Application of physical force is essential to enrich for epidermal stem cells in primary human keratinocyte isolation

We present an improved method to isolate epidermal cells and to enrich stem cell populations. The new method utilizes magnetic stirring during digestion of skin with trypsin-EDTA (magnetic stirring method). The magnetic stirring method significantly improves both cell yield and colony-forming efficiency (CFE) relative to conventional epidermal cell isolation methods, such as those involving trypsin-EDTA only, thermolysin, or dispase. The cell yield and the total number of colony-forming cells per square centimeter of adult foreskin were 7,064,000 +/- 95,000 cells and 96,030 +/- 31,990 cells by the magnetic stirring method, 1,128,000 +/- 111,000 cells and 10,192 +/- 2941 cells by trypsin-EDTA only, 3,279,000 +/- 842,000 cells and 5851 +/- 2989 cells by thermolysin, and 1,443,000 +/- 507,000 cells and 8843 +/- 3388 cells by dispase, respectively. The CFE was 1.15 +/- 0.32% by magnetic stirring, 0.95 +/- 0.35% by trypsin-EDTA only, 0.28 +/- 0.12% by thermolysin, and 0.83 +/- 0.30% by dispase. The levels of surface beta(1)-integrin expression skewed to the right with the magnetic stirring method, implying increased proportion of beta(1)-integrin-bright cells (putative stem cells). Isolated keratinocytes developed fully differentiated epidermis when they were grafted onto nude mice. In conclusion, cell isolation by the magnetic stirring method is advantageous over conventional epidermal cell isolation methods, by which full range of basal epidermal cells including stem cells, transiently amplifying cells, and differentiating cells can be isolated and allow shorter expansion time for maximal coverage of wound sites after biopsies are taken from patients.