Method for constructing tissue-engineered pulsating tissue
A technology of pulsating tissue and tissue engineering, applied in the field of biomedical engineering technology and regenerative medicine
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Embodiment 1
[0048] Example 1: Inducing mouse brown adipose tissue-derived adipose-derived stem cells into pacemaker cells
[0049] 1. Isolation and culture of adipose-derived stem cells
[0050] (1) Cut the skin between the scapula of the mouse, remove the white fat layer covered by the brown adipose tissue between the scapula, take about 0.4 g of brown fat, wash it in PBS at 4°C for 3 times, transfer it to a 5ml centrifuge tube, and add 0.8ml0 .1% type II collagenase in high-sugar DMEM (containing 1% BSA), shredded.
[0051] (2) Transfer the shredded adipose tissue to another centrifuge tube, add 10ml of 0.1% type II collagenase high-sugar DMEM (containing 1% BSA), digest at 37°C for 45min, and shake and mix repeatedly during this time. Centrifuge at 3000rpm for 1min, suck the supernatant through a 100-mesh filter, and enter (4).
[0052] (3) Add 5ml of 0.1% type II collagenase high-sugar DMEM (containing 1% BSA) to the above centrifuge tube, digest at 37°C for 20min, and shake and mix...
Embodiment 2
[0064] Example 2: Implantation of induced pacemaker cells into collagen sponges to construct tissue-engineered beating tissues
[0065] 1. Construction of pacemaker cell-collagen sponge complex
[0066] (1) Preparation of collagen sponge: Collagen sponge was directly purchased from a commercial company (Wuxi Beidi Biological Co., Ltd.), the collagen sponge was cut into a size of 0.8×0.6×0.2cm, 60 Irradiate for more than 12 hours for disinfection.
[0067] (2) Preparation of pacemaker cells: 0.25% trypsin digested adipose-derived stem cells (more than 90% had been differentiated into pacemaker cells), 5min, centrifuged at 1500rpm for 5min. Discard the supernatant, make a cell suspension, and adjust to a cell density of 1.0×10 7 pieces / ml.
[0068] (3) Construction of pacemaker cell-collagen sponge complex: under a stereomicroscope, use a micro-syringe to evenly inoculate the cell suspension on the collagen sponge at 4 points, the total amount is about 100 μl, and the cell d...
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