Carrier-free cell sheet and preparation method and application thereof
A cell sheet, carrier-free technology, applied in the field of tissue engineering, can solve the problems of cell sheet residue, difficult operation, cell damage, etc., and achieve the effect of maintaining biological characteristics, improving product quality, and good biological functions
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[0033] Example 1: Carrier-free neonatal rat cardiomyocyte sheet
[0034] (1) Transfection of the suicide gene into Sertoli cells:
[0035] Mouse cardiac fibroblasts were selected as Sertoli cells, and the thymidine kinase gene (TK gene) was transfected into mouse cardiac fibroblasts by calcium phosphate co-precipitation transfection method; fibroblasts stably expressing TK gene were screened Cells, used after expansion;
[0036] (2) Plant the supporting cells that have been transfected with the suicide gene at the bottom of the culture vessel:
[0037] 1×10 6 A mouse fibroblast stably expressing TK gene was planted on the upper surface of a 3 cm diameter invasion chamber (Transwell), and 3 mL of culture medium was added and cultured for 2 to 3 days until the fibroblasts were completely confluent;
[0038] (3) Plant the target cells on the supporting cells:
[0039] 3-day-old neonatal rat cardiomyocytes were isolated by collagenase digestion, and 3 × 10 cells were obtained ...
Example Embodiment
[0043] Example 2: Carrier-free mouse bone marrow mesenchymal stem cell sheet
[0044] (1) Transfection of the suicide gene into Sertoli cells:
[0045] Rat embryonic fibroblasts were selected as supporting cells, and cytochrome P450 gene was introduced into rat embryonic fibroblasts by lipofection method; fibroblasts stably expressing cytochrome P450 gene were screened and expanded backup;
[0046] (2) Plant the supporting cells that have been transfected with the suicide gene at the bottom of the culture vessel:
[0047] 1×10 6 Rat fibroblasts stably expressing cytochrome P450 gene were planted on the upper surface of a 6 cm diameter petri dish, and 10 mL of culture medium was added and cultured for 2 to 3 days until the fibroblasts were completely confluent;
[0048] (3) Plant the target cells on the supporting cells:
[0049] Take the femur of 4-week-old mice and flush out the bone marrow from the bone marrow cavity. After primary culture of mesenchymal stem cells for 2...
Example Embodiment
[0053] Example 3: Carrier-free human limbal stem cell sheets
[0054] (1) Transfection of the suicide gene into Sertoli cells:
[0055] Using human corneal stromal cells as supporting cells, the cytosine deaminase gene (CD gene) and the thymidine kinase gene (TK gene) were simultaneously introduced into human corneal stromal cells by adenovirus vector transfection; screening Human corneal stromal cells stably expressing CD gene and TK gene are used after expansion;
[0056] (2) Plant the supporting cells that have been transfected with the suicide gene at the bottom of the culture vessel:
[0057] Will 1x10 6 Human corneal stromal cells with CD gene and TK gene were planted on the upper surface of a 3 cm diameter invasion chamber (Transwell), and then cultured for 2 to 3 days until the corneal stromal cells were completely confluent;
[0058] (3) Plant the target cells on the supporting cells:
[0059] Limbal stem cells were isolated from human limbal tissue obtained from ...
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