① The blastocoel of the cytoplasm-labeled spore embryo is excised with a surgical needle.
② Collect the blastocyst portion and place it in a 1.5 ml test tube filled with Ringer's solution.
③ When the blastula is submerged in the bottom of the test tube, remove the upper Ringer's solution.
④ Add Ringer's solution into the blastocyst, and remove the supernatant when the blastocyst is submerged.
⑤ This is repeated three times.
⑥ Remove Ringer's solution on the blastoderm as much as possible.
⑦ Ringer's solution containing 0.25% sodium citrate is added to the blastocyst, and the cells are dissociated with a yellow tip.
⑧ Centrifuge at 300 rpm for 1 minute to sediment the cells, and remove the supernatant.
⑨ Repeat steps 7-8 several times to remove cell debris.
⑩ Add about 200 μl of Ringer's solution of Ca2+ free to the precipitate of the cells, and suspend the cells with a yellow tip.
⑪ Centrifuge at 300 rpm for 1 minute to sediment the cells, and remove the supernatant.
⑫ Repeat steps 10-11 several times.
⑬ A cell suspension containing a slight Ca2+ free of Ringer's solution is placed in an implant needle without a stenosis.
⑭ Gently attach the needle to the injector holder.
⑮ Turn the injector knob clockwise to move the cell suspension to the needle tip. At this time, only the solvent moves first, and the cells collect and move toward the air side going from the rear. Bring the tip of the solvent to the needle tip.
⑯ The tip of the implantation needle is lowered into a petri dish for implantation, and the solvent (containing a small amount of cells) is extruded into the solution. Discard the fluid until the cell mass reaches the tip.
⑰ Once the cells have migrated to the tips, the needles are inserted into the host embryo and the cells are transplanted little by little.
