hPSC 诱导分化全脑类器官试剂盒

Neuroectodermal differentiation (th 2-6 (sky)
Return the culture dish, medium and reagents to room temperature before use ( 15-25°C ）。
①Day2-6
1 , will induce well EBs Transfer to ultra-low adsorption 6 In the hole plate, add to each hole 10 one EBs Use later DPBS Wash once to remove any medium residue from the previous stage, then discard. DPBS Then add to each well 2ml basic culture medium 2 , every day 2 The fluid is changed every other day to induce co- 4 heaven.
2 Neuroectodermal expansion occurs EB The edges become transparent, and the diameter is > 400um The interior is dense and cavity-free, and the overall shape is similar to that of a primary mouse neurosphere.

Nerve budding (No. 6-25 (sky)
Return the culture dish, medium and reagents to room temperature before use ( 15-25°C ）。
①Day6-25
1 ,when EBs When the diameter meets the relevant standards 400-600um Can be embedded with matrix rubber, 4 Low growth factor matrix glue for freezing and dispensing 2ml 。
2 Prepare a sheet of equal length and width. 10cm The sealing film, in 200ul Press a dent into the gun barrel and place it. 10cm The dish was soaked in alcohol. 30 Seconds later, place in a safe cabinet for ultraviolet drying. 30min The above can be used.
3 Use a wide mouth 10-100uL pipetting gun tip ( measuring range 50uL) Absorption of the end of neuroectodermal differentiation EB On the concave points pressed, it is recommended to operate once. 10 One is preferable. Afterwards, a pipette was used to carefully absorb any remaining culture medium from the pits, and each pit and... EBs join 30uL Careful embedding of low growth factor matrix gel EBs , if EBs At the edge of the colloid, use 10uL The little spearhead was gently prodded EBs The surrounding colloid places it in the center. ( Be careful not to form bubbles. ) 。
4 The buried EBs Cover it and place it in 37 degree gel 15min Then, use sterile tweezers to lift the sealing membrane. 1mL basic culture medium 3 Each row EBs The colloid is blown against the bottom of the concave point until the adsorption is ultra-low. 6 In the orifice plate, each orifice can hold a maximum of 10 For organoids, the volume of culture medium per well should not exceed 3mL 。
5 If too many are made at once EBs You can also directly pre-cool it. 30min The above 12mL basic culture medium 3 supplement 10% Low growth factor matrix gel, (i.e. 1 、 2mL After the colloid is mixed evenly in a vortex. Aspiration of the basal medium in the original well plate 2 Replenish each well 3mL Basic medium 3+ Low growth factor matrix gel, induced 48 After an hour, the medium was aspirated and used... DPBS Wash twice and add again. 3mL Base medium without matrix gel per well 3 Conduct the first 6 Step-by-step operation.
6 Place the ultra-low six-well plate on a horizontal shaker. (80rpm/s) inside 37 degree cultivation 18 The expansion of the neuroepithelium was completed every two days, and the fluid was changed every two days. EBs Generally in the neuroepithelial dilation stage 4 Early appearance during the day VZ 、 SVZ Such a structure, 12 The sprouts gradually converge and turn black.

Neurological and Brain Maturation (Part 1) 25 (Power Queen)
Return the culture dish, medium and reagents to room temperature before use ( 15-25°C ）。
①Day25+
1 ,when EBs The center is dense, the outer layer forms a loose epithelial structure with a diameter greater than 1mm This indicates that the nerves have expanded to the mature stage and the basal culture medium has been completely abandoned. 3 Add to each well 3mL basic culture medium 4 Continuous training until 38 Pop Queen ( Change the fluid every two days /80rpm/s) ， EBs The diameter is greater than 2mm At this time, nutrients may not penetrate the interior, requiring the use of... 1mL The syringe will EBs Carefully remove the surrounding colloid and add it to each well. 3mL Basic medium 5( Change the fluid daily ) And increase the shaker speed to 120rpm/s Improving nutrient circulation allows brain organoids to be cultured for a long time. 100 heaven.