ImmunoSolv is committed to developing reagents and devices that mimic in vitro the efficient dead-cell removal mechanisms that are so important for the health of tissues in vivo, thereby improving the quality of cells in the laboratory.
Importance of dead-cell removal in vivo and in vitro cell populations
Programmed cell death (apoptosis) is a feature of all tissues. Apoptotic cells are swiftly, and in inflammatory terms quietly, removed by phagocytes, a homeostatic process that keeps tissues healthy. Necrotic (dead) cells cause tissue damage. In vitro, the phagocytic clearance mechanism is absent, dead cells persist, and their inhibitory effects lead to sub-optimal cell culture conditions with accumulation of dead cells and debris.
It is well known that, when cells engage their apoptosis ‘suicide’ programme, or when their membranes lose integrity through post-apoptotic necrosis or through primary necrosis caused, for example, by traumatic physical, or noxious chemical stimuli, they lose the phospholipid asymmetry that characterises the plasma membrane of the viable state. Notably, during apoptosis, the anionic phospholipid, phosphatidylserine (PS) that, in viable cells, is located on the inner leaflet of the plasma-membrane bilayer, becomes exposed on the outer leaflet while the membranes retain their integrity, excluding vital dyes such as trypan blue, propidium iodide and ethidium bromide. When the membranes of necrotic cells lose their integrity, they too expose PS that becomes accessible to PS-binding proteins such as annexin V. Annexin V is commonly used to monitor apoptosis in cell suspensions by flow cytometry, but its interaction with exposed PS and the maintenance of its binding requires a binding buffer containing high Ca2+concentration. This has numerous drawbacks, not least of which is the tendency of such Ca2+ levels to induce clumping of cells and the inconvenience of removing cells from their preferred medium.