CN115261326A - 建立乳腺癌及癌旁类器官模型的培养基及培养方法 - Google Patents
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Abstract
本发明公开了一种建立乳腺癌类器官和癌旁类器官模型的培养基和培养方法,所述培养基为加入如下组分的DMEM/F12培养基:L‑谷氨酰胺、HEPES、R‑Spondin 1、Neuregulin 1、FGF‑7、FGF‑10、EGF、Noggin、A83‑01、Y‑27632、SB202190、B27补充剂、N‑乙酰半胱氨酸、烟酰胺、GlutaMax、青霉素‑链霉素‑庆大霉素。本发明提供的类器官培养方法不仅培养成功率高,形成的类器官细胞球球形规则,培养周期短,且能很好地保留患者源组织的异质性;另外,本发明培养基可同时适用于癌组织及癌旁组织类器官的培养,有助于进行药物筛选中的对照分析,提高药物筛选有效性。
Description
技术领域
本发明涉及类器官模型建立领域,具体涉及一种用建立乳腺癌及癌旁类器官模型的培养基及培养方法。
背景技术
乳腺癌(Breast Cancer,BC)是导致女性恶性肿瘤死亡的主要原因,研究显示,乳腺癌是一种高度异质性的疾病,表现为肿瘤内和肿瘤间的异质性。根据雌激素受体(ER)、孕激素受体(PR)以及人表皮生长因子受体2(HER2)的表达情况,世界卫生组织根据遗传学、病理学和临床上的差异将乳腺肿瘤分为了20多种不同亚型。尽管人们对乳腺癌复杂性的认识在不断加深,但目前的治疗方法主要是基于乳腺癌的临床病理特征,标准治疗方法并不能适用于每个患者,因此从合理的靶向治疗到实现个体化治疗成为了科研人员的研究目标,从而需要一种更加接近于临床肿瘤状态的体外模型来进行深入研究。
目前有多种运用于乳腺癌研究的细胞系或动物模型,但均不能准确反映患者的癌症特征,导致临床前试验有非常高的失败率。如最早使用2D培养出的永生化乳腺癌细胞系,该技术操作简单,可轻松解剖特定细胞类型的特征,但其存在形态和结构等方面与体内细胞有巨大差异,导致开发的新药很难进入临床使用的困难;后来,研究者们将患者来源的肿瘤细胞或组织注射于免疫缺陷的小鼠体内,建立了人源肿瘤异体移植模型(patient-derived xenografts,PDX),该模型保留了原始样本分子的异质性,也能使肿瘤生长的环境更加接近原发瘤,有助于自身特有生物标志物的检出和抗肿瘤药物的研发,从而部分实现肿瘤患者的个体化治疗。但是PDX模型也面临着各种各样的问题:(1)不同肿瘤类型的移植成功率差异很大,部分移植物即使是在免疫缺陷的小鼠内仍会出现抵抗,不能存活,导致乳腺癌移植成功率很低。尽管有研究者提出雌激素预暴露的方法,使得luminal型乳腺癌移植成功率有所提高,但平均也仅为10-25%左右;(2)由于成功率低,导致PDX模型需要耗费大量免疫缺陷小鼠。而免疫缺陷小鼠无论是造价还是饲养都非常昂贵,将会耗费大量人力财力;(3)由于PDX模型是基于免疫系统高度缺乏的小鼠建立的,并不能模拟人类免疫环境,因此,该模型并不能满足于肿瘤细胞或微环境中其他组份与免疫细胞之间的相互作用的研究。后期虽然也研究者提出免疫系统人源化小鼠模型,即将人来源免疫体系导入小鼠体内用于免疫疗法的研究,但是该模型发育出的T细胞功能较弱,且移植成功率更低,小鼠耗费量更大。
近年来,患者来源的肿瘤类器官(patients derived organoid,PDO)的3D培养在研究乳腺癌肿瘤方面提供了一个更加有效和可靠的模型。该模型主要是将取自患者的肿瘤组织在体外经消化、分散、过滤、离心后分离出肿瘤细胞,接着以基质胶作为3D培养的细胞外基质,加入维持类器官生长和分化所需因子通过模拟肿瘤微环境进行培植,最后获得类器官模型。该方法培育的肿瘤类器官与源肿瘤组织具有高度相似的生物学特性。但目前研究报道的乳腺癌类器官模型制备方法及培养体系尚不统一,模型成功率存在较大差异,且大都立足于肿瘤组织类器官模型的建立,很少有研究报道同时适用于癌组织及其癌旁正常组织类器官的制备方法及培养体系。
因此,本领域亟需寻找一种培养方法简单、培养成功率高且同时适用于癌组织及其癌旁正常组织类器官建立的培养体系及制备方法。
发明内容
本发明的目的是针对现有技术中的不足,提供一种更为简洁、廉价、高效且同时适用于癌组织及其癌旁正常组织类器官建立的培养体系及制备方法。
为解决上述问题,本发明首先提供了一种建立乳腺癌类器官和癌旁类器官模型的培养基,所述培养基为加入如下组分的DMEM/F12培养基:L-谷氨酰胺、HEPES、R-Spondin 1、Neuregulin 1、FGF-7、FGF-10、EGF、Noggin、A83-01、Y-27632、SB202190、B27补充剂、N-乙酰半胱氨酸、烟酰胺、GlutaMax、青霉素-链霉素-庆大霉素。
优选地,所述组分的浓度如下:
L-谷氨酰胺:2.50mM;HEPES:15mM;R-Spondin 1:2.5nM;Neuregulin 1:5nM;FGF-7:5ng/mL;FGF-10:20ng/mL;EGF:5ng/mL;Noggin:100ng/mL;A83-01:500nM;Y-27632:5mM;SB202190:500nM;B27补充剂:1×;N-乙酰半胱氨酸:1.25mM;烟酰胺:5mM;GlutaMax:1×;青霉素-链霉素-庆大霉素:1%。
本发明另一方面还提供了一种乳腺癌类器官和癌旁类器官模型的培养方法,乳腺癌类器官和癌旁类器官平行培养,均采取以下步骤获得:
S1,将获得的样本组织进行物理预处理,得到样本组织碎块;
S2,将所述样本组织碎块进行剪碎、酶解消化处理,得到单细胞悬液;
S3,采用基质胶重悬步骤S2制备得到的单细胞悬液,并进一步将重悬液接种于培养板中,培养箱中倒置培养20~30min,使得所述重悬液固化;
S4,向培养孔内加入前面任一项所述的培养基进行培养,获得原代类器官;
其中,步骤S1中,所述的样本组织为乳腺癌组织或癌旁正常组织。
优选地,步骤S1中,所述样本组织碎块的大小为1~3mm3。
优选地,步骤S2中,采用I型胶原酶进行酶解消化处理。
优选地,步骤S4中,培养过程中,每4天更换一次所述的培养基。
优选地,步骤S4之后还包括传代培养或冻存操作,具体如下:
步骤S5:当培养板中原代类器官长至80%~90%密度时,回收基质胶中的原代类器官;
步骤S6,采用预冷的前面任一项所述的培养基重悬步骤S5收获的原代类器官,并对原代类器官进行分离处理;
步骤S7,离心、去上清后,加入基质胶重新接种后进行传代培养;或加入组织冻存液进行保存。
优选地,步骤S6中,通过移液枪吹吸或加入TrypLE Express酶消化使原代类器官分离。
本发明另一方面还提供了一种采用前面任一项所述的培养方法制得的乳腺癌类器官和癌旁类器官。
本发明另一方面还提供了一种根据前述培养方法制得的乳腺癌类器官和癌旁类器官在筛选抗乳腺癌药物中的应用。
相对于现有技术,本发明的有益效果是:
(1)本发明提供的培养基可同时适用于乳腺癌组织及癌旁组织类器官的培养,可在同一条件下同时平行培养,不仅简化操作,提高制备效率,还有助于进行药物筛选中的对照分析,提高药物筛选的有效性。
(2)本发明提供的类器官制备方法不仅能实现乳腺癌及癌旁类器官的快速生长,且培养成功率高,形成的肿瘤类器官细胞球球形规则,大小较为均一,还能在体外很好地保留患者肿瘤组织的异质性。
(3)本发明提供的类器官培养基和制备方法,对于冻存标本也能成功培养。
附图说明
图1为乳腺癌及癌旁组织类器官制备流程图。
图2为实施例2中培养3天、7天、11天后光显微镜下乳腺癌及癌旁类器官的形态图。
图3为实施例2制得的乳腺癌类器官、癌旁类器官和源组织的组织学特点和相关分子表达情况(标尺:100μm);其中,
A为HE染色对比结果;
B为免疫组化对比结果。
图4为实施例2制得的乳腺癌类器官、癌旁类器官和源组织CK 18、PR和ER的免疫荧光结果图(标尺:100μm);其中,
A为类器官和源组织CK 18和PR免疫荧光结果图;
B为类器官和源组织CK 18和ER免疫荧光结果图。
图5为冻存组织制备的类器官培养14天左右后显微镜下形态图。
其中,T表示乳腺癌类器官,T-O表示乳腺癌源组织,N表示癌旁类器官,N-O表示癌旁源组织。
具体实施方式
以下结合附图和实施例对本发明的技术方案做进一步的说明。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也视为本发明的保护范围。
术语说明
本发明所述的“平行培养”为:使用同一种培养基、同一培养条件同时分别培养乳腺癌类器官和癌旁类器官,而不是将组织混合在一起培养。
本发明所述的“BT-PDO(patient derived organoids of breast tissue)培养基”为本发明所述的建立乳腺癌类器官和癌旁类器官模型的培养基。
乳腺癌发病率逐年上升,并呈年轻化趋势,对女性身心健康带来巨大威胁。作为一种高异质性疾病,个体化精准治疗是未来乳腺癌治疗的发展方向,但现有模型并不能满足这种需求。基于此种现状,本发明申请人经过大量研究,首先提供了一种建立乳腺癌类器官和癌旁类器官模型的培养基,所述培养基(又称BT-PDO培养基)为加入如下组分的DMEM/F12培养基:L-谷氨酰胺、HEPES、R-Spondin 1、Neuregulin 1、FGF-7、FGF-10、EGF、Noggin、A83-01、Y-27632、SB202190、B27补充剂、N-乙酰半胱氨酸、烟酰胺、GlutaMax、青霉素-链霉素-庆大霉素。
优选地,所述组分的浓度如下:
L-谷氨酰胺:2.50mM;HEPES:15mM;R-Spondin 1:2.5nM;Neuregulin 1:5nM;FGF-7:5ng/mL;FGF-10:20ng/mL;EGF:5ng/mL;Noggin:100ng/mL;A83-01:500nM;Y-27632:5mM;SB202190:500nM;B27补充剂:1×;N-乙酰半胱氨酸:1.25mM;烟酰胺:5mM;GlutaMax:1×;青霉素-链霉素-庆大霉素:1%。
需要说明的是:DMEM/F12培养基适于克隆密度的培养。F12培养基成分复杂,含有多种微量元素,和DMEM以1:1结合,称为DMEM/F12培养基。
L-谷氨酰胺是一种必需氨基酸,不仅可作为培养细胞的能量来源,而且参与蛋白质的合成和核酸代谢。
HEPES缓冲液的主要成分是羟乙基哌嗪乙硫磺酸(2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonicacid),HEPES是一种非离子两性缓冲液,其在pH7.2~7.4范畴内具备不错的缓冲能力,且对细胞没有任何毒性。
R-Spondin 1属于Wnt调节物的Rspo家族,在乳腺导管发生和形成中发挥重要作用。
Neuregulin 1简称神经调节蛋白-1(NRG-1),属于表皮生长因子家族,在乳腺癌中促进癌细胞的浸润和迁移。
FGF-7也叫做角质细胞生长因子(KGF),来自基质的FGF-7能作为一种上皮生长和形态发生的间充质/基质的媒介,可作用于乳腺上皮细胞的有丝分裂原及调节上皮的形态发生,在乳腺发育中起到重要的作用。
FGF-10也叫做角质细胞生长因子2(KGF 2),在靠近上皮末端的基质中表达可促进乳腺上皮细胞增殖及维持腺泡形态对乳腺结构的重建有重要作用。
EGF是一种强效的生长因子,可刺激多种表皮和上皮细胞的增殖分化及细胞外基质形成,并有促进乳腺上皮增生的作用。
Noggin是骨形态发生蛋白抑制剂的分泌蛋白,其以不同的亲和力与骨形态发生蛋白4、骨形态发生蛋白7等结合,在类器官的长期培养中可能通过对Lgr5表达和Wnt信号传导产生影响从而发挥作用,以此促进干细胞的扩增。
A83-01是转化生长因子β1型受体ALK5、ALK4以及ALK7的有效抑制剂;可以促进细胞增殖,防止细胞分化,维持干细胞干性,抑制细胞凋亡和衰老。
Y-27632:Rho Kinase(ROCK)Inhibitor,是一种巢凋亡抑制剂,可以通过和ATP竞争与催化位点的结合,以此来达到抑制ROCK1和ROCK2的效果;作为培养辅助试剂在多种类器官中得以应用,以达到防止细胞凋亡、促进类器官形成的效果。
SB 202190是一种高效的p38 MAPK抑制剂,靶向作用于p38α/β。可用于胃肠道、乳腺类器官的培养。
B27补充剂是一种较常见的培养基补充剂,可以替代血清,常用于培养神经祖细胞和神经干细胞,而且在三维立体的类器官培养中不会引起分化
N-乙酰半胱氨酸(N-Acetylcysteine)是谷胱甘肽的前体,是一种有效的抗氧化剂和自由基清除剂,同时可以激活PI3K/Akt信号通路,以此发挥对细胞增殖、分化以及凋亡等的调控作用。
烟酰胺(Nicotinamide)是维生素B家族一员,其参与细胞代谢、氧化反应、线粒体功能以及能量产生过程。烟酰胺对肝癌干细胞和造血干细胞的自我更新发挥重要作用。
GlutaMax可减少细胞代谢过程中的氨积累,改善细胞活力和生长情况。
P/S/G(青霉素-链霉素-庆大霉素)是常用抗生素混合液,可防止类器官培养过程中的污染。
本发明另一方面还提供了一种乳腺癌类器官和癌旁类器官模型的培养方法,如图1所示,乳腺癌类器官和癌旁类器官平行培养,均采取以下步骤获得:
S1,将获得的样本组织进行物理预处理,得到样本组织碎块;
S2,将所述样本组织碎块进行剪碎、酶解消化处理,得到单细胞悬液;
S3,采用基质胶重悬步骤S2制备得到的单细胞悬液,并进一步将重悬液接种于培养板中,培养箱中倒置培养20~30min,使得所述重悬液固化;
S4,向培养孔内加入BT-PDO培养基进行培养,获得原代类器官;
其中,步骤S1中,所述的样本组织为乳腺癌组织或癌旁正常组织。
一些实施例中,步骤S1中,所述样本组织碎块的大小为1~3mm3。
一些实施例中,步骤S2中,采用I型胶原酶进行酶解消化处理。
一些实施例中,步骤S4中,培养过程中,每4天更换一次所述的培养基。
一些实施例中,步骤S4之后还包括传代培养或冻存操作,具体如下:
步骤S5:当培养板中原代类器官长至80%~90%密度时,回收基质胶中的原代类器官;
步骤S6,采用预冷的BT-PDO培养基重悬步骤S5收获的原代类器官,并对原代类器官进行分离处理;
步骤S7,离心、去上清后,加入基质胶重新接种后进行传代培养;或加入组织冻存液进行保存。
一些实施例中,步骤S6中,通过移液枪吹吸或加入TrypLE Express酶消化使原代类器官分离。
本发明另一方面还提供了一种采用前面任一项所述的培养方法制得的乳腺癌类器官和癌旁类器官。
本发明另一方面还提供了一种根据前述培养方法制得的乳腺癌类器官和癌旁类器官在筛选抗乳腺癌药物中的应用。
下面通过实施例和附图对本发明作详细说明。
本发明实施例中无特别说明外,所用试剂及耗材均为市售商品。本发明实施例中,R-Spondin 1、FGF-10、EGF、Noggin、购自R&D;Neuregulin 1、FGF-7购自Peprotech;A83-01购自Tocris;Y-27632购自Abmole;SB202190、B27补充剂、N-乙酰半胱氨酸、烟酰胺购自Sigma;GlutaMax购自Thermo;青霉素-链霉素-庆大霉素购自Beyotime。
实施例1建立乳腺癌类器官和癌旁类器官模型的培养基
按下述浓度将各组分加入到DMEM/F12(1:1)培养基中,得到本发明所述的建立乳腺癌类器官和癌旁类器官模型的培养基,即BT-PDO培养基:
L-谷氨酰胺:2.50mM;HEPES:15mM;R-Spondin 1:2.5nM;Neuregulin 1:5nM;FGF-7:5ng/mL;FGF-10:20ng/mL;EGF:5ng/mL;Noggin:100ng/mL;A83-01:500nM;Y-27632:5mM;SB202190:500nM;B27补充剂:1×;N-乙酰半胱氨酸:1.25mM;烟酰胺:5mM;GlutaMax:1×;青霉素-链霉素-庆大霉素:1%。
实施例2乳腺癌类器官和癌旁类器官模型的培养
选取22对来自复旦大学附属中山医院青浦分院病理科的乳腺癌组织及癌旁正常组织样本。
样本入选标准:
①2019.12.01-2021.12.31于复旦大学附属中山医院青浦分院外科就诊的年龄在20到70岁之间女性原发乳腺癌患者;②于本院行手术切除肿瘤,签署知情同意书,有完整的病史及病理资料。
样本排除标准:
①发生远处转移;②同时患有其他肿瘤;③同时患有其他显著影响患者生存结局的疾病(包括严重的心脑血管疾病,呼吸系统疾病,肝衰,肾衰等)。
基于上述22对样本,使用实施例1制得的BT-PDO培养基制备乳腺癌类器官和癌旁类器官,乳腺癌类器官和癌旁类器官平行培养,均采取以下步骤获得:
S1,用含1%P-S-G(青霉素-链霉素-庆大霉素)的DMEM/F12无血清培养基将新鲜的前述符合入选和排除标准的乳腺癌组织、乳腺癌旁正常组织从病理科取回,在无菌的条件下用含1%P-S-G的DMEM/F12无血清培养基进行多次清洗。在生物安全柜中用灭菌的剪刀镊子将组织剪成多个1-3mm3小块。两种组织各随机选取2块组织置于-80℃冰箱中保存用于后期基因组学的检测,另外各随机选取2块置于组织冻存液中于-80℃冰箱中保存用于研究冻存组织能否用于类器官的培养,各选择1块组织置于福尔马林溶液中固定用于病理学分析和免疫组化,剩余组织用于制备单细胞悬液;
S2,用灭菌后的剪刀镊子,在无菌的条件下将组织尽可能的剪碎,收集于15mL离心管中,4℃条件下、1000rpm离心10min,弃去上清加入PBS溶液清洗2~3遍,最后一次离心后,加入含有1mg/mL I型胶原酶和1%P-S-G的DMEM/F12培养基重悬细胞沉淀,并移入10cm培养皿中,培养箱中消化过夜,制得单细胞悬液;
S3,次日,将消化好的组织移入15mL离心管中,4℃条件下、1000rpm离心10min,弃去上清后加入PBS溶液清洗2~3遍,离心弃上清后加入10mg/mL预冷的基质胶(BME)重悬上述沉淀,取30μl重悬液置于24孔低粘附培养板(提前放入培养箱中预热)中,接着将培养板倒置于培养箱中固化20-30min左右;
S4,固化完成后,向培养孔内加入500μl BT-PDO培养基,置于37℃培养箱中培养,每4天换一次BT-PDO培养基,观察细胞的形态,光学显微镜下拍照记录,获得原代乳腺癌类器官和癌旁类器官。
图2为培养3天、7天、11天后光显微镜下乳腺癌及癌旁类器官的形态图,由图可知,本实施例培养得到的乳腺癌类器官和癌旁类器官均形成致密、松散的规则球形细胞团,且培养过程中,随着培养时间的增加,类器官逐渐增大,生长状态良好。
本实施例共培养22对组织类器官,最终成功培养20对类器官(其中两对在培养过程出现了污染使得培养过程被迫终止),培养成功率至少达90%以上。
实施例3乳腺癌类器官和癌旁类器官的传代培养和冻存
实施例2中,当原代类器官培养至14天左右时,可进行传代培养和冻存等,需要说明的是,为促进类器官3D结构的生长,所以将其培养在BME中,但在传代、冻存和分析之前必须去除,具体操作如下:
S5:培养两周左右,当培养板中原代类器官长至80%~90%密度时,用PBSB(含0.1%BSA的PBS)润湿的一次性吸管将培养物(包括原代类器官和培养基)全部转到15mL无菌的离心管中;沿离心管壁,缓慢加入预冷的PBSB至13mL,将离心管于冰上静置10~15min,让原代类器官在重力的作用下自由沉降;接着4℃条件下、1000rpm离心10min,用一次性吸管缓慢去除上清后,加入13mL预冷的PBSB清洗基质胶-原代类器官混合物2~3次,清洗结束后加入2~4mL细胞回收液(康宁:354253),上下颠倒数次,将离心管于冰上静置30min左右,将原代类器官从BME中释放出来;
S6,采用2mL预冷的BT-PDO培养基重悬步骤S5收获的原代类器官,用PBSB润湿的无菌一次性吸管和1mL枪头反复吹打数次,以分离类器官;一些实施中,对于致密的类器官,还可用TrypLE Express酶消化1~5min,以分离类器官;
S7,继续加入10mL BT-PDO培养基,4℃、1000rpm离心10min,弃上清,加入新鲜的BME重新接种后继续传代培养;或者加入组织冻存液于-80度短期保存,长期保存可将其转移至液氮罐中。
实施例4乳腺癌类器官和癌旁类器官的相关分析
(1)HE染色和免疫组化对比
实验过程:取实施例3中步骤S5收集得到的从BME中释放出的原代类器官,加入10mL新鲜的4%多聚甲醛,置于冰上孵育30min;孵育结束后弃上清,加入10mL PBS冰上沉降10min让类器官自由沉降,弃上清(小心不要吸到下面的类器官);重复PBS清洗2-3次;弃去PBS,加入70%乙醇重悬;固定好的类器官可和源组织按照常规方法进行脱水透明,浸蜡包埋制成比较硬的蜡块;将包埋好的蜡块用切片机制成石蜡切片,即可进行HE及免疫组化染色;HE染色方法与常规石蜡切片处理方法相同,在此不做赘述;
免疫组化主要步骤为:脱蜡水化,抗原修复,阻断内源性过氧化物酶,血清封闭,一抗孵育,二抗孵育,显色,苏木素复染,最后脱水封片,晾干后即可在显微镜下观察并进行图片采集及分析。
实验结果:图3为实施例2制得的乳腺癌类器官、癌旁类器官和源组织的组织学特点和相关分子表达情况。HE染色结果显示,本发明方法制得的乳腺癌类器官、癌旁类器官与源组织具有高度的形态和病理特征一致性(图3的A);免疫组化结果显示,本发明方法制得的乳腺癌类器官、癌旁类器官的相关分子(CK 18、ER、PR、HER 2、Ki 67和α-SMA)表达与源组织高度一致(图3的B)。
(2)免疫荧光检测
实验过程:首先将制备好的石蜡切片进行脱蜡水化,抗原修复,血清封闭,一抗孵育过夜,荧光二抗避光孵育,DAPI复染细胞核,最后加自发荧光淬灭剂等切片稍甩干后用抗荧光淬灭封片剂封片,切片应于荧光显微镜下观察并采集图像,与常规染色方法相同,在此不做赘述。
实验结果:图4为实施例2制得的乳腺癌类器官、癌旁类器官和源组织CK 18、PR和ER的免疫荧光结果图。结果也进一步证明了,无论是来源于乳腺癌组织的类器官还是来源于癌旁正常组织的类器官,两者在空间结构和ER、PR以及CK18的表达水平与源组织均具有一致性(图4的A和B)。
以上结果表明,本申请建立的乳腺癌及癌旁类器官与源组织有较好的一致性。
另外,本发明还进行了以冻存组织为样本、使用本发明培养基和方法进行类器官培养的研究:将两对冻存8个月左右的标本复苏后,按照实施和2所述的培养基和培养方法制备类器官,结果如图5所示:使用本发明提供的培养基和培养方法,依旧可以成功培养冻存组织的类器官。
综上所述,本发明提供了一种建立乳腺癌类器官和癌旁类器官模型的培养基和培养方法,本发明提供的类器官制备方法不仅能实现乳腺癌及癌旁类器官的快速生长,且培养成功率高,形成的肿瘤类器官细胞球球形规则,还能在体外很好地保留患者肿瘤组织的异质性;另外,本发明提供的培养基可同时适用于乳腺癌组织及癌旁组织类器官的培养,可在同一条件下同时平行培养,不仅简化操作,提高制备效率,还有助于进行药物筛选中的对照分析,提高药物筛选的有效性。
尽管本发明的内容已经通过上述优选实施例作了详细介绍,但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后,对于本发明的多种修改和替代都将是显而易见的。因此,本发明的保护范围应由所附的权利要求来限定。
Claims (10)
1.一种建立乳腺癌类器官和癌旁类器官模型的培养基,其特征在于,所述培养基为加入如下组分的DMEM/F12培养基:L-谷氨酰胺、HEPES、R-Spondin 1、Neuregulin 1、FGF-7、FGF-10、EGF、Noggin、A83-01、Y-27632、SB202190、B27补充剂、N-乙酰半胱氨酸、烟酰胺、GlutaMax、青霉素-链霉素-庆大霉素。
2.如权利要求1所述的建立乳腺癌及癌旁类器官模型的培养基,其特征在于,所述组分的浓度如下:
L-谷氨酰胺:2.50mM;HEPES:15mM;R-Spondin 1:2.5nM;Neuregulin 1:5nM;FGF-7:5ng/mL;FGF-10:20ng/mL;EGF:5ng/mL;Noggin:100ng/mL;A83-01:500nM;Y-27632:5mM;SB202190:500nM;B27补充剂:1×;N-乙酰半胱氨酸:1.25mM;烟酰胺:5mM;GlutaMax:1×;青霉素-链霉素-庆大霉素:1%。
3.一种乳腺癌类器官和癌旁类器官模型的培养方法,其特征在于,乳腺癌类器官和癌旁类器官平行培养,均采取以下步骤获得:
S1,将获得的样本组织进行物理预处理,得到样本组织碎块;
S2,将所述样本组织碎块进行剪碎、酶解消化处理,得到单细胞悬液;
S3,采用基质胶重悬步骤S2制备得到的单细胞悬液,并进一步将重悬液接种于培养板中,培养箱中倒置培养20~30min,使得所述重悬液固化;
S4,向培养孔内加入如权利要求1或2所述的培养基进行培养,获得原代类器官;
其中,步骤S1中,所述的样本组织为乳腺癌组织或癌旁正常组织。
4.如权利要求3所述的乳腺癌类器官和癌旁类器官模型的培养方法,其特征在于,步骤S1中,所述样本组织碎块的大小为1~3mm3。
5.如权利要求3所述的乳腺癌类器官和癌旁类器官模型的培养方法,其特征在于,步骤S2中,采用I型胶原酶进行酶解消化处理。
6.如权利要求3所述的乳腺癌类器官和癌旁类器官模型的培养方法,其特征在于,步骤S4中,培养过程中,每4天更换一次所述的培养基。
7.如权利要求3所述的乳腺癌类器官和癌旁类器官模型的培养方法,其特征在于,步骤S4之后还包括传代培养或冻存操作,具体如下:
步骤S5:当培养板中原代类器官长至80%~90%密度时,回收基质胶中的原代类器官;
步骤S6,采用预冷的权利要求1或2所述的培养基重悬步骤S5收获的原代类器官,并对原代类器官进行分离处理;
步骤S7,离心、去上清后,加入基质胶重新接种后进行传代培养;或加入组织冻存液进行保存。
8.如权利要求7所述的乳腺癌类器官和癌旁类器官模型的培养方法,其特征在于,步骤S6中,通过移液枪吹吸或加入TrypLE Express酶消化使原代类器官分离。
9.一种采用权利要求3-8中任一项所述的培养方法制得的乳腺癌类器官和癌旁类器官。
10.权利要求9所述的乳腺癌类器官和癌旁类器官在筛选抗乳腺癌药物中的应用。
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