An experimental model of affinity cell separation.
Cell affinity separations are based on the selective attachment of cell phenotype using antibody or lectins specific for cell surface markers. The major physicochemical factors which influence ligand-mediated cell adhesion dynamics and the efficiency of cell affinity separation have been examined. Uniform cell detachment forces were generated with a parallel-plate flow cell (plate separation 100 microns, surface area 3 cm2). Hydrodynamic shear stress was used to measure cell adhesion strength and to separate cells on the basis of surface affinity. Human cell lines grown in tissue culture were separated on a flat derivatised glass immunoadsorbent which formed the floor of the flow chamber. Flow-cell residence time, detachment shear stress, temperature, and ligand density were shown to influence cell attachment probability. An understanding of the physical basis of ligand-mediated cell adhesion provided a rationale for optimisation of affinity cell separation. At room temperature attachment of positive cells was rapid (< 2 min) and adhesion strength was directly related to immunoadsorbent ligand density. Purity and recovery of enriched fractions were dependent on the separation shear stress and could be optimised using this parameter. Enrichment factors were greater than 100-fold, with at least 90% of positive cells recovered in enriched fractions. Enrichment purity and yields did not decline at higher loading densities (10(5) cells/cm2). Selective immunoadsorbent surface chemistry is a prerequisite for efficient affinity cell separation. Purity and recovery may be optimised by fractionating enriched and depleted cell populations with uniform fluid shear stress.
Nordon
R E
RE
Centre for Biomedical Engineering, University of New South Wales, Kensington, Sydney, Australia.
Milthorpe
B K
BK
Schindhelm
K
K
Slowiaczek
P R
PR
D000915:Antibody Affinity; D002448:Cell Adhesion; D002469:Cell Separation / Q000379:methods*; D006801:Humans; D016130:Immunophenotyping; D008024:Ligands; D014407:Tumor Cells, Cultured
D008024:Ligands