A preparation method of self-assembled prevascularized stem cell membrane

[0027] Example 1 Construction of vascularized EC-BMSC membrane

[0028] 1. Isolation, culture and identification of hBMSC

[0029] Use the whole bone marrow adherent method to isolate and culture hBMSC: Take fresh aseptic bone marrow, add an equal amount of PBS to mix, centrifuge at 800 rpm for 5 minutes, discard the supernatant, wash three times, add 15% fetal bovine serum and 0.4mg / mL of glutamine-based α-MEM medium, inoculated in 75 cm 2 in a culture bottle. After 3 days, half of the medium was changed for the first time, and then a new medium was replaced the next day to remove unadhered cells, and the adherent cells were continued to be cultured. The medium was changed every 2 days. When the cells grew to cover about 80% of the culture flask, they were digested with 0.25% trypsin and subcultured. Cells of passage P4-P5 were selected for subsequent operations.

[0030] 2. Isolation and culture of hUVEC

[0031] Take a sterile free umbilical cord, separate the umbilic...

[0040] Example 2 In vitro and in vivo functional evaluation of vascularized EC-BMSC composite membrane

[0041] 1. Construction of vascularized EC-BMSC composite membrane

[0042] The construction of the vascularized EC-BMSC composite membrane is the same as that in Example 1.

[0043] 2. Protein microarray analysis of supernatant of co-cultured cells

[0044] The cell supernatants of the co-culture system at different time points (3 days, 7 days, 10 days) were collected, and protein microarrays were used to analyze the differences in the expression of related secretory proteins (Table 2) in the co-culture supernatants. The results are as follows image 3 Shown: EGF, HGF, FGF, VEGF, Ang-1, Ang-2, MMP-9, IL-8 were highly expressed in the supernatant of group 1# added with exogenous growth factors. On the one hand, the high content of exogenous VEGF factors will transform vascular endothelial cells into a large number of tip cells, resulting in excessive sprouting of blood ves...