Freeze-dried powder used for promoting healing of diabetic wound and preparing method thereof
A technology of wound healing and freeze-dried powder, which is applied in the direction of freeze-dried delivery, metabolic diseases, skin diseases, etc., can solve the problems of slow effect, high cost and unfavorable promotion and application, and achieve reduction of deterioration, low cost, and promotion of angiogenesis and neurogenesis. The effect of regeneration
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Embodiment 1
[0022] Example 1 EPC cell culture fluid freeze-dried powder (I)
[0023] 1) Extraction of human umbilical cord blood endothelial progenitor cells: extract 60ml of umbilical cord blood from healthy newborns with a syringe, anticoagulate with sodium heparin, dilute with PBS at a volume ratio of 1:1, and slowly add Ficoll (1.077) lymphocyte separation solution containing a volume ratio of 2:1 In the centrifuge tube, use the density gradient centrifugation method, centrifuge at 2000rpm in a horizontal centrifuge for 25 minutes, and carefully absorb the mononuclear cell layer with a pipette. Then wash the cells with an equal volume of PBS, centrifuge at 2000rpm for 8 minutes, and inoculate the cells on a 25cm cell coated with human fibronectin. 2 Add an appropriate amount of endothelial cell culture medium (containing 10% fetal bovine serum) to the culture flask for culture, and culture at a saturated humidity, 5% carbon dioxide concentration, and a constant temperature of 37°C.
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Embodiment 2
[0028] Example 2 EPC cell culture medium freeze-dried powder (Ⅱ)
[0029] The endothelial progenitor cells were subcultured according to the steps of 1) and 2) in Example 1. After culturing the cells to the third generation, when the cells grow to 70-80% confluence, place the cells in 0.5% oxygen concentration, saturated humidity, and 5% carbon dioxide concentration conditions to induce hypoxia for 36 hours, after that, replace the cell culture medium with no The serum basal culture medium was restored to 21% oxygen concentration, and cultured for 72 hours under saturated humidity and 5% carbon dioxide concentration. The medium was centrifuged, concentrated and lyophilized.
Embodiment 3
[0030] Example 3 EPC cell culture medium freeze-dried powder (Ⅲ)
[0031] The endothelial progenitor cells were subcultured according to the steps of 1) and 2) in Example 1. After culturing the cells to the 5th generation, when the cells grow to 70-80% confluence, place the cells in 1.5% oxygen concentration, saturated humidity, and 5% carbon dioxide concentration conditions to induce hypoxia for 24 hours, after that, replace the cell culture medium with no The serum basal culture solution was restored to 21% oxygen concentration, and cultured for 48 hours under saturated humidity and 5% carbon dioxide concentration. The medium was centrifuged, concentrated and lyophilized.
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