Overcoming clonal expansion barriers in primary human keratinocytes in a chemically defined and animal component-free culture system.

Summary: The expansion of single-cell clones from primary human keratinocytes is inherently challenging due to their limited proliferative capacity, early differentiation, and senescence. These constraints hinder the use of primary keratinocytes for applications that rely on single cell cloning, such as genome editing, <i>ex vivo</i> gene therapy, or lineage tracing. Consequently, researchers often resort to immortalized keratinocyte cell lines. Such cell lines support extended culture but exhibit drawbacks such as incomplete differentiation and tumorigenic potential. We report the successful long-term expansion of single cell clones derived from primary non-transformed human adult keratinocytes. A total of 120 keratinocytes were plated as single cells by dilution in passage 2. After two weeks, 96 single cells (80 %) exhibited successful clonal expansion. Of 21 clones harvested and propagated until growth stop, 18 clones sustained proliferation beyond 20 population doublings (PD) post-plating, while three of these clones exceeded 40 PD and 50 days of culture, which corresponds to a potential yield of 1.1 × 10^12 (over a trillion) cells derived from one single cell. Epidermal models constructed in passage 8 confirmed full stratification into all four epidermal layers in three out of four clones, indicative of functional differentiation capacity. Preliminary data from single-cell clones of primary keratinocytes from a second donor confirmed reproducibility. We established a robust cell culture system using CELLnTEC’s fully defined, animal and human component-free CnT-NX-EX Epithelial Extended Proliferation Medium without the need for surface coating or feeder cells, making it highly suitable for clinical translation. The system allows for expansion of single cell clones from primary keratinocytes, which makes it a valuable enabling tool for genetic modification, such as transfection, transduction, and genome editing. This underlines its versatility for applications in basic and translational research, gene therapy, and regenerative medicine. Elias Imahorn¹, Elena Baiardi¹, Stefan Baertschi¹ 1. CELLnTEC Advanced Cell Systems AG, Bern, Switzerland. Epidermal Structure and Barrier Function