The Development of an Advanced Model for Multilayer Human Skin Reconstruction In Vivo.
The Development of an Advanced Model for Multilayer Human Skin Reconstruction In Vivo.
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DOI：
10.21769/bioprotoc.4919
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发表时间：
2024-01-20
期刊：
影响因子：
0.8
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中科院分区：
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在免疫缺陷小鼠上进行人类皮肤重建已成为基础研究和转化实验室中进行的体内研究不可或缺的。在制造可持续的、长期的皮肤等同物以研究新的治疗干预方面的进一步进展依赖于利用患者来源的细胞和模仿活体皮肤结构的天然三维培养条件的可再生模型。在这里，我们提出了一种新的分步方案，用于将人类皮肤细胞移植到免疫功能低下的小鼠上，该方案需要低起始细胞数，这在原代患者细胞有限时建模皮肤状况是必不可少的。我们的方法的核心要素是成纤维细胞的顺序移植，然后将角质形成细胞接种到硅胶室中的纤维蛋白基水凝胶中。我们优化了纤维蛋白凝胶制剂、体内凝胶聚合的时间、细胞培养条件和接种密度，以制定稳健且有效的移植方案。使用这种方法，我们可以成功地植入少至1.0 × 106新鲜和2.0 × 106冷冻后解冻角质形成细胞每1.4平方厘米的伤口面积。此外，得出的结论是，可以在没有劳动密集型和昂贵的方法，如生物打印或工程化复杂的皮肤等效物的情况下获得皮肤细胞在体内的成功逐层植入。 ·扩展了传统的皮肤室分析方法，使用最少数量的培养皮肤细胞生成高质量的皮肤移植物。·所提出的方法允许在外科手术中使用冷冻然后解冻的角质形成细胞和成纤维细胞。·该系统有望评估来自诱导多能干细胞的皮肤细胞的功能性并复制各种皮肤表型。整个过程，从解冻皮肤细胞到建立移植物，需要54天。 图形概览
Human skin reconstruction on immune-deficient mice has become indispensable for in vivo studies performed in basic research and translational laboratories. Further advancements in making sustainable, prolonged skin equivalents to study new therapeutic interventions rely on reproducible models utilizing patient-derived cells and natural three-dimensional culture conditions mimicking the structure of living skin. Here, we present a novel step-by-step protocol for grafting human skin cells onto immunocompromised mice that requires low starting cell numbers, which is essential when primary patient cells are limited for modeling skin conditions. The core elements of our method are the sequential transplantation of fibroblasts followed by keratinocytes seeded into a fibrin-based hydrogel in a silicone chamber. We optimized the fibrin gel formulation, timing for gel polymerization in vivo, cell culture conditions, and seeding density to make a robust and efficient grafting protocol. Using this approach, we can successfully engraft as few as 1.0 × 106 fresh and 2.0 × 106 frozen-then-thawed keratinocytes per 1.4 cm2 of the wound area. Additionally, it was concluded that a successful layer-by-layer engraftment of skin cells in vivo could be obtained without labor-intensive and costly methodologies such as bioprinting or engineering complex skin equivalents. Key features • Expands upon the conventional skin chamber assay method to generate high-quality skin grafts using a minimal number of cultured skin cells. • The proposed approach allows the use of frozen-then-thawed keratinocytes and fibroblasts in surgical procedures. • This system holds promise for evaluating the functionality of skin cells derived from induced pluripotent stem cells and replicating various skin phenotypes. • The entire process, from thawing skin cells to establishing the graft, requires 54 days. Graphical overview