Method for freeze preservation of tissue-derived cell

[0038] Example 1

[0039] Three adult females (aged 33, 27, and 35 years old) and one adult male (aged 50 years old), with my consent, took the skin and performed the following experiments. After the subjects were anesthetized, a full-thickness skin of 1 mm to 3 mm was taken from the back of the auricle. Enzyme treatment (dispase 2000 units/ml, contract alcohol) was carried out immediately to separate the full-thickness skin into the epidermis and dermis. After discarding the epidermis, the dermis (diameter about 0.5 mm) was cut into pieces to make 10 specimens. The 10 specimen sections obtained were divided into 2 groups for the following experiments.

[0040] Group D (direct group): The specimen sections are immersed in cryopreservation solution (10% DMSO, 80% αMEM, 10% human serum) or the commercially available cell cryopreservation solution cellbanker (registered trademark) 1 (Shici) without any treatment. Field Co., Ltd.), after storage at -80°C for 12 hours, it was frozen with liquid nitrogen for 2 weeks.

[0041] Group C (culture group): The specimens were sliced ​​in αMEM medium containing 10% human serum, and incubated at 5% carbon dioxide concentration at 37°C for 1 week. Subsequently, the sections were collected in a centrifuge tube, the supernatant was removed, and then immersed in a cryopreservation solution in the same manner as described above. After storage at -80°C for 12 hours, it was frozen in liquid nitrogen for 2 weeks.

[0042] After 2 weeks, the specimens of group D and group C were taken out from liquid nitrogen and thawed, and incubated in αMEM medium containing 10% human serum at 5% carbon dioxide concentration and 37°C. Use a microscope to observe the conditions of each specimen just after thawing, the first day of culture, and the seventh day of culture. Representative examples of each figure 1 , figure 2 as well as image 3 Shown. Such as figure 1 As shown, there is no difference between the D group and the C group of the specimens just after thawing. From figure 2 It can be seen that there was no special change in group D immediately after thawing, but the cells in group C began to swim to the vicinity of the tissue fragments. In group D on the 7th day of culture, cell migration was found in 2 skin slices (reference image 3 D-1), but in the other 3 skin slices, no cells were found to migrate and die (refer to image 3 D-2). In addition, in group C on the 7th day of culture, a good and large amount of cell migration was found in all 5 skin slices (refer to image 3 C-1 and c-2).

[0043] As described above, in the D group, the number of cell migration was significantly less, and more than half of the skin pieces in the dermis where the cells died and there was no cell migration at all. In contrast, in group C, the number of cells migrating was significantly greater, and there was no skin patch in the dermis where the cells died and there was no cell migrating at all.