CN113801838A - 原发性肝细胞癌类器官的持续培养方法和培养基 - Google Patents
原发性肝细胞癌类器官的持续培养方法和培养基 Download PDFInfo
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Abstract
本发明公开了一种原发性肝癌类器官的培养基及其培养方法。通过调整培养中各种组分的含量,能够实现对异质性巨大的原发性肝癌细胞进行类器官培养,为揭示肝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,具体的涉及一种原发性肝癌类器官的培养基及其培养方法。
背景技术
原发性肝癌(primary liver cancer)是世界上最常见的恶性肿瘤之一,在全球所有恶性肿瘤相关死因中高居第2位,中国是肝癌发病率和死亡率最高的国家,每年肝癌新发人数和死亡人数都占到全球50%-55%,是我国第2位的肿瘤杀手。原发性肝癌包括肝细胞癌、肝内胆管癌和混合型三种不同病理类型,其中肝细胞癌占到85%-90%以上。肝细胞癌体外长期原代2D培养比较困难,成功率较低,而成功建立的2D肝细胞癌又与体内生理状态差别巨大,其体外药物敏感性效应与临床病人的反应一致性较差。因此建立一种高效稳定且能与体内生理状态相一致的肝细胞癌长期培养体系对原发性肝癌的治疗和药物研发至关重要。
过去10年,干细胞研究领域取得的关键进展之一就是类器官的发展。类器官(Organoid)是将具有干性潜能的细胞进行3D培养,从而形成相应器官的类似器官。类器官属于三维细胞培养物,与对应的器官拥有类似的空间组织,包含其代表器官的一些关键特性并能重现对应器官的部分功能。2018年Science发表Georgios Vlachogiannis关于使用胃肠消化道人源性肿瘤类器官(PDTO)测试抗癌药物,从而预测患者临床预后的文章。研究人员对比PDTOs和临床实验中患者对抗癌药物的反应,分析结果显示:PDTO表现出100%的敏感性,93%的特异性,88%的阳性预测值和100%的阴性预测值。
肝细胞癌类器官目前成功培养的报道极少,2017年Nature medicine(NM)报道3例肝细胞癌类器官成功培养后再无后续报道,国内科研机构也反映原代肝细胞癌类器官持续培养的困难,成功率极低(5%-10%),根本原因是肝细胞癌的瘤间和瘤内异质性相比其它肿瘤更大,导致单一的肝细胞癌类器官培养基培养成功率低下,因此多样性的培养基对肝细胞癌类器官的成功培养至关重要。
发明内容
本发明的目的在于,提供一种适宜原发性肝癌类器官培养的培养基,进而获得原发性肝癌类器官,以便能够研究肝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,本发明提供一种原发性肝癌的类器官培养基,所述培养基成分包括如下成分:
DMEM/F12培养基,L-谷胱甘肽(1-100mg/L),R-spondin 1(50-500ng/ml),N2:B27(0.1-2),GlutaMAX Supplement(1X),HEPES(1X),烟酰胺(1-100mM),NormocinTM(1X),N-乙酰半胱氨酸(0.1-10mM),A83-01(0.1-50μM),胃泌素I(0.1-50nM),EGF(1-200ng/ml),Y27632(1-200μM)。
优选的,所述培养基中还包括抗坏血酸,进一步优选的,所述抗坏血酸浓度为1-100mg/L。
优选的,所述培养基中还包括Holo-Transferrin,进一步优选的,所述Holo-Transferrin浓度为5-200mg/L。
优选的,所述培养基中还包括Insulin,进一步优选的,所述Insulin浓度为1-200mg/L。
优选的,所述培养基中还包括Sodium selenite,进一步优选的,所述Sodiumselenite acid浓度为0.002-0.5mg/L。
优选的,所述培养基中还包括乙醇胺盐酸盐,进一步优选的,所述乙醇胺盐酸盐浓度为2-200mg/L。
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50-500ng/ml。
优选的,所述培养基中还包括CHIR99021,进一步优选的,所述CHIR99021浓度为1-100μM。
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0.01-2%。
本发明另一方面,提供了一种培养基培养原代肝细胞癌获得肝癌类器官的培养方法,包括使用上述培养基以及优选的组分选择性的添加,能够对多种的原发性肝细胞癌实现类器官培养。
具体的,所述培养方法如下:
1).获得肝癌组织样本,切成碎片,酶解消化;
2).终止酶解;
3).将酶解后的细胞团加入Matrigel重悬接种入24孔板,待Matrigel凝固后加入类器官培养基,定期观察并换液;
4).肝癌类器官培养14-21天后,去除培养液获得肝癌类器官;
5).将步骤4获得的类器官酶解消化获得单细胞;
6).将步骤5)获得的单细胞使用Matrigel重悬,接种入24孔板,待Matrigel凝固后加入类器官的培养基;
7).培养7-14天后,重复步骤4)-6),持续培养5代后,冻存肝癌类器官。
优选的,步骤1)中,先将肝癌组织样本,切成1cmx1cm大小,冰PBS反复清洗5次后再进行酶解;进一步优选的,肿瘤组织块切成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,终止酶解的具体操作如下: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;进一步优选的,低温低速离心后重悬细胞团,显微镜计数细胞团数量,根据需要接种的孔数转移相应的细胞量至另一15ml离心管;
优选的,步骤3)中,300g离心10min,去除上清,加入冰Matrigel重悬接种入24孔板,待Matrigel凝固后加入肝癌类器官培养基。进一步优选的,每3-4天观察更换培养基;
优选的,步骤4)中,去除培养液的操作为:每孔加1ml冰PBS,将胶转移至15ml离心管,反复吹打,300g离心10min,去除上清;
优选的,步骤5)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肝癌类器官模型的制备,通过制备得到的肝癌类器官模型,能够更直观的展示肝癌原发病理机制以及癌细胞的侵袭机制。并且,所述模型还能够用于筛选治疗肝癌的药物。
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,能够对异质性差异巨大的原发性肝细胞癌的类器官进行培养,并且能够利用所述培养基制备肝癌类器官模型,为后续了解肝癌发病机制以及筛选药物奠定了基础。
附图说明
图1为不同培养基培养原代肝细胞癌类器官比较图。
图2为三种改良培养基培养原代肝细胞癌获得的类器官。
图3为NM培养基培养原代肝细胞癌类器官。
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:
DMEM/F12培养基(L310KJ,源培),抗坏血酸(A103539,Aladdin),Holo-Transferrin(T0665,Sigma),L-谷胱甘肽(G6013,Sigma),Insulin(91077C,Sigma),Sodiumselenite(S5261,Sigma),乙醇胺盐酸盐(E120635,Aladdin)。R-spondin 1(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),胃泌素I(Sigma),EGF(Peprotech),HGF(Peprotech),FGF7(Peprotech),FGF10(Peprotech),DMSO(Sigma),Y27632(Sigma),CHIR99021(Tocris)。
实施例1、改良培养基1
DMEM/F12培养基,抗坏血酸(10mg/L),L-谷胱甘肽(1-50mg/L),Insulin(20mg/L),Sodium selenite(0.05mg/L),R-spondin 1(500ng/ml),N2(1X),B27(1X),GlutaMAXSupplement(1X),HEPES(1X),烟酰胺(20mM),NormocinTM(1X),N-乙酰半胱氨酸(2mM),A83-01(10μM),胃泌素I(10nM),EGF(20ng/ml),HGF(50ng/ml),FGF7(20ng/ml),FGF10(200ng/ml),Y27632(50μM)。
实施例2、改良培养基2。
DMEM/F12培养基,抗坏血酸(10mg/L),Holo-Transferrin(50mg/L),L-谷胱甘肽(20mg/L),乙醇胺盐酸盐(30mg/L),R-spondin 1:(200ng/ml),N2(1X),B27(2X),GlutaMAXSupplement(1X),HEPES(1X),烟酰胺(5mM),NormocinTM(1X),N-乙酰半胱氨酸(2mM),A83-01(5μM),胃泌素I(10nM),EGF(50ng/ml),FGF7(100ng/ml),FGF10(100ng/ml),DMSO(1%),Y27632(50μM)。
实施例3、改良培养基3
DMEM/F12培养基,Holo-Transferrin(10mg/L),L-谷胱甘肽(1-10mg/L),Insulin(10mg/L),R-spondin 1:(100ng/ml),N2(0.5X):B27(1X),GlutaMAX Supplement(1X),HEPES(1X),烟酰胺(10mM),NormocinTM(1X),N-乙酰半胱氨酸(1.25mM),A83-01(0.5μM),胃泌素I(1nM),EGF(100ng/ml),Y27632(50μM),CHIR99021(50μM)。
实施例4、不同培养基培养原代肝细胞癌类器官比较
为了验证改良后的培养基对类器官的培养效果,将上述实施例1-3中改良的培养基与现有技术中的NM(Nature Medicine)培养基进行比较,除培养基组成不同外,其他培养条件完全一致,观察肝细胞癌类器官的生长状况。
其中,NM(Nature Medicine)培养基组成如下:
Advance DMEM/F12培养基,R-spondin 1(200ng/ml),Noggin(25ng/ml),N2(1X),B27(1X),GlutaMAX Supplement(1X),HEPES(1X),烟酰胺(10mM),NormocinTM(1X),N-乙酰半胱氨酸(1mM),A83-01(5μM),胃泌素I(10nM),EGF(50ng/ml),HGF(25ng/ml),Forskolin(10μM),Dexamethasor(3nM),Y27632(10μM)。
取10例肝细胞癌样本(Patient1-10),分别用肝癌培养基(1,2,3)和NatureMedicine培养基(NM培养基)培养14-21天,具体操作如下:
1.肝细胞癌样本1cmx1cm大小,冰PBS反复清洗5次;
2.低温状态下将肿瘤组织块切成1mm3碎片,37℃胶原酶消化30-60min;
3.加入等体积冰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;
4.100g低温离心10min,去除上清,冰PBS重悬,次步骤重复3-5次;
5.1-5ml冰PBS重悬,显微镜计数细胞团数量,根据需要接种的孔数转移相应的细胞量至另一15ml离心管;
6.300g离心10min,去除上清,加入冰Matrigel重悬接种入24孔板,待Matrigel凝固后加入上述实施例1-3所述的改良的肝癌类器官培养基和NM培养基,每3-4天观察更换培养基;通过比较类器官生长情况比较培养效果优劣。
结果如图1-3所示,培养基1成功培养4例肝癌类器官(P1,P4,P5,P7),培养基2成功培养2例肝癌类器官(P7,P10),培养基3成功培养3例肝癌类器官(P4,P5,P10),NM培养基成功培养2例肝癌类器官(P1,P7)。单一改良后培养基的培养成功率分别为40%(4/10),20%(2/10)和30%(3/10),NM培养基成功率则为20%(2/10)。相比已报道培养基,改良后的单一培养基成功率有较大提高或持平。考虑到肿瘤的异质性,三种培养基同时培养的成功率达到50%(5/10),远高于NM培养基20%的成功率,说明对于异质性较大的肝细胞癌,一种培养基是不充分的。
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)
1.一种原发性肝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:DMEM/F12培养基,L-谷胱甘肽1-100mg/L,R-spondin-1 50-500ng/ml,N2:B27 0.1-2,GlutaMAXSupplement(1X),HEPES(1X),烟酰胺1-100mM,NormocinTM(1X),N-乙酰半胱氨酸0.1-10mM,A83-01 0.1-50μM,胃泌素I 0.1-50nM,EGF 1-200ng/ml和Y27632 1-200μM。
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:抗坏血酸、Holo-Transferrin、Insulin、Sodium selenite和乙醇胺盐酸盐。
3.根据权利要求2所述的培养基,其特征在于,所述抗坏血酸浓度为1-100mg/L;所述Holo-Transferrin浓度为5-200mg/L;所述Insulin浓度为1-200mg/L;所述Sodiumselenite acid浓度为0.002-0.5mg/L;所述乙醇胺盐酸盐浓度为2-200mg/L。
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HGF、FGF7和FGF10,优选的,三种物质的浓度,所述HGF浓度为5-500ng/ml;所述FGF7浓度为1-200ng/ml;所述FGF10浓度为50-500ng/ml。
5.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CHIR99021,优选的,所述CHIR99021浓度为1-100μM。
6.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二甲亚砜(DMSO),优选的,所述二甲亚砜浓度为培养基体积比的0.01-2%。
7.一种利用上述任一权利要求所述培养基培养原发性肝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).获得肝癌组织样本,切成碎片,酶解消化;
2).终止酶解;
3).将酶解后的细胞团加入Matrigel重悬接种入24孔板,待Matrigel凝固后加入类器官培养基,定期观察并换液;
4).肝癌类器官培养14-21天后,去除培养液获得肝癌类器官;
5).将步骤4获得的类器官酶解消化获得单细胞;
6).将步骤5)获得的单细胞使用Matrigel重悬,接种入24孔板,待Matrigel凝固后加入类器官的培养基;
7).培养7-14天后,重复步骤4)-6),持续培养5代后,冻存肝癌类器官。
8.根据权利要求7所述的方法,其特征在于,步骤1)消化使用的是胶原蛋白酶,步骤5)消化使用的是胰蛋白酶。
9.权利要求1-6任一所述培养基或权利要求7或8任一项所述的方法在制备筛选抗肝癌药物的类器官模型中的应用。
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