FAQ

Table of Contents

1. Introduction and Basics

  • Nanovials are hydrogel particles that act as millions of suspendable wells for individual cells. They are made from biomaterials that can be easily modified with different biomolecules such as antibodies and antigens. Learn more at: partillion.com/technology.

  • Nanovials are sized just larger than a cell (holding less then a nanoliter of volume) allowing single-cell isolation and improved sensitivity for biomolecule detection. Learn more at: partillion.com/technology.

  • Nanovials are manufactured with a biotinylated surface. The end user functionalizes the Nanovial by first coating it with streptavidin, followed by the addition of specific capture reagents in our kitted formats, or any biotinylated biomolecules they choose.

  • There are approximately 10⁸ binding sites available on each Nanovial.

  • Our standard Nanovial formulation is uniformly coated with biotin on the surface. Our extracellular matrix (ECM) coated Nanovials have biotin and ECM localized to the cavity, which can improve capture efficiency and improve localized signal.

  • Cells of interest can be captured in the Nanovials using antibodies (e.g. anti-CD138), antigens or peptide-MHC monomers. Nanovials coated with extracellular matrix (ECM) are also available for working with adherent cells (Contact us directly for availability: https://www.partillion.com/custom-requests).

2. Benefits and Applications

  • Nanovial technology can provide previously unknown functional data about single-cell secretions. Some common applications include antibody discovery, cell therapy development, cell therapy quality control, cytokine profiling, and cell line development.

  • We have found that most users prefer to coat Nanovials with antibodies themselves to maintain longer stability and give flexibility to modify Nanovials with their own components such as antigens. Please contact us directly for custom precoated Nanovials: https://www.partillion.com/custom-requests.

  • We have two formulations: biotin-coated (BT), and biotin- and extracellular matrix-coated (BG). Both come in 35 or 50 μm diameter sizes. The 35 μm size is used in most applications. We can also produce larger sizes for customized applications.

  • Biotin-coated Nanovials are biotinylated all over the Nanovial and have a slightly smaller inside cavity.

    Biotin and ECM-coated Nanovials have an ECM gelatin coating and are biotinylated only on the inner cavity. This formulation also has as slightly larger cavity.

    Biotin-coated Nanovials may be a better option for larger flow panels (6+) due to less autofluorescence.

    Biotin-ECM may be a better option for larger cells (ie. hybridoma, CHO, HEK, MSC) because of the slightly larger inside cavity. These Nanovials have higher autofluorescence.

  • 35 μm Nanovials are used for most applications, as they are compatible with most instruments and cell types, including leukocytes and smaller cells.

  • 50 μm Nanovials may be applied for larger cells and assays characterizing interactions between two or more cells.

  • Yes! One feature of having the Nanovials partially open is that additional stains may be added to your samples after secretion incubation.

    For example in this Nature Communications paper, researchers used a panel of one secretion marker, five cell surface markers and one viability dye. If you have a large panel in mind (5+) we recommend using our standard Nanovial formulation which features reduced autofluorescence.

    For viability dyes we recommend using calcein-AM family of dyes (suggested: Biolegend 425203) for live staining and PI or DAPI (suggested: Millipore Sigma D9542) for dead staining. We don't recommend fixable dyes as they will also stain proteins attached to the Nanovial surface.

  • With Nanovials, you can directly screen antibody secreting cells such as plasma B cells and hybridomas using FACS, increasing throughput and saving you time and resources.

    • Direct B cell screening: Unlock screening of affinity matured plasma cells without the need for complex microfluidics. If you are already using antigen baiting, our assay can seamlessly integrate into your current workflow.

    • Hybridoma workflows: Enable high-throughput functional screening of hundreds of thousands to millions of hybridoma clones upfront reducing laborious culturing and subculturing steps.

  • Nanovials allow users to functionally screen and sort CAR-T cells based on both CAR binding and functional cytokine secretion. Isolated cells can be sequenced to recover unique gene expression information or recover unique CAR constructs in the case of pooled screening.

3. Usage and Protocols

  • Nanovials are guaranteed to be stable for six months from the date of shipment when stored at 4°C, and retain stability for one year from the date of manufacture. Following streptavidin modification, user-functionalized Nanovials can be stored at 4°C for two weeks prior to use.

  • "Cross-talk" is prevented in three ways:

    1. Secreted cytokines are captured locally by the capture antibodies near the loaded cell.

    2. The shape of the Nanovial helps prevent convective flows, which is a large source of cross-talk for other systems.

    3. The incubation period should be timed respective to the target cytokine secretion rate (e.g. for rapidly secreted cytokines, shorter incubation times are recommended to avoid saturation and cross-talk).

  • Cells remain viable and grow in the Nanovials. Since they are not fully encapsulated, there is sufficient nutrient exchange and cells will behave normally. Whether this means they will maintain a desired function depends on the biology. In some cases, such as adherent cells, being attached to the Nanovials may be more biologically relevant then having them in suspension.

  • Yes. Cells can be removed by one of two ways:

    1. Continuing to culture the cell-loaded Nanovials until the cells expand out of the Nanovials and then straining.

    2. Degrading the linker that binds cells to Nanovials with collagenase or trypsin followed by straining.

  • Each user should establish best practices for their specific application and samples. The following are suggested starting points based on internal assay development:

    Antibody Secretion - 30 minutes

    IFNγ - 3 hours

    TNFα- 30 minutes

  • The best Cell to Nanovial ratio will depend on the user's application and sample. In general, higher Nanovial ratios (Cell:Nanovial ratio of 1:5 to 1:20) are recommended to maximize cell recovery (e.g. for antibody discovery workflows).

    When using the Nanovials to enrich target populations, for example enrichment of antigen specific T cells, higher Cell:Nanovial ratios should be used (1:1 to 10:1).

    Please consult specific application guides or reach out to our team directly if you have questions for your specific application.

  • In general, we recommend targeting loading percentages (5-20%) to minimize the fraction of multiple cells. We have found that choosing a Nanovial size closer to the cell size will improve single-cell loading beyond Poisson statistics, but will reduce the cell recovery efficiency.

  • Cell-loaded Nanovials can be observed by brightfield microscopy or imaging cytometry. If a high instance of multiplets is observed, we recommend lowering the ratio of cells to Nanovials added in the system.

    For some cells, inclusion of a viability dye (i.e. calcein AM) can allow for exclusion gating of multiple-loaded Nanovials by flow cytometry.

  • To avoid multiplets, the assay is designed with a target Nanovial occupancy of 5-20%. Should it still be necessary, the cell loading percentage can be increased by increasing the cell to Nanovial ratio, with the trade off that some Nanovials will have more than one cell loaded per Nanovial.

    If you are observing very low loading (<3%), the cells may be too large for the Nanovial size being used. This can be inspected using brightfield microscopy. Alternatively, the cells may not be expressing high enough abundance of the surface protein that the cell capture antibody is targeting. Expression of the surface protein can be inspected using standard flow cytometry to determine if alternative capture antibodies should be used.

  • Add your surface marker antibodies at the same time as your secretion detection antibodies.

  • Yes!

    Our SEC-seq workflow enables users to directly link secretion information with transcriptome analysis. To do this you can either utilize an oligo-barcoded secondary against your secretion target, or add an oligo-barcoded antibody against the fluorophore of your standard fluorescent secondary antibody.

    Examples of successful cases are listed below:

    Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells, Udani, S., Plath, K., Di Carlo, D. (2023). bioRxiv. doi: https://doi.org/10.1101/2023.01.07.523110

    SEC-seq: association of molecular signatures with antibody secretion in thousands of single human plasma cells, Cheng, R., de Rutte, J…Di Carlo, D., James, R. (2023). Nature Communications. doi: https://doi.org/10.1038/s41467-023-39367-8

4. Compatibility and Integration

  • For new targets we typically recommend looking at clones that form sandwich pairs previously validated for ELISpot or ELISA and making sure there is not cross-species reactivity with the cell capture antibodies.

    Additionally, we have certain clones we have pre-qualified, as well as kits for IgG, IFNγ, TNFα, and IL2 assays available for direct order.

  • Similar to the Nanovial assay, ELISpot is used to assay cellular characteristics at the single-cell level whereas ELISA measures bulk analytes in solution. ELISpot clones often display reduced non-specific binding and thus are more aligned to the Nanovial format.

  • Nanovials are compatible with a large range of flow cytometry instruments. Some validated instruments are listed below:

    Sorter Compatibility

    • SONY SH800S*, MA900

    • BD FACSAriaII/III

    • NanoCellect WOLF Cell Sorter

    • On-Chip Sort

    • Union Biometric Biosorter

    Analyzer Compatibility:

    • Amnis ImageStreamX Mk II

    • LSRII/Fortessa*

    • Attune Flow Cytometers

    • Beckman CytoFLEX

    *Detailed technical instrument guides can be found at https://www.partillion.com/protocols.

  • Cells remain fully viable in the Nanovials. Since they are not fully encapsulated there is sufficient fluid exchange for them to grow normally.

5. Capacity and Throughput

  • Yes, we have performed successful secretion assays with up to 3 targets. It is possible to increase the plex, at the cost of diminishing sensitivity.

    Refer to research by Koo et al. for successful cases of functionalizing with multiple secretion capture antibodies:

    Sorting single T cells based on secreted cytokines and surface markers using hydrogel nanovials, Koo, D., Mao. Z…Witte, O.N., Di Carlo, D. (2023). bioRxiv. doi: https://doi.org/10.1101/2023.01.17.524440

  • We have validated a single-plex workflow, but it is possible to do higher plex, with the trade-off of decreased quantitative capability.

  • Yes. In most cases, not all of the marker of interest on the membrane will be bound to capture antibodies on the Nanovial and surface staining can be easily analyzed.

6. Ordering and Pricing

  • Some products may not be listed for direct sale on our website. Please send a request using our custom order form: https://www.partillion.com/custom-requests.

  • Yes. No blue ice or dry ice is needed for shipment.

7. Feedback and Collaboration

  • Please reach out to us through the contact form: https://www.partillion.com/contact-us.

  • We are always looking to collaborate with innovative scientists pushing the boundaries of their field. Please reach out to us through the contact form: https://www.partillion.com/contact-us.