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纤维真皮胶原制备:胶原来自植皮手术剩余的皮片,经0.1％戊二醛处理,并用氰丙烯树酯胶固定于直径为5cm的塑料培养皿底部。使用前至少用细胞培养液平衡7天。角质细胞培养方法:中厚皮片(取自17个月婴儿和51岁男性)用0.25％的胰酶(含0.1％葡萄糖)消化(37℃,45分钟),使表皮剥脱。将角质细胞打散和离心后混悬于含10％小牛血清的Dulbecco MEM/Hams F12培养液(3:1)中保存。3T3饲养层用丝裂霉素(20μg/ml培养液)处理(37℃,2小时),使其停止增殖。种植前用胰酶(0.25％)/EDTA(0.02％)/GS(0.1％)将角质细胞自保存瓶内分离出,
Preparation of fibroblasts collagen: Collagen from the skin graft skin graft, treated with 0.1% glutaraldehyde, and cyanoacrylate glue fixed to the bottom of a plastic dish 5cm in diameter. Equilibrate with cell culture medium for at least 7 days before use. Cultured keratinocytes: Exfoliated epidermis was dissected from medium-skinned pieces (from a 17-month old baby and a 51-year-old male) with 0.25% trypsin (containing 0.1% glucose) for 45 minutes at 37 ° C. The keratinocytes were disrupted and centrifuged and then resuspended in Dulbecco MEM / Hams F12 medium (3: 1) supplemented with 10% calf serum. The 3T3 feeder layer was treated with mitomycin (20 μg / ml of culture medium) (37 ° C, 2 hours) to stop proliferation. Before planting, keratinocytes were separated from the preserved bottle by trypsin (0.25%) / EDTA (0.02%) / GS (0.1%),