Microfluidic Squeezing Enables MHC Class I Antigen Presentation by Diverse Immune Cells to Elicit CD8+ T Cell Responses with Antitumor Activity.

CD8+ T cell responses are the foundation of the recent clinical success of immunotherapy in oncologic indications. Although checkpoint inhibitors have enhanced the activity of existing CD8+ T cell responses, therapeutic approaches to generate Ag-specific CD8+ T cell responses have had limited success. Here, we demonstrate that cytosolic delivery of Ag through microfluidic squeezing enables MHC class I presentation to CD8+ T cells by diverse cell types. In murine dendritic cells (DCs), squeezed DCs were ∼1000-fold more potent at eliciting CD8+ T cell responses than DCs cross-presenting the same amount of protein Ag. The approach also enabled engineering of less conventional APCs, such as T cells, for effective priming of CD8+ T cells in vitro and in vivo. Mixtures of immune cells, such as murine splenocytes, also elicited CD8+ T cell responses in vivo when squeezed with Ag. We demonstrate that squeezing enables effective MHC class I presentation by human DCs, T cells, B cells, and PBMCs and that, in clinical scale formats, the system can squeeze up to 2 billion cells per minute. Using the human papillomavirus 16 (HPV16) murine model, TC-1, we demonstrate that squeezed B cells, T cells, and unfractionated splenocytes elicit antitumor immunity and correlate with an influx of HPV-specific CD8+ T cells such that >80% of CD8s in the tumor were HPV specific. Together, these findings demonstrate the potential of cytosolic Ag delivery to drive robust CD8+ T cell responses and illustrate the potential for an autologous cell-based vaccine with minimal turnaround time for patients.

Flexible Octopus-Shaped Hydrogel Particles for Specific Cell Capture.

Multiarm hydrogel microparticles with varying geometry are fabricated to specifically capture cells expressing epithelial cell adhesion molecule. Results show that particle shape influences cell-capture efficiency due to differences in surface area, hydrodynamic effects, and steric constraints. These findings can lead to improved particle design for cell separation and diagnostic applications.

Synthesis of Nonspherical Microcapsules through Controlled Polyelectrolyte Coating of Hydrogel Templates.

We report a simple approach to fabricate custom-shape microcapsules using hydrogel templates synthesized by stop flow lithography. Cargo-containing microcapsules were made by coating hydrogel particles with a single layer of poly-l-lysine followed by a one-step core degradation and capsule cross-linking procedure. We determined appropriate coating conditions by investigating the effect of pH, ionic strength, and prepolymer composition on the diffusion of polyelectrolytes into the oppositely charged hydrogel template. We also characterized the degradation of the templating core by tracking the diffusivity of nanoparticles embedded within the hydrogel. Unlike any other technique, this approach allows for easy fabrication of microcapsules with internal features (e.g., toroids) and selective surface modification of Janus particles using any polyelectrolyte. These soft, flexible capsules may be useful for therapeutic applications as well as fundamental studies of membrane mechanics.

Synthesis of colloidal microgels using oxygen-controlled flow lithography.

We report a synthesis approach based on stop-flow lithography (SFL) for fabricating colloidal microparticles with any arbitrary 2D-extruded shape. By modulating the degree of oxygen inhibition during synthesis, we achieved previously unattainable particle sizes. Brownian diffusion of colloidal discs in bulk suggests the out-of-plane dimension can be as small as 0.8 µm, which agrees with confocal microscopy measurements. We measured the hindered diffusion of microdiscs near a solid surface and compared our results to theoretical predictions. These colloidal particles can also flow through physiological microvascular networks formed by endothelial cells undergoing vasculogensis under minimal hydrostatic pressure (∼5 mm H2O). This versatile platform creates future opportunities for on-chip parametric studies of particle geometry effects on particle passage properties, distribution and cellular interactions.

Mesoporous organohydrogels from thermogelling photocrosslinkable nanoemulsions.

We report the formation of mesoporous organohydrogels from oil-in-water nanoemulsions containing an end-functionalized oligomeric gelator in the aqueous phase. The nanoemulsions exhibit an abrupt thermoreversible transition from a low-viscosity liquid to a fractal-like colloidal gel of droplets with mesoscale porosity and solid-like viscoelasticity with moduli approaching 100 kPa, possibly the highest reported for an emulsion-based system. We hypothesize that gelation is brought about by temperature-induced interdroplet bridging of the gelator, as shown by its dependence on the gelator chemistry. The use of photocrosslinkable gelators enables the freezing of the nanoemulsion's microstructure into a soft hydrogel nanocomposite containing a large fraction of dispersed liquid hydrophobic compartments, and we show its use in the encapsulation and release of lipophilic biomolecules. The tunable structural, mechanical and optical properties of these organohydrogels make them a robust material platform suitable for a wide range of applications.

Synthesis of biomimetic oxygen-carrying compartmentalized microparticles using flow lithography.

We report a microfluidic approach for lithographically photo-patterning compartmentalized microparticles with any 2D-extruded shape, down to the cellular length scale (~10 microns). The prepolymer solution consists of a UV crosslinkable perfluorodecalin-in-water nanoemulsion stabilized by Pluronic(®) F-68. The nanoemulsions are generated using high-pressure homogenization and are osmotically stabilized by the trapped species method. The presence of PFC droplets increases the solubility and diffusivity of oxygen in the prepolymer solution, thereby enhancing the rate of O2 inhibition during microparticle synthesis. We develop a simple model that successfully predicts the augmented O2 mass transport, which agrees well with experimental data. Informed by our analytical results, cell-sized composite microgels are generated by controlling the oxygen environment around the polydimethylsiloxane (PDMS) microfluidic synthesis device. These nanoemulsion composites are functionally similar to red blood cells as oxygen carriers. Such bio-inspired polymeric particles with controlled physical properties are promising vehicles for drug delivery and clinical diagnostics.