CN114574443A - 一种培养复发肾细胞癌类器官的培养基及其应用 - Google Patents
一种培养复发肾细胞癌类器官的培养基及其应用 Download PDFInfo
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Abstract
本发明公开了一种复发性肾细胞癌肿瘤类器官的培养基及其培养方法。通过调整培养基各组分的含量,能够实现对复发性肾细胞癌肿瘤样本的类器官培养,大大提高了肾细胞癌类器官培养成功率,缩短培养周期,降低培养成本,为揭示复发性肾细胞癌发病机理和筛选抗癌药物奠定了基础。
Description
技术领域
本发明涉及细胞培养领域,具体的涉及一种复发性肾细胞癌类器官的培养基及其培养方法。
背景技术
肾癌是较为常见的恶性肿瘤之一。2020年全球癌症统计中,肾癌年新增病例数为43.1万多,年死亡病例数约18万。肾癌的发病率仅次于前列腺癌和膀胱癌,位居泌尿系统肿瘤第三位。传统上一般将肾癌分为三种病理类型:透明细胞癌,乳头状肾细胞癌和嫌色细胞肾细胞癌。其中肾癌最常见的病理类型为肾透明细胞癌(clear cell carcinoma),约占肾癌总数的80-90%。肾癌目前的治疗方案为手术、放疗、化疗、靶向和免疫治疗联合的综合治疗。经过多种手段综合治疗肾癌能获得较好的治疗效果,例如肾透明细胞癌的10年肿瘤特异生存率为约为71%。
肾癌初治后约有30%左右比例最终会复发,复发性肾癌往往对药物治疗耐受,治疗难度加大,预后较差,因此在肾癌治疗中,复发性肾癌是重点和难点。肿瘤精准治疗(个体化)是指利用各种疗效预测工具,在患者开始治疗前预测出有效治疗手段,为每一位不同患者寻找最佳治疗方案,最终达到改善患者生存的目的。因此精准治疗有望为复发性肾癌的治疗提供新方法和新思路。目前应用的疗效预测工具大体包括各类生物标记物和功能性试验两类。生物标记物中使用最多的是特定的基因变异和基因(或转录蛋白)表达水平;然而能从中获益的肿瘤患者较少。功能性试验是指利用患者来源的肿瘤模型进行药物或治疗方法的筛选测试,选择最优的方案进行治疗。以往有一定研究基础的模型主要是患者来源细胞系(PDC)和人源性组织异种移植模型(PDX),但存在预测准确性欠佳(PDC)和检测时间过长、所需经费过多、成功率低(PDX)等问题。
患者来源肿瘤类器官(PDO)是指对肿瘤患者的新鲜肿瘤组织经过处理提取肿瘤细胞后进行三维培养形成的类器官。大量研究已经证实肿瘤类器官能够较为忠实地模拟所来源肿瘤组织的病理形态学、基因变异和基因表达谱等特征,且在培养过程中其基因组能够保持相对稳定。最重要的是,患者来源肿瘤类器官在体外对抗肿瘤治疗疗法的反应与患者体内肿瘤对治疗的反应基本一致。与PDC或PDX相比,肿瘤类器官具有培养成功率较高、检测周期较短和疗效预测较为准确等优点,是目前肿瘤精准治疗所需的最佳疗效预测工具之一。目前原发肾癌类器官已被成功培养,最高培养成功率约在70%左右,但已有研究均是小样本量研究,确切的培养成功率尚待较大样本量研究明确。相比未经治疗的原发肾癌类器官培养,复发性肾癌类器官的培养难度理论上将大大增加,目前尚无复发性肾癌类器官培养相关研究。
如上所述,复发性肾癌的体外类器官模型对复发性肾癌的精准治疗具有很大潜力,可作为患者的“试药替身”为患者寻找最佳的治疗方案。鉴于目前尚无复发性肾癌类器官培养相关研究报道,本专利首次研究设计了复发性肾癌培养的改良培养基方案,对于复发性肾癌的精准治疗具有重要意义,也具有明显的商业化价值。
发明内容
本发明的目的在于,提供一种适宜复发性肾细胞癌类器官培养的培养基,进而获得复发肾细胞癌类器官,以便能够研究复发肾细胞癌发病机制和病程以及筛选相关抗癌药物。
为解决上述技术问题,本发明第一方面提供一种复发肾细胞癌的类器官培养基,所述培养基成分包括三个重要组成部分,即常规培养组分、常规添加因子和必须添加因子三个部分。
将所述三个部分进行组合,选择合适的组分进行配比。
其中,
常规培养组分如下所示:
F12K培养基:1640培养基(0.5-5),L-Glutathine(1-100mg/L),亚硒酸钠(0.002-0.5mg/L),乙醇胺盐酸盐(2-200mg/L),胰岛素(1-20mg/L),β-巯基乙醇(0.01-50mM)。
常规添加因子为:
R-spondin 1(50-1000ng/ml),Noggin(10-200ng/ml),N2:B27(0.1-2),GlutaMAXSupplement(1×),HEPES(1×),烟酰胺(1-100mM),NormocinTM(1×),N-乙酰半胱氨酸(0.1-10mM)。
必须添加因子为下述因子的一种或多种的组合:
优选的,所述培养基中还包括A83-01,进一步优选的,所述A83-01浓度为0.1-50μM。
优选的,所述培养基中还包括EGF,进一步优选的,所述EGF浓度为1-200ng/ml。
优选的,所述培养基中还包括DMSO,进一步优选的,所述DMSO浓度为培养基体积比的0.01-5%。
优选的,所述培养基中还包括BMP2,进一步优选的,所述BMP2浓度为5-100ng/ml。
优选的,所述培养基中还包括FGF10,进一步优选的,所述FGF10浓度为50-1000ng/ml。
优选的,所述培养基中还包括FGF9,进一步优选的,所述FGF9浓度为50-500ng/ml。
优选的,所述培养基中还包括BMP4,进一步优选的,所述BMP4浓度为50-1000ng/ml。
优选的,所述培养基中还包括BMP7,进一步优选的,所述BMP7浓度为50-1000ng/ml。
优选的,所述培养基中还包括肝素,进一步优选的,所述肝素浓度为10-200IU/ml。
本发明另一方面,提供了一种培养复发肾细胞癌肿瘤样本的培养方法,包括使用上述培养基以及优选的组分选择性的添加,能够对多种的复发肾细胞癌肿瘤样本实现类器官培养。
具体的,所述培养方法如下:
1.复发肾细胞癌穿刺样本,低温保存,24小时内转移至操作室;
2.样本低温破碎,酶解消化;
3.样本消化完全后,添加同体积的冰PBS,吹打均匀;
4.低温离心弃上清,裂解残留红细胞;
5.重悬显微镜下计数细胞;
6.低温离心弃上清,依据细胞计数加入适量冰Matrigel重悬接种于多孔板,加入肾癌培养液,每3天更换一次相应培养液;
7.肿瘤类器官培养6-9天后,加冰PBS,反复吹打;
8.低温离心,去上清,冰PBS重悬,此步骤重复3-5次,直至Matrigel全部清除;
9.离心结束后,冰Matrigel重悬,1:3接种扩增,待肿瘤类器官稳定传代后,重复步骤8,一半类器官做药物敏感性实验,一半类器官用细胞冻存液冻存。
优选的,步骤2中,先将肾细胞癌肿瘤样本切成3mm×3mm大小,冰PBS反复清洗5次后再进行酶解;进一步优选的,肿瘤样本块切成1mm3碎片,
优选的,酶解消化使用的是胶原酶进行消化;进一步优选的,酶解消化条件为37℃,胶原酶消化30-60min;
优选的,步骤2中,酶解消化后终止酶解,具体操作如下:加入与酶解液等体积冰PBS,移液管反复吹打直至组织碎片消失,镜下观察为均匀的细胞团;
优选的,步骤2中,酶解消化使用的是TrypLE Express,进一步优选的,酶解消化的操作为:根据类器官数量加1-5ml不等的TrypLE Express,消化10-15min,加入AdvanceDMEM/F12培养液终止,300g离心10min;
优选的,步骤4-9中,低温离心的参数为4℃,70-300g离心5-10min;
优选的,步骤6中,Matrigel重悬调整细胞浓度,1000-2000/50μl接种入24孔板,待Matrigel凝固后加入相应的培养基。
第三方面,本发明公开了一种利用上述培养基进行复发肾细胞癌类器官模型的制备,通过制备得到的复发肾细胞癌类器官模型,能够更直观的展示复发肾细胞癌病理机制以及癌细胞的侵袭机制。并且利用制备获得的所述类器官模型用于筛选治疗复发性肾细胞癌的药物。
本发明和现有技术相比,具有以下有益效果:
本发明公开的培养基可以进行组分的调整,能够大大缩短培养时间和提高类器官培养成功率,并且降低了对国外培养基的依赖,降低了培养基成本,为后续了解复发肾细胞癌发病机制以及筛选药物奠定了基础。
附图说明
图1为不同培养基培养复发肾细胞癌肿瘤样本类器官比较图。
图2为两种改良培养基与NC培养基培养复发肾细胞癌获得的类器官。
具体实施方式
下面将结合示意图对本发明公开的培养基以及培养方法进行更详细的描述,其中表示了本发明的优选实施例,应该理解本领域技术人员可以修改在此描述的本发明,而仍然实现本发明的有利效果。因此,下列描述应当被理解为对于本领域技术人员的广泛知道,而并不作为对本发明的限制。
从市场购买各种培养基组分,规格和来源具体如下:
(1)F12K培养基,1640培养基,L-Glutathine(G6013,Sigma),亚硒酸钠(S5261,Sigma),乙醇胺盐酸盐(E120635,Aladdin),胰岛素(91077C,Sigma),β-巯基乙醇(Sigma),R-spondin1(SinoBiological),Noggin(SinoBiological),N2(Gibco),B27(Gibco),GlutaMAX Supplement(Gibco),HEPES(Gibco),烟酰胺(Sigma),NormocinTM(Invivogen),N-乙酰半胱氨酸(Sigma),A83-01(Tocris),EGF(Peprotech),BMP2(Peprotech),BMP4(Peprotech),BMP7(Peprotech),FGF9(Peprotech),FGF10(Peprotech),肝素(Sigma),DMSO(Sigma)。
根据发明内容对培养基组分进行优化,所述培养基需要有三个部分组成,包括基本培养组分以及常规添加因子和必须添加因子,其中:
基本培养组分包括以下成分:
F12K培养基,1640培养基,L-Glutathine,亚硒酸钠,乙醇胺盐酸盐,胰岛素,β-巯基乙醇。
常规添加因子选择以下成分的一种或多种的组合:
R-spondin 1,Noggin,N2,B27,GlutaMAX Supplement,HEPES,烟酰胺,NormocinTM,N-乙酰半胱氨酸。
必须添加的因子组分主要有以下组分的一种或多种,具体如下所示:
A83-01,EGF,BMP2,BMP4,BMP7,FGF9,FGF10,肝素,DMSO。
依据前期实验(数据未显示),将培养基进行基本培养组分和因子的配合优化,设计了多组不同组成的培养基组成,下述实施例为具体培养基组成。
实施例1、改良培养基1
F12K培养基:1640培养基(4:1),L-Glutathine(50mg/L),亚硒酸钠(0.1mg/L),乙醇胺盐酸盐(100mg/L),胰岛素(5mg/L),β-巯基乙醇(30mM);
R-spondin 1(400ng/ml),Noggin(100ng/ml),N2:B27(1:2),GlutaMAXSupplement(1×),HEPES(1×),烟酰胺(15mM),NormocinTM(1×),N-乙酰半胱氨酸(1mM);
A83-01(2μM),EGF(100ng/ml),DMSO(0.5%),BMP2(50ng/ml),FGF10(150ng/ml)。
实施例2、改良培养基2
F12K培养基:1640培养基(2:1),L-Glutathine(50mg/L),亚硒酸钠(0.2mg/L),乙醇胺盐酸盐(150mg/L),胰岛素(15mg/L),β-巯基乙醇(40mM);
R-spondin 1(250ng/ml),Noggin(50ng/ml),N2:B27(2:1),GlutaMAX Supplement(1×),HEPES(1×),烟酰胺(20mM),NormocinTM(1×),N-乙酰半胱氨酸(5mM);
EGF(50ng/ml),FGF9(100ng/ml),BMP4(250ng/ml),BMP7(150ng/ml),肝素(20IU/ml)。
实施例3、本发明的2种培养基和原发肾细胞癌培养基培养复发肾细胞癌肿瘤样本类器官培养比较和药物敏感筛选。
将上述实施例1和2所述的培养基和传统的原发肾细胞癌培养基(NatureCommunications,NC)培养不同的复发肾细胞癌肿瘤样本,观察类器官成功率以及类器官形态。
具体操作方法如下所示:
1.复发肾细胞癌穿刺样本12例,4℃保存于专用保存液中,24小时内低温转移至操作室;
2.样本冰PBS清洗3-5次,每次5min;
3.样本低温切成1mm碎片入37℃预热胶原酶消化液中,消化时间30-60min.
4.样本消化完全后,添加与消化液同体积的冰PBS,反复吹打直至不见碎片组织;
5.100g低温离心5-10min,去除上清,无菌红细胞裂解1ml冰上5min,5ml冰PBS终止裂解;
6.70g低温离心5-10min,去除上清,冰PBS重悬,此过程重复3-5次;
7.200g低温离心10min,去除上清,依据获得细胞情况,1-5ml冰PBS重悬后显微镜下计数细胞;
8.300g低温离心10min,去除上清,依据细胞计数加入适量冰Matrigel重悬接种于24孔板,加入肾癌培养液,每3天更换一次相应培养液;
9.肿瘤类器官培养6-9天后,每孔添加1ml冰PBS,反复吹打,移入15ml离心管;
10.300g低温离心10min,去上清,冰PBS重悬,此步骤重复3-5次,直至Matrigel全部清除
11.离心结束后,冰Matrigel重悬,1:3接种扩增,待肿瘤类器官稳定传代后,重复步骤10,一半类器官做药物敏感性实验,一半类器官用细胞冻存液冻存。
实施例3中所使用的原发肾细胞癌(Nature Communications)培养基:
Advance DMEM/F12培养基,10%R-spondin 1conditioned medium,EGF(50ng/ml),N-乙酰半胱氨酸(1.25mM),B27(1×),A83-01(5μM),FGF10(100ng/ml),Y27632(10μM)。
结果
12例肾癌穿刺样本(Patient1-12),分别用复发肾癌培养基(1,2)和原发肾癌培养基(NC培养基)培养6-14天后,培养基1和2成功培养9例(P2-3,P4-5,P7-9,P11-12)和10例(P1-5,P7-9,P10-11)复发肾癌类器官,相同培养时间段,NC培养基成功培养5例(P4-5,P8-9,P11)复发肾癌类器官。复发肾癌培养基形成原代类器官数目多,生长快。在复发肾癌培养基(1,2)中,穿刺样本一般培养6-9天即可首次传代,且能持续稳定传代3代以上;在NC培养基中,穿刺样本需要14天左右才可以进行首次传代,但稳定持续传代的较少。复发肾癌培养基(1,2)的培养成功率分别为75%(9/12)和83.3%(10/12),NC培养基成功率则为41.7%(5/12)。已报道的原发肾癌的培养基不太适合复发肾癌类器官的培养,本发明肾癌培养基针对复发肾癌有较高的类器官培养成功率和相对较短的类器官培养时间(如图1,图2)。
Claims (9)
1.一种复发肾细胞癌类器官培养基,其特征在于,所述培养基包含如下组分:F12K培养基:1640培养基(0.5-5),L-Glutathine(1-100mg/L),亚硒酸钠(0.002-0.5mg/L),乙醇胺盐酸盐(2-200mg/L),胰岛素(1-20mg/L),β-巯基乙醇(0.01-50mM),R-spondin 1(50-1000ng/ml),Noggin(10-200ng/ml),N2:B27(0.1-2),GlutaMAX Supplement(1×),HEPES(1×),烟酰胺(1-100mM),NormocinTM(1×),N-乙酰半胱氨酸(0.1-10mM)。
2.根据权利要求1所述的培养基,其特征在于,所述培养基还包括以下物质的一种或多种:A83-01,EGF,DMSO,BMP2,FGF10。
3.根据权利要求2所述的培养基,其特征在于,所述A83-01浓度为0.1-50μM;所述EGF浓度为1-200ng/ml;所述DMSO浓度为0.1-5%,所述BMP2浓度为5-100ng/ml;所述FGF10浓度为50-1000ng/ml。
4.根据前述任一权利要求所述的培养基,其特征在于所述培养基中还包括下述物质的一种或多种的组合:FGF9,BMP4,BMP7,肝素;优选的,所述FGF9的浓度为50-500ng/ml,所述BMP4浓度为50-1000ng/ml,所述BMP7浓度为50-1000ng/ml,所述肝素的浓度为10-200IU/ml。
5.一种利用上述任一权利要求所述培养基培养复发肾细胞癌类器官的方法,其特征在于,所述方法包括如下步骤:
1).复发肾细胞癌穿刺样本,低温保存,24小时内转移至操作室;
2).样本低温破碎,酶解消化;
3).低温离心弃上清,裂解残留红细胞;
4).重悬显微镜下计数细胞;依据细胞计数加入适量冰Matrigel重悬接种于多孔板,加入肾癌培养液,每3天更换一次相应培养液;
5).肿瘤类器官培养6-9天后,低温离心,去上清,冰PBS重悬,此步骤重复3-5次,直至Matrigel全部清除;
6).离心结束后,冰Matrigel重悬,1:3接种扩增。
6.根据权利要求5所述的方法,其特征在于,步骤2)消化使用的是胶原蛋白酶。
7.根据权利要求5-6任一项所述的方法,低温离心去除上清的操作中,温度为4℃,离心转速为70-300g,时间5-10min。
8.权利要求1-4任一所述培养基或权利要求5-7任一项所述的方法在制备复发性肾细胞癌类器官模型中的应用。
9.由前述任一权利要求所述培养基或培养方法获得的类器官在筛选靶向复发性肾细胞癌药物中的应用。
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CN115820560A (zh) * | 2023-01-09 | 2023-03-21 | 山东第一医科大学附属肿瘤医院(山东省肿瘤防治研究院、山东省肿瘤医院) | 一种复发性呼吸道乳头状瘤病类器官的构建方法及其应用 |
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CN115322948B (zh) * | 2022-07-20 | 2023-08-29 | 创芯国际生物科技(广州)有限公司 | 一种微量原代组织类器官培养前的扩增培养方法 |
CN115820560A (zh) * | 2023-01-09 | 2023-03-21 | 山东第一医科大学附属肿瘤医院(山东省肿瘤防治研究院、山东省肿瘤医院) | 一种复发性呼吸道乳头状瘤病类器官的构建方法及其应用 |
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