A kind of preparation method of skin ulcer repair matrix
A skin ulcer and matrix technology, applied in medical science, prostheses, animal cells, etc., can solve problems such as high storage conditions and transportation requirements, limited wound repair effect, and inability to meet adjustments, so as to promote wound healing and reduce scar formation , the effect of prolonging the time of action
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[0052] The preparation method of the skin ulcer repair matrix specifically adopts the decellularized, de-antigenized and thickened natural extracellular matrix as a biological scaffold, and the composite seed cells form a multi-layered cell membrane (having 2-10 cells) on the surface of the natural extracellular matrix. The skin ulcer repair matrix is obtained after freeze-drying treatment; the seed cells involved are fibroblasts or embryonic or stem cells derived from autologous or allogeneic sources.
[0053] According to the preparation process, the preparation method of the skin ulcer repair matrix mainly includes the steps of preparation of extracellular matrix, preparation of culture medium, separation and in vitro culture of seed cells, inoculation of cells and three-dimensional culture, freeze-drying, etc. The specific preparation process is as follows:
[0054] Step 1. Preparation of extracellular matrix:
[0055] Decellularization: The collagen membrane tissue used...
Embodiment 1
[0074] After the bovine pericardium was decellularized and washed, it was soaked in 1M sodium hydroxide solution for 5 min at room temperature, and then washed with a large amount of 0.01M PBS solution until the pH was about 7. At this time, the thickness of bovine pericardium increased by 3 to 4 times. The bovine pericardium was then immersed in PBS solution for 2 h. The bovine pericardium was cut into a circle of 10 cm, which was sterilized by irradiation with 10 kGy of cobalt 60 for use.
[0075] Mix DMEM / F12 commercial medium with 10% fetal bovine serum as the basal fluid at a volume ratio of 9:1; then add 50 ng of insulin, 30 mg of transferrin, 10 μg of sodium selenite, and hydrocortisone according to the standard of 500 ml of basal fluid. 60μg, epidermal growth factor 20μg, basic fibroblast growth factor 40ng, transforming growth factor beta 20ng, adenine 10mg, vitamin C 30mg.
[0076] The sterilized bovine pericardium was spread on a sterile petri dish with a diameter...
Embodiment 2
[0079] After the bovine pericardium was decellularized and washed, it was soaked in 1‰ sodium hypochlorite solution at room temperature for 2 hours, and then washed with a large amount of 0.05M PBS solution until the pH was about 7. At this time, the thickness of bovine pericardium increased by 3 to 6 times. The bovine pericardium was then immersed in PBS solution for 6 h. The bovine pericardium was cut into a circle of 10 cm, which was sterilized by irradiation with 25 kGy of cobalt 60 for use.
[0080] The DMEM / F12 commercial medium and 10% fetal bovine serum were mixed as the basal solution according to the volume ratio of 9:1; then 60 ng insulin, 50 mg transferrin, 20 μg sodium selenite, and hydrocortisone were added according to the standard 500 ml basal solution. 100μg, epidermal growth factor 20μg, basic fibroblast growth factor 50ng, transforming growth factor beta 40ng, bovine pituitary extract 5mg, vitamin C 50mg.
[0081] The sterilized bovine pericardium was spre...
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