The basal keratinocyte progenitor cells in cultured epithelial autografts (CEAs) regenerate human epidermis after transplantation, a curative therapy for severe burns and, recently, diseases with epidermal loss, such as junctional epidermolysis bullosa (EB). Although a culturing technique for skin keratinocytes was developed four decades ago, the xenogeneic nature of that conventional CEA culture system restricts its use to the treatment of critical and life-threatening cases, such as severe burns on >30% of total body surface area and EB. In the present protocol, we describe how to implement a defined, xeno-free culture system that supports long-term ex vivo expansion of functional human epidermal keratinocytes. Skin-specific basement membrane proteins called laminins play important roles in the maintenance of phenotypic integrity and in supporting the survival of keratinocytes that are adhered to them. This fully human keratinocyte culture system is ‘regulatory friendly’ and increases the potential of epithelial cellular therapy, which can be expanded to treat less severe burns and other skin defects, such as chronic diabetic wounds. It takes between 7 and 14 d to obtain an initial culture. Conservatively, a secondary culture from the primary culture can be expanded up to 20-fold within 4–5 d once cells reach confluency.

培养的自体上皮移植物 (CEA) 中的基底角质形成细胞祖细胞在移植后可再生人表皮，这是一种治疗严重烧伤的治疗方法，以及最近治疗表皮缺失的疾病，例如大疱性交界性表皮松解症 (EB)。尽管 40 年前开发了一种皮肤角质形成细胞的培养技术，但传统 CEA 培养系统的异种特性限制了其用于治疗危及生命的严重病例，例如超过 30% 的全身表面积的严重烧伤和乙。在本协议中，我们描述了如何实施一个定义的、无异种的培养系统，该系统支持功能性人类表皮角质形成细胞的长期体外扩张。称为层粘连蛋白的皮肤特异性基底膜蛋白在维持表型完整性和支持粘附在其上的角质形成细胞的存活方面发挥着重要作用。这种完全的人类角质形成细胞培养系统是“监管友好的”，并增加了上皮细胞治疗的潜力，可以扩展到治疗不太严重的烧伤和其他皮肤缺陷，如慢性糖尿病伤口。获得初始培养需要 7 到 14 天。保守地，一旦细胞达到汇合，来自原代培养的二次培养可以在 4-5 天之内扩大至 20 倍。它可以扩展到治疗不太严重的烧伤和其他皮肤缺陷，例如慢性糖尿病伤口。获得初始培养需要 7 到 14 天。保守地，一旦细胞达到汇合，来自原代培养的二次培养可以在 4-5 天之内扩大至 20 倍。它可以扩展到治疗不太严重的烧伤和其他皮肤缺陷，例如慢性糖尿病伤口。获得初始培养需要 7 到 14 天。保守地，一旦细胞达到汇合，来自原代培养的二次培养可以在 4-5 天之内扩大至 20 倍。