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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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' laboratory reported the derivation of the first ASC-derived organoid, human intestinal organoid, in 2009^1,2. Afterward, ASC-derived organoids have been established for a variety of human organs and tissues, including prostate^3,4, liver^5,6, stomach^7,8,9, 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 culture^14,15.

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' lab^12,13. 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.

Protokół

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

1. Preparation of experimental materials
   1. Prepare basal medium by supplementing advanced DMEM/F12 medium with 2 mM glutamine, 10 mM HEPES, 100 U/mL of penicillin, and 100 µg/mL of streptomycin.
   2. 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 µM of Y-27632, 500 nM of A-83-01, 1 µM of SB202190, 5 ng/mL of fibroblst growth factor (FGF)-7, 20 ng/mL of FGF-10, and 100 µg/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).
   3. 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 °C. Thaw basement matrix in a 4 °C fridge. Keep the basement matrix and culture medium on ice during experimentation.
2. Cell isolation from human lung tissues for 3D organoid culture
   1. 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.
   2. 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 at 4 °C.
   3. 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 °C.
   4. Pipet up and down for 20x to shear the digested tissue pieces using a 10 mL serological pipette. Stack a 100 µm strainer on a 50 mL centrifuge tube and filter the suspension.
   5. 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., <0.5 cm) is procured.
   6. 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 °C.
   7. Resuspend the cell pellet in 10 mL of basal medium, followed by centrifugation at 400 x g for 5 min at 4 °C. Discard the supernatant.
   8. (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 °C. Discard the supernatant.
   9. Resuspend the pellet in cold basement matrix and keep on ice. Add 80-160 µL 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.
   10. Dispense 40 µL of suspension to each well of a pre-warmed 24-well suspension culture plate. Incubate the culture plate at 37 °C for 10-15 min. Let the basement matrix solidify to form a droplet.
   11. Add 500 µL 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.
   12. 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.
   13. 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.

2. Expansion of human lung organoids

1. 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.
2. Lung organoids passage with mechanical shearing
   1. 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 °C
   2. 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.
   3. Supplement with basal medium to a total volume of 10 mL, followed by centrifugation at 300 x g for 5 min at 4 °C. Resuspend the organoid fragments with cold basement matrix sufficient to enable a 1:3 to 1:5 expansion. Keep on ice.
   4. Place 40 µL of organoid suspension in each well of a pre-warmed 24-well plate. Incubate the culture plate at 37 °C for 10-15 min. Let the basement matrix solidify.
   5. Add 500 µL 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.
3. 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.
   1. 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 °C water bath for 3-5 min.
   2. 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 µL 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.
   3. Top up with basal medium to a final volume of 10 mL, followed by centrifugation at 300 x g for 5 min at 4 °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.
   4. Place 40 µL of organoid suspension in each well of a pre-warmed 24-well plate. Incubate the culture plate at 37 °C for 10-15 min. Let the basement matrix solidify.
   5. Supplement with 500 µL 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.
      ​NOTE: Around 100 organoids are mounted within a 40 µL 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.

3. Proximal differentiation to generate mature airway organoids

1. 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 µg/mL of heparin, 1 µM of hydrocortisone, 10 µM of Y-27632, and 10 µM of DAPT (see Table of Materials).
2. 3D airway organoids
   1. 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.
   2. 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.
   3. Alternatively, incubate the organoids at 37 °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.
3. 2D airway organoid
   1. 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.
   2. 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.
   3. Pre-incubate the inserts with the basal medium overnight in a cell culture incubator. Add 250 µL and 500 µL of basal medium in the top and bottom chamber of a 24-well plate, respectively. For a 12-well plate, add 500 µL and 1,000 µL of basal medium in the top and bottom chamber, respectively.
   4. 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 °C water bath for 3-5 min.
   5. 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 µL of FBS to terminate digestion.
   6. Filter the cells through a 40 µm 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 °C.
   7. Resuspend the pellet, collected from 24 droplets (40 µL), 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).
   8. Remove the basal medium from the top and bottom chambers. Add 500 µL and 1,000 µL of expansion medium in the bottom chamber of the 24-well insert and 12-well insert, respectively. Seed 100 µL and 500 µL of cell suspension prepared in step 3.3.7 onto the apical chamber of the 24-well insert and 12-well insert, respectively.
   9. 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.
   10. Measure trans-epithelial electrical resistance (TEER) every other day using an electrical resistance measurement systemaccording to a standard protocol described in¹⁷.

Wyniki

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 ...

Dyskusje

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...

Materiały

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