hPSC诱导分化结肠类器官试剂盒

Note: Kit can be completed 24 orifice plate 20 One hole hPSC From DE For the first differentiation of the hindgut, it is recommended to use... 2-3 Try each hole.

（ 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 Take the culture medium as an example: thaw the supplement 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)

Add to passage cells to 24 The operation of the orifice plate, when the convergence degree reaches 85%-90% Proceed at DE Differentiation, recorded as the first 0 heaven.

（ 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 Absorbed into cells Complete training. 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 2+ 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 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 30 µL supplements B and 30 µL supplements C join 2.94 mL cold (2 - 8°C) Basic medium 2 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 - 8 Day: Every 24 Complete media replacement was performed every hour and spheroids were evaluated, culturing the midgut. / Posterior intestinal sphere.

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 - 8 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: The timing of peak midgut spheroid production may vary. hPSC It varies by cell line. To obtain reproducible experimental results, middle and hindgut spheroids generated on the same day of differentiation should always be used to harvest and initiate human colon 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 70-100 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 colon organoid medium (HCO) Preparation of

（ 1 Prepare the required colon HCO Culture medium (basic medium 3+ supplements D+ supplements E+ supplements F ) volume ( 2 mL/ hole).

（ 2 ) to prepare 100 mL Colon HCO Taking the culture medium as an example:

Thaw replenishment on ice D , supplements E , supplements F 。 After mixing in basic culture medium 3 Previously kept on ice. Mix thoroughly.

Note: If not used immediately, repackage and store in 2-8°C Store below, you can save 6 Zhou. Do not exceed the shelf life of the supplement. After thawing aliquots, use immediately, do not refreeze.

will 100μL supplements D 、 50 µL supplements E and 2mL supplements F join 98 mL cold (2 - 8°C) Basic medium 3 middle. Mix well and store in 2 - 8°C 。

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 Growth factor reduced 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 ) Ice-thawed colon organoid supplement G Keep on ice and mix well. take 5 mL Colon ( HCO Culture medium addition 5 μL colon organoid supplement G Mix thoroughly. Per hole 500 μL Add to the edge of each hole, taking care not to disturb the hole cover. 37°C 、 5% CO₂ and 95% Cultivation was carried out under humidity.

Note: Colon organoid supplements G It needs to be configured and used immediately, paying attention to the amount used each time before configuring.

8 Replace human colon organoid culture medium ( HCO ）

（ 1 ） 3 Queen, do it once HCO Culture medium (without supplement G Replace ) and add new ones to each culture well. 500μL HCO Culture medium (without supplement G ), in  37°C 、 5% CO₂ and  95% Cultivation was carried out under humidity.

（ 2 ) Weekly 3 The culture medium is replaced secondarily, and every other 10-14 Transfer the organoid culture to a new medium containing matrix gel. When replacing, the appropriate passage ratio should be followed (approximately [number missing] per dish). 80-100 (The number of cells in each organ) is used to perform the operation.

Note: After the first passage, antibiotics (e.g. 50μg/ml Gentamicin) was added to the medium

（ 3 ) After the 3 After the second passage, the colorectal tissue cell mass should have been completely formed before identification and related experiments can be carried out.

Note: After multiple passages, the cell growth rate may decrease, which may be due to a decrease in the number of mesenchymal cells and stem cells.