Organizing leukocytes into high-density arrays makes these cells amenable to rapid optical characterization and subsequent sorting, pointing to clinical and basic science applications. The present paper describes development of a cytometry platform for creating high-density leukocyte arrays and demonstrates retrieval of single cells from the array. Poly(ethylene glycol)
(PEG) photolithography was employed to fabricate arrays of microwells composed of PEG hydrogel walls and glass attachment pads 20 µm × 20 µm and 15 µm × 15 µm in size. PEG micropatterned glass surfaces were further modified with cell-adhesive ligands, poly-L-lysine, anti-CD5 and anti-CD19 antibodies, in order to engineer specific cell–surface interactions within the individual wells. Localization of the fluorescently-labeled proteins in the glass attachment pads of PEG microwells was visualized by fluorescence microscopy. Glass slides micropatterned with PEG and cell-adhesive ligands were exposed to T-lymphocytes for 30 min. These anchorage-independent cells became selectively captured in the ligand-modified microwells forming high-density cell arrays. Cell occupancy in the microwells was found to be antibody-dependent, reaching 94.6 ± 2.3% for microwells decorated with T-cell specific anti-CD5 antibodies. Laser capture microdissection (LCM) was investigated as a method for sorting cells from the array and retrieval of single selected cells was demonstrated.