Microwell cell selection printer for single cell isolation and high throughput screening of secreted proteins: Sensitively sensing the secretions from single cells

This work introduces and applies nanofluidic chip technology (McSPRinter), a single-cell analysis platform designed for antibody-secreting cells (ASCs) and cancer biomarker profiling. Chapter 1 underscores the importance of detecting proteins at the single-cell level, reviewing various single-cell technologies. Chapter 2 introduces McSPRinter and its use in evaluating antibody secretion from individual CHO (anti-Her2) and hybridoma (VU1D9) cells using nanowell chips and FLUOROspot membranes. The platform revealed heterogeneous secretion patterns between and within cell types.

In Chapter 3, McSPRinter is used to isolate high-producing CHO clones, leading to a 1.8–4.5-fold increase in anti-Her2 monoclonal antibody (mAb) production compared to unscreened cells. This improved productivity was sustained in Fed-Batch cultures. The sterile, animal-component-free workflow completes the selection-to-expansion cycle in 4–6 weeks.

Chapter 4 explores antibody secretion from human tonsillar B-lymphocyte subpopulations. Plasmablasts and germinal center B cells produced IgM and IgG spontaneously, with class switching observed in some cells. Memory B cells responded to in vitro stimulation with varied antibody secretion, revealing significant functional heterogeneity.

In Chapter 5, McSPRinter assessed prostate cancer cells secretion of Prostate Specific Antigen (PSA) in response to drugs. PSA levels increased with androgen stimulation (R1881) and decreased under antiandrogens (enzalutamide, abiraterone), showing variable responses across single cells. Chapter 6 extended this to clinical samples from 18 untreated metastatic castration-naïve prostate cancer (mCNPC) patients. PSA secretion was detected in half the patients’ circulating tumor cells (CTCs), with substantial intra-patient variability, suggesting PSA may be an unreliable universal biomarker.

Chapter 7 demonstrates the integration of Surface Plasmon Resonance Imaging (SPRi) with nanowell technology, enabling real-time, high-resolution tracking of antibody production. This combined platform enhances the isolation and analysis of high-producing or functionally distinct cells for therapeutic and research applications.