Improve Research Reproducibility A Bio-protocol resource
  • search
  • 提交稿件
  • 订阅
  • CN change language
bpdetail_icon_lock
Peer-reviewed

Establishing Bipotential Human Lung Organoid Culture System and Differentiation to Generate Mature Alveolar and Airway Organoids

建立双潜能的人肺类器官培养系统及分化产生成熟的肺泡和气道类器官

MC Man Chun Chiu
CL Cun Li
YY Yifei Yu
XL Xiaojuan Liu
JH Jingjing Huang
ZW Zhixin Wan
KY Kwok Yung Yuen
JZ Jie Zhou

发布: 2023年04月20日第13卷第8期 DOI: 10.21769/BioProtoc.4657 浏览次数: 2749

评审: Vivien Jane Coulson-ThomasSudhir VermaAnonymous reviewer(s)

download pdf 下载 PDF 下载 PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Cell Discovery, featuring study using the protocol.

Jun 2022

Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

Abstract

A robust in vitro model of the human respiratory epithelium, including the alveolar and the airway epithelium, is essential for understanding the biology and pathology of the human respiratory system. We previously described a protocol to derive human lung organoids from primary lung tissues. We now describe a protocol to induce bidirectional differentiation to generate mature alveolar or airway organoids. The lung organoids are consecutively expanded for over one year with high stability, while the differentiated alveolar and airway organoids morphologically and functionally simulate the human alveolar and airway epithelium to a near-physiological level. Thus, we establish a robust organoid culture system of the entire human respiratory epithelium, the first two-phase bipotential organoid culture system that enables long-term expansion and bidirectional differentiation of respiratory epithelial cells. The long-term expandable lung organoids and differentiated organoids generate a stable and renewable source of respiratory epithelial cells, enabling scientists to reconstruct and expand the human respiratory epithelium in culture dishes. The respiratory organoid system provides a unique and physiologically active in vitro model of the human respiratory epithelium for various applications, including studying respiratory viral infection, disease modeling, drug screening, and pre-clinical testing.


Graphical overview


Keywords: Lung organoids (肺类器官) search Airway organoids (气道类器官) search Alveolar organoids (肺泡类器官) search Long-term expansion (长期扩张) search Proximal differentiation (近端分化) search Distal differentiation (远端分化) search

Background

Recent advances in stem cell technology enable the generation of mini-organs or organs-in-a-dish, known as organoids. By definition, organoids are three-dimensional cultures derived from stem cells; they mimic the in vivo architecture and functionality of the corresponding tissue or organ (Lancaster and Knoblich, 2014; M. Li and Izpisua Belmonte, 2019; Schutgens and Clevers, 2020). Organoids have become a robust and innovative tool for modeling developmental biology, physiology, and pathology.

Organoids can be derived from either pluripotent stem cells or adult stem cells (ASC). When provided with appropriate niche factors, ASCs isolated from tissues can self-renew and self-organize into organ-like multicellular clusters composed of multiple tissue-specific cell types, which morphologically and functionally simulate the in vivo counterparts. The generation of the first ASC-derived organoid, the human intestinal organoid, was reported in 2009 (Sato et al., 2009 and 2011). Afterward, ASC-derived organoids were established for a variety of human organs and tissues, including the prostate (Chua et al., 2014; Karthaus et al., 2014), liver (Huch et al., 2013b;Hu et al., 2018), stomach (Wroblewski et al., 2015; Schlaermann et al., 2016), pancreas (Huch et al., 2013a), mammary gland (Sachs et al., 2018), and lung (Zhou et al., 2018; Sachs et al., 2019). These ASC-derived organoids retain the fundamental cellular, structural, and functional properties of the native organ and maintain genotypic and phenotypic stability during long-term culture.

The human respiratory tract is lined with two distinct types of epithelia: the airway and the alveolar epithelium. We established the long-term expandable ASC-derived human lung organoid from lung tissues in collaboration with Clevers’ lab (Zhou et al., 2018; Sachs et al., 2019). We further developed a proximal differentiation protocol and generated 3D and 2D airway organoids that morphologically and functionally phenocopy the airway epithelium to a near-physiological level (Zhou et al., 2018). A detailed protocol describing the derivation of lung organoids and the generation of differentiated airway organoids was published recently (C. Li et al., 2022). However, derivation of the alveolar epithelium, which consists of type 1 and type 2 alveolar epithelial cells (AT1 and AT2, respectively), from the lung organoids remained elusive.

We recently reported a bipotential lung organoid culture system that can generate both airway and alveolar epithelial cells (Chiu et al., 2022). Here, we provide a detailed protocol to generate 3D alveolar organoids from long-term expanding human lung organoids, the same source for generating airway organoids as previously reported. We also describe a protocol to generate optimized 2D airway organoids from lung organoids, which mimic the airway epithelium more favorably. Altogether, we established a bipotential lung organoid culture system that could enable bidirectional differentiation into alveolar organoids upon distal differentiation or airway organoids upon proximal differentiation. The lung organoids serve as a stable source for long-term expansion, while differentiated airway and alveolar organoids faithfully phenocopy the human airway and alveolar epithelium, respectively. These organoids are robust and physiologically active tools that are applicable to various experimental manipulations, to explore the biology and pathology of the human lungs.

Materials and Reagents

  1. Nunc 15 and 50 mL conical sterile polypropylene centrifuge tubes (Thermo Scientific, catalog numbers: 339650, 339652)

  2. Surgical scalpel blade No. 22 (Swann-Morton, catalog number: 0508)

  3. 100 mm TC-treated culture dish (Corning, catalog number: 430167)

  4. T175 cell culture flask (Greiner Bio-One, catalog number: 661175)

  5. Nunc non-treated 24-well plate (Thermo Scientific, catalog number: 144530)

  6. Nunclon Sphera 24-well plate (Thermo Scientific, catalog number: 174930)

  7. Costar 6.5 and 12 mm Transwell®, 0.4 μm pore polyester membrane inserts (Stem Cell Technologies, catalog numbers: 38024, 38023)

  8. 40 and 100 μm cell strainer (Falcon, catalog numbers: 352340, 352360)

  9. Steritop threaded bottle top filter (0.22 μm) (Merck Millipore, catalog number: SCGPS01RE)

  10. 10/100/1,000 μL QSP low retention filtered pipette tips (Thermo Scientific, catalog numbers: TFLR102-10-Q, TFLR113-100-Q, TFLR1121000-Q)

  11. 5/10/15 mL Stripette serological pipettes (Corning, catalog numbers: 4487, 4488, 4489)

  12. Pasteur pipettes length ca. 225 mm (Brand, catalog number: 747720)

  13. 5 mL round bottom polystyrene test tube (Falcon, catalog number: 352052)

  14. β-BODIPYTM FL C12-HPC (Invitrogen, catalog number: D3792)

  15. LysoTrackerTM red DND-99 (Invitrogen, catalog number: L7528)

  16. Matrigel growth factor–reduced (GFR) basement membrane matrix, phenol red–free, LDEV-free (Corning, catalog number: 356231)

  17. Advanced DMEM/F-12 (Gibco, catalog number: 12634010)

  18. DMEM (Gibco, catalog number: 10569010)

  19. ZeocinTM selection reagent (100 mg/mL) (Gibco, catalog number: R25005)

  20. GeneticinTM selective antibiotic (G418 sulfate) (50 mg/mL) (Gibco, catalog number: 10131035)

  21. HEPES (Gibco, catalog number: 15630056)

  22. GlutaMAX supplement (Gibco, catalog number: 35050061)

  23. Penicillin-Streptomycin (Gibco, catalog number: 15140122)

  24. Recombinant human Rspondin1 (Stem Cell Technologies, catalog number: 78213; Peprotech, catalog number: 120-38)

  25. Rspondin1 expressing 293T cell line (Sigma-Aldrich, catalog number: SCC111)

  26. Recombinant human Noggin (Stem Cell Technologies, catalog number: 78060; Peprotech, catalog number: 120-10C)

  27. B-27 supplement (50×), serum-free (Gibco, catalog number: 17504044)

  28. N-Acetyl-L-cysteine (Sigma-Aldrich, catalog number: A9165)

  29. Nicotinamide (Sigma-Aldrich, catalog number: N0636)

  30. Y-27632 dihydrochloride (Tocris, catalog number: 1254)

  31. A 83-01 (Tocris, catalog number: 2939)

  32. SB 202190 (Sigma-Aldrich, catalog number: S7067)

  33. Recombinant human KGF (FGF-7) (Peprotech, catalog number: 100-19)

  34. Recombinant human FGF-10 (Peprotech, catalog number: 100-26)

  35. Recombinant human heregulin β-1 (Peprotech, catalog number: 100-03)

  36. Primocin (InvivoGen, catalog number: ant-pm-1)

  37. Dexamethasone (Tocris, catalog number: 1126)

  38. 8-Bromo-cAMP, sodium salt (Tocris, catalog number: 1140)

  39. IBMX (Tocris, catalog number: 2845)

  40. CHIR 99021 (Tocris, catalog number: 4423)

  41. Recombinant murine WNT3A (Peprotech, catalog number: 315-20)

  42. L WNT3A cell line (ATCC, catalog number: CRL-2647)

  43. PneumaCult-ALI medium (StemCell Technologies, catalog number: 05001)

  44. Heparin solution (StemCell Technologies, catalog number: 7980)

  45. Hydrocortisone stock solution (StemCell Technologies, catalog number: 7925)

  46. DAPT (Tocris, catalog number: 2634)

  47. PIPES (Sigma-Aldrich, catalog number: P1851)

  48. Collagenase from Clostridium histolyticum (Sigma-Aldrich, catalog number: C9407)

  49. TrypLE select enzyme (10×), no phenol red (Gibco, catalog number: A1217701)

  50. Phosphate buffered saline (PBS) (Gibco, catalog number: 10010023)

  51. Fetal bovine serum (FBS), qualified, heat inactivated (Gibco, catalog number: 10082147)

  52. Buffer EL erythrocyte lysis buffer (Qiagen, catalog number: 79217)

  53. UltraPureTM 0.5 M EDTA, pH 8.0 (Invitrogen, catalog number: 15575020)

  54. Formaldehyde solution [i.e., 37% paraformaldehyde (PFA)] (Sigma-Aldrich, catalog number: 252549)

  55. Triton X-100 (Sigma-Aldrich, catalog number: X100)

  56. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418)

  57. DAPT (Sigma-Aldrich, catalog number: D9542)

  58. Phalloidin - Atto 647N (Sigma-Aldrich, catalog number: 65906)

  59. ProLongTM glass Antifade mountant (Invitrogen, catalog number: P36980)

  60. Glutaraldehyde (purchased from electron microscope unit, HKU)

  61. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 320331)

  62. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465)

  63. Basal medium (see Recipes)

  64. Expansion medium (see Recipes)

  65. Distal differentiation medium (DD medium) (see Recipes)

  66. Proximal differentiation medium (PD medium) (see Recipes)

  67. Rspondin1 conditioned medium (see Recipes)

  68. Noggin conditioned medium (see Recipes)

  69. WNT3A conditioned medium (see Recipes)

Equipment

  1. SterilGARD e3 Class II Type A2 biosafety cabinet (Baker Co, catalog number: SG404-INT)

  2. Forma Steri-Cycle i160 CO2 165 L incubator (Thermo Fisher Scientific, catalog number: 51030301)

  3. New Brunswick Innova® 44/44R stackable incubator shaker (Eppendorf, catalog number: M1282-0002)

  4. Centrifuge (Eppendorf, model: 5810R)

  5. FE20-Kit FiveEasy benchtop pH meter (Mettler Toledo, model: FE20-KIT)

  6. LightCycler 96 Instrument (Roche, catalog number: 381711)

  7. Inverted routine microscope (Nikon, model: Eclipse TS100)

  8. Confocal microscope (Zeiss, model: LSM 800)

  9. Transmission electron microscope (FEI, model: Tecnai G2 20 S-TWIN)

  10. Cell analyzer (BD Biosciences, model: FACSCanto II & LSRFortessa)

  11. Midi Plus pipetting controllers (Sartorius, model: 710931)

  12. Research Plus mechanical pipette (Eppendorf, catalog number: 3123000900)

Procedure

English
中文翻译

文章信息

版权信息

© 2023 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).

如何引用

Chiu, M. C., Li, C., Yu, Y., Liu, X., Huang, J., Wan, Z., Yuen, K. Y. and Zhou, J. (2023). Establishing Bipotential Human Lung Organoid Culture System and Differentiation to Generate Mature Alveolar and Airway Organoids. Bio-protocol 13(8): e4657. DOI: 10.21769/BioProtoc.4657.

ris ris Download Citation in RIS Format

分类

干细胞 > 类器官培养

细胞生物学 > 细胞分离和培养

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

0/150

tip 提问指南

+ 问题描述

写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。

post 发布问题
0 Q&A
pen 提交稿件