CN114561340A - 一种培养复发乳腺癌类器官的培养基及其应用 - Google Patents
一种培养复发乳腺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乳腺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,能够实现对复发性乳腺癌肿瘤样本的类器官培养,大大提高了乳腺癌类器官培养成功率,缩短培养周期,为揭示复发性乳腺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乳腺癌类器官的培养基及其培养方法。
背景技术
乳腺癌影响全球女性健康和生存,加重家庭和国家医疗经济负担。目前乳腺癌新发病例数全球每年约226万余例,位居所有肿瘤第1位;全球每年因乳腺癌致死病例数约68万余例,位居所有肿瘤第4位。经过综合治疗后的乳腺癌患者5年生存率高达90%,初次治疗后的肿瘤复发是影响乳腺癌患者生存和预后的最重要因素之一。乳腺癌初次治疗后4年的复发率约为18%,最常见的复发部位依次为骨(27.4%)、局部复发(27.2%)、肺(16.0%)和肝(12.5%)。中位复发时间约为2.3年且复发时间与患者预后密切相关,复发越早预后越差。乳腺癌局部复发、淋巴结转移复发和远处转移复发患者的5年生存率仅为41%、20%和13%。复发性乳腺癌对放射治疗、化学治疗和靶向治疗等治疗手段相当耐受,严重影响患者的治疗效果和预后。如何进一步提升复发性乳腺癌的治疗效果是乳腺癌治疗领域的难点和重点,肿瘤个体化治疗(精准治疗)是重要的潜在方向之一,而可靠的疗效预测工具对肿瘤精准治疗极其重要。在肿瘤精准治疗领域,目前普遍应用的疗效预测工具是基因检测发现的基因变异和蛋白表达水平检测。例如HER2(人类表皮生长因子受体2)基因扩增阳性的乳腺癌患者,可以使用曲妥珠单抗靶向HER2的胞外结构域,抑制PI3K和MAPK信号通路,诱导凋亡和抑制血管生成等机制最终抑制肿瘤生长。然而复发性乳腺癌对现有相关检测所对应的治疗药物基本已经耐受,患者面临无药可用的境地。因此依据基因变异或蛋白表达水平指导复发性乳腺癌治疗的价值非常有限。复发性乳腺癌的精准治疗需要新的可靠的疗效预测工具。
类器官(organoid)是一种3D细胞培养物,具有其所代表器官的核心特征,在空间组织和功能上与所代表器官类似。类器官技术被Nature Methods杂志评为2017年年度技术。肿瘤类器官(tumor organoid)则一般是指由肿瘤患者肿瘤组织体外培养而来的类器官。肿瘤类器官能在病理学形态、基因变异、染色体稳定性和肿瘤异质性等方面忠实模拟所对应患者体内肿瘤,因此肿瘤类器官是指导肿瘤精准治疗的绝佳工具。
目前原发乳腺癌类器官培养体系已被众多研究人员成功建立。Hans Clevers教授研究组乳腺癌类器官的培养成功率约为80%,但仍有20%左右失败率,培养失败的主要原因是培养基不完善。通常复发肿瘤的生物学行为和肿瘤微环境更为复杂,相关研究也不透彻,导致复发肿瘤类器官培养难度增加,成功率也相应大大下降。因此复发性乳腺癌类器官培养是一大挑战,目前尚未见到相关成功报道,对肿瘤类器官指导复发性乳腺癌精准治疗造成明显障碍,因此研发针对复发性乳腺癌高度异质性的类器官培养基对复发性乳腺癌患者的精准治疗至关重要。
发明内容
本发明的目的在于,提供一种适宜复发性乳腺癌类器官培养的培养基,进而获得复发乳腺癌类器官,以便能够研究复发乳腺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乳腺癌的类器官培养基,所述培养基成分包括如下成分:
高糖DMEM培养基:1640培养基(0.5-5),全转铁蛋白(5-50mg/L),胰岛素(1-20mg/L),β-巯基乙醇(0.01-50mM),BSA(0.1-10%),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),Wnt3a(50-1000ng/ml)。
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Gastrin I,进一步优选的,所述Gastrin I浓度为0.1-20nM。
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0.01-10%。
优选的,所述培养基中还包括17α-estradiol,进一步优选的,所述17α-estradiol浓度为1×10-9-50×10-6M。
优选的,所述培养基中还包括氢化可的松,进一步优选的,所述氢化可的松浓度为1×10-10-80×10-7M。
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HGF,进一步优选的,所述HGF浓度为5-500ng/ml。
优选的,所述培养基中还包括FGF7,进一步优选的,所述FGF7浓度为1-200ng/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。
本发明另一方面,提供了一种培养复发乳腺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,能够对多种的复发乳腺癌肿瘤样本实现类器官培养。
具体的,所述培养方法如下:
1.复发乳腺癌样本条状或绿豆大小,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℃预热特定消化液中,消化;
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,细胞筛网过滤;
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,加入乳腺癌培养液,每3天更换一次相应培养液;
9.肿瘤类器官培养12-18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。
优选的,步骤3中,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8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乳腺癌类器官模型的制备,通过制备得到的乳腺癌类器官模型,能够更直观的展示乳腺癌原发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乳腺癌的药物。
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,能够对复发乳腺癌肿瘤样本进行培养获得乳腺癌类器官,并且能够利用所述培养基制备乳腺癌类器官模型,大大缩短了培养时间和提高了类器官成功率,并且降低了对国外培养基的依赖,降低了培养基成本,为后续了解乳腺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乳腺癌肿瘤样本类器官比较图。
图2为两种改良培养基与Cell培养基培养复发乳腺癌获得的类器官数目比较。
图3为本申请乳腺癌肿瘤样本类器官培养基和Cell培养基培养获得的类器官药物筛选实验。
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:
高糖DMEM培养基,1640培养基,全转铁蛋白(T0665,Sigma),胰岛素(91077C,Sigma),β-巯基乙醇(Sigma),BSA(Sigma),R-spondin 1(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),Wnt3a(RD),A83-01(Tocris),Gastrin I(Tocris),EGF(Prerotech),HGF(Prerotech),FGF7(Prerotech),FGF10(Prerotech),Dimethyl Sulfoxide(Sigma),17α-estradiol(E2,Sigma),氢化可的松(Sigma)。
实施例1、改良培养基1
高糖DMEM培养基:1640培养基(3:1),全转铁蛋白(10mg/L),胰岛素(5mg/L),β-巯基乙醇(10mM),BSA(1%),R-spondin 1(1000ng/ml),Noggin(200ng/ml),N2:B27(1:2),GlutaMAX Supplement(1×),HEPES(1×),烟酰胺(10mM),NormocinTM(1×),N-乙酰半胱氨酸(1mM),Wnt3a(100ng/ml),A83-01(10μM),Gastrin I(5nM),DMSO(5%),17α-estradiol(50×10-9M),氢化可的松(60×10-8M)。
高糖DMEM培养基:1640培养基(3:1),全转铁蛋白(10mg/L),胰岛素(5mg/L),BSA(1%),R-spondin 1(1000ng/ml),Noggin(200ng/ml),N2:B27(1:2),GlutaMAX Supplement(1×),HEPES(1×),烟酰胺(10mM),NormocinTM(1×),N-乙酰半胱氨酸(1mM),A83-01(10μM),Gastrin I(5nM),DMSO(5%),17α-estradiol(50×10-9M),氢化可的松(60×10-8M)。
高糖DMEM培养基:1640培养基(3:1),全转铁蛋白(10mg/L),胰岛素(5mg/L),β-巯基乙醇(10mM),BSA(1%),R-spondin 1(1000ng/ml),Noggin(200ng/ml),N2:B27(1:2),GlutaMAX Supplement(1×),HEPES(1×),烟酰胺(10mM),NormocinTM(1×),N-乙酰半胱氨酸(1mM),Wnt3a(100ng/ml),A83-01(10μM),Gastrin I(5nM),DMSO(5%)。
实施例2、改良培养基2
高糖DMEM培养基:1640培养基(1:1),全转铁蛋白(40mg/L),胰岛素(15mg/L),β-巯基乙醇(25mM),BSA(10%),R-spondin 1(250ng/ml),Noggin(50ng/ml),N2:B27(2:1),GlutaMAX Supplement(1×),HEPES(1×),烟酰胺(30mM),NormocinTM(1×),N-乙酰半胱氨酸(5mM),Wnt3a(500ng/ml),EGF(100ng/ml),HGF(300ng/ml),FGF7(150ng/ml),FGF10(500ng/ml),17α-estradiol(50×10-7M),氢化可的松(1×10-9M)。
高糖DMEM培养基:1640培养基(1:1),全转铁蛋白(40mg/L),胰岛素(15mg/L)BSA(10%),R-spondin 1(250ng/ml),Noggin(50ng/ml),N2:B27(2:1),GlutaMAX Supplement(1×),HEPES(1×),烟酰胺(30mM),NormocinTM(1×),N-乙酰半胱氨酸(5mM),EGF(100ng/ml),HGF(300ng/ml),FGF7(150ng/ml),FGF10(500ng/ml),17α-estradiol(50×10-7M),氢化可的松(1×10-9M)。
高糖DMEM培养基:1640培养基(1:1),全转铁蛋白(40mg/L),胰岛素(15mg/L),β-巯基乙醇(25mM),BSA(10%),R-spondin 1(250ng/ml),Noggin(50ng/ml),N2:B27(2:1),GlutaMAX Supplement(1×),HEPES(1×),烟酰胺(30mM),NormocinTM(1×),N-乙酰半胱氨酸(5mM),EGF(100ng/ml),FGF7(150ng/ml),17α-estradiol(50×10-7M),氢化可的松(1×10-9M)。
将上述实施例1和2所述的培养基和传统的原发乳腺癌培养基(Cell)培养不同的复发乳腺癌肿瘤样本,观察初始类器官形成数目、大小,类器官首次传代时间,稳定传代3次以上的成功率。
具体操作方法如下所示:
1.复发乳腺癌样本1-2条或绿豆大小,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℃预热特定消化液中,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,100μm细胞筛网过滤;
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,加入乳腺癌培养液,每3天更换一次相应培养液;
9.肿瘤类器官培养12-18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。
上述实施例中所使用的原发乳腺癌(Cell)培养基如下所示:
Advance DMEM/F12培养基,10%R-spondin 1conditioned medium,B27(1×),Noggin(100ng/ml),GlutaMAX Supplement(1×),HEPES(1×),Primocin(1×),烟酰胺(5mM),N-乙酰半胱氨酸(1.25mM),Neuregulin 1(5nM),EGF(5ng/ml),FGF7(5ng/ml),FGF10(20ng/ml),A83-01(500nM),SB202190(0.5μM),Y27632(5μM)。
结果
本发明采用目前公认的原发乳腺癌类器官培养基(Cell)作为对照培养基,11例复发乳腺癌穿刺样本经培养基1和2成功培养(稳定传3代后能冻存建系)8例(P1-3,P5,P7-9,P11)和7例(P1-2,P4-5,P7-9)复发乳腺癌类器官,相同培养时间段,Cell培养基成功培养2例(P2,P8)复发乳腺癌类器官。在复发乳腺癌培养基(1,2)中培养6天,类器官形成数目是Cell培养基的2.12倍和1.75倍,类器官大小是Cell培养基中的3.63倍和2.76倍。穿刺样本一般培养基1和2中培养14-16天即可首次传代,在Cell培养基中,穿刺样本需要21天以上才可以进行首次传代。复发乳腺癌培养基(1,2)的培养成功率分别为72.7%(8/11)和63.6%(7/11),Cell培养基成功率则为18.2%(2/11)。已报道的原发乳腺癌的培养基不太适合复发乳腺癌类器官的培养,本发明乳腺癌培养基针对复发乳腺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(图1,2,3)。同时我们比较发现组分中β-巯基乙醇,Wnt3a,对复发乳腺癌类器官形成至关重要,而17α-estradiol,氢化可的松,FGF10,HGF对类器官的快速生长和传代后活性有重要影响。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (9)
1.一种复发乳腺癌细胞类器官培养基,其特征在于,所述培养基包含如下组分:高糖DMEM培养基:1640培养基(0.5-5),全转铁蛋白(5-50mg/L),胰岛素(1-20mg/L),β-巯基乙醇(0.01-50mM),BSA(0.1-10%),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),Wnt3a(50-1000ng/ml)。
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,Gastrin I,DMSO,17α-estradiol,氢化可的松。
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Gastrin I浓度为0.1-20nM;所述DMSO浓度为0.1-10%(v/v);所述17α-estradiol浓度为1×10-9-50×10-6M,所述氢化可的松浓度为1×10-10-80×10-7M。
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:EGF,HGF,FGF7,FGF10,17α-estradiol,氢化可的松,优选的,所述EGF的浓度为5-500ng/ml,所述FGF7浓度为1-200ng/ml,所述FGF10浓度为50-1000ng/ml。
5.一种利用上述任一权利要求所述培养基培养复发乳腺癌细胞类器官的方法,其特征在于,所述方法包括如下步骤:
1).取复发乳腺癌肿瘤样本,4℃保存于专用保存液中,24小时内低温转移至操作室;
2).样本低温破碎,酶解消化;
3).低温离心去除上清,冰PBS重悬,此过程重复3-5次;
4).低温离心去除上清,依据获得细胞情况,1-5ml冰PBS重悬后显微镜下计数细胞;
5).低温离心去除上清,依据细胞计数加入适量冰Matrigel重悬接种于24孔板,加入乳腺癌培养液,每3天更换一次相应培养液;
6).肿瘤类器官培养6-12天后,每孔添加1ml冰PBS,反复吹打,移入15ml离心管;
7).低温离心去除上清,冰PBS重悬,此步骤重复3-5次,直至Matrigel全部清除;
8).离心结束后,冰Matrigel重悬,1:3接种扩增;
9).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。
6.根据权利要求7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。
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乳腺癌类器官模型中的应用。
9.由前述任一权利要求获得的类器官模型在筛选靶向复发性乳腺癌药物中的应用。
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