Benchmarking human skin organotypic cultures toward high-fidelity excellence

Summary: Abstract Body: Over decades, extensive efforts have been devoted to developing in vitro 3D skin models, with a particular focus on skin organotypic cultures (OTCs). These models are valued for their cost-effectiveness, straightforward setup, time efficiency, and ability to closely replicate native human skin. Despite significant progress, systematic optimization remains a formidable challenge, and the fidelity of current skin OTC models has yet to be fully defined. In this study, we systematically benchmarked existing skin OTC protocols by evaluating critical parameters, including culture media composition, submerged culture duration, culture inserts, oxygen concentration, culture temperature, and calcium levels. Furthermore, we investigated keratinocytes and fibroblasts derived from diverse sources to identify optimal culture conditions that enhance model performance. To quantitatively assess variation and fidelity in skin organoids, we performed single-cell RNA sequencing (scRNA-seq) on our optimized OTCs and integrated publicly available skin organoid scRNA-seq datasets. Comparative mapping to the human skin atlas identified cell types and states generated in vitro, highlighting developmental stage- and anatomical site-specific characteristics. Additionally, we estimated transcriptomic similarities between primary tissues and their organoid counterparts across various skin organoid models. In conclusion, our findings establish a comprehensive framework for evaluating and improving the fidelity of skin OTC models. This work provides critical insights into human skin development and serves as a valuable resource for advancing organoid-based disease modeling and protocol optimization. Yunlong Y. Jia¹, Scott Atwood¹ 1. Developmental and Cell Biology, University of California Irvine, Irvine, CA, United States. Stem Cell Biology, Tissue Regeneration and Wound Healing