Magnetic cell separation for purification of human oral keratinocytes: an effective method for functional studies without prior cell subcultivation

Abstract

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.

Cytokine Expression of Human Oral Keratinocytes

“…Previous studies showed that MACS enrichment did not alter the viability and functional abilities of different cell types [19]. This corresponds to our own data where oral keratinocytes of different origin showed unaltered cytokine expression before and after MACS [17].…”

“…For purification of oral mucosa-derived human keratinocytes we used an immunomagnetic separation technique (MACS) to avoid further subcultivation [17]. Previous studies showed that MACS enrichment did not alter the viability and functional abilities of different cell types [19].…”

“…The magnetic cell separation system (MACS; Miltenyi Biotec, Bender, Vienna, Austria) used for the purification of human oral keratinocytes from a heterogeneous cell suspension has been described in detail elsewhere [17]. In brief, before the magnetic separation procedure, phenotypic characterization by FACS (fluorescence-activated cell sorting) analyses revealed that 84 B 1.5% of cells were positive for the pancytokeratin monoclonal antibody (mAb, lu-5; Dianova, Hamburg, Germany).…”

Überexpression von β1-Integrin in oralen Keratinozyten korreliert mit deren hohen Proliferationskapazität

“…Enriching of epithelial stem cells is a desirable goal in approaches for tissue engineering of oral mucosa. Many methods exist for facilitating such a separation [18]: nonimmunological methods such as velocity sedimentation [19], and immunological procedures such as fluorescenceactivated cell sorting (FACS) [20], complement lysis [21], rosetting [22], and magnetic cell separation (MACS) [23,24]. These methods are very successful for this purpose but have disadvantages caused by the high degree of physical stress acting on the cells during these procedures.…”

Invasive Capacity of Heterotopic Endometrium

“…Separated cells were obtained by using selective enzymatic digestion [4] in conjunction with physical and immunological separation procedures [5]. In vitro procedures, using separated endometrial cell types, present the experimental approach to study the uPA and uPAR expression at the cellular level.…”