CN110042080A - 原发性肝癌肿瘤浸润淋巴细胞的分离、培养及体外扩增方法 - Google Patents
原发性肝癌肿瘤浸润淋巴细胞的分离、培养及体外扩增方法 Download PDFInfo
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Abstract
本发明公开了原发性肝癌肿瘤浸润淋巴细胞的分离、培养及体外扩增方法,发明人取无菌新鲜肿瘤癌巢和癌旁组织,通过混合酶溶液进行消化分离,并接种于共刺激培养基中,从而实现淋巴细胞的培养和体外扩增。本发明在提取上解决了TIL提取数量和纯度低的关键问题,在培养上可使TIL往具有免疫活性的CD8+TIL亚型分化扩增,在扩增上,创造性地使用肝癌患者的外周血血浆和PBMC作饲养进行体外培养,并有效扩增TIL细胞,解决自体TIL过继免疫治疗引起的免疫排斥问题。在分选上,使用StemCell磁珠法分选出具有特异识别能力的CD8+TIL细胞,为下一步免疫治疗的临床研究奠基良好基础,具有显著的应用前景。
Description
技术领域
本发明属于生物工程领域,涉及原发性肝癌肿瘤浸润淋巴细胞的分离、培养及体外扩增方法。
背景技术
原发性肝细胞肝癌(Hepatocellular carcinoma,HCC)是高度恶性的肿瘤,每年我国因肝癌死亡的人数超过12万,占全世界肝癌死亡人数的44%左右,病死率高居各类肿瘤的第三位。手术治疗虽然是肝癌治疗的首选方法,但由于肝癌血供丰富,易转移和复发,绝大多数病人的5年生存率很低。原发性肝癌术后患者生存质量低下和预后不良的问题依然十分突出,目前临床靶向药物索拉菲尼(Sorafenib),也只能延长病人生存期3-4个月。由此可见,手术和化疗不能从根本上治愈肝癌患者,新型高效的治疗手段成为医学界的诉求。
免疫治疗登上了人类抗癌历史的舞台。20世纪70年代,免疫学家发现通过阻断免疫细胞(主要是T淋巴细胞)表面的抑制分子以解除免疫系统的抑制,从而更有效的对抗肿瘤。2011年,第一个肿瘤免疫检查点CTLA-4(细胞毒性T淋巴细胞相关抗原-4)阻断剂伊匹单抗(Ipilimumab),获美国食品药品监督管理局(FDA)批准上市,用于晚期黑色素瘤治疗;2014年,程序性死亡受体PD-1阻断剂纳武单抗OPDIVO(Nivolumab)在日本获得批准,这一新药的上市,让“癌症免疫疗法”备受世界瞩目,该疗法同时也被《Science》评选为2013年度十大科技突破之首。2018年10月,诺贝尔生理学或医学奖颁给了免疫学家James P.Allision和TasukuHonjo,以表彰他们发现了抑制免疫负调节(上述两个免疫检查点)机制治疗肿瘤,为人类健康及癌症治疗开辟了一个崭新领域。这些均可认为是肿瘤免疫治疗的里程碑式事件,同时标志着肿瘤治疗进入了免疫治疗的时代。免疫检查点抑制剂不断深入研究,从最初转移性黑色素瘤的治疗,随后其适应症扩展到了肺癌、转移性肾细胞癌、霍奇金淋巴瘤、头颈癌以及局部晚期或转移性尿路上皮癌等肿瘤等,在临床应用中有明显的疗效。但尽管如此,免疫检查点抑制剂仍具有非常突出的局限性,对很多癌症患者尤其是实体瘤患者没有明显疗效或者副作用远超于治疗效果,主要原因包括肿瘤细胞表面缺少可以供免疫细胞进行识别的新抗原、肿瘤组织中浸润的T细胞数量减少、编码抗原加工或呈递组分的编码基因受损、干扰素γ信号通路异常、严重的T细胞耗竭、炎症或代谢调节以及其他免疫检查点的作用,因此亟需一种更有效的治疗方法。
细胞免疫治疗的出现给人们带来了希望。近年来,DC细胞免疫治疗,NK细胞免疫治疗,尤其是嵌合抗原受体T细胞(ChimericAntigen ReceptorT-Cell,CAR-T)免疫治疗,目前已经发展到了第四代,由于能特异性识别结合肿瘤抗原,不受肿瘤细胞MHC分子下调导致的免疫抑制,并能在体内激活扩增,在血液系统恶性疾病和黑色素瘤治疗方面取得了良好的效果。但是由于其副作用脱靶效应、细胞因子释放综合征(Cytokine Release Syndrome,CRS)以及肿瘤微环境(TumorMicroenvironment,TME)的屏障和肿瘤免疫逃避的复杂机制,限制了该疗法在肝癌这种实体性肿瘤的临床应用。因此肝癌细胞免疫疗法亟需构建一种具有更高安全性和有效性的免疫细胞。
肿瘤浸润的淋巴细胞(TumorInfiltrating Lymphocyte,TIL),是存在于肿瘤间质内的以淋巴细胞为主的异质性淋巴细胞群体,包括T淋巴细胞,B淋巴细胞,NK细胞,但以T淋巴细胞为主,而T淋巴细胞根据细胞表型可分为CD4+T细胞亚群(又叫辅助T细胞,外文名helperT cell,即Th)和CD8+T细胞亚群(又叫细胞毒性T淋巴细胞,外文名Cytotoxiclymphocyte,即CTL,能释放颗粒酶、穿孔素和细胞因子等,具有杀伤肿瘤细胞活性。以下从TIL中分选的CD8+T细胞简写为CD8+TIL。),大多聚集在肿瘤周围或其间质呈套状围绕癌巢。TIL来自肿瘤组织区域,其主要成分T淋巴细胞共同特征为表达T细胞受体(T cellreceptor,TCR),可特异识别自体肿瘤抗原,具有特异的MHC限制性溶解肿瘤活性。相对于外周血淋巴细胞来说,它除了具有免疫功能外,还能表达黏附分子和选择素浸润进肿瘤间质中。
目前针对肝癌肿瘤浸润淋巴细胞的常规分离,主要有机械分离法、组织块培养法和酶消化法。其中,机械分离法是最早用于提取TIL的方法,但由于机械作用力容易使细胞死亡,得率低逐渐被淘汰;组织块培养法主要适用于肝穿刺活检标本,体积小而且肿瘤微环境相对完整,但得率也较低,同时不适用体积大的肝癌手术标本,机械剪碎难以达到肝穿尺寸且会破坏细胞;酶消化法是较普遍采用的提取方法,但提取效果受很多因影响,比如组织切碎程度、消化酶的组成、消化温度与消化时间等。除外,在培养上,普遍采用两步法扩增,第一步为活化阶段,用中等剂量的IL-2和人AB血清培养,第二步为扩增阶段,加入共刺激抗体(anti-CD3、anti-CD28抗体),同时用经辐照灭活的健康供者的PBMC作饲养细胞培养扩增。缺点是用人AB血清,供者的PBMC这些异体的血液成分给后续免疫治疗的临床研究带来GVHD(移植物抗宿主病)、HIV及其他病毒感染的潜在风险。
本发明在提取上为改良的酶消化法,课题组经过42例的标本提取研究,摸索出了高效消化的混合酶组成以及提出的完整有效的提取方案,解决了TIL数量和纯度低的关键问题。在培养上,使用临床人注射用重组白介素-2(rIL-2)、共刺激抗体(anti-CD3、anti-CD28抗体)培养细胞,摸索出了加入的剂量与时间,使TIL往具有免疫活性的CD8+TIL分化扩增。同时,在扩增上,本发明在人重组白介素-2(rIL-2)、共刺激抗体(anti-CD3、anti-CD28抗体)基础上,用对应肝癌患者的外周血血浆和PBMC作饲养进行体外培养扩增,能有效扩增TIL细胞,同时解决自体TIL过继免疫治疗引起的免疫排斥问题,为自体TIL过继免疫治疗提高安全保障。
发明内容
本发明的目的之一是提供原发性肝癌肿瘤浸润淋巴细胞的分离方法,所述方法包括:
1)取无菌新鲜肿瘤癌巢和/或癌旁组织,浸泡于含有10%~15%热灭活FBS的RPMI1640培养基中;
2)用PBS清洗两遍,剔除血块、脂肪及坏死组织,PBS再清洗一遍;
3)剪成1mm3~3mm3大小,按组织重量:混合酶溶液体积=1g:10ml~20ml的比例,将组织浸入混合酶溶液,置于恒温摇床消化,37±1℃消化30min~2h;
4)加入HBSS液稀释,进行不连续密度梯度离心,800±100g离心30±10min。
5)收集1±0.15密度层的淋巴细胞,加入PBS(含1%~5%FBS)稀释重悬,800±100g离心10±5min,去上清;
6)加入含有1%~5%热失活FBS进行洗涤,1600±300rpm离心10±5min,去上清,重复一次。
在其中一个实施例中,所述混合酶溶液的成分包括:0.5±0.2mg/ml IV型胶原酶、0.1±0.05mg/ml透明质酸酶和/或0.02±0.01mg/ml I型DNA酶。
在其中一个实施例中,所述混合酶溶液的成分具体为:0.5mg/ml IV型胶原酶、0.1mg/ml透明质酸酶和/或0.02mg/ml I型DNA酶。
本发明的另一个目的是获得上述分离后的原发性肝癌肿瘤浸润淋巴细胞进行培养和体外扩增的方法,所述方法包括:
1)将淋巴细胞接种于六孔板,使用1±0.5μg/ml抗CD3单克隆抗体OKT3,4℃共培养过夜;
2)次日用PBS洗涤后,接种细胞于共刺激培养基上,CO2培养箱中培养;
在其中一个实施例中,所述共刺激培养基含有:10±5%热失活FBS和10000±2000IU/ml rIL-2的RPMI1640。
在其中一个实施例中,所述共刺激培养基还含有2±1μg/ml的抗CD28单克隆抗体。
在其中一个实施例中,共刺激培养2天后,更换新的共刺激培养基,如此循环,至淋巴细胞密度生长至起始密度的3~10倍。
在其中一个实施例中,淋巴细胞达到目标浓度后,更换培养基,使用含10000±2000IU/ml rIL-2的培养基,培养8~15天,获得培养和体外扩增后的淋巴细胞。
本发明的有益效果:
本发明在提取上为改良的酶消化法,课题组经过42例的标本提取研究,摸索出了高效消化的混合酶组成以及提出的高效可行的提取方案,解决了TIL数量和纯度低的关键问题。在培养上,使用临床人注射用重组白介素-2(rIL-2)、共刺激抗体(anti-CD3、anti-CD28抗体)培养细胞,摸索出了加入的剂量与时间点,使TIL往具有免疫活性的CD8+TIL亚型分化扩增。同时,在扩增上,本发明在人重组白介素-2(rIL-2)、共刺激抗体(anti-CD3、anti-CD28抗体)基础上,用对应肝癌患者的外周血血浆和PBMC作饲养进行体外培养扩增,能有效扩增TIL细胞,同时解决自体TIL过继免疫治疗引起的免疫排斥问题,为自体TIL过继免疫治疗提高安全保障。在分选上,使用StemCell磁珠分选法分选出具有特异识别能力的CD8+TIL细胞,为下一步免疫治疗的临床研究奠基良好基础。
附图说明
图1.步骤4淋巴细胞培养和体外扩增显微镜观察图,40倍;
图2.步骤4淋巴细胞培养和体外扩增显微镜观察图,100倍;
图3.步骤4淋巴细胞培养和体外扩增显微镜观察图,200倍;
图4.步骤4淋巴细胞培养和体外扩增显微镜观察图,400倍;
图5.步骤6流式检测结果;
图6.步骤7,磁珠分选CD8+T细胞显微镜观察图,40倍;
图7.步骤7,磁珠分选CD8+T细胞显微镜观察图,100倍;
图8.步骤7,磁珠分选CD8+T细胞显微镜观察图,200倍;
图9.步骤7,磁珠分选CD8+T细胞显微镜观察图,400倍;
具体实施方式
下面对本发明的实施例进行详细说明。
1.试剂与仪器
Ⅳ型胶原酶,透明质酸酶,DNA酶购自Sigma公司;RPMI1640培养基购自Gibico公司;磷酸盐缓冲液PBS粉购自博士德公司;胎牛血清购自Gibico公司;40um滤网购自Falcon公司;人用注射重组白介素2(rIL-2)购自北京四环生物公司(商品名:新德路生);抗CD3单克隆抗体(OKT3)、抗CD28单克隆抗体购自ebioscience公司;淋巴细胞分离液购自StemCell公司;HBSS液购自康宁公司;cocktail流式抗体(PerCP-CD3、FITC-CD4、PE-CD8)购自ACEABio公司;六孔细胞培养板购自Thermo公司;CO2细胞培养箱购自美国FormaScientific公司;荧光倒置显微镜购自德国Leica公司;二甲基亚枫(DMSO)购自Sigma公司;水浴箱为上海一恒DK-8D,流式细胞仪是ACEA Novocyte;电子自动分析天平购自MettlerToledo pl402-L。
2.肝癌组织标本来源
取自广州医科大学附属第二医院,外科手术切除的新鲜肝癌组织,选取癌巢和癌旁组织,所有肿瘤组织均有明确的病理诊断。
3.分离方法
取手术切除的无菌新鲜肿瘤癌巢和癌旁组织,并浸泡于含有10%FBS的RPMI1640培养基中。电子天平称重(以下按取9g组织为例说明),先PBS清洗两遍,剔除周围的血块、脂肪及坏死组织,PBS再清洗一遍,剪成1mm3大小,按组织重量:混合酶溶液体积=1g:10ml~20ml的比例,浸入30ml混合酶(IV型胶原酶,0.5mg/ml,透明质酸酶,0.1mg/ml,Ⅰ型DNA酶,0.02mg/ml),置于恒温摇床消化,37℃,消化30min,加入适量的HBSS液稀释,然后缓慢加入淋巴分离液中,作不连续密度梯度离心,800g离心30min。离心结束后收取1.0~1.15密度层的淋巴细胞,加入PBS(含2%FBS)稀释重悬,800g离心10min,去上清;往离心管加入适量含有2%热失活FBS的进行洗涤,1600rpm离心5min,去上清,重复一次。最后按2×106/m L的浓度悬浮于含10%热失活的FBS和rIL-2(10000IU/m L)的RPMI1640培养基内,接种于T25培养瓶,置于37℃、5%CO2培养箱内培养。第二天,吸取细胞悬液,950rpm离心5min,按6×105/m L的浓度悬浮于含10%热失活的FBS和r IL-2(10000IU/m L)的RPMI1640培养基内,接种于六孔板,培养板每3~5d换液一次。
4.培养与扩增方法(共刺激)
抗CD3单克隆抗体(OKT3),浓度为1μg/ml,体系为800μl PBS,4℃共培养过夜。第二天用1ml PBS洗涤六孔板两次,按5×105/m L的浓度接种细胞,培养基为含10%热失活的FBS和rIL-2(10000IU/ml)的RPMI1640,培养体系为1ml,同时加入抗CD28单克隆抗体,浓度为2μg/ml,轻轻晃动培养板以混匀。共刺激培养2天后,加500μl含10%热失活的FBS和r IL-2(10000IU/ml)的RPMI1640培养基,于CO2培养箱中继续培养,如此循环,直至孔里淋巴细胞生长至1.5×106时,更换含同等浓度rIL-2的新鲜培养基,培养10天左右,可获得扩增的淋巴细胞。每次换液保留上清液,以后续研究。
5.细胞冻存与复苏
5.1细胞冻存
细胞悬液950rpm离心5min,去掉上清,计数细胞,按1×107/ml细胞浓度加入热失活的FBS重悬,每根冻存管体系为1ml,其中90%体积为细胞悬液,10%体积为DMSO。将冻存管细胞转移至渐冻盒于-80℃过夜,再将冻存管转至液氮长期保存。
5.2细胞复苏
从液氮中取出冻存细胞后,迅速放入37℃水浴中,轻轻摇动冻存管,待其内部全部融化。解冻后将细胞转到含有完全培养基的离心管,离心去上清,加入含rIL-2(10000IU/mL)的完全培养基,接种于培养瓶培养,第二天更换新鲜培养基。
6.流式细胞术检测TIL的免疫表型
取扩增后的TIL细胞,约1×106,经PBS洗涤2次,100μL的PBS重悬,加入3μlcocktail流式抗体(PerCP-CD3、FITC-CD4、PE-CD8),混匀,室温避光孵育30min,PBS液洗2次,500g离心5min,去掉上清,加入200μl PBS,重悬上机检测。
7.磁珠分选CD8+T细胞
准备细胞1x107/ml,500μl培养基重悬,收集于一次性流式管中;在流式管中加入CD8-cocktail 10μl/ml,混合孵化3min后加入Rapidspheres 5μl/ml,混合孵化3min。加入含有2%FBS的PBS至2.5ml,上下吹打混匀。将流式管插进磁铁中孵化3min,拿起磁铁,倒出上清液,重复该步骤2次。最后用完全培养基重悬管子细胞以培养。
8.CD8+T细胞培养与扩增
方法同步骤4。
Claims (8)
1.原发性肝癌肿瘤浸润淋巴细胞的分离方法,其特征在于,所述方法包括:
1)取无菌新鲜肿瘤癌巢和/或癌旁组织,浸泡于含有10%~15%FBS的RPMI1640培养基中;
2)用PBS清洗两遍,剔除血块、脂肪及坏死组织,PBS再清洗一遍;
3)剪成1mm3~3mm3大小,按组织重量:混合酶溶液体积=1g:10ml~20ml的比例,将组织浸入混合酶溶液,置于恒温摇床消化,37±1℃消化30min~2h;
4)加入HBSS液稀释,进行不连续密度梯度离心,800±100g离心30±10min。
5)收集1±0.15密度层的淋巴细胞,加入PBS(含1%~5%FBS)稀释重悬,800±100g离心10±5min,去上清;
6)加入含有1%~5%热失活FBS进行洗涤,1600±300rpm离心10±5min,去上清,重复一次。
2.根据权利要求1所述的分离方法,其特征在于,所述混合酶溶液的成分包括:0.5±0.2mg/ml IV型胶原酶、0.1±0.05mg/ml透明质酸酶和/或0.02±0.01mg/ml I型DNA酶。
3.根据权利要求2所述的分离方法,其特征在于,所述混合酶溶液的成分具体为:0.5mg/ml IV型胶原酶、0.1mg/ml透明质酸酶和/或0.02mg/ml I型DNA酶。
4.使用权利要求1至3获得的原发性肝癌肿瘤浸润淋巴细胞进行培养和体外扩增的方法,其特征在于,所述方法包括:
1)将淋巴细胞接种于六孔板,使用1±0.5μg/ml抗CD3单克隆抗体OKT3,4℃包被过夜;
2)次日用PBS洗涤后,接种细胞于共刺激培养基上,CO2培养箱中培养。
5.根据权利要求4所述的培养和体外扩增方法,其特征在于,所述共刺激培养基含有:10±5%热失活FBS和10000±2000IU/ml rIL-2的RPMI1640。
6.根据权利要求5所述的培养和体外扩增的方法,其特征在于,所述共刺激培养基还含有2±1μg/ml的抗CD28单克隆抗体。
7.根据权利要求4至6任一所述的培养和体外扩增方法,其特征在于,共刺激培养2天后,更换新的共刺激培养基,如此循环,至淋巴细胞密度生长至起始密度的3~10倍。
8.根据权利要7所述的培养和体外扩增方法,其特征在于,淋巴细胞达到目标浓度后,更换培养基,使用含10000±2000IU/ml rIL-2的培养基,培养8~15天,获得体外扩增后的淋巴细胞。
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110540960A (zh) * | 2019-09-11 | 2019-12-06 | 苏州大学 | 结合有免疫检查点阻断剂的t淋巴细胞及其在制备抗肿瘤药物中的应用 |
WO2022105816A1 (zh) * | 2020-11-19 | 2022-05-27 | 苏州沙砾生物科技有限公司 | 肿瘤浸润淋巴细胞的培养方法及其用途 |
CN114686430A (zh) * | 2020-12-31 | 2022-07-01 | 上海赛比曼生物科技有限公司 | 一种制备til的方法 |
CN117192115A (zh) * | 2023-11-03 | 2023-12-08 | 赛德特(北京)生物工程有限公司 | 检测t淋巴细胞免疫活性的方法 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108753718A (zh) * | 2018-06-20 | 2018-11-06 | 淮安诺康生物科技有限公司 | 肿瘤浸润淋巴细胞til的体外扩增方法 |
-
2019
- 2019-04-23 CN CN201910330195.9A patent/CN110042080A/zh active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108753718A (zh) * | 2018-06-20 | 2018-11-06 | 淮安诺康生物科技有限公司 | 肿瘤浸润淋巴细胞til的体外扩增方法 |
Non-Patent Citations (3)
Title |
---|
江珊珊等: "肝癌肿瘤浸润淋巴细胞(TIL)体外扩增及特性研究", 《中国细胞生物学学报》 * |
秦鹏等: "体外大量扩增原发性肝癌肿瘤浸润淋巴细胞及其生物学特性的初步分析", 《中国医药生物技术》 * |
黄镇辉等: "人肝癌浸润淋巴细胞的分离培养及其CD8 阳性亚群特性研究", 《中华普通外科学文献( 电子版)》 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110540960A (zh) * | 2019-09-11 | 2019-12-06 | 苏州大学 | 结合有免疫检查点阻断剂的t淋巴细胞及其在制备抗肿瘤药物中的应用 |
WO2022105816A1 (zh) * | 2020-11-19 | 2022-05-27 | 苏州沙砾生物科技有限公司 | 肿瘤浸润淋巴细胞的培养方法及其用途 |
CN114686430A (zh) * | 2020-12-31 | 2022-07-01 | 上海赛比曼生物科技有限公司 | 一种制备til的方法 |
WO2022143785A1 (en) * | 2020-12-31 | 2022-07-07 | Shanghai Cellular Biopharmaceutical Group Ltd. | Methods for preparing tumor-infiltrating lymphocytes |
CN117192115A (zh) * | 2023-11-03 | 2023-12-08 | 赛德特(北京)生物工程有限公司 | 检测t淋巴细胞免疫活性的方法 |
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