A kind of slow-release exosome composite material and its preparation method and application
A composite material and exosome technology, applied in the field of biomaterials and biomedical engineering, can solve the problems of easy loss of exosomes and inability to play a long-term effect, so as to achieve long-term effective treatment methods without destroying the integrity of the membrane structure, The effect of prolonging the duration of action
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Embodiment 1
[0013] 1) Preparation of scaffold material: 7% gelatin and 0.25% glutaraldehyde mixture were placed at -20 °C for reaction for 24h, transferred to a freeze dryer for freeze-drying for 24h, and gelatin porous scaffolding with porous structure was obtained;
[0014]2) Acquisition of exosome suspension: culture of fat mesenchymal stem cells to the third generation, add 20ng / mLIFN-γ stimulate cells to obtain inflammatory mesenchymal stem cells for 48h, replace serum-free medium for 24h to collect cell culture supernatants; remove cell debris and larger vesicles by gradient centrifugation; use ultracentrifugation method 100 000g centrifugation for 70min, discard supernatant, add PBS again 100 000g centrifugation for 30min, discard supernatant, add 200 μL PBS resuspends the pellet i.e. exosome suspension;
[0015] 3) Mix the exosome suspension with 2% (w / v) TG2 enzyme and inject it into the complex scaffold to saturation, and the reaction at 37 °C for 30 min can load the exosomes into t...
Embodiment 2
[0017] 1) Preparation of scaffold material: Weigh the silk dissolved in 9.3M lithium bromide solution to prepare a 20% (w / v) silk solution, placed in a water bath pot at 60 °C for 4h, using a 3500D dialysis bag dialysis for 48h; subsequently, replace the 20% g / mL PEG solution to continue dialysis for 6-10h to obtain a concentrated silk protein solution; use 10000rpm, 4 °C, centrifuge for 20 minutes to remove impurities, and determine the concentration of concentrated silk protein by weighing method. After adding an appropriate amount of distilled water, the concentration is 6% and after adding 0.25% glutaraldehyde, it is placed at -20 °C for 24h, transferred to a freeze dryer for freeze-drying for 24h, and a gelatin porous scaffold with a porous structure is obtained;
[0018] 2) Culture bone marrow mesenchymal stem cells to the third generation, culture cells in a 2% oxygen environment for 48 h, replace serum-free medium culture for 24 h to collect cell culture supernatant; remov...
Embodiment 3
[0021] 1) Scaffold material preparation: 2% collagen and 0.135 μM hydroxide nano-mixed liquid placed at -20 °C reaction for 24 h, transferred to freeze dryer for 24 h, to obtain collagen porous scaffold with porous structure;
[0022]2) Acquisition of exosome suspension: culture of fat mesenchymal stem cells to the third generation, add 20ng / mLIFN-γ stimulate cells to obtain inflammatory mesenchymal stem cells for 48h, replace serum-free medium for 24h to collect cell culture supernatants; remove cell debris and larger vesicles by gradient centrifugation; use ultracentrifugation method 100 000g centrifugation for 70min, discard supernatant, add PBS again 100 000g centrifugation for 30min, discard supernatant, add 200 μL PBS resuspends the pellet i.e. exosome suspension;
[0023] 3) Mix the exosome suspension with 2% (w / v) TG2 enzyme and inject it into the complex scaffold to saturation, and the reaction at 37 °C for 30 min can load the exosomes into the porous scaffold.
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