Label-free microfluidic isolation of functional and viable lymphocytes from peripheral blood mononuclear cells.

The separation of peripheral blood mononuclear cells (PBMCs) into constituent blood cell types is a vital step to obtain immune cells for autologous cell therapies. The ability to separate PBMCs using label-free microfluidic techniques, based on differences in biomechanical properties, can have a number of benefits over other conventional techniques, including lower cost, ease of use, and avoidance of animal-derived labeling antibodies. Here, we report a microfluidic device that uses compressive diagonal ridges to separate PBMCs into highly pure samples of viable and functional lymphocytes. The technique utilizes the differences in the biophysical properties of PBMC sub-populations to direct the lymphocytes and monocytes into separate outlets. The biophysical properties of the monocytes and lymphocytes from healthy donors were first characterized using atomic force microscopy. Lymphocytes were found to be significantly stiffer than monocytes, with a mean cell stiffness of 1495 and 931 Pa, respectively. The differences in biophysical properties resulted in distinct trajectories through the microchannel terminating at different outlets, resulting in a lymphocyte sample with purity and viability both greater than 96% with no effect on the cells' ability to produce interferon gamma, a cytokine crucial for innate and adaptive immunity.

Heterofunctional Particles as Single Cell Sensors to Capture Secreted Immunoglobulins and Isolate Antigen-Specific Antibody Secreting Cells.

Isolating cells based on their secreted proteins remain a challenge. The authors demonstrate a capacity for high throughput single-cell protein secretion analysis and isolation based on heterofunctional particles combined with fluorescence activated cell sorting (FACS). The workflow shows that antibody secreting cells (ASCs) specific for the H1 protein from influenza virus can be isolated from B cells. The workflow consists of incubating anti-CD27 particles with the ASCs, capturing locally secreted immunoglobulins with Protein G on the particles, and identifying immunoglobulins specific to H1 via fluorescent labeled antigens followed by FACS to enrich antigen-specific ASCs. Two particles designs, Janus and mixed, are tested with hybridoma cells. Mixed particles are found to improve antibody collection, while Janus particles are found to bind target cells more effectively. Targeted hybridoma cells in coculture with non-specific hybridoma cells are identified with a sensitivity of 96% and specificity of 98%. Heterofunctional particles are used to capture ASCs that secrete antibodies specific for influenza virus from B cells from healthy adults isolated from blood after vaccination. Positive H1-tetramer sorted ASCs are validated using single ASC cultures and identify 23/56 cells specific for H1 demonstrating 164-fold enrichment from total B cells and 14.6-fold enrichment from total ASCs.

Tuning Antibody Presentation to Enhance T-Cell Activation for Downstream Cytotoxicity.

Cytotoxic effector cells are an integral component of the immune response against pathogens and diseases such as cancer and thus of great interest to researchers who wish to enhance the native immune response. Although researchers routinely use particles to stimulate cytotoxic T cells, few studies have comprehensively investigated: (1) beyond initial activation responses (i.e., proliferation and CD25/CD69 expression) to downstream cancer-killing effects and (2) how to drive cytotoxic T-cell responses by adjusting biomolecular and physical properties of particles. In this study, we designed particles displaying an anti-CD3 antibody to activate cytotoxic T cells and study their downstream cytotoxic effects. We evaluated the effect of antibody immobilization, particle size, molecular surface density of an anti-CD3 antibody, and the inclusion of an anti-CD28 antibody on cytolytic granule release by T cells. We found that immobilizing the anti-CD3 antibody onto smaller nanoparticles elicited increased T-cell activation products for an equivalent delivery of the anti-CD3 antibody. We further established that the mechanism behind increased cancer cell death was associated with the proximity of T cells to cancer cells. Functionalizing particles additionally with the anti-CD28 antibody at an optimized antibody density caused increased T-cell proliferation and T-cell binding but we observed no effective increase in cytotoxicity. Meaningfully, our results are discussed within the context of commercially available and widely used anti-CD3/28 Dynabeads. These results showed that T-cell activation and cytotoxicity can be optimized with a molecular presentation on smaller particles and thus, offer exciting new possibilities to engineer T-cell activation responses for effective outcomes.

Optimization of Microparticle Reagents to Collect and Detect Antibody.

Bead reagents are used in a large number of assays in bioscience and biotechnology to collect and purify antibodies by immobilization. Bead-based immunoassays offer high-throughput analysis of multiple antibodies in a single sample. Although a variety of antibody-binding moieties on the collection beads have been studied, the physical and material properties of collection beads have not been optimized to isolate specific antibodies over a broad range of concentrations from complex environments containing cells. We present a study of how to optimally use microparticles coated with protein G to collect low concentrations of IgG antibodies from complex solutions. We study the impact of bead material, bead size, incubation time, and protein G density to more efficiently collect antibodies and detect specific antibodies via fluorescent antigen labeling. The minimum detectable limit and the minimum incubation time for antibody collection are used as metrics to evaluate the collection parameters. We found that larger silica beads can capture more antibodies from a low concentration of sample, with a minimum incubation time of 60 min to equilibrium binding, resulting in a minimum detectable concentration of antibodies of 26 nM. We show that simple biophysical optimization of antibody collection reagents can be used to improve the collection of low concentrations of antibodies in complex environments. We demonstrate that the technology may be useful for monitoring antibody secretions from hybridoma cultures.