Jakić-Razumović J, Zekusić M, Vladović-Relja T, Boranić M
Department of Pathology, Zagreb University Hospital Center, Kispaticeva 12, HR-10000 Zagreb, Croatia.
Croat Med J. 1998 Dec;39(4):401-3.
To produce organotypic skin cultures using human skin samples as a source of keratinocytes and fibroblasts.
Keratinocytes and fibroblasts from human skin samples were separated by warm trypsine and collagenase, respectively. Keratinocytes were plated in tissue culture dishes in keratinocyte serum free medium supplemented with epidermal growth factor and bovine pituitary extract, and were grown until confluence. Fibroblasts were cultured in Dulbecco's medium (DMEM) supplemented with fetal bovine serum and hydrocortisone. A mixture of fibroblasts, rat dermal collagen type I, E tissue culture medium, and reconstitution buffer were used as a dermal equivalent. Keratinocytes were plated on the top of the dermal equivalent and cultured for 10 days in organotypic culture dishes on stainless steel grids in the supplemented DMEM medium. The cultures were fixed in formaline, embedded in paraffin, stained with hematoxylin and eosin, and immunohistochemically stained with a nti-cytokeratin and anti-HLA-DR antibody.
Cultured keratinocytes in organotypic skin cultures expressed the majority of the cytokeratins seen in the normal stratified epithelium. Consistent with previous studies, organotypic skin cultures did not show antigen-presenting Langerhans cells.
Human skin from patients who underwent thoracic surgery can be used to produce organotypic skin cultures. This artificial skin can serve as a basis for future basic science studies and as a skin transplantation model.
利用人类皮肤样本作为角质形成细胞和成纤维细胞的来源，制备器官型皮肤培养物。
分别用温胰蛋白酶和胶原酶从人类皮肤样本中分离角质形成细胞和成纤维细胞。将角质形成细胞接种于添加表皮生长因子和牛垂体提取物的无血清角质形成细胞培养基的组织培养皿中，培养至汇合。成纤维细胞在添加胎牛血清和氢化可的松的杜尔贝科培养基（DMEM）中培养。将成纤维细胞、大鼠I型真皮胶原、E组织培养基和重组缓冲液的混合物用作真皮替代物。将角质形成细胞接种在真皮替代物顶部，在添加了DMEM培养基的不锈钢网格上的器官型培养皿中培养10天。将培养物用福尔马林固定，石蜡包埋，苏木精和伊红染色，并用抗细胞角蛋白和抗HLA-DR抗体进行免疫组织化学染色。
器官型皮肤培养物中培养的角质形成细胞表达了正常复层上皮中所见的大多数细胞角蛋白。与先前的研究一致，器官型皮肤培养物未显示抗原呈递朗格汉斯细胞。
接受胸外科手术患者的人类皮肤可用于制备器官型皮肤培养物。这种人工皮肤可作为未来基础科学研究的基础和皮肤移植模型。
