CN111718895A - 一种人工心肌组织纤维化模型、制备方法、制备装置及其应用 - Google Patents
一种人工心肌组织纤维化模型、制备方法、制备装置及其应用 Download PDFInfo
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Abstract
本发明属于生物医学工程技术领域,尤其涉及一种人工心肌组织纤维化模型、制备方法、制备装置及其应用。本申请推出首个基于人源心肌细胞的人工心肌组织纤维化模型,一方面通过组织工程技术构建具备人体心肌结构特征的人源化人工心肌组织,经均一化、标准化的冷冻损伤处理后,可产生与人体心梗后相似的、以成纤维细胞活化、细胞外基质沉积产生“瘢痕化”区域为代表的纤维化表型并对药物做出响应。另一方面由于采用成熟的hiPSC‑CM分化体系与心肌组织构建体系,可低成本、批量化构建人工心肌组织纤维化模型,满足以96孔板药物筛选为主的测试需求,以达到高通量药物测试的目的,弥补现有药物筛选系统没有人工心肌纤维化模型进行相关药物研发的困境。
Description
技术领域
本发明属于生物医学工程技术领域,尤其涉及一种人工心肌组织纤维化模型、制备方法、制备装置及其应用。
背景技术
缺血性心脏病每年导致约900万人死亡,已发展成为全球致死人数最高的疾病。心脏发生缺血性梗死后,由于成体心脏心肌细胞再生能力有限,免疫浸润促使梗死区域周边成纤维细胞增殖、活化并迁移到梗死区域,同时分泌细胞外基质诱导心梗区域发生病理性重构,这一过程称为心肌纤维化。心肌纤维化形成的顽固性瘢痕组织不具备电-收缩特性,进而导致心功能下降,临床表现为心律失常或心力衰竭。
针对缺血性心脏病,以心脏搭桥、心脏支架为代表的外科手段存在着风险高、花费高的缺点,而常依赖的药物治疗也仅适用于溶栓、血管扩张等心梗症状的缓解,无法达到促进心梗区域心肌细胞再生进而恢复心功能的目的。近20多年来,相应的心血管药物研发也处于停滞不前的状态,给心梗治疗带来极大挑战。新药研发是一个漫长且昂贵的过程,数万种化合物经过数年的细胞筛选、动物测试,却有可能因为人体模型无效或潜在的心脏毒性面临退出临床实验甚至退市的风险。因此,亟需开发有别于传统药物筛选模型的人源心肌纤维化模型,该模型一方面旨在缩短药物研发进程,为心梗药物的快速研发铺平道路,另一方面也为体外研究人体心肌再生的分子机理提供新思路。
现有技术的缺点:传统药物筛选模型包括细胞模型和动物模型,细胞模型以单层平面培养的细胞为对象,hiPSC-CM的出现虽然解决了人源心肌细胞来源不足的问题,但单层培养心肌细胞成熟度较差且不具备心肌组织特征,因此无法模拟心肌纤维化表型。以小鼠、兔子为代表的动物模型,虽然可通过冠状动脉左前降支短期结扎来模拟心梗,但该操作也存在着操作复杂、均一性差的问题。此外由于动物体内生理状况复杂且存在物种差异,因此无法准确判断药物作用分子机理和潜在的人体毒性。动物模型也还面临着人力物力成本高、无法进行高通量药物筛选的问题。
发明内容
针对现有技术存在的问题,本发明提供了一种人工心肌组织纤维化模型、制备方法及其应用,目的在于解决人源心肌组织纤维化模型缺失带来的心梗药物研发及人体心肌再生研究受阻的问题。
本申请即是一种以人多能干细胞衍生心肌细胞(hPSC-CM)与天然细胞外基质纤维蛋白为基础开发出的新型人工心肌组织(EHT)纤维化模型,该体外模型一方面再现了成体心肌的结构、功能特征,另一方面经冷冻损伤诱导后,可展现出与动物体内相似的心肌损伤-纤维化过程,并具备高通量开发与药物筛选的良好潜力。
本发明是这样实现的,一种人工心肌组织纤维化模型的制备方法,包括:
将人多功能干细胞或人胚胎干细胞通过小分子分阶段调控WNT信号通路,诱导其分化为心肌细胞;
使用细胞外基质纤维蛋白原与Matrigel作为心肌细胞之间的骨架结构,构建工程化心肌组织片;
制备柱状损伤器,冷冻后插入工程化心肌组织片中,得到损伤EHT并诱导其体外自我修复产生以非心肌细胞为主体的纤维化区域,即人工心肌组织纤维化模型。
人工心肌组织纤维化模型在ROCK/Rho信号通路抑制剂Y27632的处理下,愈合区域中纤维化程度降低、心肌细胞占比增多。
进一步地,所述小分子包括CHIR99021和IWR1。
进一步地,将心肌细胞消化、胰酶酶解后得到单细胞,用水凝胶培养A液重悬并加入包含Fibronectin和Matrigel水凝胶培养B液,组成水凝胶心肌组织液,将水凝胶心肌组织液转移至EHT成型模具中,培养箱培养、固化后得到工程化心肌组织片,至于EHT培养基中培养。
进一步地,所述水凝胶培养A液包括A液Ⅰ:Low Glucose DMEM、10%FBS、1%PS、终浓度2μg/mL维生素B12、终浓度1mg/mL氨基乙酸,将58μL A液Ⅰ与2.4μL thrombin(50U/mL)混合即可;
所述水凝胶培养B液包括B液Ⅰ:Low Glucose DMEM、20%FBS、1%PS、终浓度4μg/mL维生素B12、终浓度2mg/mL氨基乙酸,将24μL B液Ⅰ与24μL Fibronectin、12μL Matrigel混合组成水凝胶培养B液。
一种高通量药物筛选人工心肌组织纤维化模型的制备方法,包括:
切去多孔PCR管每个管的底部,并在每个切口处固定中空的纸环,制得多孔EHT支型支架装置;
制备PDMS EHT成型模具,所述成型模具包括与所述多孔PCR管对应的圆形凹槽;
在EHT成型模具的凹槽中央插入损伤针,制成EHT损伤装置;
在另外的EHT成型模具的凹槽中加入水凝胶心肌组织液,所述水凝胶心肌组织液的制备方法如权利要求3;
将多孔EHT支型支架装置放在EHT成型模具上,并使纸环浸入凹槽的水凝胶心肌组织液中,培养,一次性得到多个相互独立的EHT;
将EHT损伤装置冷冻后,使损伤针穿过多孔EHT,每根损伤针对应每个孔的EHT中心,得到高通量药物筛选人工心肌组织纤维化模型。
进一步地,所述PDMS EHT成型模具的制备方法包括:首先制备EHT成型装置阳模,所述阳模上分布有与多孔PCR管对应的多个圆柱;将PDMS原液与交联剂混合后,倒入放有所述阳模的器皿中,固化后,分离阳模,去除多于的PDMS,即得带有圆形凹槽的PDMS EHT成型模具。
如上述的制备方法制备的人工心肌组织纤维化模型在心肌组织纤维化治疗或诊断的药物筛选或药物检测中的应用。
进一步地,应用方法包括将所得人工心肌组织纤维化模型至于含有待测药物的培养基中培养,通过观察损伤愈合情况判断药物效果。
ROCK/Rho信号通路抑制剂在制备由冷冻损失引起的心肌组织纤维化的药物中的应用。
本发明还提供了一种高通量组织纤维化模型制备装置,包括插入式培养支架,所述插入式培养支架上设有多个端部开口的中空管,每个所述中空管的开口端设有纸环,所述纸环内径小于所述中空管的开口端内径;
还包括与所述插入式培养支架配套使用的组织片成型装置阴模,所述成型装置阴模上设有供多个所述中空管插入的凹槽,所述凹槽内承装有组织液,所述中空管插入所述凹槽内,所述纸环浸没在组织液中,组织液固化后,组织片被支撑在纸环内;
还包括与所述插入式培养支架配套使用的组织片损伤装置,所述损伤装置上设有供多个所述中空管插入的损失凹槽,所述损伤凹槽中部竖直设有柱状的损伤器,所述插入式培养支架的中空管插入所述损伤装置中,所述损伤器穿过被支撑在纸环内的组织片。
综上所述,本发明的优点及积极效果为:
本技术推出首个人源工程化心肌组织纤维化模型,一方面通过组织工程技术,构建了具备人体心肌结构特征的人源工程化心肌组织,经均一化、标准化的冷冻损伤处理后,可产生与人体心梗后相似的、以成纤维细胞活化、细胞外基质沉积产生“瘢痕化”区域为代表的纤维化表型并对药物做出响应。另一方面由于采用成熟的hiPSC-CM分化体系与心肌组织构建体系,可低成本、批量化构建人源心肌组织纤维化模型,满足以96孔板药物筛选为主的测试需求,以达到高通量药物测试的目的,进而弥补现有药物筛选系统没有人源心肌纤维化模型进行相关药物研发的困境。
本发明还提供了一种高通量组织纤维化模型制备装置,该装置可以一次性制备大量纤维化模型
附图说明
图1是不同多能干细胞系向心肌细胞分化效率检测;
图2是工程化心肌组织片构建示意图;
图3是工程化心肌组织片;
图4是工程化心肌组织片标准化冷冻损伤;其中,A.损伤器尺寸图;B.损伤底座阴模尺寸图;C.损伤器实物图与EHT标准化冷冻损伤;
图5是心肌细胞纯度影响EHT损伤愈合速度;A.低心肌细胞纯度EHT与高心肌细胞纯度EHT损伤后愈合光镜图;B.低心肌细胞纯度EHT与高心肌细胞纯度EHT损伤后每日相对损伤面积统计图;C.低心肌细胞纯度EHT与高心肌细胞纯度EHT损伤后愈合速率统计;
图6是损伤EHT修复区域主体为成纤维细胞与以Ⅰ型胶原、纤连蛋白为代表的细胞外基质;
图7是小分子介导EHT损伤修复过程;A.小分子介导的低心肌细胞纯度EHT损伤愈合光镜图;B.小分子介导的低心肌细胞纯度损伤EHT每日相对损伤面积统计图;
图8是Y27632促进损伤EHT心肌再生实验结果图;A.不同小分子处理下的损伤EHT最终愈合区域免疫荧光图;B.损伤愈合区域三维重构;C.不同小分子处理下的EHT损伤愈合区域心肌细胞占比统计图;
图9是Y27632减少损伤愈合区域成纤维细胞活化;
图10是96孔人工心肌组织纤维化模型构建示意图;
图11是96孔人工心肌组织。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。
本发明披露了一种人工心肌组织纤维化模型、制备方法、制备装置及其应用,具体如下各实施例所示。
实施例1多能干细胞制备心肌细胞
本实施例所用细胞为人源诱导性多能干细胞或人胚胎干细胞,通过使用小分子分阶段调控WNT信号通路诱导其分化为心肌细胞,具体操作如下;
将人多能干细胞接种到用Matrigel(Corning)包被的6孔板中,接种密度为3.0×106个/mL/孔,使用mTeSR培养基(STEMCELL Technologies)在5%CO2、37℃恒温培养箱(Thermo)中培养3天,至细胞长到70%-80%满。为防止细胞死亡,培养第一天加入10μMY27632(Tocris)。
吸弃旧培养基,换用细胞培养基Ⅰ起始分化,在5%CO2、37℃恒温培养箱(Thermo)中培养2天。之后,吸弃旧培养基,换用细胞培养基Ⅱ,在5%CO2、37℃恒温培养箱(Thermo)中培养1天。之后,吸弃旧培养基,换用细胞培养基Ⅲ,在5%CO2、37℃恒温培养箱(Thermo)中培养2天。之后,吸弃旧培养基,换用细胞培养Ⅱ继续培养7-9天,得到心肌细胞。
本实施例中涉及的细胞培养基Ⅰ按照如下方法配置得到:RPMI 1640(Gibco)+50X不含胰岛素的B27(Gibco)+终浓度200μg/mL抗坏血酸+5-10μM的CHIR99021(Tocris)。
细胞培养基Ⅱ按照如下方法配置得到:RPMI1640(Gibco)+50X不含胰岛素的B27(Gibco)+终浓度200μg/mL抗坏血酸)。
细胞培养基Ⅲ按照如下方法配置得到:RPMI1640(Gibco)+50X不含胰岛素的B27(Gibco)+终浓度200μg/mL抗坏血酸+5-10μM的IWR1(Tocris)。
以SIRPα直标抗体标记心肌细胞,用流式细胞仪(BD)检测心肌细胞纯度(Miltenyi),结果如图1所示,表明利用该分化体系可促使人诱导性多能干细胞(WTC)及人胚胎干细胞(H9、H9-cTNT)定向分化为高纯度心肌细胞(SIRPα+>80%),进而能稳定得到高纯度心肌细胞用于后续工程化心肌组织片构建。
实施例2工程化心肌组织片构建
使用天然细胞外基质纤维蛋白原与Matrigel作为心肌细胞之间的骨架结构,进而构建工程化心肌组织,具体操作如下:
使用熔融沉积型3D打印机(极光尔沃),以聚乳酸(PLA)为材料打印EHT成型模具阳模。称取72克PDMS基底液并加入8克配套交联剂(SYLGARDTM184Silicone Elastomer Kit)充分搅拌混匀,倒入放有所得阳模的玻璃缸中,65℃固化PDMS 4小时。分离阳模,去掉多余固化PDMS,留下EHT成型模具阴模,如图2A所示。阴模121℃高压蒸汽灭菌20min,用1‰的F-127浸润PDMS阴模30min,以增加阴模表面亲水性;后用超纯水清洗3次,完全吸干水分,备用。
使用激光雕刻机,将尼龙纸(Cerex)切割成外边长8.0mm,内边长7.0mm的EHT支撑纸框,用以支撑EHT。将所得到的支撑纸框121℃高压蒸汽灭菌20min,用无菌水清洗三次,完全吸干水分后,放置在EHT成型模具中,如图2B。使用标本针将支撑纸框的四角固定在成型模具上,备用。
向实施例1中所得心肌细胞中加入Ⅰ型胶原酶(Sigma),使心肌细胞团脱离细胞板。消化条件为:温度37℃、5%CO2,消化时间1h。将消化所得细胞及酶液转移至15mL离心管中,静置沉积,吸去上层清液,保留下层细胞。向细胞中直接加入10mL高糖DMEM(Gibco),稀释终止胶原酶反应,静置沉积;吸去上层清液,保留下层细胞。向细胞中直接加入10mL高糖DMEM,继续洗去残余酶液,静置沉积;吸去上层清液,保留下层细胞。向所得到的细胞直接加入1mL胰酶(Thermo Fisher),期间不断摇晃离心管以分散细胞团。消化条件:温度37℃(水浴锅),消化时间5min。向所得到的细胞悬液中等比例加入消化终止液,并用枪头轻轻吹打细胞悬液,使心肌细胞团充分分离成单细胞。离心所得细胞悬液,离心条件为:1000rpm,3min;吸去上层清液,保留下层细胞待用。
将所得到的细胞用水凝胶培养A液重悬,一份水凝胶培养A液重悬1.0-1.5×106细胞。向所得到的细胞悬液中加入等体积的水凝胶培养B液,组成水凝胶心肌组织液。将所得到的水凝胶心肌组织液迅速转移到EHT成型模具中,细胞接种密度为1.0-1.5×106/EHT,置于5%CO2,37℃恒温培养箱中固化30min,如图2中C、D所示。
将所得到的EHT用镊子转移到24孔板中,使用EHT培养基,放置在摇床上培养3天,如图2中E、F。为防止细胞死亡,培养第一天加入10μM Y27632(Tocris),培养第1天即可明显观察到EHT自发跳动,如图3。
本实施例中涉及的消化终止液按照如下方法配置得到:1mL高糖DMEM+1mL胎牛血清(ABW)+终浓度20μg/mL DNA酶(Magen)。
水凝胶培养A液按照如下方法配置得到:配置A液Ⅰ,Low Glucose DMEM(Gibco)+10%FBS(ABW)+1%PS(Gibco)+终浓度2μg/mL维生素B12(Sigma)+终浓度1mg/mL氨基乙酸(Sigma)。将58μL A液Ⅰ与2.4μL thrombin(50U/mL)(Sigma)混合,组成一份水凝胶培养A液。
水凝胶培养B液按照如下方法配置得到:配置B液Ⅰ,Low Glucose DMEM+20%FBS+1%PS+终浓度4μg/mL维生素B12+终浓度2mg/mL氨基乙酸(sigma)。将24μL B液Ⅰ+24μLFibronectin(Sigma)+12μL Matrigel(Corning)混合组成一份水凝胶培养B液。
EHT培养基按照如下方法配置得到:RPMI1640+50X不含胰岛素B27+1%PS+终浓度0.4mg/mL抗坏血酸+终浓度2mg/mL氨基乙酸(sigma)+终浓度0.45μM 1-thioglycerol(Gibco)+100X Non-Essential Amino Acid(Gibco)+100X Sodium Pyruvate(Gibco)。
实施例3工程化心肌组织片冷冻损伤与自我修复
使用3D打印机(Photon),以光固化树脂为材料制作损伤器,如图4A,具体为设置在基座上的、带有尖端的柱状损伤器。使用3D打印机,以光固化树脂为材料制作损伤底座阳模。
称取72克PDMS原液并加入8克交联剂充分搅拌混匀,倒入放有损伤底座阳模的玻璃缸中,65℃固化PDMS 4小时。分离损伤底座阳模,去掉多余固化PDMS,留下损伤底座阴模,如图4B。将损伤底坐阴模在121℃高压蒸汽灭菌20min,用无菌水清洗三次,完全吸干,待用。
将PDMS倒入24孔板的板孔中,覆盖底部,在65℃固化。将所得到的PDMS 24孔板用75%酒精浸泡30min;之后,用无菌水清洗3次,待用。
将实施例2中所得到的EHT用镊子转移至前述得到的损伤底座阴模中。
将损伤器尖端浸润在液氮中15s,获得冷冻损伤器。将冷冻损伤器尖端迅速垂直刺入损伤底座阴模中的EHT中心,上下按压,保证EHT被刺穿,如图4C,得到损伤EHT。
将损伤EHT转移至前述所得PDMS 24孔板中,并用标本针(FST)固定EHT,使用愈合培养基,培养4-6天,每天用倒置显微镜观察、记录损伤EHT愈合情况并统计每天损伤区域面积,将当日损伤区域面积比上初始损伤区域面积记为当日相对损伤面积。
愈合培养基按照如下方法配置得到:EHT培养基+5%FBS。培养条件为:温度37℃、二氧化碳浓度5%,摇床培养,每两天换一次液。
经过多组多批次实验,发现EHT在损伤后可在愈合培养基中自我愈合至物理损伤区域完全修复。通过分析实施例1中得到的心肌细胞纯度与本实施例中EHT损伤愈合相对速度,发现愈合速度与EHT中心肌细胞纯度有关,如图5,即EHT中心肌细胞纯度越高(>90%),损伤愈合速度越慢。
以cTNT(abcam)、VIM(abcam)、Fibronectin(abcam)、collagenⅠ(Invitrogen)、为一抗,对愈合的EHT进行免疫荧光检测,发现愈合区域主体为成纤维细胞,且存在以Ⅰ型胶原、纤连蛋白为主的细胞外基质,如图6。
重复损伤及愈合培养步骤,换用分别添加终浓度为10μM Y27632(Tocris)、10ng/mL Rho Activator Ⅱ(Cytoskeleton)的愈合培养基培养同批损伤EHT。观察到添加了Y27632的损伤EHT愈合速度相较未添加Y27632组更快,而Rho Activator Ⅱ组愈合速度变慢,如图7。
按上述方法进行检测,发现Y27632处理的损伤EHT能够更快愈合,同时愈合区域纤维细胞占比减少,心肌细胞占比增加,说明Y27632干预可以减轻纤维化,如图8。以αSMA(abcam)为一抗,对愈合的EHT进行免疫荧光检测,结果如图9所示,Y27632作为Rock信号通路抑制剂,通过减少损伤愈合区域成纤维细胞的活化来促进心肌细胞再生,相反,RhoActivator Ⅱ作为Rock信号通路激活剂则导致损伤愈合区域成纤维细胞过度活化进而导致愈合受阻。
在利用模型进行其他药物筛选或检测时,方法同上。
实施例4高通量药物筛选的人工心肌组织纤维化模型制作
将96孔PCR管置于96孔PCR板架上,并用刀片切去PCR板露出管架的底端部分,单个96孔管口切面直径为4.5mm,如图10A。使用激光雕刻机在96孔管口切面对应位置切割尼龙纸(Cerex),在每个切面处留下外圈直径5.0mm、内圈直径4.2mm的尼龙纸圆环,圆环用于支撑固化后的EHT,如图10B。
将加热式磁力搅拌器温度设为165℃,通过热压将尼龙纸圆环固定到切割后的96孔PCR板上,制成96孔EHT支型支架装置,如图10C-D,装置用75%酒精浸泡30min、超纯水清洗3次后备用。
使用熔融沉积型3D打印机制成96孔EHT成型装置阳模,阳模具体尺寸为120mm×75mm×5mm底座,其上均匀分布96个直径5.0mm、高1.5mm圆柱,每个相邻圆柱中心距离为9.0mm。
称取117克PDMS原液并加入13克交联剂充分搅拌混匀,倒入放有阳模的玻璃缸中,65℃固化PDMS 4小时。分离阳模,去掉多余固化PDMS,留下96孔EHT成型装置PDMS阴模,阴模上有96个直径5.0mm、深1.5mm的圆形凹槽。阴模121℃高压蒸汽灭菌20min后备用。
将96根高4mm、直径1mm无菌钢针分别插入PDMS阴模各圆形凹槽正中央,制成EHT损伤装置。
用1‰的F-127另外浸润无钢针的PDMS阴模30min,后用超纯水清洗3次。按实施例1中的步骤准备水凝胶心肌组织液,迅速滴入PDMS阴模凹槽内,将96孔EHT支架装置放在PDMS阴模上,如图10E,保证每个尼龙纸圆环充分浸入PDMS阴模每个凹槽中的水凝胶心肌组织液里,连同PDMS阴模一起放入37℃、5%CO2培养箱20min,使96孔EHT成型。
将所得到的96孔EHT连同支架装置一同放入96孔培养板中,使用如实施例1中的EHT培养基,置于摇床上培养72h。
将96孔EHT损伤装置浸入液氮15s,取出后钢针尖端朝上立刻平放于实验台上,迅速将96孔EHT垂直穿过钢针,确保每根液氮钢针正对每个96孔EHT中心,上下平移96孔EHT支架装置使钢针完全穿透EHT,如图10F。
将所得损伤后96孔EHT放回96孔培养板,如图11,使用愈合培养基以及前述分别添加终浓度为10μM Y27632(Tocris)、10ng/mL Rho Activator Ⅱ(Cytoskeleton)的小分子干预培养基培养4-6天,期间观察愈合情况。
本实施例中借助96孔PCR管以及切割的纸环制备EHT支型支架,其他实施例中也可以直接定制带纸环的插入式培养支架,培养支架上具有多个端部开口的中空管,纸环位于每个中空管的端部,且纸环内径小于中空管的内径;并制备与插入式培养支架配合的EHT成型装置阴模,阴模上包括若干个供中空管插入的凹槽,凹槽内倒入水凝胶心肌组织液,中空管插入后,纸环完全浸没在组织液中,固化后的EHT组织片被支撑在纸环内;另外,还需准备EHT损伤装置,损伤装置包括若干个供中空管插入的凹槽,且在凹槽中部竖直设置有柱状的损伤器,带有EHT组织片的插入式培养支架的中空管插入冷冻后的损伤装置的凹槽内,同时,冷冻后的损伤器刺穿EHT组织片,获得损伤模型。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种人工心肌组织纤维化模型的制备方法,其特征在于,包括:
将人多能干细胞或人胚胎干细胞通过小分子分阶段调控WNT信号通路,诱导其分化为心肌细胞;
使用细胞外基质纤维蛋白原与Matrigel作为心肌细胞之间的骨架结构,构建工程化心肌组织片;
制备柱状损伤器,冷冻后插入工程化心肌组织片中,得到损伤EHT并诱导其体外自我修复产生以非心肌细胞为主体的纤维化区域,即人工心肌组织纤维化模型。
2.根据权利要求1所述的一种人工心肌组织纤维化模型的制备方法,其特征在于:所述小分子包括CHIR99021和IWR1。
3.根据权利要求1所述的一种人工心肌组织纤维化模型的制备方法,其特征在于:将心肌细胞消化、胰酶酶解后得到单细胞,用水凝胶培养A液重悬并加入包含Fibronectin和Matrigel水凝胶培养B液,组成水凝胶心肌组织液,将水凝胶心肌组织液转移至EHT成型模具中,培养箱培养、固化后得到工程化心肌组织片,至于EHT培养基中培养。
4.根据权利要求3所述的一种人工心肌组织纤维化模型的制备方法,其特征在于:所述水凝胶培养A液包括A液Ⅰ:Low Glucose DMEM、10%FBS、1%PS、终浓度2μg/mL维生素B12、终浓度1mg/mL氨基乙酸,将58μL A液Ⅰ与2.4μL thrombin(50U/mL)混合即可;
所述水凝胶培养B液包括B液Ⅰ:Low Glucose DMEM、20%FBS、1%PS、终浓度4μg/mL维生素B12、终浓度2mg/mL氨基乙酸,将24μL B液Ⅰ与24μL Fibronectin、12μL Matrigel混合组成水凝胶培养B液。
5.一种高通量药物筛选人工心肌组织纤维化模型的制备方法,包括:
切去多孔PCR管每个管的底部,并在每个切口处固定环状纸环,制得多孔EHT支型支架装置;
制备PDMS EHT成型模具,所述成型模具包括与所述多孔PCR管对应的圆形凹槽;
在EHT成型模具的凹槽中央插入损伤器,制成EHT损伤装置;
在另外的EHT成型模具的凹槽中加入水凝胶心肌组织液,所述水凝胶心肌组织液的制备方法如权利要求3;
将多孔EHT支型支架装置放在EHT成型模具上,并使纸环浸入凹槽的水凝胶心肌组织液中,培养,一次性得到多个相互独立的EHT;
将EHT损伤装置冷冻后,使损伤器穿过多孔EHT,每根损伤针对应每个孔的EHT中心,得到高通量药物筛选人工心肌组织纤维化模型。
6.根据权利要求5所述的一种高通量药物筛选人工心肌组织纤维化模型的制备方法,其特征在于:所述PDMS EHT成型模具的制备方法包括:首先制备EHT成型装置阳模,所述阳模上分布有与多孔PCR管对应的多个圆柱;将PDMS原液与交联剂混合后,倒入放有所述阳模的器皿中,固化后,分离阳模,去除多于的PDMS,即得带有圆形凹槽的PDMS EHT成型模具。
7.如权利要求1-6任一所述的制备方法制备的人工心肌组织纤维化模型在心肌组织纤维化治疗或诊断的药物筛选或药物检测中的应用。
8.根据权利要求7所述的应用,其特征在于:应用方法包括将所得人工心肌组织纤维化模型至于含有待测药物的培养基中培养,通过观察损伤愈合情况判断药物效果。
9.ROCK/Rho信号通路抑制剂在制备治疗冷冻损失引起的心肌组织纤维化的药物中的应用。
10.一种高通量组织纤维化模型制备装置,其特征在于:包括插入式培养支架,所述插入式培养支架上设有多个端部开口的中空管,每个所述中空管的开口端设有纸环,所述纸环内径小于所述中空管的开口端内径;
还包括与所述插入式培养支架配套使用的组织片成型装置阴模,所述成型装置阴模上设有供多个所述中空管插入的凹槽,所述凹槽内承装有组织液,所述中空管插入所述凹槽内,所述纸环浸没在组织液中,组织液固化后,组织片被支撑在纸环内;
还包括与所述插入式培养支架配套使用的组织片损伤装置,所述损伤装置上设有供多个所述中空管插入的损失凹槽,所述损伤凹槽中部竖直设有柱状的损伤器,所述插入式培养支架的中空管插入所述损伤装置中,所述损伤器穿过被支撑在纸环内的组织片。
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CN117159210A (zh) * | 2023-08-02 | 2023-12-05 | 四川大学 | 一种冰冻损伤诱导的口颌肌纤维化模型的建立方法 |
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