New embryonic stem cell culture system and embryonic stem cell line establishment method
A technology for embryonic stem cells and culture systems, applied in the field of improving the efficiency of embryonic stem cell line establishment, can solve the problems of low line establishment efficiency, slow cell proliferation, and no significant improvement in line establishment efficiency
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Embodiment 1
[0075] The preparation of embodiment 1 culture system:
[0076] Ingredients:
[0077]
[0078]
[0079] Operating procedures:
[0080] The first step is to prepare "N2B27 basal culture medium", the ingredients are as follows:
[0081]
[0082] Operation method: Mix the above ingredients and dosage to prepare.
[0083] The second step is to prepare the concentrated stock solution of Stemness factor, the ingredients are as follows:
[0084]
[0085]
[0086] Operation method: dissolve each component with sterile deionized water, prepare according to the above ratio, and store at -20°C.
[0087] In the third step, the N2B27 basal culture solution was mixed with the Stemness factor concentrated stock solution at a ratio of 100:1 to obtain the "2i+KoSR" system.
Embodiment 2
[0088] Example 2 Establishment of Mouse Embryonic Stem Cell Lines
[0089] 1) Take E3.5-day mouse blastocysts, inoculate them in a four-well plate with a feeder layer, 5 to 6 embryos per well, and place them at 37°C in 5% CO 2 In the incubator, hatch and cultivate with culture system (prepared in embodiment 1);
[0090] 2) After 6-7 days of culture, the blastocysts adhere to the wall and form outgrowths. Take the outgrowths with a pipette, place them in 0.25% trypsin drops preheated at 37°C, and digest them at 37°C for 5 Minutes; the growth halo (outgrowths) was sucked out with a mouth pipette, and directly blown into the new hole containing the medium (prepared in Example 1) and the growth halo (outgrowths) was blown away, placed in 37 ° C 5% CO 2 Continue to culture in the incubator, which is recorded as P1 generation;
[0091] 3) After culturing for 3 to 4 days, discard the supernatant after the clonal colony grows up, and then directly digest the whole well with 0.25% tr...
Embodiment 3
[0094] The in vitro differentiation method of embodiment 3 embryonic stem cells
[0095] a) Get overgrown mouse embryonic stem cells (prepared according to Example 2), digest with 0.25% trypsin, suspend the cells on a petri dish covered with 0.2% gelatin and cultivate for 30 minutes, so as to remove feeder cells (feeder cells) );
[0096] b) Collect the suspension cells, and use the differentiation medium (the components of the differentiation medium include: DMEM / F12 (GIBCO), 10% Knock-out serum (GIBCO) and 1× double antibody solution) to suspend and culture the mouse embryonic stem cells, so that It forms aggregates (aggregates), recorded as day 0 (day0);
[0097] c) After 2 to 3 days of differentiation culture, embryoid bodies (EBs) that have formed spheroids can be observed under a microscope;
[0098] d) After 6 days of differentiation culture, the embryoid bodies (EBs) were collected, and the embryoid bodies (EBs) were attached to the culture for growth. At this time, ...
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