Multifunctional Microwell Arrays for Single Cell Level Functional

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Multi-functional microwell arrays for single cell level functional analysis of lymphocytes Junsang Doh, HyoungJun Park, and HyeMi Kim Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00620 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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Bioconjugate Chemistry

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Multi-functional microwell arrays for single cell

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level functional analysis of lymphocytes HyoungJun Park‡∥, HyeMi Kim§∥, and Junsang Doh*†‡

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†

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Science and Technology (POSTECH), 77 Cheongam-Ro. Nam-Gu. Pohang, Gyeongbuk 790-

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784, Republic of Korea

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‡

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(POSTECH), 77 Cheongam-Ro. Nam-Gu. Pohang, Gyeongbuk 790-784, Republic of Korea

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§

School of Interdisciplinary Bioscience and Bioengineering (I-Bio), Pohang University of

Department of Mechanical Engineering, Pohang University of Science and Technology

Division of Integrative Biosciences and Biotechnology (IBB), Pohang University of Science

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and Technology (POSTECH), 77 Cheongam-Ro. Nam-Gu. Pohang, Gyeongbuk 790-784,

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Republic of Korea

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∥

These authors are equally contributed to this work.

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KEYWORDS: Microwell arrays, single cell analysis, functional analysis of lymphocytes

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1 Environment ACS Paragon Plus

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ABSTRACT

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Functional analysis of lymphocytes is important for development of vaccines and

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diagnosis/treatment of various immune-related diseases. In this review, we describe multi-

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functional microwell arrays that enable functional analysis of lymphocytes in single cell level.

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We first discuss key parameters for microwell array design. Then, we describe how different

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types of multi-functional microwell arrays was developed for various applications, including live

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cell imaging of lymphocyte activation, proliferation, and differentiation, and analyses of effector

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functions such as cytokine secretion and target cell lysis. Incorporation of novel surface

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chemistries and functional materials into microwell arrays for enhancing sensing capabilities will

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widen applications of this technology. Multi-functional microwell arrays will be a powerful tool

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for the development of novel therapeutics against immune-related diseases, in particular for

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cancer immunotherapy.

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1. Introduction

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Lymphocytes are subsets of white blood cells commonly found in lymph. They are originated

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from a common lymphoid progenitor during haematopoiesis.1 Classically, T cells, B cells, and

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natural killer (NK) cells are classified as three major types of lymphocytes, whereas innate

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lymphoid cells (ILCs), which include NK cells, have recently been identified as new subsets.2

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Lymphocytes exert diverse functions to eliminate foreign invaders and transformed cells. For

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example, CD4+ T cells secrete a number of cytokines to modulate inflammatory responses,

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CD8+ T cells and NK cells directly kill transformed cells, and B cells produce antibodies.

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Various immune related diseases, including infectious diseases, cancer, autoimmunity, and

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allergy/asthma, are closely related with dysfunctions of lymphocytes, thus functional analysis of

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lymphocytes are important for the diagnosis and therapy of the immune-related diseases.

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One of the challenges in lymphocyte function analysis is single cell level heterogeneity.

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Adaptive lymphocytes such as T cells and B cells express diverse antigen receptors, more than

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107 different T cell receptors for humans,3 to mount antigen-specific immune responses against

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wide variety of potentially harmful antigens. In addition to diversity in antigen receptors,

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lymphocytes under the same conditions also exhibit single cell level functional heterogeneity

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during expansion and differentiation.4 Flow cytometry has been extensively used to analyze

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single cell level heterogeneity in molecular expression patterns.5 However, molecular expression

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patterns may not fully recapitulate functional phenotypes of lymphocytes.6 Most conventional

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functional analyses of lymphocytes, including proliferation, cytokine secretion, migration, and

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cytolysis, have been performed in bulk population levels.



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Multiwell plates such as 96- or 384-well plates have been used in conjunction with limited

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serial dilution methods to analyze and isolate lymphocyte clones with desirable functions.

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However, sensitivity of the analysis has been limited by excess amounts of media in the well

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plates because the smallest media volume for 384-well plate (~ 10 µL) is still ~ 108 times larger

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than the size of a single lymphocyte. Microwells with dimensions 10 ~ 100 µm, which is

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comparable to the sizes of lymphocytes, would be desirable for single cell level functional

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analysis of lymphocytes. Advances in soft lithography techniques enabled facile fabrication of

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microwells in labs.7 Moreover, by incorporating various bioconjugation strategies, we can build

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multi-functional microwells that enable us to manipulate cell-cell interactions and measure

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various functions of lymphocytes in single cell levels. In this review, we will describe various

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types of multi-functional microwells developed for functional analysis of lymphocytes.

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2. Design and fabrication of microwell arrays

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There are three key design parameters to be determined prior to microwell array fabrication

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(Figure 1): 1) lateral dimensions and geometry, 2) depth, and 3) open vs. closed, (Figure 1). In

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this section, we will describe factors determining each parameter.


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Figure 1. Key design parameters for microwell array fabrication.

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Lateral dimensions and geometry of microwells are critical parameters determining the

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number of cells that can be contained within a microwell. It is particularly important for the case

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of single cell arrays. A diameter of lymphocyte varies between 5 and 20 µm, depending on types

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and activation states of lymphocytes.8-10 Typically, primary lymphocytes directly isolated from



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mice and humans have relatively small diameters (5 ~ 10 µm), whereas cell lines derived from

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leukemia and lymphoma have much larger diameters (10 ~ 20 µm). Importantly, the diameter of

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a lymphocyte can dramatically increase during activation process, as much as twice within 24 h.

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In addition to the diameter of individual lymphocytes, the field-of-view of the imaging system is

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another important factor to be considered because most assays based on microwell arrays are

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performed using a microscope equipped with a motorized stage, or a microarray scanner

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developed for DNA microarrays. The lateral dimensions of microwells need to be designed in

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such a way that each imaging field-of-view contains integer number of microwells to maximize

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data acquisition throughputs and analysis efficiency. In case of geometry, spherical microwells

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are mostly used for single cell arrays because they have higher probability of capturing a single

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cell in each microwell than square one. In contrast, square/rectangular microwells are widely

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used for microwells containing multiple cells to minimize void spaces.

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Depth of microwell is important for the entrapment of cells within microwells. Lymphocytes

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do not adhere strongly on the substrates, thus can float out of microwells unless microwells are

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deep enough. Typically, microwells with depth of 50 ~ 100 µm, which is 5 ~ 10 times greater

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than the diameter of lymphocytes, are used to constrain lymphocytes within microwells.11-13 In

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some cases, microwells with depth smaller than the diameter of the lymphocytes are intentionally

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used to allow paracrine-mediated crosstalk between cells in adjacent microwells.10, 14 In that case,

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immobilization of lymphocytes on microwell bottoms is necessary.

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In ‘open’ microwells, lymphocytes in each microwell are located under identical bulk media.

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In contrast, lymphocytes in ‘closed’ microwells are isolated from lymphocytes in other

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microwells, thus crosstalk between microwells are completely blocked. Additionally, in closed


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microwells, molecules secreted by lymphocytes will be accumulated in the microwells, thus

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detection sensitivity can be enhanced.15 Closed microwells can be simply prepared by sealing the

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microwells using flat substrates. Flat substrates used as ‘lids’ of microwells can be functionalized

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to detect molecules secreted by lymphocytes in individual microwells.16-17

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Microwell arrays can be fabricated with various materials. The first microwells for

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lymphocyte analysis, reported by Muraguchi group, were made of polystyrene (PS), a commonly

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used material for tissue cultureware, using injection molding.18 While injection molding is the

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best method for mass production, it requires expensive instruments, thus may not be a suitable

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method for typical labs. Therefore, microwell arrays used for lymphocyte analysis are more

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commonly fabricated by various soft lithography techniques, which can be easily performed in

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most labs.7 Poly (dimethyl siloxane) (PDMS) is one of the most frequently used materials for

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microwell arrays. Inverse structures of the microstructures fabricated on silicon wafers by

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standard photolithography can readily replicated by casting PDMS resin on silicon wafers and

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curing it.19 PDMS can form liquid-tight reversible sealing with glass substrates, thus PDMS-

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based microwell arrays are suitable for closed microwell arrays.11,

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excellent biocompatibility and gas permeability, thus has been widely used for microchip or

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microfluidic-based long-term (~ week) cell cultures.21 Another type of materials widely used for

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the microwell fabrication is hydrogels, polymer networks containing large amounts of water.22

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Hydrogels are also biocompatible materials widely used for in vivo implantable devices as well

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as in vitro cell cultures, but some monomers and initiators used for hydrogel fabrication are toxic,

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thus unreacted chemicals need to be extensively washed out prior to use. In comparison to

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PDMS-based microwell arrays, diverse functional monomers/crosslinkers can be integrated into

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hydrogel-based microwell arrays to incorporate multiple functions.23-24 In addition, sidewalls of


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In addition, PDMS has


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hydrogel microwells may have size-dependent molecular permeability depending on the

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crosslinking density, which may affect crosstalk between lymphocytes in neighboring

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microwells.25

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3. Simple microwell arrays for live cell imaging of cell-cell interactions

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Dynamic cell-cell interactions and synapse formations are essential for lymphocyte activation

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and effector functions.26-27 In vitro, lymphocytes are either floating or rapidly migrating, thus live

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cell imaging of lymphocytes for prolonged duration is technically challenging.28 Microwell

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arrays containing spatially constrained lymphocytes can be a powerful tool to perform long-term

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live cell imaging of cell-cell interactions. Two different types of microwells were fabricated and

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used for this application as schematically shown in Figure 2A: in the first type of microwells,

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two different type of cells are vertically positioned to interact with each other with the lower

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cells confined within the microwells forming single cell arrays (Figure 2A(i)). In the second type

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of microwells, two different type of cells contained in microwells can freely migrate and interact

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laterally within microwells (Figure 2A(ii)).


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Figure 2. Microwell arrays for the improvement of live cell imaging. A. Schematic illustration of cell-cell interactions in two different types of microwell arrays. B. High-throughput imaging of calcium mobilization in T cells during activation. Representative pseudo-color steel image showing calcium level of individual T cells within microwell arrays (left), and time-dependent average calcium level of T cells in response to various amounts of antigen (right) (Reprinted with permission from Ref 31. Copyright 2006 John Wiley and Sons) C. Long term tracking of NK cell dynamics in a microwell. Blue: NK cells, green: live target cells, and red: dead target cells. (Reprinted with permission from Ref 39. Copyright 2015 American Association of Immunologists)



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For the first type of microwells, single cell arrays of lymphocytes were first fabricated, and

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another type of lymphocytes were added on top (Figure 2A(i)). Biggs et al. fabricated PDMS-

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based circular microwells with 20 µm diameter and 40 µm deep for high-resolution imaging of

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the immunological synapse formation.29 They first filled engineered K562 cells as artificial

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antigen presenting cells (APCs), and subsequently added Jurkat T cells. Since the diameters of

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K562, a human myelogenous leukemia cell line, and Jurkat, a human T cell leukemia, cells were

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slightly smaller than 20 µm, each microwell was filled with a vertically aligned K562-Jurkat cell

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pair. In this way, immunological synapse between T-APC were formed on a plane parallel to the

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optical imaging plane so that high-resolution time-lapse images of molecules in immunological

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synapse could be obtained. Similar approach was used to visualize high-resolution dynamics of

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PD-1 in immunological syanspe.30 Kim et al. fabricated hydrogel-based circular microwell arrays

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with 12 µm diameter and 6 µm deep to monitor intracellular calcium levels of T cells triggered

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by different amounts of antigens loaded on APCs (Figure 2B).31 They first added 5C.C7 murine

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primary T cell blasts, in vitro activated CD4+ T cells from 5C.C7 T cell receptor (TCR)

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transgenic mice, into the microwells. Since the depth of microwells < the diameter of 5C.C7 T

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cells, the bottoms of the microwells were coated with an antibody to immobilize T cells. CH27 B

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cell lymphoma cells were used as APCs. CH27 cells were seeded on microwell arrays containing

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single cell arrays of 5C.C7 T cells. CH27 cell density was optimized to form near monolayers so

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that 5C.C7 cells will be in contact with at least one CH27 cells. In this setting, antigenic peptide

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in the media was presented on CH27 cells, and 5C.C7 T cells were activated by the antigen-

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presenting CH27 cells. By measuring the intracellular calcium levels of T cells, which correlates

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with the strength of TCR signaling,32 using calcium sensing fluorescence dye fura-2, the amounts


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of antigenic peptide concentration in media could be determined. In this application, single cell

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array form of T cells was also helpful for high-throughput automated data processing: since

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individual cells were isolated from each other with defined positions, time-dependent calcium

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levels of individual T cells could be automatically tracked for single cell-level analysis.

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For the second type of microwells, square microwell arrays that can contain multiple cells

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within each microwell are fabricated and used (Figure 2A(ii)). Importantly, the size of each

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microwell is smaller than the field-of-view of the microscope and lymphocytes are constrained

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within the microwells, thus this type of microwell provides ideal platform to monitor long-term

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behaviors of lymphocytes, including proliferation, differentiation,33-37 and cytolysis of target

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cells.38-41 Duffy el. al. used PDMS-based square microwells with side lengths of 50 µm and

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depth of 60 µm33 to observe single B cells for prolonged period of time (typically ~ 60 h).37 The

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size of each microwell was large enough to allow cell divisions, thus fates of individual B cells,

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including proliferation, differentiation, and cell death, could be tracked. To directly monitor B

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cell differentiation to plasmablasts, Blimp1-GFP mice was used. By quantitatively analyzing

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single cell data, they demonstrated that B cell fate decision followed internal stochastic processes.

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Önfelt group used various sizes of microwells to assess dynamic behaviors of NK cells

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encountering tumor cells (Figure 2C).38-40 In this case, a critical factor determining the

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dimensions of the microwells is whether the target cells are adhering or floating in in vitro cell

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culture environments. For adhering cells such as HEK-293 cells, transformed human embryonic

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kidney cell-line, square microwells with side lengths of 450 µm were used whereas for floating

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cells, or most leukemia and lymphoma such as K562 cells, square microwells with side lengths

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of 50 µm were used. Using the microwell arrays containing tumor cells and NK cells, dynamics



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of NK cells, including migration, conjugate formation with tumor cells, and target cell killing,

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were observed and quantitatively analyzed.

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For optimal data acquisition, not only types of microwells, but also various parameters for live

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cell imaging need to be adjusted. Large areas of microwell arrays can be scanned using a

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motorized stage to maximize data acquisition throughputs, but in this case, intervals for imaging

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each field of view will be increased, or temporal resolution will be decreased. Therefore, for

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molecular level imaging of immune synapses where ~ seconds of temporal resolution is required,

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scanning should be minimized. In contrast, for applications requiring long-term observation (>

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10 h) such as cell proliferation and differentiation, long intervals for each image acquisition, or

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low temporal resolution, is desirable to minimize photobleaching and phototoxicity.

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4. Microwell functionalization for lymphocyte immobilization, activation, and cytokine detection

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Microwell can be modified to add additional functionalities to the microwell arrays. Various

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antibodies have been attached on the microwell bottoms to immobilize or activate lymphocytes,

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or detect cytokines secreted by lymphocytes. For capture/immobilization and activation,

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antibodies should be attached on the bottom of the microwells so that cells will not adhere

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outside of microwells, whereas for cytokine detection, antibodies or capturing reagents can be

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uniformly coated on microwells (Figure 3A).


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Figure 3. Microwell functionalization methods and their applications. A. Scheme of two different types of functionalized microwells and their applications. B. Schemes of (i) dualfunctional surface preparation, and (ii) bottom functionalized hydrogel-based microwell array fabrication.

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PS or PDMS-based Microwells can be functionalized by simply incubating in capturing

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antibody/antigen solution15, 42 because PS and PDMS have good protein adsorption properties.

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Air or oxygen plasma treatment can substantially enhance coating by activating surfaces. Using

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this system, Muraguchi group successfully detected and isolated rare antigen-specific antibody-

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secreting cells.42 Circular microwells with 10 µm in diameter, 12 µm in depth, and 30 µm in

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pitch were used to make single cell arrays of B cells.18 Relatively small microwell dimensions to



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capture single primary lymphocytes were also beneficial for the rapid detection of secreted

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antibodies by minimizing diffusion time prior to capture. Capturing antibodies were immobilized

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on microwells after cell plating, and antigen-specific antibodies were detected using labeled

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antigens. Typically, doughnut-shaped fluorescence staining was observed outside of microwells

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containing antigen-specific antibody-secreting cells. More than 2×105 cells could be screened

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within 7 h. Interestingly, if the sizes of microwells were increased and capturing antibodies were

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coated prior to cell plating, secreted antibodies were mostly captured on the bottom and

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sidewalls of microwells.15

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In contrast to uniform functionalization, methods for microwell bottom functionalization

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depend on what materials the microwells are made of. Importantly, microwell bottom should be

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made of different materials from microwell itself for selective chemical modification. In case of

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PDMS microwells, large amounts of antibodies can be bound on PDMS by non-specifically

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adsorption, thus bottom and side wall parts of the microwells need to be prepared separately.

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Friedman group fabricated antibody-coated microwells by attaching PDMS thin membranes

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containing defined sizes of microholes on antibody-coated glass substrates.12 They coated the

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bottom of the microwells using anti-CD3 and anti-CD28 for activation of T cells. Importantly,

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antibody coating on side walls of microwells caused T cells climbing up and leaving microwells.

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In case of hydrogel microwells, functionalization of substrates is necessary for stable

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attachment of hydrogels on underlying substrates. As schematically shown in Figure 3B(i), thin

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glass coverslips, ideal for high-resolution optical imaging, can be modified to present primary

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amine by electrostatic interaction-mediated layer-by-layer (LbL) films31 or silane-based

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chemicals.10 Then, primary amine group can be further functionalized to introduce acrylate group,

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which will be used to anchor hydrogels by forming covalent bonding with methacrylate groups 14 Environment ACS Paragon Plus

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in poly(ethylene glycol) dimethacrylate (PEGDMA), a precursor for hydrogel, and biotin, which

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will be used to attach biotinylated antibody via streptavidin linkage. Hydrogel microwells were

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fabricated on dual-functional surfaces by perfusing hydrogel precursor solution in the PDMS

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mold and curing it by photopolymerization (Figure 3B(ii)). Microwell bottom could be

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selectively functionalized after microwell formation because PEG-based hydrogels exhibit

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minimal protein adsorption. Using these schemes, we fabricated shallow microwells with depth

Multifunctional Microwell Arrays for Single Cell Level Functional

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