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Using high-content Screening Analysis System to Analyse the Expression and Distribution of Certain Protein in Stomach Organoid   

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摘要:类器官 (Organoid) 是源自多能干细胞或器官祖细胞的体外衍生3D细胞聚集体,有自我更新、自我组织能力且具有完整器官结构和功能 (Lancaster and Knoblich, 2014)。自报道来(Sato and Vries, 2009),类器官得到了广泛关注,培养技术日趋成熟,正成为药物筛选、个性化治疗和理解人体器官的重要模型。与2D细胞相比,3D类器官保留了原器官组织结构和生物信息,已被证明为目前最接近体内生理环境和临床相似度最高的体外研究模型。建立成熟的类器官模型,并验证类器官模型的可靠性,是类器官相关研究的重要步骤,本文简要介绍了人胃类器官模型的构建过程,并利用高内涵成像分析系统成像和分析F-actin蛋白的极性分布,该蛋白的极性分布是胃类器官建立成功的重要标记之一。

关键词: 高内涵, 类器官, F-actin, 3D分析

材料与试剂

  1. 96孔板 (PerkinElmer,96 CellCarrier ultra)
  2. 人源组织保存液 (京创和益, catalog number: CH-0001A)
  3. 人胃类器官培养基 (京创和益, catalog number: CH-0302A)
  4. Advanced DMEM/F12 (Thermo Fisher Scientific, catalog number: 12634010)
  5. HEPES (Thermo Fisher Scientific, catalog number: 15630080)
  6. UltraPure 0.5M EDTA, pH 8.0 (Thermo Fisher Scientific, catalog number: 15575020)
  7. Penicillin-Streptomycin (Thermo Fisher Scientific, catalog number: 15140122)
  8. Primocin (InvivoGen, catalog number: ant-pm-1)
  9. GlutaMAX Supplement (Thermo Fisher Scientific, catalog number: 35050061)
  10. Matrigel BD (Biosciences, catalog number: 356231)
  11. B-27 Supplement (Thermo Fisher Scientific, catalog number: 17504001)
  12. N-Acetyl-L-cysteine (Sigma-Aldrich, catalog number: A9165)
  13. [Leu15]-Gastrin I human (Sigma-Aldrich, catalog number: G9145)
  14. Recombinant Human EGF (R&D, catalog number: 236-EG-200)
  15. Recombinant Human FGF10 (R&D, catalog number: 345-FG-25)
  16. A83-01 (Tocris, catalog number: 2939/10)
  17. Y-27632 (Tocris, catalog number: 1254/10
  18. Wnt3A conditioned medium (京创和益, catalog number: CH-M001A)
  19. R-spondin1 conditioned medium (京创和益, catalog number: CH-M002A)
  20. Noggin conditioned medium (京创和益, catalog number: CH-M003A)
  21. Alexa FluorTM 488 Phalloidin (Thermo Fisher Scientific, catalog number: A12379)
  22. Hoechst 33342 (Sigma-Aldrich, catalog number: 14533)

仪器设备

  1. 细胞培养箱 (Eppendorf, model: C170i)
  2. 离心机 (Eppendorf, model: 5810R)

软件

  1. Operetta CLS高内涵成像分析系统 (PerkinElmer, USA)
  2. Harmony分析软件 (PerkinElmer, USA)

实验步骤

一、人源胃类器官的培养
手术切除人正常胃样品保存于组织储存液中,EDTA法分离人源胃干细胞 (具体方法此处不再赘述,可通过邮箱交流),用Matrigel重悬后 (每孔40 μL),种植于24孔板 (低吸附) 中,放置于细胞培养箱中,待15 min后,Matrigel凝固,加入500 μL人源胃类器官培养基。细胞培养箱中培养4-6天,期间换液一次。
人源胃类器官培养基为本公司产品,可邮箱询价购买,或用以下试剂部分替代:Advanced DMEM/F12, 1x GlutaMax, 1x HEPES, 1x P/S, 50% Wnt3A conditioned medium, 10% R-spondin1 conditioned medium, 10% Noggin conditioned medium, 1x B27, 50 ng/mL EGF, 200 ng/mL FGF10, 1 mM N-Acetylcystenine, 1 nM Gastrin, 2 μM A83-01, 10 μM Y-27632 and 1 μg/μL primocin

二、人源胃类器官免疫荧光染色

  1. 培养结束后,用PBS轻柔的重悬清洗类器官,加入4% paraformaldehyde (PFA)室温固定1 h。
  2. PBS轻柔清洗类器官4次,200 × g室温离心2 min收集类器官,0.5% Triton X100室温孵育30 min以透膜。
  3. PBS轻柔清洗类器官4次,200 × g室温离心2 min收集类器官,加入PBS稀释的Phalloidin (F-actin蛋白荧光染料) 和Hoechst 33342 (细胞核荧光染料) (终浓度为试剂说明书推荐工作浓度),室温孵育1 h。
  4. PBS轻柔清洗类器官4次,200 × g室温离心2 min收集类器官。

三、高内涵系统成像
  1. 将类器官转移到96孔板 (PerkinElmer,96 CellCarrier ultra) 中,每个孔中放置5-20个类器官。
  2. 高内涵成像:打开高内涵,打开Harmony软件,首先设置5倍空气镜成像条件,对整孔进行成像,non-confocal模式,通道为Alex 405 + Alex 488 + brightfield,每个视野之间设置5% overlap,保存5倍成像条件并运行一遍;成像后,在Image analysis中编写分析算法,使得高内涵分析软件可以根据5倍成像结果找到类器官所在位置,具体算法为:Input Image (create global image,individual plane,FFC none)——Find Image Region (Alex 405 (global))——Calculate Morphology Properties (Area + Roundness)——select population (Area > 100 & Area < 8000 & Roundness > 0.8)——Determine Well Layout (Rescan 20x)——Define Results,保存该算法,将该算法与5倍成像条件关联,重新保存5倍成像条件;而后设置20倍水浸式物镜成像条件,confocal模式,通道为Alex 405 + Alex 488 + brightfield,多层成像(-10 — +488 μm,每隔2 μm一层,共250层),保存20倍成像条件。设置完成后,采用precision scan拍摄模式,载入5倍成像条件和20倍成像条件,对样品进行先后两次成像,最终获得Alex 405 + Alex 488 + brightfield三个通道下,250层共聚焦对多个类器官成像的图片结果。

四、高内涵结果分析
  1. 在Harmony软件中,选择Image analysis界面,Input Image模块中,选择“3D analysis”,对类器官进行立体重构
  2. 使用Find Image Region模块找到类器官
  3. 使用Select Region模块分别识别类器官内层和外层
  4. 使用Calculate Intensity Properties模块分布计算内层和外层的Alex 488通道荧光强度平均值和总和值 (mean & sum)
  5. 在Define Results模块选择要导出的整孔与单个类器官分析结果
  6. 保存分析程序,在Evaluation界面运行该分析程序


    图1. 高内涵结果分析流程


结果与分析

  1. 成像结果(图2):


    图2. 人胃类器官高内涵成像结果,左:最大亮度投影视图,右:3D重构视图

  2. 单个类器官分析结果见图3,由此可知,在这个类器官中,F-actin蛋白的分布,即绿色荧光信号 (Alex 488通道) 的荧光强度,内层要显著高于外层,这与预期与报道一致,即胃类器官生长过程中需要维持F-actin蛋白的极性分布:


    图3. 人胃类器官分层分析F-actin分布情况及结果

  3. 整孔分析数据可通过Harmony软件作图,得出以下结果图,由此可知内层的平均荧光强度 (mean) 显著高于外层,但内层的总荧光强度 (sum) 显著低于外层,这主要是由于外层体积要比内层大很多导致的,该结果也与预期相符(图4):


    图4. 人胃类器官F-actin极性分布统计作图结果

致谢

感谢中科院过程所生化工程国家重点实验室PerkinElmer转化医学工程共建实验室提供高内涵成像分析系统及PerkinElmer技术人员李想的支持和辅助。

参考文献

  1. Lancaster, M. A., Knoblich, J. A. (2014). Organogenesis in a dish: modeling development and disease using organoid technologies. Science 345(6194): 1247125.
  2. Sato, T., Vries, R. G. (2009). Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459(7244): 262-5.
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Copyright: © 2022 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:李鹏飞. (2022). 高内涵成像分析系统用于类器官蛋白表达分析. // 高内涵成像及分析实验手册. Bio-101: e1010889. DOI: 10.21769/BioProtoc.1010889.
How to cite: Li, P. F. (2022). Using high-content Screening Analysis System to Analyse the Expression and Distribution of Certain Protein in Stomach Organoid. // High-Content Imaging and Analysis Protocol eBook. Bio-101: e1010889. DOI: 10.21769/BioProtoc.1010889.
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