hPSC-Derived Small Intestine Organoid Differentiation Kit
hPSC-Derived Small Intestine Organoid Differentiation Kit
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Product Specification
| Description |
This product is a kit for inducing differentiation of small intestinal organoids from hPSCs. Human pluripotent stem cells (hPSCs) can be induced and differentiated with this kit to generate small intestinal organoids. These organoids are composed of intestinal cells. After maturation, they consist of polarized intestinal epithelium patterned into villus-like structures, together with surrounding mesenchymal cells that secrete niche factors.
Operators must have hands-on experience in hPSC culture and basic understanding of organoid manipulation. Product Composition:
Notice: For research use only. All components are shipped separately under dry ice conditions. |
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| Usage |
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), 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 Contents and Methods: 1. Matrigel Preparation (1) Matrigel for hPSC plating shall be aliquoted according to the batch instructions. Thaw Matrigel at 4 ℃, mix evenly with DMEM/F12 supplemented with 15 mM HEPES, then store for later use. Table 1 Recommended Matrigel Volume for Culture Vessel Coating
2. Preparation of Definitive Endoderm (DE) Medium (1) Prepare sufficient DE Medium (Basal Medium 1 + Supplement A) for Day 0, Day 1 and Day 2 at a working volume of 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 ℃ if unused; do not exceed shelf life. Use immediately after thawing, avoid repeated freeze-thaw cycles. Thaw intact Basal Medium 1 at room temperature (15–25 ℃) for 1 h or overnight at 4 ℃, mix thoroughly. Note: Unused medium can be stored at 2–8 ℃ for up to 2 months, or aliquoted and frozen at -20 ℃. Do not exceed expiry date on label. Thawed aliquots can be stored at 2–8 ℃ for maximum 2 weeks; do not refreeze. Add 20 μL Supplement A into 1.98 mL cold (2–8 ℃) Basal Medium 1, mix well and store at 2–8 ℃. 3. Differentiation to Definitive Endoderm (DE) (24-well plate as example) (1) Day 0: Pre-warm required DE Medium to 37 ℃ at 0.7 mL per well. Reserve remaining medium at 2–8 ℃. Aspirate complete medium from hPSC cultures. Tilt plate and slowly add 0.7 mL DE Medium along each well wall. Incubate at 37 ℃, 5% CO₂, 95% humidity for up to 24 h. (2) Day 1: Pre-warm required DE Medium to 37 ℃ at 0.5 mL per well. Reserve remaining medium at 2–8 ℃. Aspirate spent medium, slowly add 0.5 mL fresh DE Medium along well wall, incubate at 37 ℃, 5% CO₂, 95% humidity for 24 h. (3) Day 2: Pre-warm remaining DE Medium. Aspirate spent medium, add 0.5 mL DE Medium per well, incubate at 37 ℃, 5% CO₂, 95% humidity for 24 h. (4) Day 3: Cells can be harvested for definitive endoderm marker detection; parallel wells can be further differentiated into mid-/hindgut spheroids (MH). Note: Mass cell death may occur during DE induction. Minimize incubation time outside the cell incubator. Cells are extremely sensitive after 24 h DE induction, handle medium exchange gently. A confluent monolayer of compact endodermal cells will form after 72 h incubation. 4. Preparation of Mid-/Hindgut (MH) Medium (1) Prepare sufficient MH Medium (Basal Medium 1 + Supplement B + Supplement C) for Day 3 to Day 8 at a working volume of 3 mL per well. (2) Protocol for preparing 3 mL MH Medium: Thaw Supplement B and C on ice, keep cold before mixing with basal medium, mix thoroughly. Note: Aliquot and store at -20 ℃ if unused; do not exceed shelf life. Use immediately after thawing, avoid repeated freeze-thaw cycles. Add 30 µL Supplement B and 30 µL Supplement C into 2.94 mL cold (2–8 ℃) Basal Medium 1, mix well and store at 2–8 ℃. 5. Differentiation from DE Cells to Mid-/Hindgut Spheroids (MH) (1) Day 3: Pre-warm sufficient MH Medium to 15–25 ℃ at 0.5 mL per well. Reserve remaining medium at 2–8 ℃. (2) Aspirate spent DE medium, add 0.5 mL MH Medium per well, incubate at 37 ℃, 5% CO₂, 95% humidity for 24 h. (3) Day 4 to Day 9: Perform full medium exchange every 24 h and monitor spheroid formation. Note: Return cultures to incubator within 30 min after removal. a. Observe monolayer under microscope; 3D structures may appear as early as Day 4. Free-floating mid-/hindgut spheroids emerge from Day 5 to Day 9. b. Use 1 mL pipette to transfer 0.5 mL spent medium into a new clear 24-well plate to quantify spheroid number and density. c. Add 0.5 mL fresh MH Medium to each well, incubate at 37 ℃, 5% CO₂, 95% humidity. Note: Spheroids harvested from Day 5–9 all generate intestinal organoids; MH exposure duration determines regional identity (shorter for duodenum, longer for ileum). Peak spheroid yield varies across hPSC lines. For reproducible results, always harvest spheroids on the same differentiation day to initiate intestinal organoid culture. Maximum budding is typically observed on Day 8. (4) Day of embedding: Transfer spheroid suspension from each well into a new 24-well plate for counting. Transfer volume containing ~50 free-floating spheroids into a 15 mL conical tube for intestinal organoid embedding. Note: Mid-/hindgut spheroids are cell aggregates ≥75 µm in diameter capable of forming human intestinal organoids. Multiple fused spheroids count as one unit. Remaining monolayers can be harvested for MH marker detection or cultured longer to generate more spheroids for embedding. 6. Preparation of Organoid Growth Medium (OGM) (1) Prepare required volume of Intestinal OGM (Basal Medium 2 + Supplement D) at 2 mL per well. (2) Protocol for preparing 2 mL Intestinal OGM: Thaw Supplement D on ice, keep cold before mixing with basal medium, mix thoroughly. Note: Aliquot and store at -20 ℃ if unused; do not exceed shelf life. Use immediately after thawing, avoid repeated freeze-thaw cycles. Add 40 µL Supplement D into 1.96 mL cold (2–8 ℃) Basal Medium 2, mix thoroughly, warm to room temperature (15–25 ℃) before use. Stored at 2–8 ℃ for maximum 2 weeks. 7. Embedding Spheroids in Matrigel (1) Aliquot low-growth-factor Matrigel and store frozen (200 μL per aliquot for 4 samples). (2) Thaw one Matrigel aliquot on ice (50 µL Matrigel per sample); pre-charge sterile 100 µL pipette tips at -20 ℃ in advance. (3) Let MH spheroids settle to the bottom of the conical tube, carefully aspirate and discard supernatant. (4) Add 1 mL DMEM/F-12 supplemented with 15 mM HEPES, centrifuge at 300 × g for 5 min at room temperature (15–25 ℃). (5) Carefully aspirate all supernatant with 1 mL pipette. Note: Remove supernatant as completely as possible. (6) Keep thawed Matrigel and cell suspension on ice. Pipette 50 μL pre-cooled Matrigel with cold tips and mix with spheroid pellet, avoid bubble formation during mixing. (7) Seed 50 μL Matrigel-spheroid mixture per pre-chilled 24-well plate well; lift pipette tip gradually while dispensing to achieve even spheroid distribution inside gel droplets. (8) Place plate in cell incubator for 20–30 min to solidify Matrigel droplets. (9) Gently add 500 μL room-temperature Intestinal OGM along well wall without touching gel droplets; refresh medium every 3–4 days. (10) Intestinal organoids fully mature within 7–10 days for passaging, staining identification and related functional assays. |
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| Storage Temp. | Different components have different storage requirements and validity periods, refer to the product composition table for details. All kit components are shipped separately with dry ice. |
