Name: establishing human lung organoids and proximal differentiation to generate mature airway organoids
Uploaded: 2022-03-23T14:15:00
Description: Watch this Scientific Journal Video about Establishing Human Lung Organoids and Proximal Differentiation to Generate Mature Airway Organoids at JoVE.com

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 p...

<ol>
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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. ...

We are introducing a method to establish the organoid culture of human airway epithelium in culture plates. We derive human lung organoid directly from primary lung tissues with high efficiency. The derived human lung organoid are stably expanded for over one year, and a proximal differentiation protocol allows the generation of mature airway organoids that can faithfully simulate the human airway epithelium to a near physiological level.Therefore, the culture system allows scientists to reconstruct and expand the human airway epithelium in culture plates. Demonstrating the procedure will be Man Chun Chiu and Yifei Yu, PhD students in our lab To begin the cell isolation from human lung tissues for 3D organoid culture, mince the lung tissues into one-millimeter small pieces with a sterile scalpel and wash the tissue pieces with 10 milliliters of cold basal medium. Centrifuge the tissues at 400 times G for five minutes at four degrees Celsius and discard the supernatant before resuspending the pellet in eight milliliters of basal medium supplemented with collagenase at a final concentration of two milligrams per milliliter.Then, digest the tissue pieces by shaking the tube at 120 RPM for 30 to 40 minutes at 37 degrees Celsius. After digestion, pipette up and down 20 times using a 10-milliliter serological pipette to shear the digested tissue pieces. Then, filter the suspension with a 100-micron strainer into a 50-milliliter tube.Recover and transfer the tissue pieces from the strainer to a 15-milliliter centrifuge tube with the basal medium. To terminate the digestion, add fetal bovine serum to the flow-through to a final concentration of 2%Then, centrifuge the tube and resuspend the cell pellet in 10 milliliters of the basal medium as described before. After centrifugation, resuspend the pellet in 80 to 160 microliters of cold basement matrix medium and place the tubes on ice.Next, dispense 40 microliters of the suspension to each well of a pre-warmed 24-well suspension culture plate. Incubate the plate at 37 degrees Celsius for 10 to 15 minutes. Allow the basement matrix to solidify and form a droplet.After incubation, add 500 microliters of human lung organoids expansion medium supplemented with five-nanomolar of heregulin-beta 1 to each well and incubate the plate in a cell culture incubator. After three days, remove the old medium while keeping the droplet intact and carefully add fresh medium. After 10 to 14 days of passaging, observe the organoids under a microscope and ensure that the organoids are not embedded with a very high cell density.To passage the lung organoids with shearing, break the droplets by pipetting up and down with a one-milliliter tip. Then, transfer the organoids along with the medium to a 15-milliliter centrifuge tube and adjust the volume to 10 milliliters with a cold basal medium. Centrifuge the tube at 300 times G for five minutes at four degrees Celsius, discard the supernatant, and wash the organoids with 10 milliliters of cold basal medium.Then, resuspend the organoids in two milliliters of cold basal medium and shear into small pieces with a Pasteur pipette. Next, add basal medium to a total volume of 10 milliliters and centrifuge before resuspending the organoid fragments with a cold basement matrix sufficient to enable a 1:3 to 1:5 expansion and place the tubes on ice. Dispense 40 microliters of organoid suspension in each well of a pre-warmed 24-well plate to incubate at 37 degrees Celsius to allow the basement matrix to solidify for 10 to 15 minutes.Later, add 500 microliters of lung organoid expansion medium to each well and incubate in a cell culture incubator. Refresh the medium every three days and passage the organoids every two weeks. To passage lung organoids with trypsinization, resuspend harvested lung organoids in one milliliter of dissociation enzyme.Incubate the organoid in a 37-degree-Celsius water bath for three to five minutes. Then, add one milliliter of basal medium to the tube and shear the organoids mechanically by pipetting up and down. Check the size of organoid pieces under a microscope at 100X magnification before terminating the digestion with 40 microliters of fetal bovine serum.Make up the volume to 10 milliliters with basal medium and centrifuge before resuspending the organoid pieces in a cold basement matrix with a volume sufficient to passage with a ratio of 1:5 to 1:10. Later, dispense 40 microliters of organoid suspension in each well of a 24-well plate and culture the plate as demonstrated before. To generate 3D airway organoids, incubate the lung organoids in the expansion medium for seven to 10 days after passaging via mechanical shearing.Then, replace the expansion medium with proximal differentiation medium and incubate the organoids in a cell culture incubator. After 14 days, discard the medium in each well and add cell lysate buffer to harvest the differentiated airway organoid for RNA extraction and detection of cellular gene expression by RT-qPCR assay. Pre-incubate the inserts in the cell culture incubator with 250 and 500 microliters of the basal medium in the top and bottom chamber of a 24-well plate, respectively.Then, add one milliliter of basal medium to the tube and pipette up and down to shear the organoids into single cells. Observe the cells under a microscope before terminating the digestion with 40 microliters of fetal bovine serum. Filter the cells through a 40-micron strainer into a 50-milliliter tube and transfer the filtered cell suspension to a 15-milliliter tube.Top up the suspension with basal medium to a total volume of 10 milliliters. After centrifugation, resuspend the pellet collected from 24 droplets in 1 to 2.5 milliliters of lung organoid expansion medium, depending on the cell density. Count the number of cells with the cell counter under a microscope, then adjust the cell concentration to 1.3 times 10 to the six per milliliter for 24-well inserts.Remove the basal medium from the top and bottom chambers before adding 500 microliters of expansion medium in the bottom chamber. Seed 100 microliters of cell suspension prepared earlier onto the apical chamber of the 24-well insert and incubate. After two days, replace the expansion medium with proximal differentiation medium in both the apical and bottom chamber of the plate.Incubate the organoids for 14 days and refresh the medium every three days. Measure the trans-epithelial electrical resistance every day using an electrical resistance measurement system. In the representative microphotograph, the freshly isolated lung cells can be seen embedded in reduced growth factor basement membrane matrix type two.The cystic organoids were appeared and grown over time. The lung organoids after the fourth passage are demonstrated here. Within two hours of mechanical shearing, the organoid fragments embedded in the basement membrane extract formed the cystic domains.The microphotograph of the same field on day five confirmed organoid growth over time. The expanding organoids harbored all the four major airway epithelial cell types, including ACCTUB-positive or FOXJ1-positive ciliated cell, P63-positive basal cell, CC10-positive club cell, and MUC5AC-positive goblet cell in a premature state. The organoids incubated in the expansion and proximal differentiation medium developed distinct morphology over time.After two weeks of differentiation in the proximal differentiation medium, motile cilia were discernible in every organoid. It was observed that the synchronously beating celia drove the cell debris inside the organoid lumen and swirled them unidirectionally to remove the inhaled particles. Further, the flow cytometry analysis demonstrated that the differentiated organoids accommodated four airway epithelial cell types in proximal differentiation and expansion medium.After two weeks of differentiation, 2D airway organoids developed an intact epithelial barrier. A dextran blockage assay was performed to assess the integrity of the epithelial barrier formed in 2D airway organoids. The 2D organoids contained abundant ciliated cells.The long-term expandable lung organoid and the differentiated airway organoid provide a physiologically active and universal model system for studying respiratory biology and pathology, including COVID-19.

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