ABSTRACTPersonalized medicine offers the promise of tailoring therapy to patients, based on their cellular biomarkers. To achieve this goal, cellular profiling systems are needed that can quickly and efficiently isolate specific cell types without disrupting cellular biomarkers. Here we describe the development of a unique platform that facilitates gentle cell capture via a secondary, surface‐anchoring moiety, and cell release. The cellular capture system consists of a glass surface functionalized with APTES, d‐desthiobiotin, and streptavidin. Biotinylated mCD11b and hIgG antibodies are used to capture mouse macrophages (RAW 264.7) and human breast cancer (MCF7‐GFP) cell lines, respectively. The surface functionalization is optimized by altering assay components, such as streptavidin, d‐desthiobiotin, and APTES, to achieve cell capture on 80% of the functionalized surface and cell release upon biotin treatment. We also demonstrate an ability to capture 50% of target cells within a dual‐cell mixture. This engineering advancement is a critical step towards achieving cell isolation platforms for personalized medicine. Biotechnol. Bioeng. 2015;112: 2214–2227. © 2015 Wiley Periodicals, Inc.