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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents for lung organoid culture</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 &#946;-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 &#181;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 &#181;M</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>5 &#181;M</td>
		</tr>
		<tr>
			<td>Proximal differentiation medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Tocris</td>
			<td>2634</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td>Heparin Solution</td>
			<td>StemCell Technology</td>
			<td>7980</td>
			<td>4 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Hydrocortisone Stock Solution</td>
			<td>StemCell Technology</td>
			<td>7925</td>
			<td>1 &#181;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 &#181;M</td>
		</tr>
		<tr>
			<td>Equipment</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>Consumables</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 &#181;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>Antibodies</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-&#946;-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&#38;D Systems</td>
			<td>MAB4218-SP</td>
			<td></td>
		</tr>
		<tr>
			<td>Other reagent</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

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JoVE Journal

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7.7K 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

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 ...

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the ...

Utilizing the Precision-Cut Lung Slice to Study the Contractile Regulation of Airway and Intrapulmonary Arterial Smooth Muscle

Smooth muscle cells (SMC) mediate the contraction of the airway and the intrapulmonary artery to modify airflow resistance and pulmonary circulation, respectively, hence playing a critical role in the homeostasis of the pulmonary system. Deregulation of SMC contractility contributes to several pulmonary diseases, including asthma and pulmonary hypertension. However, due to limited tissue access and a lack of culture systems to maintain in vivo SMC phenotypes, molecular mechanisms underlying the deregulated SMC contractility in these diseases remain fully identified. The precision-cut lung slice (PCLS) offers an ex vivo model that circumvents these technical difficulties. As a live, thin lung tissue section, the PCLS retains SMC in natural surroundings and allows in situ tracking of SMC contraction and intracellular Ca2+ signaling that regulates SMC contractility. Here, a detailed mouse PCLS preparation protocol is provided, which preserves intact airways and intrapulmonary arteries. This protocol involves two essential steps before subjecting the lung lobe to slicing: inflating the airway with low-melting-point agarose through the trachea and infilling pulmonary vessels with gelatin through the right ventricle. The PCLS prepared using this protocol can be used for bioassays to evaluate Ca2+-mediated contractile regulation of SMC in both the airway and the intrapulmonary arterial compartments. When applied to mouse models of respiratory diseases, this protocol enables the functional investigation of SMC, thereby providing insight into the underlying mechanism of SMC contractility deregulation in diseases.

The present protocol describes preparing and utilizing mouse precision-cut lung slices to assess the airway and intrapulmonary arterial smooth muscle contractility in a nearly in vivo milieu.

Smooth muscle cells (SMC) mediate the contraction of the airway and the intrapulmonary artery to modify airflow resistance and pulmonary circulation, ...

A High-Throughput Platform for Culture and 3D Imaging of Organoids

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.g., spinning disk, point scanner confocal, wide field, and brightfield). Moreover, they can be used with light-sheet modalities such as the single-objective, single-plane illumination microscopy (SPIM) technology (soSPIM). 
The protocol described here gives detailed steps for the preparation of the microfabricated culture chips and the culture and staining of organoids. Only a short length of time is required to become familiar with, and consumables and equipment can be easily found in normal biolabs. Here, the 3D imaging capabilities will be demonstrated only with commercial standard microscopes (e.g., spinning disk for 3D reconstruction and wide field microscopy for routine monitoring).

This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm2, in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed.

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by ...

Establishing 3D Endometrial Organoids from the Mouse Uterus

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of luminal and glandular epithelium, a stromal compartment, a vascular network, and a complex immune cell population. Mouse models have been a powerful tool to study the endometrium, revealing critical mechanisms that control implantation, placentation, and cancer. The recent development of 3D endometrial organoid cultures presents a state-of-the-art model to dissect the signaling pathways that underlie endometrial biology. Establishing endometrial organoids from genetically engineered mouse models, analyzing their transcriptomes, and visualizing their morphology at a single-cell resolution are crucial tools for the study of endometrial diseases. This paper outlines methods to establish 3D cultures of endometrial epithelium from mice and describes techniques to quantify gene expression and analyze the histology of the organoids. The goal is to provide a resource that can be used to establish, culture, and study the gene expression and morphological characteristics of endometrial epithelial organoids.

This protocol describes methodologies to establish mouse endometrial epithelial organoids for gene expression and histological analyses.

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of ...

Establishment, Maintenance, Differentiation, Genetic Manipulation, and Transplantation of Mouse and Human Lacrimal Gland Organoids

The lacrimal gland is an essential organ for ocular surface homeostasis. By producing the aqueous part of the tear film, it protects the eye from desiccation stress and external insults. Little is known about lacrimal gland (patho)physiology because of the lack of adequate in vitro models. Organoid technology has proven itself as a useful experimental platform for multiple organs. Here, we share a protocol to establish and maintain mouse and human lacrimal gland organoids starting from lacrimal gland biopsies. By modifying the culture conditions, we enhance lacrimal gland organoid functionality. Organoid functionality can be probed through a "crying" assay, which involves exposing the lacrimal gland organoids to selected neurotransmitters to trigger tear release in their lumen. We explain how to image and quantify this phenomenon. To investigate the role of genes of interest in lacrimal gland homeostasis, these can be genetically modified. We thoroughly describe how to genetically modify lacrimal gland organoids using base editors-from guide RNA design to organoid clone genotyping. Lastly, we show how to probe the regenerative potential of human lacrimal gland organoids by orthotopic implantation in the mouse. Together, this comprehensive toolset provides resources to use mouse and human lacrimal gland organoids to study lacrimal gland (patho)physiology.

This protocol describes how to establish, maintain, genetically modify, differentiate, functionally characterize, and transplant lacrimal gland organoids derived from primary mouse and human tissue.

The lacrimal gland is an essential organ for ocular surface homeostasis. By producing the aqueous part of the tear film, it protects the eye from ...

Rat Intestinal Crypts Isolation and Intestinal Organoids Generation

Generation and Manipulation of Rat Intestinal Organoids

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. Accordingly, patient-derived organoids are used for disease modeling, drug discovery, and personalized treatment screening. Mouse intestinal organoids are commonly utilized to understand aspects of both intestinal function/physiology and stem cell dynamics/fate decisions. However, in many disease contexts, rats are often preferred over mice as a model due to their greater physiological similarity to humans in terms of disease pathophysiology. The rat model has been limited by a lack of genetic tools available in vivo, and rat intestinal organoids have proven fragile and difficult to culture long-term. Here, we build upon previously published protocols to robustly generate rat intestinal organoids from the duodenum and jejunum. We provide an overview of several downstream applications utilizing rat intestinal organoids, including functional swelling assays, whole mount staining, the generation of 2D enteroid monolayers, and lentiviral transduction. The rat organoid model provides a practical solution to the need of the field for an in vitro model which retains physiological relevance to humans, can be quickly genetically manipulated, and is easily obtained without the barriers involved in procuring human intestinal organoids.

Author Spotlight: Generation and Manipulation of Rat Intestinal Organoids  

Here, we present a protocol to generate rat intestinal organoids and use them in several downstream applications. Rats are often a preferred preclinical model, and the robust intestinal organoid system fills the need for an in vitro system to accompany in vivo studies.