hPSC诱导分化小肠类器官试剂盒

2 , endoderm ( DE Preparation of culture medium

（ 1 ) in the 0 God, prepare the first 0 、 1 and 2 Daily requirements DE Culture medium (basic medium 1 + supplements A ) volume ( 1.7 mL/ hole).

（ 2 ) to prepare 2 mL DE Taking the culture medium as an example:

Thaw replenishment on ice A 。 Mix thoroughly.

Note: If not used immediately, repackage and store in -20°C Lower storage. Do not exceed the shelf life of the supplement. After thawing aliquots, use immediately, do not refreeze.

At room temperature (15 - 25°C) thaw 1h or In 4°C Thaw the entire bottle of basic culture medium overnight in the refrigerator. 1 Mix thoroughly.

Note: If not used immediately, it can be used in 2 - 8°C Store for up to 2 months. Alternatively, repackage and -20°C Lower storage. Do not exceed the expiration date indicated on the label. After thawing the aliquot, use immediately or in 2 - 8°C Store for up to 2 Zhou, do not refreeze.

will 20 μL supplements A join 1.98 mL cold (2 - 8°C) Basic medium 1 middle. After thorough mixing, store in 2 - 8°C 。

3 Differentiation into endoderm cells ( DE )(with 24 (Taking orifice plates as an example)

（ 1 )th 0 Day: Preheating  (37°C) No 0 Daily requirements DE medium volume (0.7 mL/ hole ) 。 The remaining DE The culture medium is stored in 2 - 8°C 。

From hPSC The cells are completely absorbed and cultured. Add dropwise to each well of the tilted culture plate along the well wall. 0.7 mL DE culture medium. In  37°C 、 5% CO₂ and  95% Most incubated under humidity  24 Hours.

（ 2 )th 1 Day: Preheating (37°C) No 1 Required for daily use DE medium volume (0.5 mL/ hole ) 。 The remaining DE The culture medium is stored in 2 - 8°C 。 The culture medium is aspirated from the cells. Add dropwise to each well of the tilted culture plate along the well wall. 0.5 mL DE culture medium. In  37°C 、 5% CO₂ and  95% Incubation under humidity  24 Hours.

（ 3 )th 2 Day: Preheating (37°C) The remaining DE culture medium. The culture medium is aspirated from the cells. Add dropwise to each well of the tilted culture plate along the well wall. 0.5 mL DE culture medium. In  37°C 、 5% CO₂ and  95% Incubation under humidity  24 Hours.

（ 4 )th 3 Day: Cells can detect the formation of definitive endoderm. Multiporous cells can continue to differentiate into middle and hindgut spheres ( MH ）。

Note: During definitive endoderm induction, cells appear to undergo massive cell death. Shorten the cell life as much as possible 37°C Time outside the incubator. pass 24 After hours of definitive endoderm induction, the cells are very sensitive and require careful medium changes. incubation 72 After hours, a confluent monolayer of closely aligned endoderm cells will form.

4 ,middle / hindgut (MH) Preparation of culture medium

（ 1 ) in the 3 God, prepare the first 3 - 8 Daily requirements MH Culture medium (basic medium 1 + supplements B + supplements C ) volume ( 3 mL/ hole).

（ 2 ) to prepare 3 mL MH Taking the culture medium as an example:

Thaw replenishment on ice B and C 。 After mixing in basic culture medium 1 Previously kept on ice. Mix thoroughly.

Note: If not used immediately, repackage and store in -20°C Lower storage. Do not exceed the shelf life of the supplement. After thawing aliquots, use immediately, do not refreeze. will 30 µL supplements B and 30 µL supplements C join 2.94 mL cold (2 - 8°C) Basic medium 1 middle. Mix well and store in 2 - 8°C 。

5 Endoderm cells differentiate into middle / hindgut (MH)

（ 1 )th 3 Day: Preheating (15 - 25°C) sufficient MH culture medium (0.5 mL/ hole ) 。 The remaining MH The culture medium is stored in 2 - 8°C 。

（ 2 Remove the culture medium from the endoderm cells and replace it with... 0.5 mL MH culture medium. In 37°C 、 5% CO2 and 95% Incubation under humidity 24 Hours.

（ 3 )th 4 - 9 Day: As described below, each 24 Complete media changes were performed every hour and spheroids were evaluated.

Note: Ensure that the culture is removed. 30 Return to the incubator within minutes.

a. The monolayers were observed under a microscope. As early as the differentiation stage 4 It may be observed in the sky. 3D Structure. The free-floating middle and hindgut spheroids will be in the first stage of differentiation. 5 - 9 The sky appeared.

b. Use 1 mL Pipette, suction from the cells 0.5 mL Culture medium, transferred to sterile 24 The wells were in clear flat-bottomed plates to assess the number and concentration of middle and posterior intestinal spheroids released from the cell monolayer.

c. Add to cells 0.5 mL fresh MH culture medium. In 37°C 、 5% CO2 and 95% Incubate under humidity.

Note: Although 5 - 9 All spheroids released on the day can produce small intestinal organoids, but the length of time the cells are exposed to the midgut culture medium will control the regional characteristics of developing small intestinal organoids, such as the duodenum (shorter exposure time) or the ileum (longer exposure time). The timing of peak midgut spheroid production may vary. hPSC It varies by cell line. To obtain reproducible experimental results, midgut spheroids generated on the same day of differentiation should always be used to harvest and initiate human intestinal organoid cultures. Usually at the stage of differentiation 8 The largest number of sprouts was observed on that day.

（ 4 On the day of spheroid embedding: Using a pipette, add the spheroid suspension from each well to a new one. 24 Counting was performed in one well of the well plate. Approximately 50 Corresponding volume addition of free floating spheres (based on previous sphere count) 15 mL In a conical tube. Culture of intestinal organoids was performed.

Note: The middle and posterior intestinal spheroid is the diameter ≥ 75 µm The cell aggregates have the potential to produce a human intestinal organoid. Multiple fused spheroids should be counted as a unit that will generate a human intestinal organoid. The remaining monolayer cultures can be used to detect the formation of the midgut or further differentiate in subsequent days to embed more spheroids.

6 Human intestinal organoid growth medium (OGM) Preparation of

（ 1 Prepare the necessary intestines OGM Culture medium (basic medium 2+ supplements D ) volume ( 2 mL/ hole).

（ 2 ) to prepare 2 mL intestinal tract OGM Taking the culture medium as an example:

Thaw replenishment on ice D 。 After mixing in basic culture medium 2 Previously kept on ice. Mix thoroughly.

Note: If not used immediately, repackage and store in -20°C Lower storage. Do not exceed the shelf life of the supplement. After thawing aliquots, use immediately, do not refreeze.

will 40 µL intestinal organoid supplement D join 1.96 mL cold (2 - 8°C) Basic medium 2 middle. Mix thoroughly. Preheat to room temperature before use (15 - 25°C) 。 In 2 - 8°C Store for up to 2 Zhou.

7 Embedding the spheroid in Matrigel middle

（ 1 The low growth factor matrix gel was subpackaged and frozen for storage (each aliquot of reagent). 200μL For 4 (a sample).

（ 2 Thaw an aliquot of low growth factor matrix gel on ice (each sample requires...). 50 µL Low growth factor matrix glue). A box of sterile 100 µL The pipette tip is placed in -20°C 。

（ 3 ) and other collected MH The spheroids settled to the bottom of the conical tube, and the supernatant was carefully aspirated and discarded.

（ 4 Add to the spheroid 1 mL Contains 15 mM HEPES  of DMEM/F-12 。 At room temperature (15 - 25°C) Below 300xg centrifuge 5 minutes.

（ 5 ) Use 1 mL Pipette, carefully aspirate and discard the supernatant.

Note: Remove the supernatant as much as possible.

（ 6 The thawed matrix gel and cell suspension were placed on an ice box and aspirated using a precooled gun tip. 50μL Mix the matrix gel with the cell suspension, taking care to avoid bubbles.

（ 7 Using a pre-cooled gun tip, press each hole. 50μL Seed the cell-containing matrix gel into 24 On the well plate, when the mixture is slowly injected, the gun tip is gradually moved upwards so that the cells are evenly distributed in the glue.

（ 8 Place the culture plate in an incubator. 20-30min Solidifying the gel droplets;

（ 9 Carefully add along the hole wall. 500μL Restoring the intestines to room temperature (OGM) Organoid medium, every other 3-4 Change the liquid every day.

（ 10 ) to 7-10 Once the intestinal organoids mature, passage, identification, and related experiments can be carried out.

This product is hPSC Induced differentiation intestinal organoid kit, human pluripotent stem cells hPSC Induction of differentiation by this kit results in intestinal organoids composed of cells of the intestinal tract, which, after maturation, consist of polarized intestinal epithelium (patterned into villous structures) and surrounding mesenchymal cells that produce microenvironmental factors.
This product requires operators to have hPSC Experience in cell culture and a certain understanding of organoids.
Product Composition: