Development and optimization of a process for automated recovery of single cells identified by microengraving

Choi, Jae‐Hyeok; Ogunniyi, Adebola O.; Du, Mindy; Du, Minna; Kretschmann, Marcel; Eberhardt, Jens; Love, J. Christopher

doi:10.1002/btpr.374

Cited by 80 publications

(53 citation statements)

References 30 publications

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“…Because microengraving is a nondestructive process, and cells have known spatial addresses within the array of wells used, we expected that microengraving would allow the efficient recovery of activated antigen-specific cells by micromanipulation for clonal expansion (Fig. S5) (14,28).…”

Section: Rapid and Efficient Cloning Of Hiv-specific Cd8mentioning

confidence: 99%

Rapid, efficient functional characterization and recovery of HIV-specific human CD8 ⁺ T cells using microengraving

Varadarajan

Kwon

Law

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The nature of certain clinical samples (tissue biopsies, fluids) or the subjects themselves (pediatric subjects, neonates) often constrain the number of cells available to evaluate the breadth of functional T-cell responses to infections or therapeutic interventions. The methods most commonly used to assess this functional diversity ex vivo and to recover specific cells to expand in vitro usually require more than 10 6 cells. Here we present a process to identify antigen-specific responses efficiently ex vivo from 10 4 –10 5 single cells from blood or mucosal tissues using dense arrays of subnanoliter wells. The approach combines on-chip imaging cytometry with a technique for capturing secreted proteins—called “microengraving”—to enumerate antigen-specific responses by single T cells in a manner comparable to conventional assays such as ELISpot and intracellular cytokine staining. Unlike those assays, however, the individual cells identified can be recovered readily by micromanipulation for further characterization in vitro. Applying this method to assess HIV-specific T-cell responses demonstrates that it is possible to establish clonal CD8 + T-cell lines that represent the most abundant specificities present in circulation using 100- to 1,000-fold fewer cells than traditional approaches require and without extensive genotypic analysis a priori. This rapid (<24 h), efficient, and inexpensive process should improve the comparative study of human T-cell immunology across ages and anatomic compartments.

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Section: Rapid and Efficient Cloning Of Hiv-specific Cd8mentioning

confidence: 99%

Rapid, efficient functional characterization and recovery of HIV-specific human CD8 ⁺ T cells using microengraving

Varadarajan

Kwon

Law

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Cell recovery from closedformat microfluidic devices (such as those developed here), however, often requires more sophisticated cell manipulation strategies--generally at the expense of device footprint area--to route cells to the outlet [for example, via multiplexer architectures (29,30), electrical manipulation (31), or optical sorting techniques (32,33)]. Here, as our microfluidic devices share essential features with open-format devices, in particular portable device operation and registration of cells at spatially defined positions, we were able to adapt a semiautomated micromanipulator-based cell recovery approach previously established for microwell arrays (34). For this purpose, we introduced a design variation that involved bonding the polydimethyl siloxane (PDMS) devices to a thin (∼10-20-μm) PDMS sheet (membrane) instead of glass slides ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Cells were retrieved from their traps using an semiautomated micromanipulator (CellCelector, AVISO GmbH) by piercing the membrane and applying suction following an adaptation of the operation procedures described before (34). Glass capillaries were pulled and broken back manually to induce an angled opening at the tip to facilitate easy membrane piercing.…”

Section: Methodsmentioning

confidence: 99%

Longitudinal multiparameter assay of lymphocyte interactions from onset by microfluidic cell pairing and culture

Dura

Servos

Barry

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

104

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Resolving how the early signaling events initiated by cell-cell interactions are transduced into diverse functional outcomes necessitates correlated measurements at various stages. Typical approaches that rely on bulk cocultures and population-wide correlations, however, only reveal these relationships broadly at the population level, not within each individual cell. Here, we present a microfluidics-based cell-cell interaction assay that enables longitudinal investigation of lymphocyte interactions at the single-cell level through microfluidic cell pairing, on-chip culture, and multiparameter assays, and allows recovery of desired cell pairs by micromanipulation for off-chip culture and analyses. Well-defined initiation of interactions enables probing cellular responses from the very onset, permitting singlecell correlation analyses between early signaling dynamics and later-stage functional outcomes within same cells. We demonstrate the utility of this microfluidic assay with natural killer cells interacting with tumor cells, and our findings suggest a possible role for the strength of early calcium signaling in selective coordination of subsequent cytotoxicity and IFN-gamma production. Collectively, our experiments demonstrate that this new approach is well-suited for resolving the relationships between complex immune responses within each individual cell.microfluidics | cell pairing | single-cell analysis | cell-cell interactions | multiparameter assay I nitiation and progression of immune responses require direct cell-cell interactions. Molecular interactions at the contact interface mediate cellular cross-talk and trigger a series of wellorchestrated downstream signaling events. The magnitude and dynamics of these signals promote and coordinate immune cell activation, and underlie a broad array of functional outcomes, such as target cell elimination, secretory activity, and proliferation (1, 2). Understanding how these early signaling events are transduced into functional responses demands correlated measurements at different stages as these responses unfold.Typically, these relationships are probed by first activating cells in bulk cocultures, often mixing cell populations and initiating contacts via a brief centrifugal cosedimentation, and then piecing together measurements from independent assays performed at different time points. This approach may determine broad outcomes of intercellular interactions, but it suffers from three major limitations. First, individual assay measurements are averaged over many different combinations of interactions due to the uncontrolled and indefinite nature of cell-cell interactions in bulk cocultures. For example, varying times of initiation and duration of contacts (1, 3), number of interacting partners (4), and differences in antigen presenting cells (5) can all modulate immune cell responses. Unpredictable variation is therefore introduced into measured responses, masking the true intrinsic cellular heterogeneity and blurring the correlations. Second, conventional as...

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“…It is also straightforward to recover specific cells by micromanipulation to rescue particular genes or generate clonal lines of interest. [102][103][104] The integration of microfluidic systems with these types of arrays can further expand their utility by enabling the modulation of microenvironments or application of stimuli. 105,106 Similar to most examples of microfluidic assays, one of the main disadvantages in these systems is the efficiency of distributing stem cells into individual wells.…”

Section: Microwell-based Analysis Systemmentioning

confidence: 99%

“…70 This dimension of time is common for monitoring cellular behaviors such as motility, morphology, and growth by microscopy, but is less commonly applied to the evolution of functions such as secretion. 104 The other implication of knowing the location of individual stem cells throughout an analytical process is that it should be possible to design strategies that conserve the available sample.…”

Section: Figmentioning

confidence: 99%

Technology Advancement for Integrative Stem Cell Analyses

Jeong

Choi

Lee

2014

Tissue Engineering Part B: Reviews

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Scientists have endeavored to use stem cells for a variety of applications ranging from basic science research to translational medicine. Population-based characterization of such stem cells, while providing an important foundation to further development, often disregard the heterogeneity inherent among individual constituents within a given population. The population-based analysis and characterization of stem cells and the problems associated with such a blanket approach only underscore the need for the development of new analytical technology. In this article, we review current stem cell analytical technologies, along with the advantages and disadvantages of each, followed by applications of these technologies in the field of stem cells. Furthermore, while recent advances in micro/nano technology have led to a growth in the stem cell analytical field, underlying architectural concepts allow only for a vertical analytical approach, in which different desirable parameters are obtained from multiple individual experiments and there are many technical challenges that limit vertically integrated analytical tools. Therefore, we propose-by introducing a concept of vertical and horizontal approach-that there is the need of adequate methods to the integration of information, such that multiple descriptive parameters from a stem cell can be obtained from a single experiment.

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Development and optimization of a process for automated recovery of single cells identified by microengraving

Cited by 80 publications

References 30 publications

Rapid, efficient functional characterization and recovery of HIV-specific human CD8 ⁺ T cells using microengraving

Rapid, efficient functional characterization and recovery of HIV-specific human CD8 ⁺ T cells using microengraving

Longitudinal multiparameter assay of lymphocyte interactions from onset by microfluidic cell pairing and culture

Technology Advancement for Integrative Stem Cell Analyses

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Development and optimization of a process for automated recovery of single cells identified by microengraving

Cited by 80 publications

References 30 publications

Rapid, efficient functional characterization and recovery of HIV-specific human CD8 + T cells using microengraving

Rapid, efficient functional characterization and recovery of HIV-specific human CD8 + T cells using microengraving

Longitudinal multiparameter assay of lymphocyte interactions from onset by microfluidic cell pairing and culture

Technology Advancement for Integrative Stem Cell Analyses

Contact Info

Product

Resources

About

Rapid, efficient functional characterization and recovery of HIV-specific human CD8 ⁺ T cells using microengraving

Rapid, efficient functional characterization and recovery of HIV-specific human CD8 ⁺ T cells using microengraving