一、Experimental Preparation

1、hPSC-Induced Hepatic Organoid Differentiation Kit (abs90127-1kit)（abs90127-1kit）

Product Composition:

Culture Stage	Code	Name	Specification	Storage
Stage 1	H01	Base Medium 1	19.798mL	-20°C, 12 months
	H01-A	Supplement A	200uL	-20°C, 6 months
	H01-B	Supplement B	2uL	-20°C, 6 months
Stage 2	H02	Base Medium 2	19.9mL	-20°C, 12 months
	H02-C	Supplement C	100uL	-20°C, 6 months
Stage 3	H03	Base Medium 3	194.1mL	-20°C, 12 months
	H03-D	Supplement D	5.9mL	-20°C, 6 months
		Matrigel	5mL	-20°C, 6 months

 

2、auxiliary reagent

	Product Name	Product Number	Specification	Storage Conditions and Shelf Life
1	DMEM/F12	abs9560	500mL	Store at 2-8°C, protect from light, 12 months shelf life
2	PBS	abs962	500mL	Store at 2-8°C or room temperature, 12 months shelf life
3	Y-27632	abs812864	10mg	Store at -20°C, 1 year shelf life (lyophilized)
4	Human Multipotent Stem Cell Dissociation Solution	abs9409	100mL	Store at -20°C, 2 years shelf life
5	Advance Human Multipotent Stem Cell Culture Medium	abs9404	100mL	Store at -20°C, 2 years shelf life
6	Trypan Blue Staining Cell Viability Assay Kit	abs50036	500T	Store at 2-8°C, 12 months shelf life
7	Organoid Media Dissociation Solution	abs9520	500mL	Store at -20°C, 12 months shelf life

 

3、laboratory consumables

	Product Name	Product Number	Specification	Storage Conditions and Shelf Life
1	Cell Culture Plate (Standard Transparent 6-well)	abs7033	1 box	Room temperature, shelf life of 3 years
2	Cell Culture Plate (Standard Transparent 12-well)	abs7034	1 box	Room temperature, shelf life of 3 years
3	Cell Culture Plate (Standard Transparent 24-well)	abs7035	1 box	Room temperature, shelf life of 3 years
4	1.5 mL Centrifuge Tube	abs7119	1 box	Room temperature, shelf life of 3 years
5	15 mL Centrifuge Tube	abs7120	1 box	Room temperature, shelf life of 3 years
6	50 mL Centrifuge Tube	abs7121	1 box	Room temperature, shelf life of 3 years
8	10 mL Disposable Transfer Pipe	abs7053	1 box	Room temperature, shelf life of 3 years
9	50 mL Disposable Transfer Pipe	abs7055	1 box	Room temperature, shelf life of 3 years
10	10 µL Tip (Boxed, Sterile, No Enzyme)	abs7072	1 box	Room temperature, shelf life of 3 years
11	200 µL Tip (Boxed, Sterile, No Enzyme)	abs7075	1 box	Room temperature, shelf life of 3 years

 

 二、Induction Process Diagram

三、Cultivation Method

1、Differentiation of hPSCs into Endoderm Cells (DE) (using two wells of a 6-well plate as an example)
(1) Prepare DE differentiation medium ① by mixing 5.938 mL of Base Medium 1, 60 µL of Supplement A, and 2 µL of Supplement B, which can culture three wells of a 6-well plate at the same time;
(2) Thaw the Matrigel at 4°C and mix it evenly with DMEM/F12, then coat the plate and set aside for later use.
(3) When the confluence of hiPSCs reaches 75%-85%, aspirate the medium, wash the hiPSCs with 2 mL of 1x room temperature PBS (without Ca2+/Mg2+), and then aspirate the PBS.
(4) Add 1 mL of room temperature hESC/iPSC Passage Solution (a solution for digesting human pluripotent stem cells) and transfer to a 37°C, 5% CO2 incubator for 5 minutes to dissociate into single cells.
(5) After 5 minutes, aspirate the hESC/iPSC Passage Solution, add an equal volume of Advance Human Pluripotent Stem Cell Culture Medium, and gently pipette up and down with a P-1000 tip to create a single-cell suspension. Count the cells using an automatic cell counter and exclude dead cells with trypan blue. Collect the single-cell suspension into a 15 mL centrifuge tube and centrifuge at 300g for 5 minutes at room temperature.
(6) Carefully aspirate the coating solution from the Matrigel-coated plate without damaging the Matrigel coating surface.
(7) After centrifugation, completely remove the supernatant, resuspend the cells in 1 mL of DE differentiation medium ①, and gently pipette up and down to ensure an even single-cell solution.
(8) Transfer the cell suspension into the coated wells at a concentration of 2.0 x 10^5 cells/mL, add 1 mL of DE differentiation medium ①, bringing the total volume in each well to 2 mL;
(9) 24 hours later (Day 1), prepare DE differentiation medium ② by mixing 13.86 mL of Base Medium 1 with 140 µL of Supplement A, which can culture two wells of a 6-well plate at the same time. Aspirate the medium and add 2 mL of DE differentiation medium ②, continue culturing for 2 days, changing the medium every 24 hours.
(10) Before changing to HS medium on Day 3, one well can be taken for endoderm marker detection.
2、Induction of DE into Hepatic Progenitor Cells (HS) (using two wells of a 6-well plate as an example)
(1) On Day 3, mix 19.9 mL of Base Medium 2 with 100 µL of Supplement C to prepare HS medium, which can culture two wells of a 6-well plate at the same time. Aspirate the medium from the culture, wash each well of the 6-well plate with 2 mL of 1x room temperature PBS (without Ca2+/Mg2+), then remove the PBS from the wells, and add 2 mL of HS medium to each well.
(2) Place the culture plate in a 37°C, 5% CO2 incubator, change the medium every 24 hours, and continue culturing for 5 days.
(3) Before embedding in Matrigel on Day 8, one well can be taken for HS marker detection.
3、Induction of HS into 3D Hepatic Organoids (using six wells of a 24-well plate as an example)
(1) On Day 8, mix 194.1 mL of Base Medium 3 with 5.9 mL of Supplement D to prepare 3D hepatic organoid medium, aliquot and store.
(2) Thaw the Matrigel on ice, and place the pipette tips in a -20°C freezer in advance.
(3) Aspirate the HS medium and wash once with 2 mL of 1x room temperature PBS (without Ca2+/Mg2+).
(4) Add room temperature hESC/iPSC Passage Solution (a solution for digesting human pluripotent stem cells) containing 10 µM Y-27632 and incubate in a 37°C, 5% CO2 incubator for 5 minutes; after 5 minutes, aspirate the hESC/iPSC Passage Solution, add 1 mL of DMEM/F12 (containing 10 µM Y-27632) back to room temperature to each well, gently pipette the cells to detach them from the bottom of the well.
(5) Count the cells using an automatic cell counter and exclude dead cells with trypan blue.
(6) Collect the cell suspension into a 15 mL centrifuge tube and centrifuge at 300g for 5 minutes at room temperature, aspirate the medium and resuspend the cells in cold Matrigel at a density of 3000-10000 cells/well embedded in 30-50 µL of Matrigel, place the Matrigel with cells on ice.
(7) Dispense 200 µL of Matrigel/cell mixture into each well of a 24-well plate at 30 µL per well.
(8) Place the plate in a 37°C, 5% CO2 incubator for 20-30 minutes to allow the Matrigel to solidify.
(9) Add 700 µL of 3D hepatic organoid medium to each well for long-term culture, change the medium every other day, and passage every 7-10 days.
4、Passage of 3D Hepatic Organoids
(1) Thaw the frozen Matrigel at 4°C and pre-cool the pipette tips at -20°C for 1 hour in advance;
(2) Aspirate the medium, add 1 mL of pre-cooled PBS to dissolve the Matrigel, transfer the mixture to a 1.5 mL EP tube, centrifuge at 300g for 5 minutes, and remove the upper layer of PBS and Matrigel;
(3) Add 1 mL of organoid passage digestion solution, pipette 5-8 times to ensure full contact between the organoids and the digestion solution, then transfer to the incubator and let it stand to dissociate for 5 minutes;
(4) After 5 minutes, remove the EP tube, centrifuge at 300g for 5 minutes, remove the supernatant, add 1 mL of DMEM/F12, pipette the mixture in the well 40-50 times to completely dissociate the organoid clusters into single cells, centrifuge, and remove the supernatant;
(5) Use pre-cooled tips to add an appropriate amount of Matrigel to the centrifuge tube at 30 µL per well, pipette 5-8 times to evenly mix the single cells with Matrigel, being careful to avoid creating bubbles during pipetting;
(6) Remove the 24-well plate that was placed in the incubator in advance, add 30 µL of gel droplets in the center of each well, and slowly pipette the mixture while gradually lifting the pipette tip to evenly distribute the cells in the gel; (7) Place the culture plate in the incubator for 20-30 minutes to allow the gel drops to solidify;
(8) Carefully add 700 µL of 3D hepatic organoid medium back to room temperature along the wall of each well, being careful not to touch the gel drops;
(9) Place the plate in the incubator and continue culturing, change the medium every day (you can change it on Monday, Wednesday, and Friday, add up to 800 µL on Friday, and do not change it on Saturday and Sunday), passage once a week.
(10) After the hepatic organoids mature, they can be stained for identification or tested for hepatic progenitor cells/hepatic stem cells. 

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