hPSC Induction Differentiation Liver Organoid Kit
hPSC Induction Differentiation Liver Organoid Kit
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Product Specification
| Usage |
I. Experimental instruments and materials: hESC/iPSC a cell culture kit, hESC （ H1 ）、 hESC/iPSC passage working fluid, Y-27632 、 DMEM/F12 Culture medium, low growth factor matrix gum, matrix gum, Accutase 、 PBS 、 6 hole /12/24 well cell culture plates, 15mL/50mL centrifuge tube, 10μL/200μL/1000μL a sterile tip, 10mL/50mL Pipette, low adsorption 24 well cell culture plate 2. Reagent preparation:
(2) In the biosafety cabinet, refer to the table 1 and table 3 Use a sterile pipette and a gun tip to mix evenly and prepare the culture medium for each stage of componentization according to the proportion. for example DE Induction medium a Composition as basic culture medium 1 、 1× concentration of supplements A If the dosage is 7mL The preparation method is 6.875mL Basic medium 1+0.125mL supplements A （ 56× ）。 (3) Differentiation medium is recommended to be prepared and used fresh, and placed in 4℃ storage, 2 Use during the week. Note: All supplements can be repackaged according to the amount used to avoid repeated freezing and thawing. E Store away from light, and avoid strong light exposure during preparation. III. hESC Cultivation and preparation See details abs90487 hESC/iPSC Instructions for use of cell culture kit hESC/iPSC Cell Culture Kit Product Instructions IV. DAY 0-4: definitive endoderm stage (1) Matrix glue in 4℃ Thawing below, with DMEM/F12 Homogeneous mixing (low growth factor matrix gum :DMEM/F12=1:100 ), lay the boards, and set them aside. (2) DAY-1: When hESC The convergence degree reaches 75%-85% Aspirate off the culture medium, use 2mL1x room temperature PBS (Excluding Ca2+/Mg2+) cleaning hESC , suck away PBS 。 (3) join 1mL Contains Y-27632 （ 10 μM ) room temperature Accutase , transfer to 37℃ 5%CO2 In the cell culture box 3-5min To dissociate them into single cells. (4) 3-5min Subsequently, the stem cell subculture medium was added directly in an equal volume. Accutase In, and use P-1000 The tip gently blows the cells up and down to create a single-cell suspension, which is then collected into... 15mL In a centrifuge tube, at room temperature 300 g centrifuge 5min 。 (5) The coating liquid was carefully aspirated from the low growth factor matrix gel coated plate without damaging the matrix gel coating surface. (6) After centrifugation is complete, the supernatant is fully removed. 1mL preheated hESC/iPSC Complete culture medium (including 10μM Y-27632 Resuspend the cells and gently pipette up and down to ensure uniformity of the single-cell solution. (7) take 0.5mL Cell suspension (or 1:1-1:2 Passaging), seeding cells to step 1 On the prepared matrix glue-coated plate, 12 The final volume of each well in the well plate is 1mL (Including 10μMY-27632 ), transfer the well plate to 37℃ 、 5%CO2 Incubation in cell culture box 24h 。 (8) DAY0: 24h The culture medium is then aspirated away (the density should be 95%-100% ), and join 1mLDE Induction medium a (Ingredients: basic culture medium) 1 ， 1× supplements A ) cultivation 24h 。 (9) DAY1: 24h Then aspirate the culture medium and use... DE Induction medium b (Ingredients: basic culture medium) 1 ， 1× supplements B Change the fluid until the first 4 During this period, the medium was changed daily or every other day depending on the color of the medium. Note: ① When using the matrix adhesive, it must be handled on ice. All items in direct contact with the matrix adhesive must be pre-cooled beforehand. If the matrix adhesive exceeds... 10℃ It will solidify rapidly, resulting in failure of the plating. ② Massive cell death on the first day of induction resulted in a density of less than 50% Solution: a Can be induced in the first 0 Add to the culture medium 10μMY-27632 cultivate 24 Remove after an hour Y-27632 ； b , can be induced respectively in the first 1 Heaven, first 2 Added by day 0.2% and 2% Fetal bovine serum, the first 3 The sky was removed; c In inducing the first 0 Tianwang culture medium (required) 4℃ Add to pre-cooling 2% The matrix glue lays a layer of matrix glue on the cells to form a matrix glue sandwich structure to prevent cell death. V. DAY 4-7:PFG cell stage (1) DAY4 ： No 4 Discard the culture medium. Use 1mL room temperature PFG Induction medium (consisting of: basic medium) 2 ， 1× supplements C Change the fluid. (2) Use daily or every other day depending on the color of the culture medium PFG Change the induction medium until the first 7 heaven. VI. DAY 7-11:PFG amplification stage (1) DAY7: No 7 Days later, the culture medium was aspirated from the culture. 12 Each hole in the hole plate is used 1mL1× room temperature PBS (Excluding Ca2+/Mg2+) Wash the culture and aspirate. PBS 。 Use 0.5mL room temperature Accutase Digestion in incubator 3-5 min Use equal volumes DMEM/F12 Stop digestion, blow up the cells, and transfer to 1.5mL In the centrifuge tube. (2) After counting 300g centrifuge 3min Discard the supernatant and use a small amount ( 5-20µL Precooling PBS or PFG Expansion medium (consisting of: basal medium 3 ， 1× supplements D ) Resuspended. (3) Based on the cell size, add a certain amount of matrix gel and mix, according to 1×105 microscopic cell /50µL matrix rubber / Proportional points of holes 50 µL In 24 The center of one hole in the orifice plate is placed in 37℃ incubator solidification 20-30min 。 (4) After the gel droplets solidify, they should form a dome-shaped structure without bubbles or collapse. Per hole 500µL Add the amount PFG Expansion medium culture, used every other day PFG Change the expansion medium to the first 11 heaven. Note: ① All operations related to the matrix gel, except for well plates, all consumables and culture media that come into contact with the matrix gel must be kept in a safe condition. 0-4℃ Otherwise, the gel will fail. VII. DAY 11-15:HepL induction stage (1) DAY11: No 11 Days later, the culture medium was aspirated from the culture, the original medium was discarded from each well, and then used... 500µL room temperature HepL Induction medium (consisting of: basic medium) 2 ， 1× supplements E Change the fluid. (2) Use the next day HepL Change the induction medium until the first 15 heaven. 8. DAY 15-25:LO mature stage (1) DAY15: No 15 During the day, discard the culture medium and add pre-cooled medium to each well. 500µL calcium-magnesium-free PBS Blow the glue droplets into pieces and transfer to 1.5mL In the centrifuge tube, 300g centrifuge 3min Discard the supernatant and the upper transparent matrix layer, and repeat the process. procedure 3 Generally, to remove the matrix glue as much as possible. (2) Based on the amount of organoids collected, add a certain amount of matrix gel and mix, according to 1:2 Proportions (e.g. 24 orifice plate 1 One hole can transmit two holes) points 50µL In 24 The low-adsorption plate with holes is placed in the center of one hole. 37℃ incubator solidification 30-40min (because The resuspended organoid originally contained liquid, at which point the matrix gum is equivalent to diluted, so the coagulation time needs to be prolonged). (3) After the gel droplets solidify, press each well. 500µL Add the amount LO Mature medium (consisting of: basic medium) 4 ， 1× supplements F Culture and use the next day. LO The mature medium was changed to the first 25 heaven. Note: ① If collected 5 The amount of pores, the centrifuged precipitation volume reaches 100µL , should use 410-420µL Resuspension of matrix adhesive (due to the viscosity of the matrix adhesive, some loss may occur; reservation is required). 10-20µL ), press 1 ： 2 Proportional transmission 10 Hole. ② Because of the use 24 The low-hole adsorption plate allows for gentle movement during gel operation to prevent the formation of irregular gels. Furthermore, gel droplets will float up during liquid change, so it can be operated according to the suspension liquid change procedure. ③ All operations related to the matrix gel, except for well plates, all consumables and culture media that come into contact with the matrix gel must be kept in a safe condition. 0-4℃ Otherwise, the gel will fail. |
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| Description | This kit is specifically designed for the efficient and targeted differentiation of human pluripotent stem cells into liver organoids. It provides a standardized, serum-free, and chemically defined culture system, suitable for disease model construction, drug screening, and cell therapy research. The obtained liver organoids can express liver parenchyma and hepatic bile duct cell markers (such as ALB 、 CYP3A4 、 CK19 etc.), has the function of secreting albumin and is suitable for in vitro research. This product requires operators to have experience and common knowledge in organoid culture. With 12 Taking orifice plates as an example, the specifications provided by this product can supply at least 6 Human pluripotent cells with multiple wells induce differentiation, and depending on the differentiation method, ultimately obtain... 90-120 Each gel droplet may contain... 50-200 An organoid.  Note: The content in parentheses is as 56× This is the mother liquor concentration; the final concentration during use should be... 1× 。 Such as supplements A （ 56× It needs to be added to the basic culture medium. 1 Dilute it 56 Times, making the supplement A The final concentration is 1× , composition DE Induction medium a 。 hPSC （ human pluripotent stem cell ): human pluripotent stem cells; DE （ definitive endoderm ): definitive endoderm; PFG （ Posterior Foregut ): posterior foregut; HepL （ Hepatic lineage ): Liver lineage; LO （ Liver organoid ): Liver organoids
Product Composition:
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| Storage Temp. | Please refer to the product composition for details. |
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Immunohistochemistry
