Pig and monkey are widely used models for exploration of human diseases and evaluation of drug efficiency and toxicity,but high cost limits their uses.Organoids have been shown to be promising models for drug test as ...Pig and monkey are widely used models for exploration of human diseases and evaluation of drug efficiency and toxicity,but high cost limits their uses.Organoids have been shown to be promising models for drug test as they reasonably preserve tissue structure and functions.However,colonic organoids of pig and monkey are not yet established.Here,we report a culture medium to support the growth of porcine and monkey colonic organoids.Wnt signaling and PGE2 are important for long-term expansion of the organoids,and their withdrawal results in lineage differentiation to mature cells.Furthermore,we observe that porcine colonic organoids are closer to human colonic organoids in terms of drug toxicity response.Successful establishment of porcine and monkey colonic organoids would facilitate the mechanistic investigation of the homeostatic regulation of the intestine of these animals and is useful for drug development and toxicity studies.展开更多
The recent advent of robust methods to grow human tissues as 3D organoids allows us to recapitulate the 3D architecture of tumors in an in vitro setting and offers a new orthogonal approach for drug discovery.However,...The recent advent of robust methods to grow human tissues as 3D organoids allows us to recapitulate the 3D architecture of tumors in an in vitro setting and offers a new orthogonal approach for drug discovery.However,organoid culturing with extracellular matrix to support 3D architecture has been challenging for high-throughput screening(HTS)-based drug discovery due to technical difficulties.Using genetically engineered human colon organoids as a model system,here we report our effort to miniaturize such 3D organoid culture with extracellular matrix support in high-density plates to enable HTS.We first established organoid culturing in a 384-well plate format and validated its application in a cell viability HTS assay by screening a 2036-compound library.We further miniaturized the 3D organoid culturing in a 1536-well ultra-HTS format and demonstrated its robust performance for large-scale primary compound screening.Our miniaturized organoid culturing method may be adapted to other types of organoids.By leveraging the power of 3D organoid culture in a high-density plate format,we provide a physiologically relevant screening platform to model tumors to accelerate organoid-based research and drug discovery.展开更多
类器官是一种来源于干细胞,在特定诱导条件下分化为与研究器官细胞组成类似的3D培养物。与传统细胞培养物相比,类器官更加接近研究器官的三维结构和特征,因此基于类器官进行研究更能体现所研究器官体内的真实反应。这一研究手段广泛应...类器官是一种来源于干细胞,在特定诱导条件下分化为与研究器官细胞组成类似的3D培养物。与传统细胞培养物相比,类器官更加接近研究器官的三维结构和特征,因此基于类器官进行研究更能体现所研究器官体内的真实反应。这一研究手段广泛应用于肿瘤药物筛选,具有广阔前景。文章目的是建立一种稳定的结肠类器官提取分离方法,使得到的类器官有良好的增殖活性;建立结肠类器官增殖活性评价方法,能够准确评价结肠类器官的增殖活性。采用方法为取大约5 cm C57BL/6小鼠的结肠,用胶原酶分离结肠隐窝,通过梯度离心纯化隐窝,最后将其用基质胶包埋接种在培养皿中,加入肠道类器官培养基培养6 d,最后使用光学显微镜拍照记录并量化类器官的出芽情况,通过qPCR检测干细胞标志物Lgr5的表达。结果显示提取得到的类器官较为纯净,通过光学显微镜可以记录类器官的出芽情况,计算平均每个类器官含有的隐窝数来量化类器官增殖活性,qPCR检测发现干细胞标志物Lgr5的表达与平均每个类器官含有的隐窝数变化一致,因此可以通过出芽情况和基因表达情况来表征类器官的增殖活性。文章建立的结肠类器官提取和分析方法可以用于研究在不同条件影响下结肠类器官的增殖变化,还可以研究药物对结肠类器官的影响机制,可以作为结肠体外研究的有力工具。展开更多
基金This work was supported by grants from the National Key Research and Development Program of China(2017YFA0103601)the National Natural Science Foundation of China(31730056 and 31988101)to YGC.
文摘Pig and monkey are widely used models for exploration of human diseases and evaluation of drug efficiency and toxicity,but high cost limits their uses.Organoids have been shown to be promising models for drug test as they reasonably preserve tissue structure and functions.However,colonic organoids of pig and monkey are not yet established.Here,we report a culture medium to support the growth of porcine and monkey colonic organoids.Wnt signaling and PGE2 are important for long-term expansion of the organoids,and their withdrawal results in lineage differentiation to mature cells.Furthermore,we observe that porcine colonic organoids are closer to human colonic organoids in terms of drug toxicity response.Successful establishment of porcine and monkey colonic organoids would facilitate the mechanistic investigation of the homeostatic regulation of the intestine of these animals and is useful for drug development and toxicity studies.
基金This research was supported by the NCI Cancer TargetDiscovery and Development(CTD^2)Network(1U01CA217875 toH.F.and 1uo1CA217851 to C.J.K.)the RAS Synthetic LethalNetwork(RSLN+4 种基金1UO1CA199241 to C.J.K.)the Emory LungCancer SPORE(NIH P5OCA217691)the Winship Cancerlnstitute(NIH 5P30CA138292)the Emory WHSC 10x SingleCell Sequencing Seed Grant(X.M.and Y.D.)Emory WoodruffHealth Sciences Center Synergy Award,and the lmagine,lnnovate and Impact(3)Funds from the Emory School ofMedicine and through the Georgia CTSA NIH award(UL1-TRO02378).
文摘The recent advent of robust methods to grow human tissues as 3D organoids allows us to recapitulate the 3D architecture of tumors in an in vitro setting and offers a new orthogonal approach for drug discovery.However,organoid culturing with extracellular matrix to support 3D architecture has been challenging for high-throughput screening(HTS)-based drug discovery due to technical difficulties.Using genetically engineered human colon organoids as a model system,here we report our effort to miniaturize such 3D organoid culture with extracellular matrix support in high-density plates to enable HTS.We first established organoid culturing in a 384-well plate format and validated its application in a cell viability HTS assay by screening a 2036-compound library.We further miniaturized the 3D organoid culturing in a 1536-well ultra-HTS format and demonstrated its robust performance for large-scale primary compound screening.Our miniaturized organoid culturing method may be adapted to other types of organoids.By leveraging the power of 3D organoid culture in a high-density plate format,we provide a physiologically relevant screening platform to model tumors to accelerate organoid-based research and drug discovery.
文摘类器官是一种来源于干细胞,在特定诱导条件下分化为与研究器官细胞组成类似的3D培养物。与传统细胞培养物相比,类器官更加接近研究器官的三维结构和特征,因此基于类器官进行研究更能体现所研究器官体内的真实反应。这一研究手段广泛应用于肿瘤药物筛选,具有广阔前景。文章目的是建立一种稳定的结肠类器官提取分离方法,使得到的类器官有良好的增殖活性;建立结肠类器官增殖活性评价方法,能够准确评价结肠类器官的增殖活性。采用方法为取大约5 cm C57BL/6小鼠的结肠,用胶原酶分离结肠隐窝,通过梯度离心纯化隐窝,最后将其用基质胶包埋接种在培养皿中,加入肠道类器官培养基培养6 d,最后使用光学显微镜拍照记录并量化类器官的出芽情况,通过qPCR检测干细胞标志物Lgr5的表达。结果显示提取得到的类器官较为纯净,通过光学显微镜可以记录类器官的出芽情况,计算平均每个类器官含有的隐窝数来量化类器官增殖活性,qPCR检测发现干细胞标志物Lgr5的表达与平均每个类器官含有的隐窝数变化一致,因此可以通过出芽情况和基因表达情况来表征类器官的增殖活性。文章建立的结肠类器官提取和分析方法可以用于研究在不同条件影响下结肠类器官的增殖变化,还可以研究药物对结肠类器官的影响机制,可以作为结肠体外研究的有力工具。
