Strategy of IPS-induced inner ear organoid culture

1. Experimental preparation  

 

hPSC-induced differentiation inner ear organoid kit (abs90132-1kit)

 

2. Induction flow

 

Cultivation stage	serial number	Name	Specifications	Save
EB induction phase	IE01	EB forming medium	30mL	-20 ℃, 12 months
	IE01-A	Supplement A	40μL	-20 ℃, 6 months
EB differentiation stage	IE02	Basal Medium 1	20mL	-20 ℃, 12 months
	IE02-B	Supplement B	250uL	-20 ℃, 6 months
	IE02-C	Supplement C	70uL	-20 ℃, 6 months
	IE02-D	Supplement D	50uL	-20 ℃, 6 months
Ear bubble formation stage	IE03	Basal Medium 2	50mL	-20 ℃, 12 months
	IE03-E	Supplement E	1.2mL	-20 ℃, 6 months
Maturation stage of inner ear organoids	IE04	Maturation medium	200mL	-20 ℃, 12 months
Organoid maturation stage		Matrigel	500uL	-20 ℃, 6 months

 

 

3. Kit usage process

 

① EB formation (2 days)

 

1) hPSC was cultured in one well of a six-well plate coated with Matrigel and proliferated with Advance human pluripotent stem cell culture medium to 70-80% confluence.

 

2) Aspirate the Advance human pluripotent stem cell culture medium.

 

3) Add 1mL PBS (no calcium and magnesium ions) to the wells, wash and aspirate.

 

4) Add 1mL of human pluripotent stem cell digestion juice to the well, and digest at 37 ℃ for 3min.

 

5) Mix 20mL of EB forming medium and 40μL of EB forming supplement evenly to obtain EB forming complete medium.

(Note: The culture medium used should be placed on the workbench for 30min in advance to restore room temperature, and the same is true for the following operations.)

 

6) After digestion, the clone can be seen white and bright under the microscope.

 

7) Aspirate the digestion juice of human pluripotent stem cells, and add 1mL of EB to each well to form a complete medium to terminate digestion.

 

8) Gently blow the cells in the well 2-3 times, and transfer 500uL of the cell suspension to a 15mL centrifuge tube with 9mLEB forming medium.

 

9) Centrifuge at 100g for 5min, and aspirate and discard the supernatant.

 

10) Flick the cell pellet, add one ml of EB to form complete medium to resuspend the cells, add 9mLEB to the cell suspension to form complete medium, and make a cell suspension again.

 

11) Transfer the cell suspension to the sample loading tank, use a multi-channel pipette to plant it into an ultra-low adsorption 96 plate, 100μL per well, inoculate 96 wells, add 100μL PBS per well of an ordinary 96-well plate, and 96 wells are used as trim.

 

12) Centrifuge in a low-speed horizontal centrifuge at 850rpm/120g for 3min, put the plate in a 37 °C, 5% CO2 incubator, and record it as D-2.

 

13) The formation of EB of uniform size can be seen on day 2 (D-1). Each well is supplemented with 100 μL of EB forming medium (without supplements) and placed in an incubator at 37 °C, 5% CO2.

(Note: After replenishing the EB-forming medium, the multi-channel pipette tip can be placed under the liquid surface and gently blown to mix the medium without affecting the EB.)

 

② EB differentiation stage (12 days)

 

14) D0, thaw supplement A, mix 9.6 mL of pre-cooled basal medium 1 evenly with supplement A and 200 μL of Matrigel. After returning to room temperature, change the medium at 100 μL per well to induce EB differentiation, and use a multi-channel pipette. Gently pipette to suspend EB. Incubation was performed in a 5% CO2 incubator at 37 °C for 4 days.

 

15) D4, thaw supplement B, 2.5 mL of basal medium 1 + supplement B and mix evenly, add it to a 96-well plate at 25 μL per well, and gently pipette 8-10 times to mix evenly, so that the final volume of medium per well is 125 μL, 37 ℃, 5% CO2 culture for 4 days.

 

16) D8, thaw supplement C, 2.5 mL of basal medium 1 + supplement C and mix evenly, add it to a 96-well plate at 25 μL per well, and gently pipette 8-10 times to mix evenly, so that the final volume of each well medium is 150 μL, and the 96-well plate is returned to the incubator and cultured at 37 °C and 5% CO2 for 4 days.

 

③ Ear bubble formation stage (6 days)

 

17) D12, after EB induction, prepare basal medium 2 (refrigerate on ice for at least 30 minutes in advance); Alternatively, basal medium 2 was stored at 4 ° C. overnight on D11. Matrigel was thawed with supplement D at 4 °C in advance, and the cold basal medium 2 was evenly mixed with supplement D. 200 uL of Matrigel was added to 20 mL of cold mixed medium, repeatedly pipetted and dissolved 20 times, and rewarmed to room temperature. The aggregates in the 96-well plate were transferred to two 100 mm dishes with 48 aggregates in each dish. The aggregates were washed twice with 1-2 mL of PBS and once again with 1 mL of Matrigel-free mixed medium, then 10 mL of Matrigel-containing mixed medium were added respectively, and the petri dishes were placed in an incubator and cultured at 37 °C and 5% CO2 for 3 days; On D15, the culture medium in the petri dish was aspirated, the mixed medium without Matrigel was readded, and the culture was continued for 3 days to induce Ear bubble formation;

(Note: Generally, 10mL of culture medium is needed for solution change of 10mm dish)

 

④ Maturation stage of inner ear organoids (42 days)

 

18) D18-60, inner ear organoids gradually mature, change the medium to mature medium, start long-term culture, change the fluid every 5 days, and obtain mature inner ear organoids with sensory hair cells in about 60 days.  

 

4. Identification map of inner ear organoids

 

 

Product Recommendations:

Item number	Product name	Specifications	Storage conditions and cycles
abs90132	hPSC-induced differentiation inner ear organoid kit	kit	Stored at-20 ℃, the validity period of the basal medium is 12 months, and the validity period of the supplement is 6 months.
abs962	PBS	500mL	Stored at 2 ~ 8 ℃ or room temperature, the validity period is 12 months.
abs9404	Advance human pluripotent stem cell culture medium	100mL	Store at-20 ℃, shelf life is 2 years.
abs9409	Human pluripotent stem cell digestive juice	100mL	Stored at-20 ℃, shelf life is 2 years.
abs7033	Cell culture plate (standard clear 6-well plate)	1 box	Room temperature, shelf life 3 years.
abs7034	Cell Culture Plate (Standard Clear 12 Well Plate)	1 box	Room temperature, shelf life 3 years.
abs7035	Cell culture plate (standard clear 24-well plate)	1 box	Room temperature, shelf life 3 years.
abs7119	1.5 mL Centrifuge tube	1 box	Room temperature, shelf life 3 years.
abs7120	15 mL Centrifuge tube	1 box	Room temperature, shelf life 3 years.
abs7121	50 mL Centrifuge tube	1 box	Room temperature, shelf life 3 years.
abs7053	10mL disposable pipette	1 box	Room temperature, shelf life 3 years.
abs7055	50mL disposable pipette	1 box	Room temperature, shelf life 3 years.
abs7072	10uL tips (boxed, sterile and enzyme-free)	1 box	Room temperature, shelf life 3 years.
abs7075	200uL tips (boxed, sterile and enzyme-free)	1 box	Room temperature, shelf life 3 years.

 

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