一、Experimental Preparation

1、hPSC-induced lung organoid differentiation kit（abs90128-1kit）

Composition of the product：

Cultivation Stage	Code	Name	Specification	Storage Conditions and Duration
Definitive Endoderm (DE) Stage	L01	Basic Medium 1	19.77mL	-20℃，12个月
	L01-A	Supplement A	30uL	-20℃，6个月
	L01-B	Supplement B	200uL	-20℃，6months
Anterior Foregut Endoderm (AFE) Stage		Basic Medium 2	20mL	-20℃，12months
Lung Progenitor Cells (LPC) Stage	L03	Basic Medium 3	39.898mL	-20℃，6months
	L03-C	Supplement C	102uL	-20℃，6months
Lung (3D) Organoid Formation Stage	L04	Basic Medium 4	19.96mL	-20℃，12months
	L04-D	Supplement D	40uL	-20℃，6months
Lung (3D) Organoid Differentiation Stage	L05	Basic Medium 5	19.95mL	-20℃，12months
	L05-E	Supplement E	50uL	-20℃，6months
Lung (3D) Organoid Maturation Stage	L06	Basic Medium 6	199.488mL	-20℃，12months
	L06-F	Supplement F	512uL	-20℃，6months
		Matrigel	5mL	-20℃，6months

 

2、Auxiliary Reagents

	Product Name	Product Number	Specification	Storage Conditions and Duration
1	DMEM/F12	abs9560	500mL	Store at 2-8℃, light protection required, valid for 12 months.
2	PBS	abs962	500mL	Store at 2-8℃ or room temperature, valid for 12 months.
3	Y-27632	abs812864	10mg	Store at -20℃, valid for 1 year (powder).
4	Cell Dissociation Solution (Stem Cell Grade) (Accutase)	abs47014935	100mL	Store at -20℃, valid for 2 years.
5	Advance Human Multipotent Stem Cell Culture Medium	abs9404	100mL	Store at -20℃, valid for 2 years.
6	Trypan Blue Staining Cell Viability Test Kit	abs50036	500T	Store at 2-8℃, valid for 12 months.
7	Organoid Metabolic Digestion Solution	abs9520	500mL	Store at -20℃, valid for 12 months.

 

3. Laboratory Consumables

	Product Name	Product Number	Specification	Storage Conditions and Duration
1	Cell Culture Plate (Standard Transparent 6-well Plate)	abs7033	1 Box	Room temperature, 3 years shelf life
2	Cell Culture Plate (Standard Transparent 12-well Plate)	abs7034	1 Box	Room temperature, 3 years shelf life
3	Cell Culture Plate (Standard Transparent 24-well Plate)	abs7035	1 Box	Room temperature, 3 years shelf life
4	1.5 mL Centrifuge Tube	abs7119	1 Box	Room temperature, 3 years shelf life
5	15 mL Centrifuge Tube	abs7120	1 Box	Room temperature, 3 years shelf life
6	50 mL Centrifuge Tube	abs7121	1 Box	Room temperature, 3 years shelf life
8	10 mL Disposable Transfer Pipe	abs7053	1 Box	Room temperature, 3 years shelf life
9	50 mL Disposable Transfer Pipe	abs7055	1 Box	Room temperature, 3 years shelf life
10	10 uL Pipette Tip (Boxed, Sterile and Enzyme-free)	abs7072	1 Box	Room temperature, 3 years shelf life
11	200 uL Pipette Tip (Boxed, Sterile and Enzyme-free)	abs7075	1 Box	Room temperature, 3 years shelf life

 

 二、Induction Process Diagram

三、Cultivation Method

1、Differentiation of hPSCs into Endoderm Cells (DE) (Calculated per well of a 6-well plate)
 (1) Thaw the Matrigel at 4°C, mix evenly with DMEM/F12, coat the plate, and set aside for later use.
(2) When the confluence of hiPSCs reaches 75%-85%, aspirate the culture medium, wash the hiPSCs with 2mL of 1x room temperature PBS (without Ca2+/Mg2+), and then aspirate the PBS.
(3) Add 1mL of room temperature Accutase containing Y-27632 (10 μM), transfer to a 37°C, 5% CO2 incubator for 20 minutes to dissociate into single cells.
(4) After 20 minutes, directly add an equal volume of complete hESC/iPSC culture medium to the Accutase, and gently pipette up and down with a P-1000 tip to create a single cell suspension. Collect the cell suspension into a 15mL centrifuge tube and centrifuge at 300 g for 5 minutes at room temperature.
(5) Carefully aspirate the coating solution from the Matrigel-coated plate without damaging the Matrigel coating surface.
(6) After centrifugation, completely remove the supernatant, resuspend the cells with 1 mL pre-warmed Advance Human Pluripotent Stem Cell Complete Medium (containing 10μM Y-27632), and gently pipette up and down to ensure an even single cell solution.
(7) Count the cells using an automatic cell counter, exclude dead cells with trypan blue, and seed the cells at a density of 2.0 * 10^5 cells/mL onto the 6-well Matrigel-coated plate prepared in step 1. The final volume per well in the 6-well plate should be 2 mL, and then incubate in a 37°C, 5% CO2 incubator for 24 hours.
(8) After 24 hours (Day 1), aspirate the culture medium and add 1.97mL of Basic Medium 1 + 10μL of Supplement A + 20μL of Supplement B.
(9) On Days 2 and 3, aspirate the culture medium, add 1.98mL of Basic Medium 1 + 20μL of Supplement B, and begin differentiation into endoderm cells.

2、Induction of DE into Anterior Foregut Endoderm (AFE) Cells

（1）On Day 4, aspirate the culture medium from the cultures, wash the cultures with 2 mL of 1x room temperature PBS (without Ca2+/Mg2+) per well in a 6-well plate, then remove the PBS from the wells, and add 2 mL of Basic Medium 2 per well.
（2）Place the culture plate in a 37°C, 5% CO2 incubator for cultivation, change the culture medium every 24 hours, and continue to culture for 3 consecutive days.

3、Induction of AFE into Lung Progenitor Cells (LPC) (Using 4 wells of a 12-well plate as an example)
(1) Prepare LPC induction medium by mixing 29.92mL of Basic Medium 3 with 80μL of Supplement C.
(2) On Day 7, aspirate the culture medium from the cultures, wash the cultures with 2 mL of 1x room temperature PBS (without Ca2+/Mg2+) per well in a 6-well plate, and then aspirate the PBS.
(3) Add 1mL of room temperature Accutase containing Y-27632 (10 μM) per well, and incubate the cultures at 37°C in a 5% CO2 incubator for 10 minutes.
(4) After 10 minutes, add 1mL of DMEM/F12 (containing 10μM Y-27632) per well, gently pipette the cells to detach them from the bottom of the wells.
(5) Collect the cell suspension into a 15 mL centrifuge tube and centrifuge at 300 g for 5 minutes at room temperature.
(6) Carefully aspirate the supernatant without disturbing the cells, resuspend the cells with 1mL of LPC induction medium (pre-warmed to room temperature and containing 10μM Y-27632), and gently pipette up and down to ensure an even single cell solution.
(7) Count the cells using an automatic cell counter, exclude dead cells with trypan blue, and seed the cells at a density of 2.0 x 10^5 cells/mL onto the pre-prepared 12-well Matrigel-coated plate, with a final volume of 1 mL of medium per well in the 12-well plate.
(8) The next day, replace the medium with LPC induction medium without Y-27632. Change the medium every other day for 11 days.

4、Induction of LPC into 3D Lung Organoids (Using 6 wells of a 24-well plate as an example)
(1) On Day 18, prepare 3D organoid induction medium by mixing 14.965mL of Basic Medium 4 with 35μL of Supplement D.
(2) Thaw the Matrigel on ice, and pre-chill the pipette tips in a -20°C freezer.
(3) Aspirate the LPC induction medium and wash once with 1 mL of 1x PBS (without Ca2+/Mg2+).
(4) Add room temperature Accutase containing 10μM Y-27632 (0.5 mL/well in a 12-well plate) and incubate at 37°C, 5% CO2 incubator for 10 minutes; after 10 minutes, add 1mL of DMEM/F12 (pre-warmed to room temperature and containing 10μM Y-27632) per well, gently pipette the cells to detach them from the bottom of the wells.
(5) Collect the cell suspension into a 15 mL centrifuge tube and centrifuge at 300 g for 5 minutes at room temperature.
(6) Carefully aspirate the supernatant without disturbing the cells, resuspend the cells with 1mL of 3D organoid induction medium, and gently pipette up and down to ensure an even single cell solution.
(7) Count the cells using an automatic cell counter, exclude dead cells with trypan blue. Note: The cell number must be optimized for each cell line; during the 18 days of differentiation, the cells should not become overly confluent.
(8) Centrifuge at 300 g for 5 minutes at room temperature, aspirate the medium and resuspend the cells in cold Matrigel, for ESCs, add 200μL of Matrigel per well with 4.010^4 cells, for iPSCs, add 200 μL of Matrigel per well with 8.010^4 cells, place the Matrigel and cells on ice together.
(9) Dispense 200μL of Matrigel/cell mixture into each well of the 24-well plate at 30μL per well.
(10) Place the plate in a 37°C, 5% CO2 incubator for 20-30 minutes to allow the Matrigel to solidify.
(11) Add 700μL of 3D organoid induction medium per well, and change the medium every other day for 6 days.
(12) On Day 23, prepare 3D organoid differentiation medium by mixing 14.96mL of Basic Medium 5 with 40μL of Supplement E, replace the medium in the wells with 700μL of pre-warmed 3D organoid differentiation medium, and change the medium every other day for 6 days.
(13) On Day 29, prepare 3D organoid maturation medium by mixing 199.48mL of Basic Medium 6 with 520μL of Supplement F, replace the medium in the wells with 700μL of pre-warmed 3D organoid maturation medium, and change the medium every other day for long-term culture.
5、Passaging of 3D Lung Organoids
(1) Take out the frozen Matrigel and thaw it at 4°C, and pre-chill the pipette tips at -20°C for 1 hour in advance;
(2) Aspirate the medium, add 1mL of pre-chilled organoid passaging digestion solution, and let it sit for 1 minute;
(3) Use a 1mL pipette to pipette the mixture in the well 40-50 times, note: avoid generating bubbles; (4) Add 1mL of pre-chilled PBS per well, transfer the mixture to a 15mL centrifuge tube;
(5) Wash the culture plate with 1mL of pre-chilled PBS, add this washing solution to the centrifuge tube from the previous step, and supplement with pre-chilled PBS to a final volume of 12mL in the centrifuge tube; centrifuge at 300g for 5 minutes, and remove the supernatant;
(6) Use a pre-chilled pipette tip to add an appropriate amount of Matrigel to the centrifuge tube at 30μL per well, pipette 5-8 times to evenly mix the single cells with Matrigel, note: avoid generating bubbles during pipetting;
(7) Take out the 24-well plate that has been pre-placed in the incubator, add 30μL of gel drop in the center of the well, and gradually move the pipette tip upward while slowly dispensing the mixture to evenly distribute the cells in the gel;
(8) Place the culture plate in the incubator for 20-30 minutes to allow the gel drops to solidify;
(9) Carefully add 700μL of pre-warmed 3D organoid maturation medium along the wall of the well, change the medium every day (you can change it on Monday, Wednesday, and Friday, add up to 800μL on Friday, and do not change it on Saturday and Sunday), note: avoid touching the gel drops, place the plate in the incubator and continue to culture.

 

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