Defining T cell receptor repertoires using nanovial-based binding and functional screening.

The ability to selectively bind to antigenic peptides and secrete effector molecules can define rare and low-affinity populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs inducing the secretion of effector molecules including IFN-γ and granzyme B that are accumulated on nanovials, allowing sorting based on both binding and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired αß-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes and secretions with oligo-barcoded detection antibodies, we could accurately link TCR sequences to specific targets and rank each TCR based on the corresponding cell's secretion level. Using the technique, we identified an expanded repertoire of functional TCRs targeting viral antigens with high specificity and found rare TCRs with activity against cancer-specific splicing-enhanced epitopes.

Defining T cell receptor repertoires using nanovial-based affinity and functional screening.

The ability to selectively bind to antigenic peptides and secrete cytokines can define populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with millions of peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs and secrete cytokines on nanovials, allowing sorting based on both affinity and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired αß-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes we could link TCR sequence to targets with 100% accuracy. We identified with high specificity an expanded repertoire of functional TCRs targeting viral antigens compared to standard techniques.

Single-cell sorting based on secreted products for functionally defined cell therapies.

Cell therapies have emerged as a promising new class of "living" therapeutics over the last decade and have been particularly successful for treating hematological malignancies. Increasingly, cellular therapeutics are being developed with the aim of treating almost any disease, from solid tumors and autoimmune disorders to fibrosis, neurodegenerative disorders and even aging itself. However, their therapeutic potential has remained limited due to the fundamental differences in how molecular and cellular therapies function. While the structure of a molecular therapeutic is directly linked to biological function, cells with the same genetic blueprint can have vastly different functional properties (e.g., secretion, proliferation, cell killing, migration). Although there exists a vast array of analytical and preparative separation approaches for molecules, the functional differences among cells are exacerbated by a lack of functional potency-based sorting approaches. In this context, we describe the need for next-generation single-cell profiling microtechnologies that allow the direct evaluation and sorting of single cells based on functional properties, with a focus on secreted molecules, which are critical for the in vivo efficacy of current cell therapies. We first define three critical processes for single-cell secretion-based profiling technology: (1) partitioning individual cells into uniform compartments; (2) accumulating secretions and labeling via reporter molecules; and (3) measuring the signal associated with the reporter and, if sorting, triggering a sorting event based on these reporter signals. We summarize recent academic and commercial technologies for functional single-cell analysis in addition to sorting and industrial applications of these technologies. These approaches fall into three categories: microchamber, microfluidic droplet, and lab-on-a-particle technologies. Finally, we outline a number of unmet needs in terms of the discovery, design and manufacturing of cellular therapeutics and how the next generation of single-cell functional screening technologies could allow the realization of robust cellular therapeutics for all patients.

Suspendable Hydrogel Nanovials for Massively Parallel Single-Cell Functional Analysis and Sorting.

Techniques to analyze and sort single cells based on functional outputs, such as secreted products, have the potential to transform our understanding of cellular biology as well as accelerate the development of next-generation cell and antibody therapies. However, secreted molecules rapidly diffuse away from cells, and analysis of these products requires specialized equipment and expertise to compartmentalize individual cells and capture their secretions. Herein, we describe methods to fabricate hydrogel-based chemically functionalized microcontainers, which we call nanovials, and demonstrate their use for sorting single viable cells based on their secreted products at high-throughput using only commonly accessible laboratory infrastructure. These nanovials act as solid supports that facilitate attachment of a variety of adherent and suspension cell types, partition uniform aqueous compartments, and capture secreted proteins. Solutions can be exchanged around nanovials to perform fluorescence immunoassays on secreted proteins. Using this platform and commercial flow sorters, we demonstrate high-throughput screening of stably and transiently transfected producer cells based on relative IgG production. Chinese hamster ovary cells sorted based on IgG production regrew and maintained a high secretion phenotype over at least a week, yielding >40% increase in bulk IgG production rates. We also sorted hybridomas and B lymphocytes based on antigen-specific antibody production. Hybridoma cells secreting an antihen egg lysozyme antibody were recovered from background cells, enriching a population of ∼4% prevalence to >90% following sorting. Leveraging the high-speed sorting capabilities of standard sorters, we sorted >1 million events in <1 h. IgG secreting mouse B cells were also sorted and enriched based on antigen-specific binding. Successful sorting of antibody-secreting B cells combined with the ability to perform single-cell RT-PCR to recover sequence information suggests the potential to perform antibody discovery workflows. The reported nanovials can be easily stored and distributed among researchers, democratizing access to high-throughput functional cell screening.

IL-2 secretion-based sorting of single T cells using high-throughput microfluidic on-cell cytokine capture.

Secreted proteins are critical for the coordination of potent immune defenses, such as in engineered T cell therapies, however, there are few widely accessible approaches to accurately analyze and sort large numbers of cells based on their secretory functions. We report a workflow for the rapid screening and sorting of single individual T cells based on IL-2 secretion accumulated at high concentrations in nanoliter droplets and encoded back onto the secreting cell's surface. In our method, droplets are used solely to partition cells, enabling rapid accumulation of signals onto cell surfaces, and eliminating diffusive crosstalk between neighbors. All downstream sorting leverages conventional high-throughput and readily accessible flow cytometry after the emulsion is disrupted. We achieve monodisperse droplet generation (CV < 10%) at flow rates up to 200 µL min-1 using step emulsification, enabling processing of entire libraries of cells within tens of minutes without significant secretion crosstalk. In comparison to our approach, strong mitogenic activation overwhelmed the conventional bulk on-cell cytokine assay, rendering labeled, non-activated cells indistinguishable from actively secreting neighbors within one hour. Processing of identical cell mixtures following droplet encapsulation yielded no apparent crosstalk even after three hours. Instead, IL-2 production spanning several orders of magnitude was observed from roughly 20% of analyzed activated lymphocytes, representing an at least 10-fold increase in dynamic range compared to unencapsulated cells. Secreting cells could also be sorted using fluorescence activated cell sorting (FACS). The approach can ultimately enable sorting of cells based on functional properties with higher accuracy in a more accessible format to life science researchers.

Sorting single-cell microcarriers using commercial flow cytometers.

The scale of biological discovery is driven by the vessels in which we can perform assays and analyze results, from multi-well plates to microfluidic compartments. We report on the compatibility of sub-nanoliter single-cell containers or "nanovials" with commercial fluorescence activated cell sorters (FACS). This recent lab on a particle approach utilizes 3D structured microparticles to isolate cells and perform single-cell assays at scale with existing lab equipment. Use of flow cytometry led to detection of fluorescently labeled protein with dynamic ranges spanning 2-3 log and detection limits down to ∼10,000 molecules per nanovial, which was the lowest amount tested. Detection limits were improved compared to fluorescence microscopy measurements using a 20X objective and a cooled CMOS camera. Nanovials with diameters between 35-85 µm could also be sorted with purity from 99-93% on different commercial instruments at throughputs up to 800 events/second. Cell-loaded nanovials were found to have unique forward and side (or back) scatter signatures that enabled gating of cell-containing nanovials using scatter metrics alone. The compatibility of nanovials with widely-available commercial FACS instruments promises to democratize single-cell assays used in discovery of antibodies and cell therapies, by enabling analysis of single cells based on secreted products and leveraging the unmatched analytical capabilities of flow cytometers to sort important clones.

Scalable Fabrication and Use of 3D Structured Microparticles Spatially Functionalized with Biomolecules.

Microparticles with defined shapes and spatial chemical modification can interface with cells and tissues at the cellular scale. However, conventional methods to fabricate shaped microparticles have trade-offs between the throughput of manufacture and the precision of particle shape and chemical functionalization. Here, we achieved scalable production of hydrogel microparticles at rates of greater than 40 million/hour with localized surface chemistry using a parallelized step emulsification device and temperature-induced phase-separation. The approach harnesses a polymerizable polyethylene glycol (PEG) and gelatin aqueous two-phase system (ATPS) which conditionally phase separates within microfluidically generated droplets. Following droplet formation, phase separation is induced and phase separated droplets are subsequently cross-linked to form uniform crescent and hollow shell particles with gelatin functionalization on the boundary of the cavity. The gelatin localization enabled deterministic cell loading in subnanoliter-sized crescent-shaped particles, which we refer to as nanovials, with cavity dimensions tuned to the size of cells. Loading on nanovials also imparted improved cell viability during analysis and sorting using standard fluorescence activated cell sorters, presumably by protecting cells from shear stress. This localization effect was further exploited to selectively functionalize capture antibodies to nanovial cavities enabling single-cell secretion assays with reduced cross-talk in a simplified format.

Wound healing with topical BRAF inhibitor therapy in a diabetic model suggests tissue regenerative effects.

Wound healing is a multi-step process to rapidly restore the barrier function. This process is often impaired in diabetic patients resulting in chronic wounds and amputation. We previously found that paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway via topical administration of the BRAF inhibitor vemurafenib accelerates wound healing by activating keratinocyte proliferation and reepithelialization pathways in healthy mice. Herein, we investigated whether this wound healing acceleration also occurs in impaired diabetic wounds and found that topical vemurafenib not only improves wound healing in a murine diabetic wound model but unexpectedly promotes hair follicle regeneration. Hair follicles expressing Sox-9 and K15 surrounded by CD34+ stroma were found in wounds of diabetic and non-diabetic mice, and their formation can be prevented by blocking downstream MEK signaling. Thus, topically applied BRAF inhibitors may accelerate wound healing, and promote the restoration of improved skin architecture in both normal and impaired wounds.

Accelerated wound healing by injectable star poly(ethylene glycol)-b-poly(propylene sulfide) scaffolds loaded with poorly water-soluble drugs.

Injectable hydrogel matrices take the shape of a wound cavity and serve as scaffold for tissue repair and regeneration. Yet these materials are generally hydrophilic, limiting the incorporation of poorly water soluble, hydrophobic drugs. Here we show this shortcoming is circumvented through a star-shaped amphiphilic block copolymer comprising poly(ethylene glycol) and poly (propylene sulfide). This star-shaped amphiphilic polymer self-assembles in an aqueous medium into a physically stable hydrogel and effectively dissolves hydrophobic molecules delivering them at therapeutic doses. The self assembled hydrogel is a robust three-dimensional scaffold in vivo effectively promoting cellular infiltration, reducing inflammation, and wound clsoure. When combined with a hydrophobic BRAF inhibitor that promotes paradoxical mitogen-activated protein kinase (MAPK) activation in keratinocytes and wound closure, our self assembled scaffold supported dermal wound closure at a reduced drug dosage compared to administering the drug in dimethyl sulfoxide (DMSO) without a polymeric matrix. This family of star-shaped amphiphilic polymers delivers poorly water soluble active agents at a fraction of generally required dosage for efficacy and supports three-dimensional cell growth at tissue wounds, showing great promise for novel uses of hydrophobic drugs in tissue repair applications.

In situ forming injectable hydrogels for drug delivery and wound repair.

Hydrogels have been utilized in regenerative applications for many decades because of their biocompatibility and similarity in structure to the native extracellular matrix. Initially, these materials were formed outside of the patient and implanted using invasive surgical techniques. However, advances in synthetic chemistry and materials science have now provided researchers with a library of techniques whereby hydrogel formation can occur in situ upon delivery through standard needles. This provides an avenue to minimally invasively deliver therapeutic payloads, fill complex tissue defects, and induce the regeneration of damaged portions of the body. In this review, we highlight these injectable therapeutic hydrogel biomaterials in the context of drug delivery and tissue regeneration for skin wound repair.

Hydrogels with precisely controlled integrin activation dictate vascular patterning and permeability.

Integrin binding to bioengineered hydrogel scaffolds is essential for tissue regrowth and regeneration, yet not all integrin binding can lead to tissue repair. Here, we show that through engineering hydrogel materials to promote α3/α5ß1 integrin binding, we can promote the formation of a space-filling and mature vasculature compared with hydrogel materials that promote αvß3 integrin binding. In vitro, α3/α5ß1 scaffolds promoted endothelial cells to sprout and branch, forming organized extensive networks that eventually reached and anastomosed with neighbouring branches. In vivo, α3/α5ß1 scaffolds delivering vascular endothelial growth factor (VEGF) promoted non-tortuous blood vessel formation and non-leaky blood vessels by 10 days post-stroke. In contrast, materials that promote αvß3 integrin binding promoted endothelial sprout clumping in vitro and leaky vessels in vivo. This work shows that precisely controlled integrin activation from a biomaterial can be harnessed to direct therapeutic vessel regeneration and reduce VEGF-induced vascular permeability in vivo.