CN116555166B - 用于制备鼠胆管类器官的组合物及鼠胆管类器官的制备方法和用途 - Google Patents
用于制备鼠胆管类器官的组合物及鼠胆管类器官的制备方法和用途 Download PDFInfo
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- CN116555166B CN116555166B CN202310833629.3A CN202310833629A CN116555166B CN 116555166 B CN116555166 B CN 116555166B CN 202310833629 A CN202310833629 A CN 202310833629A CN 116555166 B CN116555166 B CN 116555166B
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Abstract
本发明涉及医药领域,特别是涉及用于制备鼠胆管类器官的组合物及鼠胆管类器官的制备方法和用途,所述组合物包括组织消化试剂,所述组织消化试剂中包括胶原酶IV和中性蛋白酶。所述组合物还包括培养基,以培养基的总体积为基准,所述培养基包括以下成分:基础培养基、L‑谷氨酰胺或其替代品、HEPES、B27、乙酰半胱氨酸、RSPO、烟酰胺、胃泌素I肽、上皮生长因子、成纤维细胞生长因子10、肝细胞生长因子。本发明的组合物和制备方法可以提高胆管细胞消化后的细胞活力,而且不需要离心和红细胞裂解液裂解红细胞,因此方法较简单,可以实现胆管类器官的高密度培养,为成体肝脏中的胆道修复提供参考。
Description
技术领域
本发明涉及医药领域,特别是涉及用于制备鼠胆管类器官的组合物及鼠胆管类器官的制备方法和用途。
背景技术
人体胆道系统的结构像树一样:一部分位于肝脏内部,即肝内胆管,常简称为肝胆管,类似于树的枝叉,有各级分支,被称为一级胆管、二级胆管、三级胆管等等;另一部分在肝脏外部,即肝外胆管,类似于树干,包括肝总管、胆囊和胆囊管、胆总管。小鼠和离体人源肝脏模型显示,肝胆管具有再生潜能。由于肝脏细胞在解离过程中易破裂,因此直接制备肝脏类器官培养成功率较低。此外,肝胆管细胞可转化生成肝实质细胞;因此胆管类器官有望作为肝实质细胞来源候选库,应用于肝实质细胞疗法,缓解临床肝脏移植供体短缺的压力。但目前鼠胆管类器官培养的报道较少,更无实现高密度培养的方法。
发明内容
鉴于以上所述现有技术的缺点,本发明的目的在于提供用于制备鼠胆管类器官的组合物及鼠胆管类器官的制备方法和用途,用于解决现有技术中的问题。
为实现上述目的及其他相关目的,本发明提供一种用于制备鼠胆管类器官的组合物,所述组合物包括组织消化试剂,所述组织消化试剂中包括胶原酶IV和中性蛋白酶。
优选的,所述组合物还包括培养基,以培养基的总体积为基准,所述培养基包括以下成分:
基础培养基
L-谷氨酰胺或其替代品 1.5~2.5 mM
HEPES 8~12 mM
B27 1.5~2.5 %
乙酰半胱氨酸 0.5~1.5 mM
RSPO 100 ~300 ng/mL
烟酰胺 8~12 mM
胃泌素 I 肽 8~12 nM
上皮生长因子 30~150 ng/mL
成纤维细胞生长因子10 50~200 ng/mL
肝细胞生长因子 30 ~150 ng/mL。
本发明还提供所述的组合物在制备鼠胆管细胞分离产品或制备鼠胆管类器官中的用途。
本发明还提供一种鼠胆管类器官的制备方法,所述方法包括如下步骤:
1)将鼠肝脏组织块与清洗液混合,静置待组织块沉降后,弃上清;
2)将步骤1)获得的产物与所述组合物中的组织消化试剂混合,消化20~30min,终止消化后静置以使胆管组织沉淀,去除消化液,重悬细胞;
3)将细胞与基质胶混合并接种至培养容器中,待基质胶凝固后,加入所述组合物中的培养基进行培养,培养3~5天即获得鼠胆管类器官。
本发明还提供所述的制备方法获得的鼠胆管类器官。
本发明还提供所述的鼠胆管类器官在制备肝实质细胞产品或胆道修复产品或胆道再生产品或肝脏移植产品中的用途。
如上所述,本发明的用于制备鼠胆管类器官的组合物及鼠胆管类器官的制备方法和用途,具有以下有益效果:对健康胆管细胞的损伤小,可以提高胆管细胞消化后的细胞活力,而且不需要红细胞裂解液裂解红细胞,能够获得活力高的单细胞和细胞团,因此方法较简单,可以实现胆管类器官的高密度培养,为开辟类器官应用于临床上进行器官修复、促成胆管系统再生提供了试验的原型。器官修复过程中,自体组织是很紧缺的,因此在供体组织较少的情况下,本发明能够进行高密度培养,就可以实现取材少而得率高的效果
附图说明
图1显示为本发明的提取的小鼠胆管细胞或细胞团。
图2显示为本发明培养5天的小鼠胆管类器官图。
图3显示为培养3天的胆管类器官图。
图4显示为上皮细胞标志物图。
图5显示为胆管标志物图。
图6显示为胆管类器官标志物融合图。
具体实施方式
本发明提供一种用于制备鼠胆管类器官的组合物,所述组合物包括组织消化试剂,所述组织消化试剂中包括胶原酶IV和中性蛋白酶。
在本发明的某些实施方式中,所述组织消化试剂为液体或固体。在本发明的某些实施方式中,所述组织消化试剂为液体。
以液体组织消化试剂的总体积为基准,所述胶原酶IV的终浓度为0.05~0.2U/mL。所述胶原酶IV的终浓度例如选自以下任一范围:0.05~0.1U/mL、0.1~0.15U/mL、0.15~0.2U/mL。优选的,所述胶原酶IV的终浓度为0.05~0.15U/mL。更优选的,胶原酶IV的终浓度为0.1U/mL。
以液体组织消化试剂的总体积为基准,所述中性蛋白酶的终浓度为0.5~1.5U/mL。所述中性蛋白酶的终浓度例如选自以下任一范围:0.5~0.7U/mL、0.7~0.9U/mL、0.9~1.1U/mL、1.1~1.3U/mL、1.3~1.5U/mL。优选的,所述中性蛋白酶的终浓度为0.6~1.0U/mL。更优选的,中性蛋白酶的终浓度为0.8U/mL。
中性蛋白酶是最早用于工业生产的蛋白酶。大多数微生物产生的中性蛋白酶是金属酶,一分子酶蛋白中含一原子锌,分子质量为35,000-40,000,等电点pI为8~9,是微生物蛋白酶中最不稳定的酶,很易自溶,即使在低温冰冻干燥,也会造成分子质量的明显减少。所述中性蛋白酶选自耐热解蛋白芽孢杆菌所产生的热解素或枯草杆菌产生的中性蛋白酶。优选的,所述中性蛋白酶选自Dispase II。
所述组织消化试剂用于消化动物组织。所述组织消化试剂用来分散组织,以达到离散细胞,获得细胞悬液的目的。所述组织消化试剂用于消化肝组织、神经元、睾丸组织。在一优选的实施方式中,适用于消化鼠源肝组织。所述鼠源肝组织来源于大鼠或小鼠。
所述组合物还包括培养基,以培养基的总体积为基准,所述培养基包括以下成分:
基础培养基
L-谷氨酰胺或其替代品 1.5~2.5 mM
HEPES 8~12 mM
B27 1.5~2.5 %
乙酰半胱氨酸 0.5~1.5 mM
RSPO 100 ~300 ng/mL
烟酰胺 8~12 mM
胃泌素 I 肽 8~12 nM
上皮生长因子 30~150 ng/mL
成纤维细胞生长因子10 50~200 ng/mL
肝细胞生长因子 30 ~150 ng/mL。
所述基础培养基选自DMEM/F12或Advanced DMEM/F12。
在本发明的某些实施方式中,所述L-谷氨酰胺的替代品为GlutaMAX。
所述L-谷氨酰胺或其替代品的终浓度选自以下范围中的任一种:1.5~1.7mM、1.7~1.9mM、1.9~2.1mM、2.1~2.3mM、2.3~2.5mM。在一种优选的实施方式中,所述L-谷氨酰胺或其替代品的终浓度为1.8~2.2mM。
所述HEPES为N-(2-羟乙基)哌嗪-N-(2-乙烷磺酸)。
在本发明的某些实施方式中,所述HEPES的终浓度选自以下范围中的任一种:8~9mM、9~10mM、10~11mM、11~12mM。在一种优选的实施方式中,所述HEPES的终浓度为9~11mM。
在本发明的某些实施方式中,所述B27的终体积浓度选自以下范围中的任一种:1.5~1.7%、1.7~1.9%、1.9~2.1%、2.1~2.3%、2.3~2.5%。在一种优选的实施方式中,所述B27的终体积浓度为1.8~2.2%。
在本发明的某些实施方式中,所述乙酰半胱氨酸(N-acetyl-L-cysteine)的终浓度选自以下范围中的任一种:0.5~0.7mM、0.7~0.9mM、0.9~1.1mM、1.1~1.3mM、1.3~1.5mM。在一种优选的实施方式中,所述乙酰半胱氨酸的终浓度为0.8~1.2mM。
在本发明的某些实施方式中,所述RSPO选自RSPO1或RSPO3。所述RSPO为重组人Rspondin 1(rhRSPO1)或重组人R spondin 3或鼠源的RSPO1或RSPO3。在本发明的某些实施方式中,所述RSPO的终浓度选自以下范围中的任一种:100~150ng/mL、150~200ng/mL、200~250ng/mL、250~300ng/mL。在一种优选的实施方式中,所述RSPO的终浓度为180~220ng/mL。
在本发明的某些实施方式中,所述烟酰胺的终浓度选自以下范围中的任一种:8~9mM、9~10mM、10~11mM、11~12mM。在一种优选的实施方式中,所述烟酰胺的终浓度为9~11mM。
所述胃泌素 I 肽为人胃泌素 I 肽。在本发明的某些实施方式中,所述胃泌素 I肽的终浓度选自以下范围中的任一种:8~9mM、9~10mM、10~11mM、11~12mM。在一种优选的实施方式中,所述胃泌素 I 肽的终浓度为9~11mM。
在本发明的某些实施方式中,所述上皮生长因子为重组人上皮生长因子(rhEGF)或鼠EGF。在本发明的某些实施方式中,所述上皮生长因子的终浓度选自以下范围中的任一种:30~40ng/mL、40~50ng/mL、50~60ng/mL、60~80ng/mL、80~100ng/mL、100~120ng/mL、120~150ng/mL。在一种优选的实施方式中,所述上皮生长因子的终浓度为30~70ng/mL。
在本发明的某些实施方式中,所述成纤维细胞生长因子10 为重组人成纤维细胞生长因子10 (rhFGF-10)或鼠FGF-10。在本发明的某些实施方式中,所述成纤维细胞生长因子10 的终浓度选自以下范围中的任一种:50~70ng/mL、70~90ng/mL、90~110ng/mL、110~130ng/mL、130~150ng/mL、150~170ng/mL、170~200ng/mL。在一种优选的实施方式中,所述成纤维细胞生长因子10的终浓度为80~120ng/mL。
在本发明的某些实施方式中,所述肝细胞生长因子为重组人肝细胞生长因子(rhHGF)或鼠HGF。在本发明的某些实施方式中,所述肝细胞生长因子的终浓度选自以下范围中的任一种:30~40ng/mL、40~50ng/mL、50~60ng/mL、60~80ng/mL、80~100ng/mL、100~120ng/mL、120~150ng/mL。在一种优选的实施方式中,所述肝细胞生长因子的终浓度为30~70ng/mL。所述肝细胞生长因子的添加更有助于肝内胆管细胞的发育。
在本发明的某些实施方式中,所述培养基中还包括抗生素。所述抗生素选自青霉素和/或链霉素。所述抗生素的浓度不做具体限定,可以为本领域常规的使用浓度,例如以培养基的总体积为基准,青霉素的工作浓度为100U/mL ,链霉素的工作浓度为0.1mg/mL,或者按照商品化试剂的说明书使用。在一种实施方式中,所述抗生素选自Primocin。
本发明还提供所述组合物在制备鼠胆管细胞分离产品或制备鼠胆管类器官中的用途。
本发明还提供一种鼠胆管类器官的制备方法,所述方法包括如下步骤:
1)将鼠肝脏组织块与清洗液混合,静置待组织块沉降后,弃上清;
2)将步骤1)获得的产物与所述组合物中的组织消化试剂混合,消化20~30min,终止消化后静置以使胆管组织沉淀,去除消化液,重悬细胞;
3)将细胞与基质胶混合并接种至培养容器中,待基质胶凝固后,加入所述组合物中的培养基进行培养,培养3~5天即获得鼠胆管类器官。
在本发明的某些实施方式中,步骤1)中鼠肝脏组织块为小鼠肝脏组织块或大鼠肝脏组织块。
步骤1)中鼠肝脏组织块通过将新鲜的鼠肝脏组织清洗后剪碎获得。
在本发明的某些实施方式中,所述步骤1)中清洗液为基础培养基。
步骤1)重复2次以上,直至上清澄清。
步骤2)中,所述组织消化试剂经过预热后使用。
步骤2)中,消化温度为35~38℃。
步骤2)中,在摇床上消化。摇床的转速为120-160rpm。
步骤2)中,用胎牛血清终止消化。
步骤2)中,静置以使胆管组织沉淀,去除消化液,该步骤重复2次完全去除消化液。
步骤2)中,用基础培养基或完全培养基重悬细胞。
步骤3)中,所述培养容器为培养板,例如六孔板、24孔板等。
步骤3)中,培养基的量以基质胶完全浸没在培养基中为准。
步骤3)中,培养条件为35~37℃、5%CO2。
步骤3)中,培养过程中每2~3天更换一次培养基。
培养过程中,第2天能看到明显的空泡结构。
本发明的制备方法使用组织消化组合物消化组织块,可以提高胆管细胞消化后的细胞活力,而且不需要离心和红细胞裂解液裂解红细胞,因此方法较简单。另外通过重力沉降方法从小鼠肝脏组织分离肝胆管组织并进行体外3D胆管类器官培养,这种鼠成体干细胞来源的胆管类器官可以实现胆管类器官的高密度培养,胆管细胞可以充当干细胞并产生肝细胞参与肝修复再生。
本发明还提供所述制备方法获得的鼠胆管类器官。
本发明还提供所述鼠胆管类器官在制备肝实质细胞产品或胆道修复产品或胆道再生产品或肝脏移植产品中的用途。
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前,应理解,本发明的保护范围不局限于下述特定的具体实施方案;还应当理解,本发明实施例中使用的术语是为了描述特定的具体实施方案,而不是为了限制本发明的保护范围;在本发明说明书和权利要求书中,除非文中另外明确指出,单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时,应理解,除非本发明另有说明,每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义,本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外,根据本技术领域的技术人员对现有技术的掌握及本发明的记载,还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
实施例1 消化液及培养基的配制
配制组织消化液:
胶原酶/中性蛋白酶工作液浓度为:Collagenase IV 0.1U/mL,中性蛋白酶:0.8U/mL
按照以下配方配制类器官完全培养基(以下浓度均为终浓度):
Advanced DMEM/F12基础培养基
青/链霉素 1%
GlutaMAX 2mM
HEPES 10mM
B27 2%
N-acetyl-L-cysteine 1 mM
rhRSPO1 200 ng/mL
烟酰胺 10 mM
Gastrin I 10 nM
rhEGF 50 ng/mL
rhFGF-10 100 ng/mL
rhHGF 50 ng/mL
缩写:
GlutaMAX:GlutaMAX 添加剂
HEPES:N-2羟乙基哌嗪-N-2-乙烷磺酸;
Pen/Strep:青/链霉素
B27:B-27 添加剂
N-acetyl-L-cysteine:乙酰半胱氨酸
rhRSPO1:重组人R-spondin-1
Gastrin I:人胃泌素 I 肽
rhEGF:重组人上皮生长因子
rhFGF-10:重组人成纤维细胞生长因子10
rhHGF:重组人肝细胞生长因子。
实施例2类器官的培养
1.取材
(1)取新鲜的小鼠肝脏组织,用含有青/链霉素的PBS中清洗3-5遍。
(2)使用眼科剪将组织剪碎,加入5mL Advanced DMEM/F12基础培养基上下吹打以清洗掉杂质,然后静置待大部分组织沉降下去,将上清弃掉。重复3次至上清澄清。
2. 消化
(1)加入5mL预热过的组织消化液,放置于37℃摇床消化20-30min。
(2)吹打组织碎片,观察组织消化状态,加入1~5%胎牛血清终止消化,静置使胆管组织沉淀。
(3)弃上清,用1mL AdvancedDMEM/F12基础培养基重悬沉淀,静置使胆管组织沉淀。
(4)重复步骤(3)2次,以彻底去除消化液。提取的小鼠胆管细胞或细胞团如图1所示。
(5)基质胶与细胞混合,通常情况下加入200μl基质胶(品牌:Corning,货号354230或354234)与细胞混匀,24孔板可播种4~5孔进行培养。
(6)待基质胶完全凝固后,加入37℃预热的小鼠肝胆管类器官完全培养基,每孔加入500μl培养基,确保基质胶完全浸在培养基中。
(7)将24孔板置于37℃,5%CO2的恒温培养箱中培养并持续观察。
(8)每3天更换一次培养基,更换培养基时应避免破坏基质胶;
(9)密切监测类器官生长状态,小鼠肝胆管类器官在第2天看到明显的空泡结构,在3-5天内建成模型,如图2示例,显微镜下能够看到明显的空泡结构,而且胆管类器官的密度较高,每个空心球是一个类器官,计数后得知为34.268个类器官/mm2。而现有技术中最高能达到20.64个类器官/mm2。
实施例3 类器官标志物的检测
委托第三方公司将类器官进行石蜡包埋并染色以检测类器官的标志物,检测流程大致如下:
包埋步骤:
1、类器官回收:在冰上溶解基质胶,吸弃上清取沉淀类器官。用D-PBS清洗2次,去除残余基质胶。
2、向沉淀中加入1mL的4%多聚甲醛固定30min(4℃),用D-PBS清洗2次,去除残余多聚甲醛。
3、将固定后的类器官包埋在水浴(70℃)融化的3 %的琼脂糖中,体系为50μl,冰上(0-4℃)静置30min凝固。
4、将脱水盒放进吊篮里于脱水机内依次梯度酒精进行脱水。75%酒精4h-85%酒精2h-90%酒精2h-95%酒精1h-无水乙醇I 30min-无水乙醇II 30min-醇苯5-10min-二甲苯I5-10min-二甲苯II 5-10min-蜡I 1h-蜡II 1h-蜡III 1h。
5、将浸好蜡的组织于包埋机内进行包埋。先将融化的蜡放入包埋框,待蜡凝固之前将组织从脱水盒内取出按照包埋面的要求放入包埋框并贴上对应的标签。于-20℃冻台冷却,蜡凝固后将蜡块从包埋框中取出并修整蜡块。
切片:
1、将修整好的蜡块置于石蜡切片机上切片,片厚5-8μm。切片漂浮于摊片机40℃温水上将组织展平,用载玻片将组织捞起,并放进45℃ 烘箱内烤片。待水烤干蜡烤化后取出常温保存备用。
2、迅速从烤箱中转移至二甲苯中,二甲苯I 10min;二甲苯II 10min;二甲苯III10min。
3、水化:高浓度到低浓度酒精,100% 5min;95% 5min;85% 5min ;75% 5min。
4、水化后纯水摇洗3次,每次洗3min。
抗原修复
加入pH=6.0的0.01M柠檬酸钠缓冲液浸泡切片,在微波炉中高火4min至沸腾后,取出自然冷却至室温,重复2次,每次补足液体避免干片。D-PBS清洗3次,每次洗3min。
血清封闭
10%二抗同源血清,37℃封闭30min,用滤纸吸去多余血清。
孵育抗体
加入20μl一抗,4℃过夜孵育。之后用PBST清洗3次,每次洗3min。
然后加入20μl二抗,37℃孵育1-2小时。之后用PBST清洗3次,每次洗3min。
滴加DAPI水溶液,常温孵育10min。
封片
用棉签棒蘸取少量抗荧光淬灭封片剂,盖玻片封片,进行观察并拍照。
结果如图4-6所示,胆管类器官能够正常表达上皮标记物(EpCAM)和胆管标记物(CK19),说明培养的类器官没有丢失原组织的特征。
以上的实施例是为了说明本发明公开的实施方案,并不能理解为对本发明的限制。此外,本文所列出的各种修改以及发明中方法的变化,在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述,但应当理解,本发明不应仅限于这些具体实施例。事实上,各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。
Claims (4)
1.一种鼠胆管类器官的制备方法,其特征在于,所述方法包括如下步骤:
1)将鼠肝脏组织块与清洗液混合,上下吹打以清洗掉杂质,静置待组织块沉降后,弃上清;
2)将步骤1)获得的产物与组织消化试剂混合,消化20~30min,终止消化后静置以使胆管组织沉淀,去除消化液,重悬细胞;所述组织消化试剂由胶原酶IV和中性蛋白酶组成,以组织消化试剂的总体积为基准,所述胶原酶IV的终浓度为0.05~0.2U/mL,所述中性蛋白酶的终浓度为0.5~1.5U/mL;
3)将细胞与基质胶混合并接种至培养容器中,待基质胶凝固后,加入培养基进行培养,培养3~5天即获得鼠胆管类器官;以培养基的总体积为基准,所述培养基由以下成分组成:
基础培养基,所述基础培养基选自DMEM/F12或Advanced DMEM/F12
L-谷氨酰胺或其替代品 1.5~2.5 mM
HEPES 8~12 mM
B27 1.5~2.5 %
乙酰半胱氨酸 0.8~1.5 mM
RSPO1或RSPO3 100 ~300 ng/mL
烟酰胺 8~12 mM
胃泌素 I 肽 8~12 nM
上皮生长因子 30~150 ng/mL
成纤维细胞生长因子10 50~200 ng/mL
肝细胞生长因子 30 ~150 ng/mL;
抗生素。
2.根据权利要求1所述的制备方法,其特征在于,步骤3)中所述培养基还包括如下特征中的任一项或多项:
1)所述L-谷氨酰胺的替代品为GlutaMAX;
2)所述RSPO为重组人R spondin;
3)所述胃泌素 I 肽为人胃泌素 I 肽;
4)所述上皮生长因子为重组人或鼠上皮生长因子;
5)所述成纤维细胞生长因子10 为重组人或鼠成纤维细胞生长因子10;
6)所述肝细胞生长因子为重组人或鼠肝细胞生长因子。
3.根据权利要求1所述的制备方法,其特征在于,所述抗生素选自青霉素和/或链霉素。
4.根据权利要求1所述的制备方法,其特征在于,还包括如下特征中的任一项或多项:
a)步骤1)中鼠肝脏组织块为小鼠肝脏组织块或大鼠肝脏组织块;
b)步骤2)中,消化温度为35~38℃;
c)步骤2)中,在摇床上消化;
d)步骤3)中,培养条件为35~37℃、5%CO2;
e)步骤3)中,培养过程中每2~3天更换一次培养基。
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