Epiderm substitute in tissue engineering and its prepn process
A technology of epidermis and epidermis, which is applied in the fields of medicine and biomedical engineering, and can solve the problems of graft loss, prone to blisters, residual wounds, etc.
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Embodiment 1
[0059] Isolation and culture of epidermal cells
[0060] 1) Take materials
[0061] The surgically removed foreskin of children is placed in the keratinocyte culture medium containing antibiotics and brought into the cell culture room.
[0062] 2) Separate the epidermis
[0063] Carefully cut off the subcutaneous fat tissue and part of the dermis with scissors, and trim them into strips with a width of about 1-2 mm, rinse twice with calcium and magnesium-free PBS, and soak the epidermis in 0.24 U / ml neutral protease (Dispase II) overnight at 4°C.
[0064] 3) digestion
[0065] After 18 hours, the epidermis was torn off from the dermis with two ophthalmic tweezers, cut into pieces, and then digested with 0.05% trypsin-0.53mM EDTA at 37°C for 15 minutes, and 10ml trypsin inhibitor (10mg / ml) to stop the digestion . Filter through a 150-mesh stainless steel filter, centrifuge at 1000r / min for 10 minutes, discard the supernatant, add 10ml of keratinocyte culture medium, mix we...
Embodiment 2
[0071] Preparation of chitosan-gelatin film
[0072] (a) the acetic acid solution (140ml) of 2% chitosan, the gelatin solution (30ml) of 4% (the ratio of chitosan and gelatin is 7: 3) mixes, stirs and mixes rapidly at room temperature for 12 hours;
[0073] Add a certain amount (12ml) of the mixed solution into the petri dish and dry it to form a film;
[0074] Soak the membrane in a 20ml petri dish containing 50mM MES and 40% ethanol for 30min;
[0075] Discard the liquid, react with 30mM EDC solution for 2 hours, wash with a large amount of distilled water for 24 hours, and dry for the second time to form a film. A chitosan-gelatin composite film material with a thickness of 120 microns was obtained.
[0076] (b) Repeat step (a), except that the weight ratio of chitosan and gelatin is changed from 7:3 to 1:9, 2:8, 3:7, 5:5, 7:3, 8:2 and 9:1, the same membrane material was prepared. The thickness ranges from 50-500 microns.
Embodiment 3
[0078] Preparation of tissue-engineered epidermal substitutes
[0079] Press 0.2×10 5 , 1×10 5 , 5×10 5 , 1×10 6 cells / cm 2 The seeding density, the epidermal cells prepared in Example 1 were respectively inoculated on the surface of various chitosan-gelatin films prepared in Example 2, cultivated in serum-free keratinocyte culture medium (GIBCO, USA), culture solution 3 Change every day.
[0080] It takes an average of 2-5 days from inoculation to obtaining the epidermal graft, and the epidermal substitute can be obtained (Fig. 1 and Fig. 2). Microscopic observation shows that in the epidermis substitute of the present invention, epidermal cells adhere and grow well on the chitosan-gelatin membrane.
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Abstract
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