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 obtain a pure population of cells without prior in vitro expansion. An immunomagnetic separation technique, or magnetic cell separation (MACS), was modified for efficient purification of human oral keratinocytes. Subsequent to two-step enzymatic digestion, the cell suspension was labelled with a mouse anti-CD45 (pan-leukocyte) monoclonal antibody (MoAb) to stain mononuclear cells. In a second step a rat anti-mouse antibody conjugated with colloidal superparamagnetic particles was used. Labelled cells were retained in the magnetic field of a permanent magnet on columns containing a ferromagnetic matrix. The unlabelled, unretained cells were further examined by flow cytometry analysis, enzyme-linked immunosorbent assay and polymerase chain reaction. After the MACS procedure, unretained cells showed a strong positivity for the lu-5 MoAb (as a marker for pan-cytokeratin) and were negative for anti-vimentin (to mark mesenchymal cells), for anti-CD45 MoAb and for melanocyte-detecting antibodies, thus representing pure keratinocytes (> 98%). Purified keratinocytes maintained full viability (> 91%) and functional capacities. [3H]thymidine uptake and epidermal growth factor (EGF) receptor 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 enrichment. Our findings show that MACS appears to be a useful tool for purification of oral keratinocytes and allows for further functional studies without prior subcultivation of cells.
Formanek
M
M
Department of Otorhinolaryngology, University of Vienna, Austria.
Temmel
A
A
Knerer
B
B
Willheim
M
M
Millesi
W
W
Kornfehl
J
J
D000818:Animals; D000911:Antibodies, Monoclonal; D002455:Cell Division / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D015603:Keratinocytes / Q000166:cytology*; D051379:Mice; D009061:Mouth Mucosa / Q000166:cytology*; D051381:Rats
D000911:Antibodies, Monoclonal