MAGNETIC CELL-SEPARATION FOR PURIFICATION OF HUMAN ORAL KERATINOCYTES- AN EFFECTIVE METHOD FOR FUNCTIONAL-STUDIES WITHOUT PRIOR CELL SUBCULTIVATION

In studying human oral keratinocytes, it would be very helpful to obta in a pure population of cells without prior in vitro expansion. An imm unomagnetic separation technique, or magnetic cell separation (MACS, w as modified for efficient purification of human oral keratinocytes. Su bsequent to two-step enzymatic digestion, the cell suspension was labe lled with a mouse anti-CD45 (pan-leukocyte) monoclonal antibody (MoAb) to stain mononuclear cells. In a second step a rat anti-mouse anti-bo dy conjugated with colloidal superparamagnetic parti cles was used. La belled cells were retained in the magnetic field of a permanent magnet on columns containing a ferromagnetic matrix. The unlabelled, unretai ned cells were further examined by flow cytometry analysis, enzyme-lin ked immunosorbent assay and polymerase chain reaction. After the MACS procedure, unretained cells showed a strong positivity for the 1u-5 Mo Ab las a marker for pan-cytokeratin) and were negative for anti-viment in (to mark mesenchymal cells), for anti-CD45 MoAb and for melanocyte- detecting antibodies, thus representing pure keratinocytes (> 98%). Pu rified keratinocytes maintained full viability (> 91%) and functional capacities. [H-3]thymidine uptake and epidermal growth factor (EGF) re ceptor expression were unaltered when compared with the non-separated cell population. Furthermore, interleukin-1 alpha was detected at the protein and RNA levels in keratinocytes immediately after MACS enrichm ent. Our findings show that MACS appears to be a useful tool for purif ication of oral keratinocytes and allows for further functional studie s without prior subcultivation of cells.