为25μg/ml。
于一具体实施例中，该培养基另包含白介素-2；于一较佳实施例中，该白介素-2的浓度介于500IU/ml～2000IU/ml；于一更佳实施例中，该白介素-2的浓度为1000IU/ml。
于一具体实施例中，该培养基另包含唑来膦酸；于一较佳实施例中，该唑来膦酸的浓度介于1μM～20μM；于一更佳实施例中，该唑来膦酸的浓度为5μM。
于一具体实施例中，该培养基另包含自体血浆、人源AB血清或胎牛血清；于一较佳实施例中，该自体血浆、人源AB血清或胎牛血清约占总培养体积的10％。
于一具体实施例中，该培养基另包含一基础培养基；于一较佳实施例中，该基础培养基选自由AIM V、X-VIV015、CellGro SCGM、KBM 501、DMEM及RPM1-1640培养基所组成的群。
前面的概述，以及本发明以下的详细描述，在结合图式一起阅读时将可以被更好地理解。为了说明本发明，所附图式示出一些，但不是所有的，可替代的具体实施例。然而，应该理解的是，本发明并不限于所示的精确安排和手段。这些图式，其被并入并构成说明书的一部分，有助于解释本发明的原理。
附图说明
图1是本发明于培养基中加入自体树突细胞外泌体后的γδ-T细胞扩增倍数分析。
图2是本发明于培养基中加入自体树突细胞外泌体后，γδ-T细胞对Daudi细胞的毒杀效率分析。
图3是本发明于培养基中加入自体树突细胞外泌体后，γδ-T细胞对A549细胞的毒杀效率分析。
具体实施方式
有鉴于上述待解决的问题，本发明提出一种用于体外扩增暨活化γδ-T细胞的培养基，该培养基是添加自体树突细胞外泌体，能够有效率地提升γδ-T细胞扩增倍数，并产出高癌细胞毒杀活性的γδ-T细胞。
定义
除非另有定义，所有在此使用的技术及科学术语，具有与本发明所属领域的通常知识者一般所理解的意义相同的意义。当有所冲突时，以本文件及其所定义者为准。
于此处所使用者，「约」、「大约」、或「大概」一般应指一特定数值或范围的20％以内，较佳10％以内，及更佳5％以内。在此所使用的数值为近似值，代表若未明示地陈述，「约」、「大约」、或「大概」等词可以被推断适用。
于本发明中，「γδ-T细胞」(gamma-delta T cell)是指细胞表面抗原呈现CD3+且表现TCR Vγ9以及TCR Vδ2的细胞。
于本发明中，「细胞扩增倍数」是以下列方式判断：「经体外培养12天后的细胞数量」除以「自周边血单核球细胞分离出的起始γδ-T细胞数量」。
于本发明中，「癌细胞毒杀效率」是以γδ-T细胞为作用细胞(effector cell)，并以Daudi细胞株或A549细胞株为目标细胞(target cell)，在作用细胞与目标细胞的比例(E:T ratio)为0.5、1或5的情况下进行毒杀试验，将目标细胞死亡的比例做为毒杀效率。
于本发明中，「外泌体(exosome)」是一种能被大多数细胞分泌的微小膜泡，具有脂质双层膜结构，直径大约40-100nm。外泌体最初在1983年就被发现，但人们一直认为它只是一种细胞的废弃物。然而最近发现这种微小膜泡中含有细胞特异的蛋白、脂质和核酸，能作为信号分子传递给其他细胞从而改变其他细胞的功能。最近的研究发现外泌体在很多生理病理上起着重要的作用，如免疫中抗原呈递、肿瘤的生长与迁移、组织损伤的修复等，且不同细胞分泌的外泌体具有不用的组成成分和功能。
材料与方法
本发明所述周边血检体，是依据伦理委员会通过的计划，自受试者手臂采集全血，置于无菌采血管，于室温存放待后续处理。
本发明所使用的基础培养基可选用自：CellGro SCGM(CellGenix公司)、KBM 501(Kohjin Bio公司)、AIM-V(Thermo Fisher公司)、X-VIV015(Lonza公司)、DMEM或RPM1-1640等市售的基础培养基。
本发明所述培养基中，有时含有适当的蛋白质、细胞激素、抗体、血清、化合物等成分。所述细胞激素有时为白介素-2(IL-2)、白介素-3(IL-3)、白介素-7(IL-7)、白介素-12(IL-12)、白介素-15(IL-15)、白介素-18(IL-18)或白介素-21(IL-21)。
自血液检体分离周边血单核球细胞的方法
抽取7.5-8ml的血液于采血管中，该采血管含有抗凝血剂-肝素，以及Ficoll-Hypaque试剂，外加一聚脂凝胶隔层以分离前述二液体。于室温下以1800g将采血管离心20分钟。离心完成后，收集血浆分层用于后续的细胞培养，并留下5至10mm的血浆层于接口上，操作时不扰动细胞层。接着以移液器收集接口的周边血单核球细胞(peripheral bloodmononuclear cell,PBMC)层至15ml圆锥形管中，以10ml磷酸盐缓冲生理食盐水(Phosphatebuffer saline；简称PBS)清洗PBMC并翻转圆锥形管5次，再以400g进行离心5分钟。重复前述清洗步骤两次之后，以5ml的PBS将细胞再悬浮。计算细胞数，通常1ml全血能够分离出1.3x106的PBMC。最后以流式细胞仪确认PBMC中γδ-T细胞的比例与表型。
体外扩增及活化γδ-T细胞的方法
于室温以400g离心15ml圆锥形管中的周边血单核球细胞悬浮液5分钟后，丢弃上清液。准备细胞培养基，培养基中加入白介素-2(IL-2)及唑来膦酸(Zometa)至最终浓度分别为1000IU/ml及5μM。其中唑来膦酸以液体形式添加，每30ml的培养基中加入50μl的唑来膦酸(浓度为4mg/5ml)。接着将细胞沉淀物重新悬浮于培养基中并调整至每毫升培养基含1x106细胞。使用24孔细胞培养盘，每孔加入1x106细胞进行培养。若需大量培养，可以每平方公分0.5x106细胞的密度为原则，根据所使用的培养盘、培养瓶表面积进行调整。接着加入自体血浆、人源AB血清、胎牛血清或自体树突细胞外泌体，使其约占总培养液体积的10％(相当于24孔细胞培养盘，每孔加入100μl细胞)。将细胞培养盘置于37℃,5％CO2的细胞培养箱中培养24～48小时。将细胞密度维持在每毫升0.5x 106～2x106个细胞，每隔2至3天加入含有1000IU/ml白介素-2的新鲜培养基，必要时根据细胞扩增程度将细胞转移至新的培养盘或培养瓶中继续培养。培养过程中维持培养基中的血清浓度至少在1％。在第12天收获细胞并以流式细胞仪确认γδ-T细胞的数量、表型及功能。
以流式细胞仪分析细胞表面抗原
以2x105细胞/200μl将扩增暨活化后的细胞置于96孔盘，并加入3μl荧光标记的抗体于4℃反应15分钟，接着以PBS清洗3次后，加入400μl的PBS悬浮细胞，再以流式细胞仪分析细胞表面的荧光标记。上述荧光标记的抗体包含anti-CD3抗体、anti-TCR Vγ9抗体以及anti-TCR Vδ2抗体。
γδ-T细胞毒杀癌细胞能力测试方式
将扩增及活化后的γδ-T细胞做为作用细胞(effector cell)，并以Daudi细胞株(淋巴癌细胞株)或A549细胞株(肺癌细胞株)做为毒杀目标细胞(target cell)。将作用细胞及目标细胞以0.5:1、1:1或5:1混合培养后，反应4小时，再以7-AAD进行细胞染色，藉此测定凋亡的细胞数量。
自体树突细胞外泌体的制备与纯化
自体树突细胞外泌体(Autologous dendritic cell exosome)的制备是以新鲜的细胞培养基取代培养至第五天的树突细胞培养基，并加入颗粒单核球群落刺激生长因子(Granulocyte-macrophage colony-stimulating factor,GM-CSF)以及白介素-4并继续培养树突细胞24小时。收集经培养后的培养基并分别以300g及1000g离心10分钟，接着以孔径0.45μm的滤膜进行过滤以移除细胞及其碎片。经过滤后的培养基再以Centricon Plus-70Millipore滤膜于1000g离心45分钟进行浓缩，接着再以100,000g进行超速离心一小时后可由培养基分离得到树突细胞外泌体，再使用PBS清洗二次，通过Amicon Ultra-15滤膜以1000g离心25分钟，最后将树突细胞外泌体再悬浮于200μL PBS中。使用BCA蛋白质分析套组(Thermo Scientific)进行树突细胞外泌体的定量。
经前述方法所获得的扩增暨活化后的γδ-T细胞，可混合于适当的赋形剂中保存，所述赋形剂可为一磷酸缓冲液，最后制备成医药组合物。
实施例
实施例1、于γδ-T细胞培养基中添加自体树突细胞外泌体的培养结果
图1为培养基中加入自体树突细胞外泌体后的γδ-T细胞扩增倍数分析。由图1发现，使用含有25μl/ml自体树突细胞外泌体的培养基，于培养第12天的细胞扩增倍数为6,236倍；对照组(不含自体树突细胞外泌体)的细胞扩增倍数为3,754倍。
实施例2、本发明培养的γδ-T细胞对Daudi细胞的毒杀试验
图2为培养基中加入自体树突细胞外泌体后，γδ-T细胞对Daudi细胞的毒杀效率分析。由图2发现，在E:T比例为5的情况下，使用含有25μl/ml自体树突细胞外泌体的培养基培养的γδ-T细胞毒杀效率为78.3％；对照组(不含自体树突细胞外泌体)的γδ-T细胞毒杀效率为64.4％。
实施例3、本发明培养的γδ-T细胞对A549细胞的毒杀试验
图3为培养基中加入自体树突细胞外泌体后，γδ-T细胞对A549细胞的毒杀效率分析。由图3发现，在E:T比例为5并添加唑来膦酸的情况下，使用含有25μl/ml自体树突细胞外泌体的培养基培养的γδ-T细胞毒杀效率为58.9％；对照组(不含自体树突细胞外泌体)的γδ-T细胞毒杀效率为43.7％。
综上，于γδ-T细胞培养基中添加自体树突细胞外泌体，可有效提升γδ-T细胞的扩增倍率及对Daudi、A549癌细胞的毒杀活性。
本发明前述实施例的描述仅为呈现与说明的目的，而非意图穷尽或限制本发明于所揭露的特定方式。按照上述教示所为的修改或变化都是可能的。
实施例的替代方案在不脱离本发明的精神与范围下，对于所属领域的技术人员而言是明确的。因此，本发明的范围是由所附权利要求而定义，而非前述说明与实施例。部份参考文献，可能包括专利、专利申请与各种公开文献，于本发明中被引述与讨论。所有于本说明书中被引述与讨论的参考文献全体皆引用作为本说明书的揭示内容，如同其被单独地引用时具有相同程度的完整性。
Claims (10)
1.一种用于体外扩增暨活化γδ-T细胞的培养基，其包含浓度范围介于5μg/ml至50μg/ml的自体树突细胞外泌体。
2.如权利要求1所述的培养基，其特征在于，其中自体树突细胞外泌体的浓度为25μg/ml。
3.如权利要求1所述的培养基，其特征在于，其另包含白介素-2。
4.如权利要求3所述的培养基，其特征在于，其中该白介素-2的浓度介于500IU/ml～2000IU/ml。
5.如权利要求4所述的培养基，其特征在于，其中该白介素-2的浓度为1000IU/ml。
6.如权利要求1所述的培养基，其特征在于，其另包含唑来膦酸。
7.如权利要求6所述的培养基，其特征在于，其中该唑来膦酸的浓度介于1μM～20μM。
8.如权利要求7所述的培养基，其特征在于，其中该唑来膦酸的浓度为5μM。
9.如权利要求1所述的培养基，其特征在于，其另包含自体血浆、人源AB血清或胎牛血清，且该自体血浆、人源AB血清或胎牛血清约占总培养体积的10％。
10.如权利要求1～9任一所述的培养基，其特征在于，其另包含一基础培养基；其中该基础培养基选自由AIM V、X-VIV015、CellGro SCGM、KBM 501、DMEM及RPM1-1640培养基所组成的群。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108103026A (zh) * | 2017-12-05 | 2018-06-01 | 四川省肿瘤医院 | 用于肿瘤免疫治疗的γδ-T细胞外泌体及其制备方法 |
Non-Patent Citations (4)
| Title |
|---|
| KATHERINE E.KUNIGELIS ET AL.: "The dichotomy of tumor exosomes(TEX) in cancer immunity: is it all in the conTEXt?", 《VACCINES(BASEL)》 * |
| NAOHIRO SEO ET AL.: "Exosome-mediated regulation of tumor immunology", 《CANCER SCI》 * |
| 冯奇等: "树突状相关外泌体在肿瘤中的研究进展", 《临床与病理杂志》 * |
| 吴振勇等: "一种gamma-delta T细胞体外扩增体系的研制", 《临床医学工程》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| TWI768357B (zh) | 2022-06-21 |
| TWI768356B (zh) | 2022-06-21 |
| TW202118858A (zh) | 2021-05-16 |
| CN112300989A (zh) | 2021-02-02 |
| CN115678847A (zh) | 2023-02-03 |
| TW202104578A (zh) | 2021-02-01 |
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