Takifugu obscurus testis cell line and construction method and application thereof
A dark-patterned pufferfish, a construction method technology, applied in the direction of artificial cell constructs, applications, animal cells, etc., to achieve the effects of stable characteristics, strong proliferation ability, and good state
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Embodiment 1
[0045] Example 1 Isolation, culture and passage of testicular cells of pufferfish
[0046] (1) Cell isolation and primary culture: Take healthy pufferfish with a weight of 80-100 g, and soak the pufferfish in 75% alcohol for 3 min. After taking it out, it was placed in an ultra-clean bench, and the testis tissue was taken out under aseptic conditions, and rinsed three times in FBS containing 400 U / ml penicillin and 400 μg / ml streptomycin. Subsequently, the testis tissue was taken out and placed in a beaker, and the tissue was cut into small pieces with sterilized scissors, and 1 ml of 0.25% trypsin was added, and the tissue was cut into mince. After standing at room temperature for 15 min, the trypsin-digested cell suspension was filtered using a 40 μm cell sieve. The filtered cell suspension was centrifuged to remove the supernatant, and the cell pellet was collected. Cell culture medium, pipet into 25 cm 2 Cell culture flasks were cultured in a 28°C incubator. After 48 h ...
Embodiment 2
[0049] Example 2 Cryopreservation and recovery of testicular cell line of pufferfish with dark streaks
[0050] (1) Cryopreservation of cells: Select cells of different passages, when the cell growth density reaches 90%, according to the conventional method, aspirate the old medium, add 1ml of trypsin digestion solution to digest; when the cells are observed to shrink, aspirate the trypsin, Add 2 ml of fresh cell culture medium. The cells were then transferred to a 5 ml centrifuge tube, centrifuged at 1000 g for 5 min, and the supernatant was removed. Add 1 ml of cell cryopreservation solution, resuspend the cells and transfer into a cryopreservation tube. The cryovials were placed in a programmed cooling box, placed at -80 °C for 24 h, and then transferred to liquid nitrogen for long-term storage.
[0051] (2) Cell recovery: Take out the cells stored in liquid nitrogen, quickly place them in a 37°C water bath, and shake the cryopreservation tube constantly to thaw the cells...
Embodiment 3
[0052] Example 3 Determination of growth characteristics of testis cell line of pufferfish with dark stripes
[0053] (1) Determination of optimal serum concentration: cell culture medium containing fetal bovine serum FBS concentration of 5%, 10%, 15%, and 20% was prepared respectively. figure 2 The growth curves of the testis cell line of the pufferfish in the dark striae under different culture conditions: A is the medium with different serum concentrations; B is the different culture temperature conditions.
[0054] The 45th passage testicular cells were taken, and cell suspensions of four serum concentrations were prepared at a density of 2.5×104 cells / mL, inoculated in a 12-well plate, and cultured in an incubator at 28°C. At the same time every day thereafter, 3 wells of cells of each serum concentration were taken, digested by conventional trypsinization method, collected and counted with a cell counting plate, and counted continuously for 7 days. Taking the culture t...
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