CN116396920A - 一种原代肝细胞共培养三维肝微球模型及其制备方法和应用 - Google Patents
一种原代肝细胞共培养三维肝微球模型及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种原代肝细胞共培养三维肝微球模型及其制备方法和应用。所述三维肝微球模型呈球体状,所述三维肝微球模型含有肝实质细胞和肝非实质细胞,所述肝非实质细胞包括肝窦内皮细胞、枯否细胞和肝星状细胞,所述三维肝微球模型中肝实质细胞与肝非实质细胞的接种浓度百分比为(67±5)%:(33±5)%。本发明构建新型三维肝微球模型,能更好地模拟体内环境和对药物反应,更长时间地共培养,维持数周细胞活性、代谢活性和相关肝细胞功能,肝非实质细胞能够稳定和功能地整合于模型中,模型中肝实质细胞高度极化和形成毛细胆管及具有功能型外排型药物转运体介导的跨膜转运,使该模型成为研究药物代谢、毒性反应等长期效应的理想模型。
Description
技术领域
本发明属于细胞微球技术领域,涉及一种原代肝细胞共培养三维肝微球模型及其制备方法和应用。
背景技术
癌症的治疗是目前医学领域备受关注的领域,近年来,纳米药物(尺寸1~100nm)在肿瘤治疗方面取得一系列重大突破,然而目前研究人员无法有效将该药物运输到患者体内发挥作用,仅有大约0.7%的化疗纳米药物才能顺利抵达患者机体患处靶向作用肿瘤细胞,剩余的药物全被机体其它细胞吸收了,包括肝脏、肾脏和脾脏等,当药物在这些器官中积累时,就会产生毒性和副作用,比如药物诱导性肝损伤(DILI)仍然是其药物研发失败的常见原因。
纳米颗粒的肝胆清除一般是通过以下几种途径:肝窦、窦周隙、肝细胞、胆管和肠道排出体外,有研究发现纳米颗粒与肝非实质细胞(例如库普弗细胞和肝窦内皮细胞)的相互作用导致纳米粒子的低内在清除和肝胆清除的主要2个重要关键障碍(参见Wilson Poonet al.Elimination Pathways of Nanoparticles.ACS Nano 2019)。因此,预测评估纳米药物在体内肝脏渗透积累或清除是其临床转化成功的关键,也是临床前阶段,对纳米药物的安全性、局限性和治疗潜力的综合体外测定必不可少的步骤。故迫切需要建立和使用一个有临床相关性的肝脏体外模型来正确预测纳米药物在肝脏中的渗透,积累和排除。
由于二维肝细胞模型在培养过程中肝细胞特异性表型的丢失,以及其本身缺乏复杂的体内生理环境,所以目前的二维肝模型系统通常无法预测化学诱导的肝毒性。对于更具预测性的体外模型,肝细胞必须维持在允许极化和细胞间接触的三维环境中。近年来肝细胞的体外三维(3D)球体模型因其具有3D空间排列,与2D模型相比能够更好地模拟生物体内细胞环境,重现肝功能,延长细胞培养时间而备受关注。然而,建立良好的三维肝脏细胞模型除了选择培养形式外,选择合适的细胞来源和细胞类型对于药物的肝毒性预测同样重要,即使有一定肝脏表征的永生化的肝源细胞已用于体外肝脏3D模型的构建,但是它们的适用性受到其相对代谢活性低和敏感性水平低的阻碍(参见:Gerets HHJ etal.Characterization of primary human hepatocytes,HepG2 cells,and HepaRG cellsat the mRNA level and CYP activity in response to inducers and theirpredictivity for the detection of human hepatotoxins.Cell Biol Toxicol,2012)。而且通常建立和使用的是简单的仅包含原代肝实质细胞或永生化的HepaRG肝细胞或HepG2肝癌细胞系的3D球体模型(Christopher R.Cox et al.2020),或采用永生化人肝癌细胞系(HepG2)和人肝星形细胞系(LX-2)共培养构建肝细胞聚球体模型(WANG Ying etal.2019)。
综上所述,目前简单型的3D肝脏模型与体内肝组织细胞组成和微环境相比依然存在很大的缺陷,不能完全反应真实体内的多细胞类型生理环境,因此,开发适用于纳米药物筛选的更复杂、更具生物相关性和预测性的体外3D肝脏细胞模型是纳米药物发展领域亟需解决的问题之一。
发明内容
针对现有技术的不足和实际需求,本发明提供一种原代肝细胞共培养三维肝微球模型及其制备方法和应用,能够有效模拟体内多细胞类型环境,为药物研发提供体外评估模型。
为达上述目的,本发明采用以下技术方案:
第一方面,本发明提供一种原代肝细胞共培养三维肝微球模型,所述三维肝微球模型呈球体状,所述三维肝微球模型含有肝实质细胞和肝非实质细胞,所述肝非实质细胞包括肝窦内皮细胞、枯否细胞和肝星状细胞,所述三维肝微球模型中肝实质细胞与肝非实质细胞的接种浓度百分比为(67±5)%:(33±5)%。
本发明中,构建一种新型的原代肝细胞共培养三维(3D)肝微球模型,由特定的肝实质细胞(HEP)和非实质细胞(NPC)共同组成构建。能够更好地模拟体内环境和对药物治疗的反应,够进行更长时间的共培养,并且能够维持数周的细胞活性、代谢活性和相关的肝细胞功能,肝非实质细胞稳定的和功能性的整合于共培养3D肝微球中,共培养肝微球体细胞高度极化和形成毛细胆管以及具有功能型外排型药物转运体介导的跨膜转运。
本发明由原代肝实质/非实质细胞构建的体外共培养3D肝微球模型可用于临床前高通量筛选评估药物诱导性肝毒性,以及预测纳米药物在肝脏组织的渗透,积累风险,以便在药物开发的早期阶段正确测试药物的有效性和局限性,具有提高药物临床应用转化价值。
可选地,所述三维肝微球模型中肝窦内皮细胞、枯否细胞和肝星状细胞的接种浓度百分比为(17±4)%:(12±4)%:(8±4)%。
可选地,所述三维肝微球模型中细胞数量为500~3000个细胞,包括但不限于501个细胞、502个细胞、510个细胞、550个细胞、1000个细胞、1500个细胞、2000个细胞、2500个细胞、2600个细胞、2800个细胞或2900个细胞。
可以理解,本领域常用的药物研究实验动物的肝脏细胞均适用于本发明。
可选地,所述肝实质细胞和肝非实质细胞的来源包括人、大鼠、小鼠、比格犬、猴等种属。
可选地,所述三维肝微球模型的球直径为150~250μm,包括但不限于151μm、152μm、160μm、170μm、180μm、190μm、220μm、240μm或249μm。
第二方面,本发明提供一种第一方面所述的原代肝细胞共培养三维肝微球模型的制备方法,所述方法包括:
按肝脏细胞体内生理比例将肝实质细胞、肝窦内皮细胞、枯否细胞和肝星状细胞配制为混合细胞悬浮液;将混合细胞悬浮液接种到超低吸附孔板中,进行培养,得到所述原代肝细胞共培养三维肝微球模型。
所述方法具体包括:
进行肝实质细胞和肝非实质细胞的富集、纯化和冻存,按2:1比例将肝实质细胞和肝非实质细胞配制为混合细胞悬浮液;将混合细胞悬浮液接种到超低吸附孔板中,进行培养,得到所述原代肝细胞共培养三维肝微球模型。
优选地,所述肝非实质细胞包括大鼠肝非实质细胞、小鼠肝非实质细胞和食蟹猴肝非实质细胞。
优选地,所述大鼠肝非实质细胞或小鼠肝非实质细胞的富集、纯化方法包括:
将含有大鼠肝非实质细胞或小鼠肝非实质细胞悬液于50×g、4℃离心3min,离心两次,每次离心后,吸取上清,弃去沉淀物,然后将每次离心后收集的上清液于360×g、4℃离心8min,收集沉淀物,用含有3%血清肝细胞培养基重悬细胞沉淀物,得到肝非实质细胞悬浮液;
在离心管中依次加入20mL 50%Percoll分离液和20mL 25%Percoll分离液,然后加入10mL肝非实质细胞悬浮液于Percoll分离液的上层表面,于600×g、4℃进行Percoll密度梯度离心8min,弃上清液和沉淀物,收集含有肝非实质细胞的位于离心管中间部分液体;
用含有3%血清肝细胞培养基重悬肝非实质细胞,于360×g、4℃离心8min,弃除上清液,重复两次,用含有10%血清的常规肝细胞培养基肝重悬肝非实质细胞。
优选地,所述食蟹猴肝非实质细胞的富集、纯化方法包括:
将含有食蟹猴肝非实质细胞悬液于100×g、4℃离心3min,离心两次,每次离心后,吸取上清,弃去沉淀物,然后将每次离心后收集的上清液于720×g、4℃离心8min,收集沉淀物,用含有3%血清肝细胞培养基重悬细胞沉淀物,得到肝非实质细胞悬浮液;
在离心管中依次加入20mL 50%Percoll分离液和20mL 25%Percoll分离液,然后加入10mL肝非实质细胞悬浮液于Percoll分离液的上层表面,于1200×g、4℃进行Percoll密度梯度离心8min,弃上清液和沉淀物,收集含有肝非实质细胞的位于离心管中间部分液体;
用含有3%血清肝细胞培养基重悬肝非实质细胞,于720×g、4℃离心8min,弃除上清液,重复两次,用含有10%血清的常规肝细胞培养基肝重悬肝非实质细胞。
可选地,所述96孔超低吸附孔板中每孔的接种量为500~3000个细胞/100±20μL混合细胞悬浮液/每孔。
可以理解,本领域通用的细胞培养的超低吸附孔板均适用于本发明。
可选地,所述超低吸附孔板为96孔U形底。
可选地,所述混合细胞悬浮液的配制方法包括:
按比例将肝实质细胞、肝窦内皮细胞、枯否细胞、肝星状细胞和培养基混合,得到混合细胞悬浮液。
优选地,所述培养基包括含有10%血清肝细胞完全培养基。
可以理解,本发明制备方法中还可包括分离原代肝细胞和肝实质细胞和肝非实质细胞的纯化和冻存的步骤。
可选地,所述培养的方法包括:将接种后混合细胞悬浮液置于37℃、5%CO2的培养箱中培养3-4天后,然后用不含血清的肝微球维持培养基替换50%的超低吸附孔板中培养基。
第三方面,本发明提供第一方面所述的原代肝细胞共培养三维肝微球模型在药物研发中的应用。
可选地,所述应用包括临床药物诱导性肝毒性的评估、纳米药物的肝脏滞留/排除的预测、药物诱导的胆汁淤积风险评估、允许长时间重复给药毒性试验、慢性药物毒性效应的预测、低药物清除率的评估或药物诱导的炎症和肝纤维化的预测中任意一种。
与现有技术相比,本发明具有以下有益效果:
本发明构建一种新型的原代肝细胞共培养三维肝微球模型,缩短二维细胞培养物与整体动物模型之间的转化差距,可以更准确、高效的筛选和预测纳米药物在肝脏中的脱靶滞留风险/用来筛选和研究纳米药物的肝脏滞留/排除,由此指导纳米医学上设计和优化,加快脱靶药物的体内清除,有效的解决抗癌纳米药物在靶向-清除和疗效-毒性间的矛盾问题,开发精确靶向的多功能纳米药物以实现有效的肿瘤治疗,具有较好的肿瘤渗透积累和低的肝脏脱靶富集等优点,促进纳米药物等的发展和转化。
附图说明
图1为从原代肝细胞分离到肝细胞体外共培养三维肝微球模型构建的流程图;
图2为三维肝微球模型制备、维持培养及活性功能测定操作流程示意图;
图3A为不同物种HEP/NPC共培养三维肝微球模型的形态图(第5和第12天);
图3B为不同物种HEP/NPC共培养3D肝微球直径与细胞数量相关性结果图(第5和第12天);
图4为小鼠HEP/NPC共培养肝微球模型中肝实质细胞和非实质细胞标志物的免疫荧光染色图,染色比例尺为100μm;
图5为不同物种HEP/NPC共培养三维肝微球模型的ATP含量百分比结果图(第5,第12和第17天);
图6A为小鼠肝细胞混合悬浮液(D0)与HEP/NPC共培养肝微球(D17)主要细胞色素P450酶代谢活性比较结果图(第0和第17天);
图6B为大鼠肝细胞混合悬浮液(D0)与HEP/NPC共培养肝微球(D17)主要细胞色素P450酶代谢活性比较结果图(第0和第17天);
图6C为食蟹猴肝细胞混合悬浮液(D0)与HEP/NPC共培养肝微球(D17)主要细胞色素P450酶代谢活性比较结果图(第0和第17天);
图7为大、小鼠HEP-NPC共培养肝微球白蛋白分泌量测定结果图(第5,第12和第17天);
图8为脂多糖诱导的小鼠HEP/NPC共培养肝微球模型的白细胞介素-6(IL-6)分泌释放结果图。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果,以下结合实施例和附图对本发明作进一步地说明。可以理解的是,此处所描述的具体实施方式仅仅用于解释本发明,而非对本发明的限定。
实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道购买获得的常规产品。
本发明具体实施例中以大、小鼠和猴来源的肝细胞为例构建原代肝细胞共培养三维肝微球模型,具体流程参考图1。
实施例1
本实施例进行胶原酶二步灌注分离原代肝细胞。
啮齿动物原位胶原酶两步灌注法
使用常规改良的Seglen两步灌流法(参考Seglen,P.O.Preparation of isolatedrat liver cells.Methods Cell.Biol.1976)用于CD1小鼠(维通利华、雄性、6-8周)和SD大鼠(维通利华、雄性、6-8周)原代肝脏细胞的分离。动物麻醉后,剪开腹腔,通过门静脉腔静脉进针,先用已预温至37℃含EDTA优化缓冲液进行肝脏灌注冲洗,然后换为预温的含有胶原酶缓冲液进行灌注酶消化分离肝细胞。中止酶消化后,通过过滤(70μm滤膜)、低速离心(50g,2min),将含有非实质细胞(NPC)的上清液收集并保存于4℃用于下一步大、小鼠肝非实质细胞制备。重悬离心管中细胞沉淀物,获得大、小鼠肝实质细胞(HEP)悬液。
实施例2
本实施例进行食蟹猴离体胶原酶两步灌流法分离原代肝细胞。
食蟹猴(雄性、3周岁以上)麻醉后,剪开腹腔,首先剥离全肝,获得的肝组织块的血管,先用已预温至37℃含EGTA缓冲液进行肝脏灌注冲洗,然后换为含有胶原酶缓冲液进行灌注分离肝细胞。中止酶消化后,通过过滤(70μm滤膜)、低速离心(100g,2min),将含有非实质细胞的上清液收集并保存于4℃用于下一步食蟹猴肝非实质细胞制备。重悬离心管中细胞沉淀物得到食蟹猴肝实质细胞悬液。
实施例3
本实施例进行肝实质细胞和肝非实质细胞的纯化和冻存。
肝实质细胞的纯化和冻存
将得到的大、小鼠和食蟹猴肝实质细胞悬液通过肝细胞的密度梯度离心纯化HEP细胞(大、小鼠使用35%percoll/100g/3min/4℃;食蟹猴使用28%percoll/200g/3min/4℃),吸弃上清液,用含有3%血清的肝培养基重悬HEP细胞沉淀物,离心洗涤HEP细胞两次(大、小鼠50g/3min/4℃;食蟹猴100g/3min),弃除上清液,用肝微球体形成培养基重悬纯化的HEP沉淀,获得大、小鼠和食蟹猴肝实质细胞悬液,然后用常规台盼蓝排除法/细胞计数仪计测定得到HEP活细胞数及活细胞率(85%-95%)。纯化后的新鲜原代肝实质细胞可以直接用于肝微球的构建,或者悬浮于冻存培养基,分装后使用全自动细胞冻存仪获得冻存原代肝实质细胞以便于后期复苏和使用相同程序构建肝微球。
肝非实质细胞的富集、纯化和冻存
将暂存于4℃的富含有NPC细胞悬液进行低速离心两次(大、小鼠50g/3min/4C;食蟹猴100g/3min/4℃),每次离心后,吸取上清(NPC),弃去沉淀物(残余的肝实质细胞,HEP),然后将每次离心后收集的上清液进行高速离心收集NPC细胞(大、小鼠360g/8min/4℃);食蟹猴720g/8min/4℃),用含有3%血清(经过了热灭活处理)肝细胞培养基重悬细胞沉淀物得到肝非实质细胞(NPC)细胞悬浮液。
将获得的不同物种肝非实质细胞(NPC)通过2个浓度Percoll密度梯度离心纯化,在一离心管中依次加入20mL Percoll(50%)和20mL Percoll(25%),然后轻缓加入10mLNPC细胞悬浮液于Percoll分离液的上层表面,Percoll密度梯度离心后(大,小鼠600g/8min/4℃);食蟹猴1200g/8min/4℃),吸弃离心管中含有细胞碎片和死细胞的上清液,收集含有NPC的位于离心管中间含有NPC部分液体,丢弃离心管下部沉淀物,然后用然后用上述相同培养基和离心速度离心洗涤两次,弃除上清液,用含有10%血清的常规肝细胞培养基肝重悬NPC细胞,然后用常规台盼蓝排除法/细胞计数仪计测定得到NPC活细胞数及活细胞率(85%-95%)。
纯化后的新鲜原代肝非实质细胞(NPC)可以直接用于肝微球的构建,或者悬浮于冻存培养基,分装后使用全自动细胞冻存仪获得冻存原代肝非实质细胞(NPC)以便于后期复苏和使用相同程序构建肝微球。
实施例4
本实施例进行无支架3D肝微球构建、培养和形态分析。
HEP/NPC共培养3D肝微球模型构建和培养
建立一个由肝实质细胞(HEP)与肝脏非实质细胞(NPC)多细胞类型共培养3D肝微球体模型,其对照模型是仅包含肝实质细胞(HEP)的单培养3D肝微球体模型,操作流程示意图如图2所示,具体为按照设定的系列细胞浓度(大、小鼠的肝细胞有2个浓度:1000和2000个细胞/100μL/每孔;食蟹猴的肝细胞有3个浓度:1000、2000和3000个细胞/100μL/每孔)和细胞类型比例(HEP 100%悬浮液,或者HEP 67%:NPC 33%,即2:1比例多细胞混合液),用含有10%血清肝细胞完全培养基制备HEP单培养悬浮液和HEP-NPC共培养细胞悬浮液,然后将不同浓度和不同类别细胞混合悬浮液接种超低附着96孔板(100μL细胞悬液/孔),并通过平板离心机离心96孔板(50g/3min)使细胞集聚在孔板中心底部,然后将细胞板置于37℃、5%CO2培养箱中培养4天,此期间无需更换培养基,在第4天,当细胞球体足够致密时,用不含血清的肝微球维持培养基替换50%的培养板中培养基(50μL/孔),以便在低血清条件培养基中长期培养,每2天更新一次直到细胞球体成熟极化。一般到第5天,细胞球体更加致密稳定,并且通常在第7天肝微球开始极化成熟,因此,本实施例在三个不同培养时间点(第5天、第7和第17天)取样观察和测试3D肝微球活性和功能(图2)。
HEP/NPC共培养3D肝微球形态表征
初始肝细胞接种密度影响肝细胞球体直径的大小,如图3A所示,通过测试比较CD1小鼠和SD大鼠每个HEP/NPC共培养3D肝球状体的初始细胞接种数量(1000-2000个细胞/每孔),或食蟹猴每个共培养3D肝微球状体的初始细胞接种数量(1000-3000个细胞),以及经过5天和12天无支架3D培养,测试结果(图3A)表明接种数量为1000-2000个大鼠或小鼠HEP-NPC肝细胞可形成直径在160-210μm范围之内最佳球状体,而接种数量为2000-3000个食蟹猴HEP-NPC肝细胞可形成直径在190-240μm范围之内最佳球状体,因为球体直径控制在200-300μm范围之内可确保球状体中心有足够的营养和氧气供应,有助于避免坏死球状体核心的扩展。
同时通过2周的低粘附培养期间可观察到共培养3D肝微球的形态和球体大小的逐渐变化,接种的所有细胞可在3天内聚集形成较松散球体,从第5天已形成的具有明显的边界3D球状体(图3A),到第12天形成具有圆滑边界和致密的球状体,在培养长达12天时间内,共培养3D球状体结构随时间推移而更加紧凑,同时球体直径会随着时间的推移而微减(图3B)。
实施例5
本实施例进行共培养3D肝微球中肝实质细胞与非实质细胞共培养的表征。
1、共培养肝微球中肝实质细胞与非实质细胞共存培养的表征
为了验证当将肝实质细胞(HEP)与非实质细胞(NPC)以2:1的比例混合并在无支架的3D细胞共培养期间,其中非实质细胞(肝星状细胞HSC、枯否细胞KC和肝窦内皮细胞LSEC)可成功聚集整合到PHH/NPC共培养3D肝球体中,并在整个测试的12天培养期间保持与肝实质细胞共培养状态。
通过特异生物标记染色对小鼠HEP/NPC共培养3D肝微球中肝实质与非实质细胞进行表征(表1),即小鼠HEP/NPC共培养肝微球中实质细胞和非实质细胞的生物标记物免疫荧光染色。
将5个小鼠HEP/NPC共培养12天球龄的肝微球汇集到1.5mL Eppendorf管中,并在25℃下用4%多聚甲醛固定液固定1h,PBS缓冲液洗涤两次之后,在4℃下用递增浓度梯度甲醇(50%、80%和100%)通透化球体,每次10min;然后,将肝微球样品在不同的溶液中依次在25℃下孵育洗涤10min:20%二甲基亚砜(DMSO)甲醇溶液→50%甲醇→80%甲醇→PBS洗涤→含有0.2%Triton的PBS的最终洗涤;随后,将共培养3D肝微球样品在HISTO渗透缓冲液(Visikol HISTO Penetration bufferTM,ref HSK-PB-30,SIGMA)中孵育30min;并在HISTO封闭缓冲液(HISTOTM Blocking Buffer,ref.HSK-BB-30,Sigma)中37℃下封闭60min;然后用HISTO抗体稀释缓冲液(/>HISTOTM Antibody Buffer,Ref.HSK-AB-30,Sigma)将一抗稀释至四档的浓度并加入肝微球样品中后于4℃孵育过夜。
次日,将肝微球样品用HISTO洗涤缓冲液(HISTOTM Washing Buffer,Ref.HSK-WB-70,Sigma)洗涤5次,每次10min;并将含有10μM赫斯特荧光染料(Hoechst33342,Ref.62249,Thermo Fisher Scientific)与相应的二抗的HISTO抗体稀释缓冲液加入肝微球样品中,25℃孵育1h;将肝微球样品在HISTO洗涤缓冲液中洗涤10次,每次5min;然后用HISTO-M透明缓冲液(Visikol HISTO-MTM Tissue Clearing Reagents,Ref.HM-30,Sigma)孵育15min,进行透明处理,最后对HEP/NPC共培养3D肝微球进行共聚焦成像和分析评估,对于共聚焦分析,将清除的球体转移到96孔玻璃底板中(PerkinElmer,Germany),使用共聚焦显微镜进行荧光成像和拍照。
表1
根据肝脏生理比例建立了共培养三维肝微球模型,以2:1的比例构建HEP/NPC共培养肝微球。12天共培养后的肝微球免疫荧光染色显示肝实质细胞(HEP)和肝非实质细胞(HSC、KC和LSEC)保持并分布在共培养肝微球中(图4),巨噬细胞特异性表面抗原F4/80作为星状细胞(HSC)特异生物标记;结蛋白(desmin)作为枯否细胞(KC)特异生物标记;透明质酸受体(LYVE-1)作为肝窦内皮细胞(LSEC)特异生物标记。
与共培养的非实质细胞相比,三维肝微球模型中肝实质细胞(Albumin,红色荧光)密度显著高于非实质细胞类型并与接种比例相符合,此共培养肝微球特异性荧光染色结果表明当将HEP(67%)与NPC(33%)混合共培养后,肝非实质细胞(HSC、KC和LSEC)在在球体聚集时成功地整合到PHH/NPC共培养3D肝球体中,并在整个12天培养期间保持稳定共存状态。
2、HEP/NPC共培养3D肝微球的功能表征
2.1、对不同物种的HEP/NPC共培养3D肝微球的模型在三周内的长期稳定性、活性和肝功能活性进行表征。
HEP/NPC共培养3D肝微球的长期稳定性和活性
由于细胞内的三磷酸腺苷(ATP)含量水平直接反映细胞的活性,ATP含量可作为测定3D肝微球活性的标志物,在HEP/NPC共培养3D肝微球构建形成的第5、第12和第17天的3个时间点,分别使用3D发光法细胞活力检测试剂盒对肝微球细胞ATP的含量进行测定,其HEP/NPC共培养3D肝微球活力表示为第5天共培养3D球状体的ATP含量百分比,结果图表2所示,表2中的/>3D细胞活力检测结果表明与第5天的共培养肝微球ATP含量相比较,共培养肝微球细胞内ATP含量相当稳定的持续到第17天的3D球体培养,表明本发明的共培养3D肝微球可以维持肝细胞活性至少超过2周体外培养时间(图5)。
表2
2.2、HEP/NPC共培养3D肝微球的代谢功能
对三个不同物种肝实质细胞(HEP)和肝非实质细胞(NPC)混合悬浮液在未接种之前(D0)和HEP/NPC共培养3D肝微球形成后第17天(D17)进行了5种主要P450代谢酶活性的测量(表3),相比之下,所有物种的HEP/NPC共培养微球模型在近3周的培养期间保持显著的和可比性的P450代谢活性,尽管共培养肝微球模型的P450代谢水平在第17天有轻微下降(图6A-图6C)。
表3
*将共培养肝微球中包含的67%肝实质细胞(HEP)代谢活性值标准化为100万肝实质细胞代谢活性值。
实施例6
本实施例进行HEP/NPC共培养3D肝微球白蛋白的分泌定量。
白蛋白的产生和分泌中是肝脏的主要功能参数之一,通过白蛋白-Elisa评估的白蛋白分泌作为肝脏功能的标志物之一。
分别在小鼠、大鼠HEP/NPC共培养3D肝微球17天培养期间,更换培养基后4天收集上清液样品,然后使用大鼠白蛋白ELISA试剂盒(Abcam)对共培养肝微球培养基上清液样品进行分泌的白蛋白定量(表4),3个时间点的白蛋白量化测定结果显示在球体生产后第5天开始,共培养肝微球显示出稳定的白蛋白分泌,尽管在培养后12天开始检测到轻微的增加(表4,图7)。
表4
实施例7
本实施例分析HEP/NPC共培养3D肝微球中的NPC功能和活化潜力。
白细胞介素-6(IL6)分泌水平的测定
用脂多糖(LPS)刺激HEP/NPC共培养3D肝微球中的枯否细胞导致白细胞介素(IL6)分泌,通过评估脂多糖(LPS)诱导的白细胞介素(IL-6)释放来评估枯否细胞(Kupffer)的存在和活化功能,3D肝微球形成12天后,在小鼠HE单培养肝微球样品(HEP,n=10个球状体)和小鼠HEP/NPC共培养肝微球样品(n=10个球状体)中加入10μM脂多糖(LPS,L4391-1MG,Sigma),48h后收集上清液,通过ELISA试剂盒(Mouse IL-6 ELISA Kit-Thermo FisherScientific)测量IL-6释放浓度,以不进行脂多糖刺激作为对照,结果如图8所示,在存在和不存在LPS(脂多糖)的刺激处理下,HEP单培养肝微球仅显示可忽略的微量IL-6释放(1.5pg/mL),表明HEP单培养肝微球不含或者微量的NPC,正如预期的那样,含有NPC的共培养球状体显示出较高的IL-6释放(60pg/mL),如果不用LPS刺激,则不会释放IL-6,这些结果不仅证实了共培养球体中NPC枯否细胞的稳定整合,还证实了它们可以在共培养3D肝微球体外系统中被脂多糖(LPS)刺激分泌白细胞介素6(IL-6)。
综上所述,本发明构建一种新型的原代肝细胞共培养三维肝微球模型,由特定的肝实质细胞(HEP)和非实质细胞(NPC)共同组成构建,能够更好地模拟体内环境和对药物治疗的反应,够进行更长时间的共培养,并且能够维持数周的细胞活性、代谢活性和相关的肝细胞功能,肝非实质细胞稳定的和功能性的整合于模型中,模型中体细胞高度极化和形成毛细胆管以及具有功能型外排型药物转运体介导的跨膜转运。为药物诱导的肝损伤和纳米药物安全性和有效性提供体外评估模型,能够更加准确地、高通量筛选和评价药物诱导的肝毒性,对药物在开发的早期阶段进行有效性和安全性的正确评估。
申请人声明,本发明通过上述实施例来说明本发明的详细方法,但本发明并不局限于上述详细方法,即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
Claims (10)
1.一种原代肝细胞共培养三维肝微球模型,其特征在于,所述三维肝微球模型呈球体状,所述三维肝微球模型含有肝实质细胞和肝非实质细胞;
所述肝非实质细胞包括肝窦内皮细胞、枯否细胞和肝星状细胞;
所述三维肝微球模型中肝实质细胞和肝非实质细胞的接种浓度百分比为(67±5)%:(33±5)%。
2.根据权利要求1所述的原代肝细胞共培养三维肝微球模型,其特征在于,所述三维肝微球模型中肝窦内皮细胞、枯否细胞和肝星状细胞的接种浓度百分比为(17±4)%:(12±4)%:(8±4)%。
3.根据权利要求1或2所述的原代肝细胞共培养三维肝微球模型,其特征在于,所述三维肝微球模型中细胞数量为500~3000个细胞。
4.根据权利要求1所述的原代肝细胞共培养三维肝微球模型,其特征在于,所述肝实质细胞和肝非实质细胞的来源包括人、大鼠、小鼠、猴或比格犬中任意一种;
优选地,所述三维肝微球模型的球直径为100~250μm。
5.一种权利要求1-4任一项所述的原代肝细胞共培养三维肝微球模型的制备方法,其特征在于,所述方法包括:
按肝脏细胞体内生理比例将肝实质细胞、肝窦内皮细胞、枯否细胞和肝星状细胞配制为混合细胞悬浮液;将混合细胞悬浮液接种到超低吸附孔板中,进行培养,得到所述原代肝细胞共培养三维肝微球模型。
6.根据权利要求5所述的原代肝细胞共培养三维肝微球模型的制备方法,其特征在于,所述方法还包括进行肝实质细胞和肝非实质细胞的富集、纯化和冻存的步骤;
优选地,所述肝非实质细胞包括大鼠肝非实质细胞、小鼠肝非实质细胞和食蟹猴肝非实质细胞;
优选地,所述大鼠肝非实质细胞或小鼠肝非实质细胞的富集、纯化方法包括:
将含有大鼠肝非实质细胞或小鼠肝非实质细胞悬液于50×g、4℃离心3min,离心两次,每次离心后,吸取上清,弃去沉淀物,然后将每次离心后收集的上清液于360×g、4℃离心8min,收集沉淀物,用含有3%血清肝细胞培养基重悬细胞沉淀物,得到肝非实质细胞悬浮液;
在离心管中依次加入20mL50%Percoll分离液和20mL25%Percoll分离液,然后加入10mL肝非实质细胞悬浮液于Percoll分离液的上层表面,于600×g、4℃进行Percoll密度梯度离心8min,弃上清液和沉淀物,收集含有肝非实质细胞的位于离心管中间部分液体;
用含有3%血清肝细胞培养基重悬肝非实质细胞,于360×g、4℃离心8min,弃除上清液,重复两次,用含有10%血清的常规肝细胞培养基肝重悬肝非实质细胞;
优选地,所述食蟹猴肝非实质细胞的富集、纯化方法包括:
将含有食蟹猴肝非实质细胞悬液于100×g、4℃离心3min,离心两次,每次离心后,吸取上清,弃去沉淀物,然后将每次离心后收集的上清液于720×g、4℃离心8min,收集沉淀物,用含有3%血清肝细胞培养基重悬细胞沉淀物,得到肝非实质细胞悬浮液;
在离心管中依次加入20mL50%Percoll分离液和20mL25%Percoll分离液,然后加入10mL肝非实质细胞悬浮液于Percoll分离液的上层表面,于1200×g、4℃进行Percoll密度梯度离心8min,弃上清液和沉淀物,收集含有肝非实质细胞的位于离心管中间部分液体;
用含有3%血清肝细胞培养基重悬肝非实质细胞,于720×g、4℃离心8min,弃除上清液,重复两次,用含有10%血清的常规肝细胞培养基肝重悬肝非实质细胞。
7.根据权利要求5或6所述的原代肝细胞共培养三维肝微球模型的制备方法,其特征在于,所述超低吸附孔板为96孔细胞培养板,每孔的接种量为500~3000个细胞/100±20μL混合细胞悬浮液/每孔。
8.根据权利要求5-7任一项所述的原代肝细胞共培养三维肝微球模型的制备方法,其特征在于,所述混合细胞悬浮液的配制方法包括:
按比例将肝实质细胞、肝窦内皮细胞、枯否细胞、肝星状细胞和培养基混合,得到混合细胞悬浮液;
优选地,所述培养基包括含有10%血清肝细胞完全培养基;
优选地,所述培养的方法包括:将接种后混合细胞悬浮液置于37℃、5%CO2的培养箱中培养3~4天,期间不更换培养基,3~4天后,用不含血清的肝微球维持培养基替换50%的超低吸附孔板中培养基。
9.权利要求1-4任一项所述的原代肝细胞共培养三维肝微球模型在药物研发中的应用。
10.根据权利要求9所述的应用,其特征在于,所述应用包括临床药物诱导性肝毒性的评估、纳米药物的肝脏滞留/排除的预测、药物诱导的胆汁淤积风险评估、允许长时间重复给药毒性试验、慢性药物毒性效应的预测、低药物清除率的评估或药物诱导的炎症和肝纤维化的预测中任意一种。
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CN117925374A (zh) * | 2024-03-21 | 2024-04-26 | 妙顺(上海)生物科技有限公司 | 一种细胞高温复苏方法及细胞复苏器 |
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