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干细胞类器官形成能力检测

Organoid Formation Efficiency Assay of Stem Cells

郭 郭旺昕*
田 田维新*
韩 韩帅
赵 赵宏伟
高 高栋

*Contributed equally to this work 发表时间: 2021年11月18日 DOI: 10.21769/BioProtoc.1010867 浏览次数: 2592

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高内涵成像及分析实验手册
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摘要：干细胞类器官形成能力的鉴定是干细胞研究领域经典的方法。随着类器官培养体系的日渐成熟，基于基质胶的3D培养的干细胞类器官形成实验也逐渐地被广泛地应用于体外干细胞潜能的鉴定。而前列腺的类器官培养体系是由小肠类器官培养体系优化而来。本文将利用基质胶3D培养体系以前列腺干细胞为例，分析前列腺干细胞体外类器官形成能力，探究前列腺干细胞是否具有更高的类器官形成能力，验证基于3D培养体系的类器官形成实验能够用于检测干细胞潜能。

Keywords: 前列腺干细胞 search 类器官培养 search 基质胶 search

材料与试剂

  1. 15 ml 离心管 (Greiner Bio-One, catalog number: 188261) 
  2. Plates, 24 well (Greiner Bio-One, catalog number: 662 102)
  3. Matrigel, growth factor reduced, Phenol Red-free (CORNING, catalog number: 356231)
  4. Advanced DMEM/F12 (adDMEM/F12; Life Technologies, catalog number: 12634-034)
  5. GlutaMAX, 100x (Life Technologies, catalog number: 35050-068)
  6. Penicillin-streptomycin (Life Technologies, catalog number: 15140-122)
  7. HEPES (Life Technologies, catalog number: 15630-056)
  8. B27 supplement, 50x (Life Technologies, catalog number: 17504-044)
  9. N-acetyl-l-cysteine (Sigma-Aldrich, catalog number: A9165)
  10. A83-01 (Tocris Bioscience, catalog number: 2939)
  11. Y-27632 dihydrochloride (Abmole Bioscience, catalog number: M1817)
  12. Human epidermal growth factor (EGF) (PeproTech, catalog number: AF-100-15)
  13. Recombinant human Noggin (Peprotech, catalog number: 120-10C)
  14. recombinant R-spondin 1 protein (R&D Systems, catalog number: 4645-RS-025)
  15. (DiHydro)testosterone (5α-Androstan-17β-ol-3-one) (Sigma-Aldrich, catalog number: A8380)
  16. DAPI (Invitrogen, catalog number: D21490, use concentration 1ng/μl)
  17. EpCAM-PE-Cy7 (Biolegend, catalog number: 118216, dilution rate1:500)
  18. Tacstd2-APC (R&D system, catalog number: FAB1122F, dilution rate1:20)
  19. Collagenase/Hyaluronidase (STEMCELL, catalog number: 7912) 
  20. 小鼠前列腺消化液
    成分终浓度
    Advanced DMEM/F12——
    Collagenase/Hyaluronidase10x稀释
    Y-2763210 μM
    HEPS100x稀释
    GlutaMAX100x稀释
    Penicillin-streptomycin100x稀释
    Primocin500x稀释

  21. 前列腺类器官培养基 (Gao et al., 2014 and 2016; Drost et al., 2016)
    细胞因子终浓度
    Advanced DMEM/F12——
    B2750x稀释
    N-acetylcysteine1.25 mM
    EGF50 ng/ml
    Noggin10%
    R-spondin 15%
    A83-01200 nM
    DHT1 nM
    Y-2763210 μM
    HEPS100x稀释
    GlutaMAX100x稀释
    Penicillin-streptomycin100x稀释
    Primocin500x稀释

仪器设备

  1. 多色干细胞流式细胞分选仪 (BD FACS Aria III) 
  2. 细胞培养箱 (Thermo) 
  3. 离心机 (Eppendorf) 
  4. 移液枪 (Eppendorf) 
  5. 细胞成像多功能微孔板检测仪 (BioTek, Cytation 5)

实验步骤

Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用格式：郭旺昕, 田维新, 韩帅, 赵宏伟, 高栋. (2021). 干细胞类器官形成能力检测. // 高内涵成像及分析实验手册. Bio-101: e1010867. DOI: 10.21769/BioProtoc.1010867.
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How to cite: Guo, W. X., Tian, W. X., Han, S., Zhao, H. W. and Gao, D. (2021). Organoid Formation Efficiency Assay of Stem Cells. // High-Content Imaging and Analysis Protocol eBook. Bio-101: e1010867. DOI: 10.21769/BioProtoc.1010867.
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