Lectin receptor sites at the cell surface employed for affinity separation of tissue culture cells. Basic requirements as realized by lens culinaris lectin (LCL) immobilized on 2tb-sepharose.

Abstract

Lens culinaris lectin (LCL) immobilized on large 2B-Sepharose beads has been used to investigate the lectin-binding capacity of cell surfaces for the separation of cells by affinity chromatography. Immobilized LCL induced the tissue culture cells studied (HeLa, SV3T3) to bind to the agarose beads. This binding could be prevented by the addition of hapten sugars such as methyl-α d -mannopyranoside (MαMP) and methyl-α d -glucopyranoside (MαGP) according to the affinity of these sugars to LCL in a concentration-dependent manner. The cell-bead linkages were sufficiently strong to resist any appreciable mechanical breakage. The binding of the cells occurred so fast that any release could be started before the cells interacted unspecifically with the beads. Immobilized LCL released most of the cells upon addition of the competing sugar MαMP in a concentration-dependent manner under physiological conditions. The cells retained viability during the separation procedure as demonstrated by subsequent growth in culture. The difficulties so far observed with procedures involving columns have been overcome by a batch technique for controlled cell binding and release. For rapid separation of free from bound cells a gauze of defined pore size is introduced. Methodological problems such as agarose concentration, cell stickiness, lectin amount, and mechanical stability of the bead-cell complex are discussed.