We thank the Center of PanorOmic Sciences and Electron Microscope Unit, Li Ka Shing Faculty of Medicine, University of Hong Kong, for assistance in confocal imaging and flow cytometry. This work was partly supported by funding from Health and Medical Research Fund (HMRF, 17161272 and 19180392) of the Food and Health Bureau; General Research Fund (GRF, 17105420) of the Research Grants Council; and Health@InnoHK, Innovation and Technology Commission, the Government of the Hong Kong Special Administrative Region.

All experimentation using human tissues described herein was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW13-364 and UW21-695). Informed consent was obtained from patients before tissue collection.
1. Derivation of human lung organoid
<ol>
	<li>Preparation of experimental materials
	<ol>
		<li>Prepare basal medium by supplementing advanced DMEM/F12 medium with 2 mM glutamine, 10 mM HEPES, 100 U/mL of penicillin, and 100 &#181;g/mL of streptomycin.</li>
		<li>Prepare human lung organoid expansion medium by supplementing basal medium with 10% R-spondin 1 conditioned medium, 10% Noggin conditioned medium, 1x B27 supplement, 1.25 mM N-acetylcysteine, 10 mM nicotinamide, 5 &#181;M of Y-27632, 500 nM of A-83-01, 1 &#181;M of SB202190, 5 ng/mL of fibroblst growth factor (FGF)-7, 20 ng/mL of FGF-10, and 100 &#181;g/mL of primocin (see Table of Materials). 
		NOTE: The R-spondin 1 and Noggin conditioned medium can be replaced with commercial recombinant R-spondin 1 (500 ng/mL) and Noggin (100 ng/mL).</li>
		<li>Prewarm a 24-well suspension culture plate in a cell culture incubator. Use a standard cell culture incubator with 5% CO2 and humidified atmosphere at 37 &#176;C. Thaw basement matrix in a 4 &#176;C fridge. Keep the basement matrix and culture medium on ice during experimentation.</li>
	</ol>
	</li>
	<li>Cell isolation from human lung tissues for 3D organoid culture
	<ol>
		<li>Procure freshly resected human lung tissues sized around 0.5 cm from patients who undergo surgical resection due to various diseases. Transport the lung tissues in 30 mL of basal medium at room temperature and process as quickly as possible inside a biosafety hood.</li>
		<li>Mince lung tissues into small pieces (0.5-1 mm) with a sterile scalpel in a 10 cm cell culture dish. Wash tissue pieces with 10 mL of cold basal medium in a 15 mL centrifuge tube, followed by centrifugation at 400 x g for 5 min&#160;at 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend the pellet in 8 mL of basal medium supplemented with collagenase at a final concentration of 2 mg/mL. Digest the tissue pieces by shaking the tube at 120 rpm for 30-40 min at 37 &#176;C.</li>
		<li>Pipet up and down for 20x to shear the digested tissue pieces using a 10 mL serological pipette. Stack a 100 &#181;m strainer on a 50 mL centrifuge tube and filter the suspension.</li>
		<li>Recover tissue pieces on the strainer with the basal medium and transfer them to a 15 mL centrifuge tube, followed by a second round of shearing and filtering. Additional shearing-filtering can be performed 1x-2x to isolate more cells, especially when a small piece of tissue (e.g., &#60;0.5 cm) is procured.</li>
		<li>Add FBS to the flow-through with a final concentration of 2% to terminate digestion, followed by centrifugation at 400 x g for 5 min at 4 &#176;C.</li>
		<li>Resuspend the cell pellet in 10 mL of basal medium, followed by centrifugation at 400 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
		<li>(Optional) If many erythrocytes are seen in the pellet (estimated by the color of the pellet), resuspend the cell pellet in 2 mL of red blood cell lysis buffer and incubate at room temperature for 5 min. Then, add 10 mL of basal medium to the tube, followed by centrifugation at 400 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Resuspend the pellet in cold basement matrix and keep on ice. Add 80-160 &#181;L of basement matrix for cells recovered from a lung tissue of size around 0.5 cm; the amount is sufficient to seed 2-4 droplets.</li>
		<li>Dispense 40 &#181;L of suspension to each well of a pre-warmed 24-well suspension culture plate. Incubate the culture plate at 37 &#176;C for 10-15 min. Let the basement matrix solidify to form a droplet.</li>
		<li>Add 500 &#181;L of human lung organoids expansion medium supplemented with 5 nM of Heregulin beta-1 to each well and incubate the plate in a cell culture incubator.</li>
		<li>Refresh the medium every 3 days. Remove the old medium while keeping the droplet intact and add fresh medium with caution. Passage the organoids after incubation for 10-14 days. Use Heregulin beta-1 only in the initial culture before the first passage.</li>
		<li>Observe the organoids under a microscope to ensure the organoids are not embedded with a very high cell density. If a droplet disintegrates due to an overly high cell density, recover and re-embed the organoids and cells with a higher volume of basement matrix to make more droplets with a lower and desirable cell density.</li>
	</ol>
	</li>
</ol>
2. Expansion of human lung organoids
<ol>
	<li>Prepare a Pasteur pipette by burning the tip of a Pasteur pipette onto a flame, such as a Bunsen burner, to narrow the opening from 1.5 mm to around 1.0 mm in diameter. Cool down the pipettes, followed by autoclaving to sterilize. Wet the pipettes with the basal medium to avoid cell attachment and loss during mechanical shearing.</li>
	<li>Lung organoids passage with mechanical shearing
	<ol>
		<li>Use a 1 mL tip and pipet up and down to break the droplets obtained above. Then, transfer the organoids along with the medium to a 15 mL centrifuge tube and adjust the volume to 10 mL with cold basal medium. Discard the supernatant after centrifugation at 300 x g for 5 min at 4 &#176;C</li>
		<li>Wash the organoids with 10 mL of cold basal medium once again.Resuspend the organoids in 2 mL of cold basal medium. Pipet up and down to shear the organoids into small pieces with a Pasteur pipette.</li>
		<li>Supplement with basal medium to a total volume of 10 mL, followed by centrifugation at 300 x g for 5 min at 4 &#176;C. Resuspend the organoid fragments with cold basement matrix sufficient to enable a 1:3 to 1:5 expansion. Keep on ice.</li>
		<li>Place 40 &#181;L of organoid suspension in each well of a pre-warmed 24-well plate. Incubate the culture plate at 37 &#176;C for 10-15 min. Let the basement matrix solidify.</li>
		<li>Add 500 &#181;L of lung organoid expansion medium to each well and incubate in a cell culture incubator. Refresh the medium every 3 days. Passage the organoids every 2 weeks with a ratio of 1:3 to 1:5.</li>
	</ol>
	</li>
	<li>Lung organoids passage with trypsinization 
	NOTE: Trypsinization is preferred when it is difficult to shear the lung organoid into small pieces using a Pasteur pipet, or the size of the organoids is highly variable, or the subsequent experimentations require organoids of more uniform size.
	<ol>
		<li>Harvest the lung organoids as shown in step 2.2.1. Resuspend the organoid in 1 mL of dissociation enzyme and incubate in a 37 &#176;C water bath for 3-5 min.</li>
		<li>Add 1 mL of basal medium to the tube. Shear the organoids mechanically by pipetting the organoids up and down into small pieces using a Pasteur pipet. Check the size of organoid pieces under a microscope at 4x magnification. Then, add 40 &#181;L of FBS to terminate digestion. 
		NOTE: Determine the size of organoid fragments under the microscope according to the experimental arrangement. If an experiment needs more organoids or organoids of more uniform size, shear organoids into smaller pieces or even single cells. Then, it takes a longer time, probably 3 weeks, before the organoids are ready for experimentation.</li>
		<li>Top up with basal medium to a final volume of 10 mL, followed by centrifugation at 300 x g for 5 min at 4 &#176;C. Resuspend the organoid pieces in cold basement matrix with a volume sufficient to passage with a ratio of 1:5-1:10. Keep on ice.</li>
		<li>Place 40 &#181;L of organoid suspension in each well of a pre-warmed 24-well plate. Incubate the culture plate at 37 &#176;C for 10-15 min. Let the basement matrix solidify.</li>
		<li>Supplement with 500 &#181;L of lung organoid expansion medium per well and incubate in a cell culture incubator. Refresh the expansion medium every 3 days. Passage the organoids after 2-3 weeks. 
		&#8203;NOTE: Around 100 organoids are mounted within a 40 &#181;L droplet of basement matrix. The lung organoids normally grow better with a relatively high cell density. Re-embed the organoids with a lower cell density if droplets disintegrate or the growing organoids attach together due to the overly high cell density.</li>
	</ol>
	</li>
</ol>
3. Proximal differentiation to generate mature airway organoids
<ol>
	<li>Prepare proximal differentiation medium (PD medium) by supplementing the air liquid interface basal medium with 1x air liquid interface supplement, 1x air liquid interface maintenance supplement, 4 &#181;g/mL of heparin, 1 &#181;M of hydrocortisone, 10 &#181;M of Y-27632, and 10 &#181;M of DAPT (see Table of Materials).</li>
	<li>3D airway organoids
	<ol>
		<li>Incubate lung organoids in the expansion medium for 7-10 days after passaging via mechanical shearing. Replace the expansion medium with the PD medium. Incubate the organoids in the PD medium for 14 days in a cell culture incubator.</li>
		<li>Discard the PD medium in each well. Add cell lysate buffer to harvest the differentiated airway organoid for RNA extraction and detection of cellular gene expression by RT-qPCR assay.</li>
		<li>Alternatively, incubate the organoids at 37 &#176;C for 60 min after addition of 10 mM EDTA to disassociate the organoids into single cells, followed by flow cytometry analysis to examine cell populations. The organoids are ready for various experimental manipulations.</li>
	</ol>
	</li>
	<li>2D airway organoid
	<ol>
		<li>Prepare sufficient 3D lung organoids for 2D differentiation culture. A total of 1.3 x 105 and 4.5 x 105 cells are required for a 24-well permeable support insert and a 12-well permeable support insert, respectively.</li>
		<li>After 3D lung organoids grow in the expansion medium for 2 weeks, digest the organoids into single cells and seed into the 24-well and 12-well inserts to generate 2D airway organoids.</li>
		<li>Pre-incubate the inserts with the basal medium overnight in a cell culture incubator. Add 250 &#181;L and 500 &#181;L of basal medium in the top and bottom chamber of a 24-well plate, respectively. For a 12-well plate, add 500 &#181;L and 1,000 &#181;L of basal medium in the top and bottom chamber, respectively.</li>
		<li>Harvest 3D lung organoids as described in step 2.2.1. Resuspend the organoids with 1 mL of dissociation enzyme and incubate in a 37 &#176;C water bath for 3-5 min.</li>
		<li>Add 1 mL of basal medium to the tube. Pipet up and down to shear the organoids into single cells with a Pasteur pipet and check the cells under a microscope. Then, add 40 &#181;L of FBS to terminate digestion.</li>
		<li>Filter the cells through a 40 &#181;m strainer into a 50 mL centrifuge tube. Transfer the filtered cell suspension to a 15 mL tube. Top up with basal medium to a total volume of 10 mL, followed by centrifugation at 300 x g for 5 min at 4 &#176;C.</li>
		<li>Resuspend the pellet, collected from 24 droplets (40 &#181;L), in 1-2.5 mL of lung organoid expansion medium depending on the cell density in the droplets. Count the number of cells with a cell counter under a microscope. Adjust the cell concentration to 1.3 x 106/mL (for 24-well inserts) or 9 x 105/mL (for 12-well inserts).</li>
		<li>Remove the basal medium from the top and bottom chambers. Add 500 &#181;L and 1,000 &#181;L of expansion medium in the bottom chamber of the 24-well insert and 12-well insert, respectively. Seed 100 &#181;L and 500 &#181;L of cell suspension prepared in step 3.3.7 onto the apical chamber of the 24-well insert and 12-well insert, respectively.</li>
		<li>Incubate in a cell culture incubator for 2 days. Replace the expansion medium with the PD medium in both apical and bottom chamber of the plates. Incubate the organoids in cell culture incubator for 14 days and refresh the PD medium every 3 days. 
		NOTE: Mobile cilia are discernible in the 3D and 2D organoids under a microscope from day 7 after incubation in the PD medium. After 14 days of differentiation culture in the PD medium, the airway organoids are mature for various experimental manipulations.</li>
		<li>Measure trans-epithelial electrical resistance (TEER) every other day using an electrical resistance measurement systemaccording to a standard protocol described in17.</li>
	</ol>
	</li>
</ol>

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J. Z., C.L., and M.C.C. are listed as inventors on the patent of airway organoids (publication No: US-2021-0207081-A1). The other authors declare no competing interests.

The human airways are lined with the airway epithelium, also known as the pseudostratified ciliated epithelium. The major cell types of the upper airway epithelium are ciliated cells that enable the coordinated movement of their apical cilia to expel mucus and inhaled particles from the airways, goblet cells that produce and secrete mucus, and basal cells that line the basement membrane and are implicated in regeneration. In the small airway such as bronchioles, the cuboidal airway epithelium contains secretory club cell...

Organoids have become a robust and universal tool for in vitro modeling of organ development and studying biology and disease. When cultured in a growth factor-defined culture medium, adult stem cells (ASC) from a variety of organs can be expanded in 3-dimension (3D) and self-assembled into organ-like cellular clusters composed of multiple cell types, termed organoids. Clevers&#39; laboratory reported the derivation of the first ASC-derived organoid, human intestinal organoid, in 20091,2. Afterward, ASC-derived organoids have been established for a variety of human organs and tissues, including prostate3,4, liver5,6, stomach7,8,9, pancreas10, mammary gland11, and lung 12,13. These ASC-derived organoids retained the critical cellular, structural, and functional properties of the native organ and maintained genetic and phenotypic stability in long-term expansion culture14,15.
Organoids can also be derived from pluripotent stem cell (PSC), including embryonic stem (ES) cells and induced pluripotent stem (iPS) cell16. While PSC-derived organoids exploit the mechanisms of organ development for their establishment, ASCs can be coerced to form organoids by rebuilding conditions that mimic the stem cell niche during physiological tissue self-renewal or tissue repair. PSC-derived organoids are favorable models to explore development and organogenesis, albeit unable to reach the comparable maturation level of ASC-derived organoids. The fetal-like maturation status of PSC-derived organoids, and complexity for establishing these organoids substantially prevent their broad applications for studying biology and pathology in mature tissues.
The human respiratory tract, from nose to terminal bronchiole, is lined with the airway epithelium, also called the pseudostratified ciliated epithelium, which consists of four major cell types, i.e., ciliated cell, goblet cell, basal cell, and club cell. We established the ASC-derived human lung organoid from human lung tissues in collaboration with Clevers&#39; lab12,13. These lung organoids are consecutively expanded in the expansion medium for over a year; the precise duration varies among different organoid lines obtained from different donors. However, compared to the native airway epithelium, these long-term expandable lung organoids are not mature enough since ciliated cells, the major cell population in the human airway, are under-represented in these lung organoids. Thus, we 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.
Here we provide a video protocol to derive human lung organoids from the primary lung tissues, expand the lung organoids and induce proximal differentiation to generate 3D and 2D airway organoids.

<table><tbody><tr><td>&lt;strong&gt;Reagents for lung organoid culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Advanced DMEM/F12</td><td>Invitrogen</td><td>12634010</td><td>-</td></tr><tr><td>A8301</td><td>Tocris</td><td>2939</td><td>500nM</td></tr><tr><td>B27 supplement</td><td>Invitrogen</td><td>17504-044</td><td>1x</td></tr><tr><td>Cultrex Reduced Growth Factor Basement Membrane Matrix, Type 2 (BME 2)</td><td>Trevigen</td><td>3533-010-0</td><td>70-80%</td></tr><tr><td>FGF-10</td><td>Peprotech</td><td>100-26</td><td>20 ng/mL</td></tr><tr><td>FGF-7</td><td>Peprotech</td><td>100-19</td><td>5 ng/mL</td></tr><tr><td>GlutaMAX (glutamine)</td><td>Invitrogen</td><td>35050061</td><td>1x</td></tr><tr><td>HEPES 1M</td><td>Invitrogen</td><td>15630-056</td><td>10 mM</td></tr><tr><td>Heregulin &amp;beta;-1</td><td>Peprotech</td><td>100-03</td><td>5 nM</td></tr><tr><td>N-Acetylcysteine</td><td>Sigma-Aldrich</td><td>A9165</td><td>1.25 mM</td></tr><tr><td>Nicotinamide</td><td>Sigma-Aldrich</td><td>N0636</td><td>10 mM</td></tr><tr><td>Noggin (conditional medium)</td><td>home made</td><td>-</td><td>10x</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Invitrogen</td><td>15140-122</td><td>1x</td></tr><tr><td>Primocin</td><td>Invivogen</td><td>ant-pm-1</td><td>100 &amp;micro;g/mL</td></tr><tr><td>Rspondin1 (conditional medium)</td><td>home made</td><td>-</td><td>10x</td></tr><tr><td>SB202190</td><td>Sigma-Aldrich</td><td>S7067</td><td>1 &amp;micro;M</td></tr><tr><td>Y-27632</td><td>Tocris</td><td>1254</td><td>5 &amp;micro;M</td></tr><tr><td>&lt;strong&gt;Proximal differentiation medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DAPT</td><td>Tocris</td><td>2634</td><td>10 &amp;micro;M</td></tr><tr><td>Heparin Solution</td><td>StemCell Technology</td><td>7980</td><td>4 &amp;micro;g/mL</td></tr><tr><td>Hydrocortisone Stock Solution</td><td>StemCell Technology</td><td>7925</td><td>1 &amp;micro;M</td></tr><tr><td>PneumaCult-ALI 10X Supplement</td><td></td><td></td><td>air liquid interface supplement</td></tr><tr><td>PneumaCult-ALI Basal Medium</td><td>StemCell Technology</td><td>05001</td><td>air liquid interface basal medium</td></tr><tr><td>PneumaCult-ALI Maintenance Supplement</td><td></td><td></td><td>air liquid interface maintenance supplement</td></tr><tr><td>Y-27632</td><td>Tocris</td><td>1254</td><td>10 &amp;micro;M</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Biological safety cabinet</td><td>Baker</td><td>1-800-992-2537</td><td></td></tr><tr><td>Carl Zeiss LSM 780 or 800</td><td>Zeiss</td><td></td><td>confocal microscope</td></tr><tr><td>CO2 Incubator</td><td>Thermo Fisher Scientific</td><td>42093483</td><td></td></tr><tr><td>Stereo-microscope</td><td>Olympus Corporation</td><td>CKX31SF</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5418BG040397</td><td></td></tr><tr><td>Serological pipettor</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Micropipette</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>ZEN black or ZEN blue software</td><td>Zeiss</td><td></td><td>analysis software</td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>12mm Trans-well</td><td>StemCell Technology</td><td>#38023</td><td></td></tr><tr><td>12-well cell culture plate</td><td>Cellstar</td><td>665970</td><td></td></tr><tr><td>15- and 50 ml conical tubes</td><td>Thermo Fisher Scientific</td><td>L6BF5Z8118</td><td></td></tr><tr><td>24-well cell culture plate</td><td>Cellstar</td><td>662160</td><td></td></tr><tr><td>6.5mm Trans-well</td><td>StemCell Technology</td><td>#38024</td><td></td></tr><tr><td>Medical Syringe Filter Unit, 0.22 &amp;micro;m</td><td>Sigma-Aldrich</td><td>SLGPR33RB</td><td></td></tr><tr><td>Microfuge tubes</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Micropipette tips</td><td>Thermo Fisher Scientific</td><td>TFLR140-200-Q21190531</td><td></td></tr><tr><td>Pasteur pipette glass</td><td>Thermo Fisher Scientific</td><td>22-378893</td><td></td></tr><tr><td>Serological pipettes(5ml, 10ml, 25ml)</td><td>Thermo Fisher Scientific</td><td>BA08003, 08004, 08005</td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Goat Anti-Mouse Alexa Fluor 594</td><td>Invitrogen</td><td>A11005</td><td></td></tr><tr><td>Goat Anti-Mouse, Alexa Fluor 488</td><td>Invitrogen</td><td>A11001</td><td></td></tr><tr><td>Goat Anti-Rabbit Alexa Fluor 488</td><td>Invitrogen</td><td>A11034</td><td></td></tr><tr><td>Goat Anti-Rabbit Alexa Fluor 594</td><td>Invitrogen</td><td>A11037</td><td></td></tr><tr><td>Goat Anti-Rat Alexa Fluor 594</td><td>Invitrogen</td><td>A11007</td><td></td></tr><tr><td>Mouse Anti-Cytokeratin 5</td><td>Abcam</td><td>ab128190</td><td></td></tr><tr><td>Mouse Anti-FOX J1</td><td>Invitrogen</td><td>14-9965-82</td><td></td></tr><tr><td>Mouse Anti-Mucin 5AC</td><td>Abcam</td><td>ab3649</td><td></td></tr><tr><td>Mouse Anti-&amp;beta;-tubulin 4</td><td>Sigma</td><td>T7941</td><td></td></tr><tr><td>Rabbit Anti-p63</td><td>Abcam</td><td>ab124762</td><td></td></tr><tr><td>Rat Anti-Uteroglobin/CC-10</td><td>R&amp;amp;D Systems</td><td>MAB4218-SP</td><td></td></tr><tr><td>&lt;strong&gt;Other reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>TrypLE Select Enzyme (10X)</td><td>Thermo Fisher Scientific</td><td>A1217701</td><td>dissociation enzyme</td></tr></tbody></table>

establishing human lung organoids and proximal differentiation to generate mature airway organoids

The lack of a robust in vitro model of the human respiratory epithelium hinders the understanding of the biology and pathology of the respiratory system. We describe a defined protocol to derive human lung organoids from adult stem cells in the lung tissue and induce proximal differentiation to generate mature airway organoids. The lung organoids are then consecutively expanded for over 1 year with high stability, while the differentiated airway organoids are used to morphologically and functionally simulate human airway epithelium to a near-physiological level. Thus, we establish a robust organoid model of the human airway epithelium. The long-term expansion of lung organoids and differentiated airway organoids generates a stable and renewable source, enabling scientists to reconstruct and expand the human airway epithelial cells in culture dishes. The human lung organoid system provides a unique and physiologically active in vitro model for various applications, including studying virus-host interaction, drug testing, and disease modeling.

This protocol enables the derivation of human lung organoids with a high success rate. Fresh human lung tissue is minced into small pieces, and then decomposed with collagenase. The resultant single cells are embedded in the basement matrix and incubated in the lung organoid expansion medium supplemented with a cocktail of niche factors for the outgrowth of epithelial stem cells (step 1.1.2). Figure 1 shows the microphotograph of freshly isolated lung cells embedded in reduced growth factor ...

Watch this Scientific Journal Video about Establishing Human Lung Organoids and Proximal Differentiation to Generate Mature Airway Organoids at JoVE.com

Establishing Human Lung Organoids and Proximal Differentiation to Generate Mature Airway Organoids

The protocol presents a method to derive human lung organoids from primary lung tissues, expand the lung organoids and induce proximal differentiation to generate 3D and 2D airway organoids that faithfully phenocopy the human airway epithelium.

The lack of a robust in vitro model of the human respiratory epithelium hinders the understanding of the biology and pathology of the respiratory system. ...

establishing-human-lung-organoids-proximal-differentiation-to

Research

JoVE Journal

Biology

8.0K Views.  The University of Hong Kong. The protocol presents a method to derive human lung organoids from primary lung tissues, expand the lung organoids and induce proximal differentiation to generate 3D and 2D airway organoids that faithfully phenocopy the human airway epithelium.

Video: Establishing Human Lung Organoids and Proximal Differentiation to Generate Mature Airway Organoids

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Bioengineering

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent stem cells (hiPSCs) can be differentiated stepwise into 3D multicellular lung organoids, made of both epithelial and mesenchymal cell populations. We recapitulate embryonic developmental cues by temporally introducing a variety of growth factors and small molecules to efficiently generate definitive endoderm, anterior foregut endoderm, and subsequently lung progenitor cells. These cells are then embedded in growth factor reduced (GFR)-basement membrane matrix medium, allowing them to spontaneously develop into 3D lung organoids in response to external growth factors. These whole lung organoids (WLO) undergo early lung developmental stages including branching morphogenesis and maturation after exposure to dexamethasone, cyclic AMP and isobutylxanthine. WLOs possess airway epithelial cells expressing the markers KRT5 (basal), SCGB3A2 (club) and MUC5AC (goblet) as well as alveolar epithelial cells expressing HOPX (alveolar type I) and SP-C (alveolar type II). Mesenchymal cells are also present, including smooth muscle actin (SMA), and platelet-derived growth factor receptor A (PDGFR&#945;). iPSC derived WLOs can be maintained in 3D culture conditions for many months and can be sorted for surface markers to purify a specific cell population. iPSC derived WLOs can also be utilized to study human lung development, including signaling between the lung epithelium and mesenchyme, to model genetic mutations on human lung cell function and development, and to determine the cytotoxicity of infective agents.

The article describes step wise directed differentiation of induced pluripotent stem cells to three-dimensional whole lung organoids containing both proximal and distal epithelial lung cells along with mesenchyme.

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent ...

Developmental Biology

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major causes of morbidity and mortality worldwide. In vitro platforms using human bronchial epithelial cells (HBECs) have been instrumental to our understanding of the airway epithelium in health and disease. Access to HBECs from individuals with rare genetic diseases or rare mutations is a bottleneck in lung research.
Induced pluripotent stem cells (iPSCs) are readily generated by "reprogramming" somatic cells and retain the unique genetic background of the individual donor. Recent advances allow for the directed differentiation of iPSCs to lung epithelial progenitor cells, alveolar type 2 cells, as well as the cells of the conducting airway epithelium via basal cells, the major airway stem cells.
Here we outline a protocol for the maintenance and expansion of iPSC-derived airway basal cells (hereafter iBCs) as well as their trilineage differentiation in air-liquid interface (ALI) cultures. iBCs are maintained and expanded as epithelial spheres suspended in droplets of extracellular matrix cultured in a primary basal cell medium supplemented with inhibitors of TGF-&#223; and BMP signaling pathways. iBCs within these epithelial spheres express key basal markers TP63 and NGFR, can be purified by fluorescence activated cell sorting (FACS), and when plated on porous membranes in standard ALI culture conditions, differentiate into a functional airway epithelium. ALI cultures derived from healthy donors are composed of basal, secretory and multiciliated cells and demonstrate epithelial barrier integrity, motile cilia, and mucus secretion. Cultures derived from individuals with CF or PCD recapitulate the dysfunctional CFTR-mediated chloride transport or immotile cilia, the respective disease-causing epithelial defects.
Here, we present a protocol for the generation of human cells that can be applied for modeling and understanding airway diseases.

Recent advances in human induced pluripotent stem cell differentiation protocols allow for the stepwise derivation of organ-specific cell types. Here, we provide detailed steps for the maintenance and expansion of iPSC-derived airway basal cells and their differentiation into a mucociliary epithelium in air-liquid interface cultures.

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major ...

Airway Model for Studying Lung Barrier Integrity, Disease, and Drug Discovery

iPSC-Derived Epithelial, Mesenchymal, Endothelial, and Immune Cell Co-Culture to Model Airway Barrier Integrity in Lung Health and Disease

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the nasopharynx to the smallest alveolar sac. Interactions between these cells are crucial in lung development and disease, acting as a barrier against harmful chemicals and pathogens. Current in vitro co-culture models utilize immortalized cell lines with different biological backgrounds, which may not accurately represent the cellular milieu or interactions of the lung. We differentiated human iPSCs into 3D lung organoids (containing both epithelium and mesenchyme), endothelial cells, and macrophages. These were co-cultured in an air-liquid interface (ALI) format to form an epithelial/mesenchymal apical barrier invested with macrophages and a basolateral endothelial barrier (iAirway). iPSC-derived iAirways showed a reduction in barrier integrity in response to infection with respiratory viruses and cigarette toxins. This multi-lineage lung co-culture system provides a platform for studying cellular interactions, signaling pathways, and molecular mechanisms underlying lung development, homeostasis, and disease progression. iAirways closely mimic human physiology and cellular interactions, can be generated from patient-derived iPSC&#39;s, and can be customized to include different cell types of the airway. Overall, iPSC-derived iAirway models offer a versatile and powerful tool for studying barrier integrity to better understand genetic drivers for disease, pathogen response, immune regulation, and drug discovery or repurposing in vitro, with the potential to advance our understanding and treatment of airway diseases.

Author Spotlight: Modeling Human Airway Remodeling and Viral Responses Using Isogenic Epithelial, Endothelial, and Immune Cells

This article describes the generation of a complex, multi-cellular airway barrier model composed of induced pluripotent stem cell (iPSC)-derived lung epithelium, mesenchyme, endothelial cells, and macrophages in an air-liquid interface culture.

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the ...

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in regenerative medicine and bioreactor technologies offer opportunities to produce lab-grown functional tissue and organ constructs that can be used to screen drugs, model disease, and engineer tissue replacements. Here, a miniaturized bioreactor coupled with an imaging modality that allows in situ visualization of the inner lumen of explanted rat trachea during in vitro tissue manipulation and&#160;culture is described. Using this bioreactor, the protocol demonstrates imaging-guided selective removal of endogenous cellular components while preserving the intrinsic biochemical features and ultrastructure of the airway tissue matrix. Furthermore, the delivery, uniform distribution, and subsequent prolonged culture of exogenous cells on the decellularized airway lumen with optical monitoring in situ are shown. The results highlight that the imaging-guided bioreactor can potentially be used to facilitate the generation of functional in vitro airway tissues.

The protocol describes an imaging-enabled bioreactor that allows the selective removal of the endogenous epithelium from the rat trachea and homogenous distribution of exogenous cells on the lumen surface, followed by long-term in vitro culture of the cell-tissue construct.

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Mucociliary Epithelial Organoids from Xenopus Embryonic Cells: Generation, Culture and High-Resolution Live Imaging

Mucociliary epithelium provides the first line of defense by removing foreign particles through the action of mucus production and cilia-mediated clearance. Many clinically relevant defects in the mucociliary epithelium are inferred as they occur deep within the body. Here, we introduce a tractable 3D model for mucociliary epithelium generated from multipotent progenitors that were microsurgically isolated from Xenopus laevis embryos. The mucociliary epithelial organoids are covered with newly generated epithelium from deep ectoderm cells and later decorated with distinct patterned multiciliated cells, secretory cells, and mucus-producing goblet cells that are indistinguishable from the native epidermis within 24 h. The full sequences of dynamic cell transitions from mesenchymal to epithelial that emerge on the apical surface of organoids can be tracked by high-resolution live imaging. These in vitro cultured, self-organizing mucociliary epithelial organoids offer distinct advantages in studying the biology of mucociliary epithelium with high-efficiency in generation, defined culture conditions, control over number and size, and direct access for live imaging during the regeneration of the differentiated epithelium.

We describe a simple protocol to develop mucociliary epithelial organoids from deep ectoderm cells isolated from Xenopus laevis embryos. The multipotent progenitors regenerate epithelial goblet cell precursors and allow live tracking of the initiation and progression of the cell transitions on the surface of organoids.