hPSC-Derived Colon Organoid Differentiation Kit
hPSC-Derived Colon Organoid Differentiation Kit
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1. Experimental Instruments and Materials: Instruments: Biological safety cabinet, cell incubator, horizontal centrifuge, inverted microscope, low-temperature refrigerator Materials: Cell culture plates (6-well, 12-well, 24-well, 96-well), centrifuge tubes (15 mL, 50 mL), pipettes (10 μL, 100 μL, 1000 μL), sterile pipette tips (10 μL, 200 μL, 1000 μL), serological pipettes (10 mL, 50 mL) 2. Experimental Protocols: 1. Preparation of Matrigel (1) Stem cell qualified Matrigel is required for hPSC plating. Aliquot Matrigel following batch instructions. Thaw Matrigel at 4 °C, mix evenly with DMEM/F12 supplemented with 15 mM HEPES, coat culture vessels and set aside. Table 1. Recommended volume of diluted Matrigel for vessel coating Tissue culture treated vessel | Volume of diluted Matrigel 24-well plate | 250 µL/well 12-well plate | 500 µL/well 6-well plate | 1 mL/well 100 mm dish | 6 mL/dish T-25 cm² flask | 3 mL/flask T-75 cm² flask | 8 mL/flask 2. Preparation of Definitive Endoderm (DE) Medium Note: This kit supports hPSC differentiation from DE to hindgut for 20 wells of a 24-well plate. It is recommended to test with 2–3 wells for the first trial. (1) On Day 0, prepare DE medium (Basal Medium 1 + Supplement A) required for Day 0, Day 1 and Day 2 (1.7 mL per well). (2) Protocol for preparing 2 mL DE medium: Thaw Supplement A on ice and mix thoroughly. Note: Aliquot and store at -20 °C if not used immediately. Do not exceed the shelf life. Use immediately after thawing aliquots; do not re-freeze. Thaw the complete bottle of Basal Medium 1 at room temperature (15–25 °C) for 1 h or overnight in a 4 °C refrigerator, then mix well. Note: Can be stored at 2–8 °C for up to 2 months if unused. Alternatively, aliquot and store at -20 °C. Do not exceed the labeled expiry date. After thawing aliquots, use promptly or store at 2–8 °C for maximum 2 weeks; do not re-freeze. Add 20 μL Supplement A into 1.98 mL cold (2–8 °C) Basal Medium 1. Mix thoroughly and store at 2–8 °C. 3. Differentiation into Definitive Endoderm (DE) (24-well plate format) Passage hPSCs into 24-well plates. Initiate DE differentiation when cell confluency reaches 85%–90% (defined as Day 0). (1) Day 0: Pre-warm required volume of DE medium to 37 °C (0.7 mL/well). Store remaining DE medium at 2–8 °C. Aspirate complete medium from hPSCs. Tilt the plate and slowly add 0.7 mL DE medium along the side of each well. Incubate at 37 °C, 5% CO₂, 95% humidity for up to 24 hours. (2) Day 1: Pre-warm required DE medium to 37 °C (0.5 mL/well). Store leftover medium at 2–8 °C. Remove spent medium. Tilt plate and add 0.5 mL fresh DE medium per well. Incubate for 24 hours at 37 °C, 5% CO₂, 95% humidity. (3) Day 2: Pre-warm remaining DE medium. Aspirate old medium and add 0.5 mL DE medium per well. Incubate for 24 hours at 37 °C, 5% CO₂, 95% humidity. (4) Day 3: Definitive endoderm formation can be examined. Replicate wells can be further differentiated into mid-hindgut spheroids (MH). Note: Substantial cell death may be observed during DE induction. Minimize incubation time outside the 37 °C incubator. Cells become very sensitive 24 h after DE induction; perform medium changes gently. After 72 h incubation, a confluent monolayer of tightly packed endodermal cells will form. 4. Preparation of Mid-Hindgut (MH) Medium (1) On Day 3, prepare MH medium (Basal Medium 2 + Supplement B + Supplement C) for Day 3–Day 8 (3 mL per well). (2) Protocol for preparing 3 mL MH medium: Thaw Supplement B and Supplement C on ice and keep cold until mixing with Basal Medium 2. Mix thoroughly. Note: Aliquot and store at -20 °C if unused. Do not exceed shelf life. Use immediately after thawing; avoid repeated freezing. Add 30 µL Supplement B and 30 µL Supplement C into 2.94 mL cold (2–8 °C) Basal Medium 2. Mix fully and store at 2–8 °C. 5. Differentiation of Endodermal Cells into Mid-Hindgut (MH) (1) Day 3: Equilibrate sufficient MH medium at 15–25 °C (0.5 mL/well). Reserve remaining MH medium at 2–8 °C. (2) Remove medium from endodermal cells and replace with 0.5 mL MH medium. Incubate for 24 h at 37 °C, 5% CO₂, 95% humidity. (3) Day 4–Day 8: Perform full medium exchange every 24 hours and monitor spheroid development. Note: Return cultures to incubator within 30 minutes after removal. a. Observe monolayers under microscope. 3D structures may emerge as early as Day 4. Free-floating mid-hindgut spheroids appear between Day 5 and Day 8. b. Use a 1 mL pipette to transfer 0.5 mL spent medium into a sterile clear 24-well plate to quantify free spheroids released from the monolayer. c. Add 0.5 mL fresh MH medium back to cells. Incubate at 37 °C, 5% CO₂, 95% humidity. Note: The peak yield of mid-hindgut spheroids varies between hPSC lines. For reproducible results, always harvest spheroids generated on the same differentiation day to initiate human colon organoid culture. Maximum budding is typically observed on Day 8. (4) Day of spheroid embedding: Transfer spheroid suspension from each well into a new 24-well plate for counting. Transfer suspension containing approximately 70–100 free-floating spheroids (based on counting) into a 15 mL conical tube for colon organoid culture. Attention: Mid-hindgut spheroids are cell aggregates ≥75 µm in diameter capable of forming human colon organoids. Multiple fused spheroids count as one unit that generates one organoid. Remaining monolayers can be used to characterize mid-hindgut formation or further differentiated to obtain more spheroids in subsequent days. 6. Preparation of Human Colon Organoid (HCO) Medium (1) Prepare required HCO medium (Basal Medium 3 + Supplement D + Supplement E + Supplement F) (2 mL per well). (2) Protocol for preparing 100 mL HCO medium: Thaw Supplement D, Supplement E and Supplement F on ice and keep cold before mixing. Mix thoroughly. Note: Prepared medium can be stored at 2–8 °C for up to 6 weeks if unused. Do not exceed reagent expiry dates. Use aliquots immediately after thawing; do not re-freeze. Add 100 μL Supplement D, 50 µL Supplement E and 2 mL Supplement F into 98 mL cold (2–8 °C) Basal Medium 3. Mix completely and store at 2–8 °C. 7. Embedding Spheroids in Matrigel (1) Aliquot low growth factor Matrigel and freeze (200 μL per aliquot for 4 samples). (2) Thaw one aliquot of low growth factor Matrigel on ice (50 µL Matrigel required per sample). Place sterile 100 µL pipette tips at -20 °C for pre-chilling. (3) Allow collected MH spheroids to settle at the bottom of the conical tube, then carefully aspirate and discard supernatant. (4) Add 1 mL DMEM/F-12 supplemented with 15 mM HEPES. Centrifuge at 300×g for 5 minutes at room temperature (15–25 °C). (5) Carefully aspirate supernatant using a 1 mL pipette. Note: Remove supernatant as completely as possible. (6) Keep thawed Matrigel and cell suspension on ice. Mix 50 μL pre-chilled Matrigel with cell suspension, avoid bubble formation. (7) Seed 50 μL cell-Matrigel mixture per well into a 24-well plate using pre-cooled tips. Slowly dispense while gradually lifting the pipette tip to ensure uniform cell distribution. (8) Place plate in incubator for 20–30 min to allow Matrigel polymerization. (9) Thaw Colon Organoid Supplement G on ice and mix well. Prepare working solution by adding 5 μL Supplement G into 5 mL HCO medium. Gently add 500 μL supplemented medium along the well wall without disturbing solidified gel droplets. Culture at 37 °C, 5% CO₂, 95% humidity. Note: Supplement G working solution must be freshly prepared according to required volume right before use. 8. Medium Exchange for Human Colon Organoids (HCO) (1) Perform medium replacement with HCO medium (without Supplement G) on Day 3. Add additional 500 μL HCO medium (without Supplement G) to each well and incubate at 37 °C, 5% CO₂, 95% humidity. (2) Refresh medium three times per week. Passage organoids into fresh Matrigel every 10–14 days with appropriate splitting ratio (~80–100 organoids per vessel). Note: Antibiotics (e.g. 50 μg/mL gentamicin) may be added after the first passage. (3) After the 3rd passage, mature colorectal organoids are fully established and ready for characterization and downstream experiments. Note: Growth rate may decrease after prolonged passaging, possibly caused by loss of mesenchymal cells and stem cell populations. |
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| Description |
This product is the hPSC-derived colon organoid induction and differentiation kit. Human pluripotent stem cells (hPSCs) can be induced and differentiated using this kit to generate colon organoids composed of colon-related cell populations. These organoids express specific human colon markers, including colon transcription factor SATB2, α-carbonic anhydrase II (CA-II), α-carbonic anhydrase IV (CA-IV), goblet cell markers Mucin-2 (MUC2) and Mucin-5B (MUC5B), as well as posterior HOX genes. Operators are required to have hands-on experience in hPSC culture and fundamental knowledge of organoid technology. Product Composition:
Notice: For research use only. Transported with dry ice. |
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| Storage Temp. | Please refer to storage conditions listed in product composition table. Shipped on dry ice. |
