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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Research

JoVE Journal

Biology

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 ...

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, ...

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.

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. ...

Establishment and Passaging of Small Intestine Organoids

To begin, place the euthanized rat onto the dissection surface with the ventral side up. Pinch the skin layer with sterile forceps and make cuts at the surface level, avoiding damage to internal organs. Using large sharp dissection scissors, make a longitudinal surface level cut in the center of the abdomen.Then stemming from that cut, make two shorter horizontal cuts one on each side. Using forceps, peel the skin away to expose the abdominal cavity. Cut through the peritoneal membrane to fully expose the internal organs in the abdominal cavity with ready access to the intestine.Using scissors and forceps, locate the stomach and identify the duodenum, approximately two to three centimeters distal to it which appears as a yellowish segment. The proximal jejunum is located approximately four to five centimeters distal to the ligament of Treitz. A landmark between the duodenum and jejunum.Place the isolated intestinal fragment into a 10 centimeter Petri dish. Flush the desired intestinal segment of mesentery with 10 milliliters of ice cold PBS until cleared of luminal contents. On a paper towel, cut the intestinal segment into two centimeter long pieces.Then open each intestinal piece longitudinally to expose the epithelium. Scrape the exposed luminal surface using a glass microscope slide to remove villi. Place the intestinal pieces into the EDTA solution on ice and rotate it four degrees Celsius for 30 minutes on a tube revolver set to 10 RPM.At the dissecting microscope, pour the contents of the tube into a 10 centimeter Petri dish. Add in an additional five milliliters of ice cold PBS. Using fine forceps, hold an intestinal segment and shake vigorously to observe the epithelium release into the PBS.Initially, the PBS will contain mostly villi. Shake the intestinal fragments continuously, periodically discard the PBS containing villi, and add 10 milliliters of fresh PBS to the intestinal fragments. Continue to shake the fragments and repeat the PBS washing step until villi are no longer released into the PBS.But instead, the PBS primarily contains crypts. Discard the remaining intestinal fragments and enrich the remaining PBS in the Petri dish for intestinal crypts. In a tissue culture hood, collect the PBS containing crypts and filter through a 70 micron cells drainer.Centrifuge the filtrate at 250 x g for five minutes. Then remove their supernatant and resuspend the pellet in five millimeters of advanced DMEM plus. Centrifuge again at 250 x g for five minutes.And remove the supernatant, leaving 50 microliters of media with the pellet. Resuspend the pellet in the remaining media and add it to the aliquot of the extracellular matrix extract or EME on ice. Gently pipet up and down to suspend the crypts evenly throughout the EME avoiding making bubbles.Place 50 microliters of the EME domes into a 35 millimeter tissue culture dish. An incubated 37 degrees Celsius with 5%carbon dioxide for 20 minutes. After incubation, add a two milliliters of rat intestinal organoid media, or RIOM, containing 10 micromolar each of Y27632 and CHIR99021.Next, to passage rat intestinal organoids, aspirate the medium from a plate containing organoids and add one milliliter of dissociation reagent to release the organoids from the EME domes. Transfer the dissociation reagent with fragmented organoids to a 15 milliliter conical tube. Wash the culture plate with two milliliters of advanced DMEM plus and add it to the 15 milliliter conical tube containing the fragmented organoids.Using a glass pasture pipette, gently pipette up and down 15 to 20 times to fragment the organoids. Centrifuge the organoids at 350 x g for two minutes and add 50 microliters of solution from the bottom of the tube into the EME. After mixing the solution, plate 50 microliters of the EME domes into a 35 millimeter tissue culture dish and incubate at 37 degrees Celsius with 5%carbon dioxide for 20 minutes.After incubation, add two milliliters of RIOM containing 10 micromolar each of Y27632 and CHIR99021. Representative images of villi fragments and crypts are shown. Crypts are smaller in size compared to villi.Plated crypts expand over the next few days and begin to bud and differentiate by days four to seven. After two days of thawing, a healthy organoid culture showed the presence of both spheroids and budded organoids. The same organoid line after passaging showed the presence of single crypt-like domains.

Optimizing Human Intestinal Enteroids for Circadian Rhythm Studies

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse repertoire of cellular and physiological processes ranging from sleep-wake cycles to metabolism. This clock mechanism entrains the organism based on environmental changes and coordinates the temporal regulation of molecular and physiological events. Previously, it was demonstrated that autonomous circadian rhythms are maintained even at the single-cell level using cell lines such as NIH3T3 fibroblasts, which were instrumental in uncovering the mechanisms of circadian rhythms. However, these cell lines are homogeneous cultures lacking multicellularity and robust intercellular communications. In the past decade, extensive work has been performed on the development, characterization, and application of 3D organoids, which are in vitro multicellular systems that resemble in vivo morphological structures and functions. This paper describes a protocol for detecting circadian rhythms using a bioluminescent reporter in human intestinal enteroids, which enables the investigation of circadian rhythms in multicellular systems in vitro.

Author Spotlight: In Vitro Investigations of Circadian Rhythms in Multicellular Systems

Circadian rhythms, which exist in most organisms, regulate the temporal organization of biological processes. 3D organoids have recently emerged as a physiologically relevant in vitro model. This protocol describes the use of bioluminescent reporters to observe circadian rhythms in organoids, enabling in vitro investigations of circadian rhythms in multicellular systems.

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse ...

Integrating Single-Cell Transcriptomics with Human Intestinal Organoids

Using Human Intestinal Organoids to Understand the Small Intestine Epithelium at the Single Cell Transcriptional Level

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid cultures provide ex vivo model systems that bridge the gap between animal models and clinical studies. The application of single cell transcriptomics to human intestinal organoid (HIO) models is revealing previously unrecognized cell biology, biochemistry, and physiology of the GI tract. The advanced single cell transcriptomics platforms use microfluidic partitioning and barcoding to generate cDNA libraries. These barcoded cDNAs can be easily sequenced by next generation sequencing platforms and used by various visualization tools to generate maps. Here, we describe methods to culture and differentiate human small intestinal HIOs in different formats and procedures for isolating viable cells from these formats that are suitable for use in single-cell transcriptional profiling platforms. These protocols and procedures facilitate the use of small intestinal HIOs to obtain an increased understanding of the cellular response of human intestinal epithelium at the transcriptional level in the context of a variety of different environments.

Author Spotlight: Integrating Single-Cell Transcriptomics with Organoid Cultures for Advanced Research and Therapeutic Insights

The protocol combines human intestinal organoid technology with single cell transcriptomic analysis to provide significant insight into previously unexplored intestinal biology.

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid ...