Min Lu, Kevin Su, Nick Asbrock, Vi Chu
Lung organoids are useful three-dimensional (3D) cell culture models for studying human lung development and respiratory diseases including viral infections (SARS-CoV, H1N1, MERS), cystic fibrosis, asthma/COPD, exposure to air pollution and the effects of smoking. Unlike traditional immortalized pulmonary cell lines and primary cells, lung organoids contain various differentiated cell types with complex tissue architecture that more closely resemble in vivo tissues and functionality. Additionally, lung organoids can be derived from small amounts of patient tissues or pluripotent stem cells to create living biobanks that facilitate personalized biomedical research.
The 3dGRO™ Human Lung Organoid Culture System is a serum-free, multi-stage culture system for efficient differentiation of human induced pluripotent stem (iPS) cells to mature lung organoids that structurally resemble the in vivo branching airway and early alveolar structures. Using the 3dGRO™ Human Lung Organoid Culture System, large numbers of mature lung organoids may be generated that express appropriate markers indicative of multiple cell types found in the mature lung and airways including SFTPB and SFTPC (surfactant protein B and surfactant protein C) found in type II alveolar epithelial (ATII) cells, MUC5AC (airway goblet cells), EpCAM, Sox9 and Nkx2.1 (pulmonary endoderm), Acetyl-α-Tubulin (ciliated cells) and the mesenchymal marker vimentin. Additionally, lung organoids also express angiotensin-converting enzyme 2 (ACE2), the receptor for the novel SARS-CoV-2 virus that causes COVID-19, along with TMPRSS2, the serine protease that enhances SARS-CoV-2 viral entry.
Figure 1. Human lung organoid differentiation workflow. Human pluripotent stem cells are differentiated into definitive endoderm cells using a 4-day induction medium. During days 4-8, induction media (SCM305, SCM306) directs human definitive endoderm cells toward anterior foregut endoderm (AFE). AFE cells may be cryopreserved (SCC301) or further differentiated into branching lung bud organoids using 3dGRO™ Lung Organoid Branching Medium (SCM307) and further matured into branching and alveolar lung organoids using 3dGRO™ Lung Organoid Maturation Medium (SCM308).
Step 1: Differentiation of Human iPS Cells into Definitive Endoderm (Day 0-4)
Note: Start with high-quality undifferentiated human ES/iPS cells (SCC271) that are ~70-80% confluent and contain <5% differentiated cells. The following protocol is for differentiation of one well of a six-well tissue culture-treated plate. Indicated volumes are for a single well. Adjust volumes as necessary.
Step 2: Differentiation of DE Cells into Anterior Foregut Endoderm Cells (Day 4-8)
Note: High cell density should be observed at both day 4 of definitive endoderm cells and also at day 8 after differentiation to anterior foregut endoderm (AFE) cells. The protocol below provides a reference seeding density in a 6-well plate format. Adjust the seeding density for other plate formats.
Figure 2. Anterior foregut endoderm (AFE) marker expression. Approximately 95-100% of AFE cells are double positive for Sox2 (A, C, AB5603A4) and Pax9 (B, C). Approximately 20-30% of AFE cells are double positive for EpCAM (D, F, MAB4444) and Pax9 (E, F).
Step 3: Differentiation of AFE Cells into Lung Bud Organoids (Day 8-25)
Figure 3. Human lung bud organoids (LBOs) in suspension cultures. Morphology of immature human iPS cell-derived LBOs on day 23 of differentiation. The lower panels show optimal organoids with folded structures (arrows) that may be selected for further maturation in Matrigel® sandwiches using 3dGRO™ Lung Organoid Maturation Medium (step 4).
Step 4: Differentiation of Lung Bud Organoids (LBOs) into Mature Lung Organoids (Day 25-60)
Figure 4. Time course for lung organoid maturation. Tracking human iPS cell-derived lung organoid differentiation in two individual wells. At day 20-25, around 4-6 immature LBOs were embedded in a Matrigel® matrix using 3dGRO™ Lung Organoid Maturation Medium. Various morphologies were observed which included branching structures and/or rounded expansion at the tips with dense materials in the center resembling alveolospheres. When the culture is maintained beyond day 60-70, some large rounded structures may rupture, releasing a mucus-like material.
Figure 5. Lung organoid marker expression. Day 70 mature lung organoids derived from human peripheral blood mononuclear cells (PBMCs) and HFF human iPS cells express markers of surfactant-producing type II alveolar epithelial cells (SFTPC and SFTPB), airway goblet cells (Muc5AC), pulmonary endoderm (EPCAM, Sox9, NKX2.1) and ciliated cells (Acetyl-α-Tubulin). Nuclei are counterstained with DAPI.
Figure 6. Lung organoids can be used to study SARS-CoV-2 respiratory viral infections. Lung bud organoids express ACE2, the SARS-CoV-2 binding receptor (A) and the serine protease, TMPRSS2 (B), that enhances SARS-CoV-2 viral entry.
Airway Epithelial Lung Organoids Troubleshooting Guide
Problem 1: After transferring lung bud organoids (LBOs) at D20-25 to Matrigel® sandwiches, no branching or alveolar structures are observed after two weeks.
Problem 2: Floating LBOs attach to the bottom of the plate during suspension culture.
Use ultra-low attachment 24-well plates rather than tissue culture treated 24-well plates or Petri dishes.
Problem 3: After changing the medium for the Matrigel® sandwiches, the top layer appears soft or collapses.
Add 75 µL of 100% Growth Factor Reduced (GFR) Matrigel® matrix to the top layer of the sandwich instead of 50 µL. Put the 24-well plates in the incubator for ≥30 minutes to ensure that the Matrigel® sandwiches have solidified.
Problem 4: The proportion of c-kit+/CXCR4+ positive cells is below 80% at day 4.
A compact c-kit+/CXCR4+ cell population should be observed at day 4 with positive cells 80-90% or higher. It is not recommended to continue the differentiation without successful definitive endoderm induction at day 4. Restart using high quality, undifferentiated human iPS cell cultures.
Problem 5: During the later stages of maturation, the medium may turn yellow quickly.
During this time, the embedded organoids may enlarge and some may extend complex processes (Figure 5). Add 750 µL of 3dGRO™ Lung Organoid Maturation Medium (SCM308) to the top of the inserts and 500 µL into the wells underneath the inserts.