Amniotic mesenchymal stem cell resuscitation culture medium and resuscitation culture method thereof
A technology of amniotic mesenchymal stem cells and culture methods, which is applied in the field of recovery medium of amniotic mesenchymal stem cells, can solve the problems of low cell proliferation rate, inability to guarantee the vitality of stem cells, and affect the clinical utilization of stem cells, so as to achieve good biological characteristics, The effect of maintaining biological characteristics
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Embodiment 1
[0048] (1) Culture of amniotic mesenchymal stem cells and collection of conditioned medium
[0049] Shred the amnion tissue to 1.0-20.0cm 3 Finally, the tissue block was digested with 0.1% trypsin for 60 minutes, and then digested with 0.5% type I collagenase. When the tissue block was basically digested, it was centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, resuspended with PBS, and passed through a 70 μm cell sieve. Centrifuge again at 2000 rpm for 5 min, and culture and resuspend the cell pellet with DMEM / F12+10% FBS. Place at 37°C, 5% CO 2 Culture the incubator statically, change the medium every 2-3 days, and when the confluence of the cell clones reaches 80%-90%, digest the cells with 0.25% trypsin, centrifuge and resuspend the pellet with complete medium, 7×10 3 / cm 2 The cell density was seeded in a culture dish to continue culturing. The P3-P5 generation cells were collected for conditioned medium collection.
[0050] Conditioned medium col...
Embodiment 2
[0065] (1) Culture of amniotic mesenchymal stem cells and collection of conditioned medium
[0066] Shred the amnion tissue to 1.0-20.0cm 3 Finally, the tissue block was digested with 0.1% trypsin for 60 minutes, and then digested with 0.5% type I collagenase. When the tissue block was basically digested, it was centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, resuspended with PBS, and passed through a 70 μm cell sieve. Centrifuge again at 2000 rpm for 5 min, and culture and resuspend the cell pellet with DMEM / F12+10% FBS. Place at 37°C, 5% CO 2 Culture the incubator statically, change the medium every 2-3 days, and when the confluence of the cell clones reaches 80%-90%, digest the cells with 0.25% trypsin, centrifuge and resuspend the pellet with complete medium, 7×10 3 / cm 2 The cell density was seeded in a culture dish to continue culturing. The P3-P5 generation cells were collected for conditioned medium collection.
[0067] Conditioned medium col...
Embodiment 3
[0080] (1) Culture of amniotic mesenchymal stem cells and collection of conditioned medium
[0081] Shred the amnion tissue to 1.0-20.0cm 3 Finally, the tissue block was digested with 0.1% trypsin for 60 minutes, and then digested with 0.5% type I collagenase. When the tissue block was basically digested, it was centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, resuspended with PBS, and passed through a 70 μm cell sieve. Centrifuge again at 2000 rpm for 5 min, and culture and resuspend the cell pellet with DMEM / F12+10% FBS. Place at 37°C, 5% CO 2 Culture the incubator statically, change the medium every 2-3 days, and when the confluence of the cell clones reaches 80%-90%, digest the cells with 0.25% trypsin, centrifuge and resuspend the pellet with complete medium, 7×10 3 / cm 2 The cell density was seeded in a culture dish to continue culturing. The P3-P5 generation cells were collected for conditioned medium collection.
[0082] Conditioned medium col...
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