Biology subjects

Mellody, M. P.

Publications and source records attributed to Mellody, M. P..

3 recordsLinked to original sources

bioRxiv2025

Sealable capped nanovials for high-throughput screening of cell growth and function

Millions of modular nanoliter-scale compartments that isolate functionally rich single-cell and cell-to-cell communication data can scale biological discovery for the age of AI. Here, we introduce capped nanovials--suspendable, sealable microscale compartments formed by the docking of hydrogel capping particles into bowl-shaped nanovials--as a versatile system for culturing, analyzing, and sorting single cells and small colonies. This two-particle architecture enables localized confinement of cells and secreted products while maintaining compatibility with standard laboratory workflows such as wash and reagent exchange steps, fluorescence microscopy, and flow cytometry. Crucially, these compartments are formed via simple pipetting and centrifugation steps, making the platform highly democratized. We demonstrate the ability of capped nanovials to compartmentalize single mammalian, bacterial, and yeast cells and support growth into colonies, enabling selection based on proliferation and bioproduction. We further show that capped nanovials enhance single-cell secretion assays by reducing molecular crosstalk and increasing signal-to-noise ratios. Importantly, we demonstrate functional co-culture assays by permitting stable confinement of cell pairs, enabling detection and enrichment of antibody-secreting cells based on the ability of their secreted antibodies to activate co-encapsulated reporter T cells, achieving a signal-to-noise ratio of >30 and up to 100% selection purity. By combining the simplicity of standard lab handling with the resolution and throughput of traditional microfluidic compartmentalization approaches, capped nanovials provide a new class of scalable, accessible test tubes for modern single-cell biology.

bioengineering↗

bioRxiv2025

Functionalizing hydrogel nanovials with vesicles mimicking antigen-presenting vesicles and cancer exosomes improves T cell capture and activation

Recent advances have demonstrated the application of microcavity-containing hydrogel microparticles, known as nanovials, for the massively parallel and high-throughput screening of therapeutic T cell populations for adoptive cell therapies. Nanovial cavities coated with peptide-MHC (pMHC) or antigen tetramers selectively bind to their cognate T cell receptor (TCR) or chimeric antigen receptor (CAR) to activate T cells and capture secreted cytokines. However, binding of tetramers or recombinantly expressed antigen by T cells is not always correlated with T cell activation or cytotoxicity as the binding interface is not fully representative of the natural immunological synapse formed between T cells and professional antigen-presenting cells (APCs). Here, we leverage the recent discovery of an ESCRT- and ALIX-binding region (EABR) sequence to generate antigen-presenting vesicles and cancer-mimicking exosomes from standard HEK293T and Expi293F cell cultures. EABR-mediated vesicles present natural, full-length oncologically-relevant membrane proteins embedded in lipid bilayers to functionalize the nanovial cavity with cell-like membranes. These hydrogel nanovials functionalized with the EABR-mediated vesicles show improved T cell capture of 1G4 T cells and enhanced activation of HER2 CAR-T cells compared to hydrogel surfaces functionalized with recombinantly-expressed soluble proteins.

bioengineering↗

bioRxiv2024

Multi-reactive hydrogel nanovials for temporal control of secretion capture from antibody-secreting cells

Antibody discovery can benefit from techniques to screen antibody-secreting cells (ASCs) at scale for the binding and functionality of a diverse set of secreted antibodies. Previously, we demonstrated the use of cavity-containing hydrogel microparticles (nanovials) coated with a single affinity agent, biotin, to capture and identify ASCs secreting antibodies against a recombinant antigen bound to the nanovial through biotin-streptavidin linkages. However, rapidly secreted antibodies from unbound cells or cells in adjacent nanovials can cause crosstalk leading to background signal. Earlier efforts address this by localizing capture sites to the nanovial cavity, emulsifying nanovials, or short secretion times to limit secreted antibodies from binding to neighboring nanovials. Here, we demonstrate a method to functionalize nanovials with moieties that impart orthogonal reactivity, enabling conjugation of cell capture antibodies and antigens at different times. We show that by using a strained alkyne moiety to attach cell-capture antibodies via click chemistry to nanovials, we can capture cells and subsequently quantify secretions via biotin-streptavidin linkages. By delaying the loading of antigens onto the nanovials until after cell capture, we were able to ensure high purity (>95%) isolation of hybridoma secreting an antigen-specific antibody in a background of other hybridoma. This approach allows tight temporal control of the secretion measurement, which is independent of the cell loading time, and requires less convective transfer steps. Click chemistry-based coupling further improved cell loading into nanovials by 58% compared to biotin-streptavidin-biotin coupling and caused no reduction in cell viability. We demonstrate an implementation of this system to improve antigen-specific hybridoma screening, yielding an 8-fold improvement in hybridoma enrichment while maintaining a similar workflow complexity. Hybridomas on nanovials sorted into well plates regrew into colonies following sorting using standard fluorescence-activated cell sorting and maintained secretion of antigen-specific antibodies with high purity ([~]90%), as validated via standard enzyme-linked immunosorbent assays. This lab-on-a-particle approach can be applied more generally to decouple cell loading, treatment, or activation, from secretion measurements for single-cell functional assays.

bioengineering↗