Efficient Replacement of Outer Leaflet Lipids of Plasma Membrane using Exogenous Lipids with Minimal Cell Damage

Recently our lab developed a technique using methyl alpha cyclodextrin to efficiently replace the outer leaflet of plasma membrane in mammalian cells using exogenous lipids at physiological temperature (37°C). At sub-optimal conditions of lipid exchange, either too much or too little lipid is transferred to the plasma membrane outer leaflet, causing cells to round up and damaging membranes sufficiently to allow proteins as big as GFP to leak out of at least some of the cells. In contrast, under optimal conditions of exchange cells maintain normal cell morphology with no significant loss of viability. This can be achieved for different cell types by changing the amount of lipid and cyclodextrin used for lipid exchange. We were able to introduce lipids supporting ordered domains such as sphingomyelin into cells at physiological temperature with minimal damage, while under these conditions exchange of pure unsaturated phosphatidylcholines, which support disordered domain formation severely perturbed cell morphology. This was overcome by reducing the temperature during exchange without a substantial loss of exchange efficiency. In addition, incubating cells in normal media immediately after exchange was found to help minimize cell damage without substantial remodeling of the exogenous lipid content. Use of optimal lipid conditions should allow application of lipid exchange method to study the lipid-dependence of membrane properties and lipid-protein interactions in living cells