Long-Term Expansion of Single-Cell Clones from Primary Human Keratinocytes

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, ex vivo 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.
 

CnT-NX-EX is a next-generation medium delivers efficient isolation and prolonged expansion of primary epithelial cells. It is crafted to overcome challenges like early senescence and limited lifespan of primary cells in defined culture conditions. We demonstrate the implications of CnT-NX-EX medium for the expansion of single-cell clones.

CnT-NX-EX

 
 

Experimental Protocol and Key Steps of Single-Cell Clones Amplification:

 

A total of 120 keratinocytes were plated as single cells by dilution in passage 2 (as shown in Fig. 2). After two weeks, 96 single cells (80 %) exhibited successful clonal expansion. Of 21 clones harvested and propagated until growth stop (as shown in Fig 3), 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.

 

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Continued Growth of Single Cell Clones and 3D Epidermal 3D Modeling:

 

We report the successful long-term expansion of single cell clones derived from primary non-transformed human adult keratinocytes.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.

 

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Proof of principle:

• Clonal expansion of adult primary human keratinocytes was achieved in an animal-component–free environment, without the necessity for surface coating, feeder cells, or immortalization
• Trillion-fold expansion from single cell
• Several passages and full stratification potential retained
• High success rates

– clonal growth in ca. 80 % of single cells seeded

– clonal growth > 20 population doublings in ca. 66 % of single cells seeded

– clonal growth > 40 population doublings in ca. 10 % of single cells seeded

 

CONCLUSION & SUMMARY:

We established a robust cell culture system using CELLnTEC’s fully defined, animal and human component-free CnT-NX-EX 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.