Nucleus pulposus cell source active micro-carrier construction method
A technology of nucleus pulposus cells and microcarriers, applied in the field of biological tissue engineering cell carrier materials, can solve the problems of reduced activity of biological materials, inability to provide the original ecological microenvironment for stem cells, and weakened ability of stem cells to orientate differentiation, and achieve the effect of avoiding the impact.
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Embodiment 1
[0020] see figure 1 , take 4×10 amplified in vitro ^5 The 3rd generation SD rat nucleus pulposus cells were resuspended in 1mL F12 medium and placed in a 15mL centrifuge tube, centrifuged at 1000rpm for 5 minutes by centrifugal enrichment method, left in F12 medium for 24h, and the nucleus pulposus cells were used Self-crimping ability to obtain nucleus pulposus cell microspheres. The culture environment of the nucleus pulposus cell microspheres was replaced: 10 ng / mL TGF-β3, 10% fetal bovine serum, and 50 nM vitamin C were added to the DMEM high-glucose medium. At 37°C, 5% CO 2 cultured in an environment for 12 days, and the solution was changed every 3 days to promote the production and deposition of matrix in the nucleus pulposus cell microspheres. The cultured nucleus pulposus cell microspheres were decellularized by combined decellularization agents 2% Triton-100 and 50 mM SB-10, and treated in a constant temperature shaker at 37°C at a rate of 100 rpm for 1 hour. Aft...
Embodiment 2
[0021] Example 2: To use 8×10 ^5 A nucleus pulposus cell-derived active microcarrier was constructed.
[0022] see figure 1 , take the 8×10 amplified in vitro ^5 The 3rd generation SD rat nucleus pulposus cells were resuspended in 2mL F12 medium, and evenly divided into 2 tubes of 15mL centrifuge tubes, so that the number of cells in each centrifuge tube was 4×10 ^5 indivual. Centrifuge at 1000rpm for 5 minutes by centrifugation enrichment method, let stand in F12 medium for 24 hours, and use the self-curling ability of nucleus pulposus cells to obtain nucleus pulposus cell microspheres. The culture environment of the nucleus pulposus cell microspheres was replaced: 10 ng / mL TGF-β3, 10% fetal bovine serum, and 50 nM vitamin C were added to the DMEM high-glucose medium. Cultivate at 37° C. in an environment of 5% CO2 for 12 days, and change the liquid every 3 days to promote the production and deposition of matrix in the nucleus pulposus cell microspheres. The cultured nuc...
Embodiment 3
[0023] Example 3: In vitro activity verification of nucleus pulposus cell-derived active microcarriers.
[0024] After the nucleus pulposus cell-derived active microcarriers were obtained through the steps described above, mesenchymal stem cells were loaded in vitro, the obtained nucleus pulposus cell-derived active microcarriers were placed in a culture dish, and 2uL containing 5*10 ^4 For the medium of mesenchymal stem cells, let mesenchymal stem cells stand at 37°C for 2 hours to fully load the mesenchymal stem cells on the surface of active microcarriers derived from nucleus pulposus cells, transfer the microspheres to DMEM low-glucose medium, and detect the proliferation activity of stem cells after 14 days of culture. And through gene level detection, it was found that the proliferative ability of mesenchymal stem cells planted on nucleus pulposus cell-derived active microcarriers was enhanced compared with planar culture and chitosan hydrogel carrier culture (see figur...
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