Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
Ann Surg. 2010 Feb;251(2):368-76. doi: 10.1097/SLA.0b013e3181c1ab5f.
We hypothesize that the pathogen-free NIKS human keratinocyte progenitor cell line cultured in a chimeric fashion with patient's primary keratinocytes would produce a fully stratified engineered skin substitute tissue and serve to deliver autologous keratinocytes to a cutaneous wound.
Chimeric autologous/allogeneic bioengineered skin substitutes offer an innovative regenerative medicine approach for providing wound coverage and restoring cutaneous barrier function while delivering autologous keratinocytes to the wound site. NIKS keratinocytes are an attractive allogeneic cell source for this application.
Mixed populations of green fluorescent protein (GFP)-labeled NIKS and unlabeled primary keratinocytes were used to model the allogeneic and autologous components in chimeric monolayer and organotypic cultures.
In monolayer coculture, GFP-labeled NIKS had no effect on the growth rate of primary keratinocytes and cell-cell junction formation between labeled and unlabeled keratinocytes was observed. In organotypic culture employing dermal and epidermal compartments, chimeric composite skin substitutes generated using up to 90% GFP-labeled NIKS exhibited normal tissue architecture and possessed substantial regions attributable to the primary keratinocytes. Tissues expressed proteins essential for the structure and function of a contiguous, fully-stratified squamous epithelia and exhibited barrier function similar to that of native skin. Furthermore, chimeric human skin substitutes stably engrafted in an in vivo mouse model, with long-term retention of primary keratinocytes but loss of the GFP-labeled NIKS population by 28 days after surgical application.
This study provides proof of concept for the use of NIKS keratinocytes as an allogeneic cell source for the formation of bioengineered chimeric skin substitute tissues, providing immediate formal wound coverage while simultaneously supplying autologous cells for tissue regeneration.
我们假设，以嵌合方式与患者原代角质形成细胞共培养的无病原体 NIKS 人角质形成细胞祖细胞系，将产生完全分层的工程化皮肤替代物组织，并将自体角质形成细胞递送至皮肤创面。
嵌合自体/同种异体生物工程皮肤替代物为提供创面覆盖和恢复皮肤屏障功能提供了一种创新的再生医学方法，同时将自体角质形成细胞递送至创面部位。NIKS 角质形成细胞是该应用的一种有吸引力的同种异体细胞来源。
使用绿色荧光蛋白（GFP）标记的 NIKS 和未标记的原代角质形成细胞的混合群体，在嵌合单层和器官型培养物中模拟同种异体和自体成分。
在单层共培养物中，GFP 标记的 NIKS 对原代角质形成细胞的生长速度没有影响，并且观察到标记和未标记的角质形成细胞之间的细胞-细胞连接形成。在使用真皮和表皮隔室的器官型培养物中，使用高达 90% GFP 标记的 NIKS 生成的嵌合复合皮肤替代物表现出正常的组织结构，并具有归因于原代角质形成细胞的大量区域。组织表达对于连续、完全分层的鳞状上皮的结构和功能至关重要的蛋白质，并表现出类似于天然皮肤的屏障功能。此外，嵌合人皮肤替代物在体内小鼠模型中稳定植入，在手术应用后 28 天内，原代角质形成细胞长期保留，但 GFP 标记的 NIKS 群体丢失。
本研究为将 NIKS 角质形成细胞用作生物工程嵌合皮肤替代物组织形成的同种异体细胞来源提供了概念验证，为即时提供正式的创面覆盖，同时为组织再生提供自体细胞。
