Microwell Array Research Articles

Selective retrieval of antibody-secreting hybridomas in cell arrays based on the dielectrophoresis

A cascade of the formation of cell arrays, the discrimination of cells secreting specific molecules, and the selective retrieval of cells has been developed to harvest antibody-secreting hybridomas in heterogeneous cell populations simply and rapidly. The microwell array device consisted of three-dimensional microband electrodes by assembling both upper and lower substrates perpendicularly. Arrays of hybridomas secreting specific antibodies were prepared by aligning hybridomas in each microwell based on the attractive force of positive dielectrophoresis (p-DEP). Antibody secreted by the hybridomas in the microwells was recognized by the antigen immobilized on the microwells or the membrane surfaces of hybridomas to discriminate hybridomas with the secretion ability. Thereafter, a repulsive force of negative dielectrophoresis (n-DEP) was applied to release the target hybridomas from the microwell array. To harvest the target hybridoma, AC signals could be modulated in the n-DEP frequency region and applied to a pair of microband electrodes located above and below each microwell containing target hybridoma. Thus, the cell-based array system described in this study allowed selective retrieval of single target hybridomas by merely switching from p-DEP to n-DEP after selecting the antibody-secreting hybridomas trapped in each microwell. The development of this high-affinity device could be useful to recover hybridomas producing antibodies in large populations of cells rapidly and effectively.

Capillary array electrophoresis imaging of biochemicals in tissue sections.

It is of great significance to reveal the molecular distribution images in biological tissues, which has led to the bloom of mass spectrometry imaging. Unfortunately, its application is encountering the resistance of high technical barriers and equipment cost, as well as the inability to image substances that cannot be desorbed or ionized, or cannot be separated by their mass-to-charge ratios. Herein presented is a complementary and cost-effective method called capillary array electrophoresis (CAE) imaging. To have the information of molecules and their spatial location, a gridding cutter was fabricated to orderly dissect a tissue section into a leakproof array of micro wells enclosed by the grid-blade arrays. After in situ extraction and fluorophore-labeling of analytes, the samples in the wells were directly subjected to CAE-LIF (laser-induced fluorescence), and the molecular distribution images were depicted with the separated peaks. The practicability was demonstrated by CAE imaging of rat brain tissue sections with amino acid neurotransmitters (e.g., glutamine, 4-aminobutyric acid, alanine, glutamic acid and aspartic acid) as targets. The resultant images showed the global differences of molecular distributions, with a spatial resolution of 1000μm that was presently determined by the well width but ultimately by the bore size of capillary (down to 10-50μm). CAE imaging can hence be promising for its low cost, low technical barriers and abundant mechanisms to separate the charged and non-charged, chiral and non-chiral substances.

Static systems to obtain 3D spheroid cell models: a cost analysis comparing the implementation of four types of microwell array inserts

3D cell culture models in the form of spheroids are undoubtedly the best approach to understand complex cellular physiological mechanisms. These structures have been used in a plethora of applications, spanning from pharmaceutical to regenerative medicine, showing potential for complete tissue and organ development. All these different applications demand large quantities of uniform spheroids regarding size and geometry and several methods have already been proposed to address this demand. Along with scientific and technical aspects, cost analysis is equally important to assess the feasibility of each system to better fit the intended scale and application with appropriate cost-benefit ratio. Herein, we describe a case study comparing the costs of four microwell arrays, three commercial systems, and a proprietary device developed in-house. The cost analysis considered three scale scenarios ranging from batches of 100, 1000 and 5000 spheroids. The main cost drivers and limitations were identified, to enable future additional process improvement. Overall, the resulting case study offered insights in terms of quality, reproducibility, feasibility, and costs for the implementation of 3D model static systems in a cell culture laboratory either for academic use or for small-scale or start-up companies.

Tunable and scalable fabrication of block copolymer-based 3D polymorphic artificial cell membrane array

Owing to their excellent durability, tunable physical properties, and biofunctionality, block copolymer-based membranes provide a platform for various biotechnological applications. However, conventional approaches for fabricating block copolymer membranes produce only planar or suspended polymersome structures, which limits their utilization. This study is the first to demonstrate that an electric-field-assisted self-assembly technique can allow controllable and scalable fabrication of 3-dimensional block copolymer artificial cell membranes (3DBCPMs) immobilized on predefined locations. Topographically and chemically structured microwell array templates facilitate uniform patterning of block copolymers and serve as reactors for the effective growth of 3DBCPMs. Modulating the concentration of the block copolymer and the amplitude/frequency of the electric field generates 3DBCPMs with diverse shapes, controlled sizes, and high stability (100% survival over 50 days). In vitro protein–membrane assays and mimicking of human intestinal organs highlight the potential of 3DBCPMs for a variety of biological applications such as artificial cells, cell-mimetic biosensors, and bioreactors.

Development of digital organ-on-a-chip to assess hepatotoxicity and extracellular vesicle-based anti-liver cancer immunotherapy

Organ-on-a-chip systems have been increasingly recognized as attractive platforms to assess toxicity and to develop new therapeutic agents. However, current organ-on-a-chip platforms are limited by a “single pot” design, which inevitably requires holistic analysis and limits parallel processing. Here, we developed a digital organ-on-a-chip by combining a microwell array with cellular microspheres, which significantly increased the parallelism over traditional organ-on-a-chip for drug development. Up to 127 uniform liver cancer microspheres in this digital organ-on-a-chip format served as individual analytical units, allowing for analysis with high consistency and quick response. Our platform displayed evident anti-cancer efficacy at a concentration of 10 μM for sorafenib, and had greater alignment than the “single pot” organ-on-a-chip with a previous in vivo study. In addition, this digital organ-on-a-chip demonstrated the treatment efficacy of natural killer cell-derived extracellular vesicles for liver cancer at 50 μg/mL. The successful development of this digital organ-on-a-chip platform provides high-parallelism and a low-variability analytical tool for toxicity assessment and the exploration of new anticancer modalities, thereby accelerating the joint endeavor to combat cancer.Graphic abstract

Inhibition of BMP signaling with LDN 193189 can influence bone marrow stromal cell fate but does not prevent hypertrophy during chondrogenesis.

SummaryBone morphogenetic protein (BMP) cascades are upregulated during bone marrow-derived stromal cell (BMSC) chondrogenesis, contributing to hypertrophy and preventing effective BMSC-mediated cartilage repair. Previous work demonstrated that a proprietary BMP inhibitor prevented BMSC hypertrophy, yielding stable cartilage tissue. Because of the significant therapeutic potential of a molecule capable of hypertrophy blockade, we evaluated the capacity of a commercially available BMP type I receptor inhibitor with similar properties, LDN 193189, to prevent BMSC hypertrophy. Using 14-day microtissue chondrogenic induction cultures we found that LDN 193189 permitted BMSC chondrogenesis but did not prevent hypertrophy. LDN 193189 was sufficiently potent to counter mineralization and adipogenesis in response to exogenous BMP-2 in osteogenic induction cultures. LDN 193189 did not modify BMSC behavior in adipogenic induction cultures. Although LDN 193189 is effective in countering BMP signaling in a manner that influences BMSC fate, this blockade is insufficient to prevent hypertrophy.

Integrated microfluidic single-cell immunoblotting chip enables high-throughput isolation, enrichment and direct protein analysis of circulating tumor cells

Effective capture and analysis of a single circulating tumor cell (CTC) is instrumental for early diagnosis and personalized therapy of tumors. However, due to their extremely low abundance and susceptibility to interference from other cells, high-throughput isolation, enrichment, and single-cell-level functional protein analysis of CTCs within one integrated system remains a major challenge. Herein, we present an integrated multifunctional microfluidic system for highly efficient and label-free CTC isolation, CTC enrichment, and single-cell immunoblotting (ieSCI). The ieSCI-chip is a multilayer microfluidic system that combines an inertia force-based cell sorter with a membrane filter for label-free CTC separation and enrichment and a thin layer of a photoactive polyacrylamide gel with microwell arrays at the bottom of the chamber for single-cell immunoblotting. The ieSCI-chip successfully identified a subgroup of apoptosis-negative (Bax-negative) cells, which traditional bulk analysis did not detect, from cisplatin-treated cells. Furthermore, we demonstrated the clinical application of the ieSCI-chip with blood samples from breast cancer patients for personalized CTC epithelial-to-mesenchymal transition (EMT) analysis. The expression level of a tumor cell marker (EpCAM) can be directly determined in isolated CTCs at the single-cell level, and the therapeutic response to anticancer drugs can be simultaneously monitored. Therefore, the ieSCI-chip provides a promising clinical translational tool for clinical drug response monitoring and personalized regimen development.

Electrofusion of cells with different diameters by generating asymmetrical electric field in the microwell array.

This paper reports a superiority of the asymmetric electric field formed in the rectangle microwell array for the electrofusion of splenocytes and myeloma cells with different diameters. The upper substrate with microband electrodes was mounted on the lower substrate with the microwell array. Two electrodes were arranged at the both sides of the microwells on the bottom surface. An attractive force of positive dielectrophoresis was employed to capture splenocytes with smaller diameter and myeloma cells with larger diameter at the right and left of microwells by applying AC electric field. The splenocytes and myeloma cells were fused by the asymmetric electric field that was generated in the microwells by applying DC electric pulse to the bottom electrode at the right side. The asymmetric field could allow to the formation of small openings on the membrane for the fusion of smaller splenocytes by experiencing higher field and the suppression for the disruption of larger myeloma cells by experiencing lower field.

Development of a device useful to reproducibly produce large quantities of viable and uniform stem cell spheroids with controlled diameters

Three-dimensional cellular aggregates can mimic the natural microenvironment of tissues and organs and obtaining them through controlled and reproducible processes is mandatory for scaling up and implementing drug cytotoxicity and efficacy tests, as well as tissue engineering protocols. The purpose of this work was to develop and evaluate the performance of a device with two different geometries fabricated by additive manufacturing. The methodology was based on casting a microwell array insert using a non-adhesive hydrogel to obtain highly regular microcavities to standardize spheroid formation and morphology. Spheroids of dental pulp stem cells, bone marrow stromal cells and embryonic stem cells showing high cell viability and average diameters of around 253, 220, and 500 μm, respectively, were produced using the device with the geometry considered most adequate. The cell aggregates showed sphericity indexes above 0.9 and regular surfaces (solidity index higher than 0.96). Around 1000 spheroids could be produced in a standard six-well plate. Overall, these results show that this method facilitates obtaining a large number of uniform, viable spheroids with pre-specified average diameters and through a low-cost and reproducible process for a myriad of applications.

Direct Dielectrophoretic Characterization of Particles in the High-Density Microwell Array Using Optical Tweezers

Direct Dielectrophoretic Characterization of Particles in the High-Density Microwell Array Using Optical Tweezers

An electroactive microwell array device to realize simultaneous trapping of single cancer cells and clusters.

The importance of circulating tumor cells (CTCs) as biomarkers has been greatly increased for early diagnosis and detection of cancer metastases. Along with a single form of CTCs, CTC clusters have recently attracted much attention due to their characteristics, such as suppression of apoptosis and survival from immune responses with high metastatic potential. Thus, it is highly necessary to investigate not only single cells but clustered cells at the same time to perform precise analysis of the current cancer state and develop suitable treatment. However, no cancer marker-free microfluidic devices have been realized to trap single cells and clusters at the same time in a single device yet. In this paper, we introduced a novel microfluidic device utilizing a microwell-on-electrode (MOE) array to realize simultaneous trapping of a single cell and clustered cells at a single cell/cluster level. Cell-sized microwells fabricated on interdigitated electrodes efficiently arrayed single cells with high trapping efficiency and single-cell occupancy (more than 90%) using dielectrophoresis (DEP). This high single cell trapping performance of MOE allows arraying of single clusters by trapping one of the cells that constitute a cluster. The feasibility of the MOE device for simultaneous arraying of single cancer cells and clusters was demonstrated by trapping a mixture of single cancer cells and clusters and measuring the size distribution of trapped clusters, which was almost identical with that of introduced cell population. Our work demonstrated that the developed MOE device can be one of the promising methods for trapping single cancer cells as well as clusters on a single device for cancer diagnosis and performing further analyses at a single cell/cluster level.

In Vitro Brain Organoids and Computational Models to Study Cell Death in Brain Diseases.

Understanding the mechanisms underlying the formation and progression of brain diseases is challenging due to the vast variety of involved genetic/epigenetic factors and the complexity of the environment of the brain. Current preclinical monolayer culture systems fail to faithfully recapitulate the in vivo complexities of the brain. Organoids are three-dimensional (3D) culture systems that mimic much of the complexities of the brain including cell-cell and cell-matrix interactions. Complemented with a theoretical framework to model the dynamic interactions between different components of the brain, organoids can be used as a potential tool for studying disease progression, transport of therapeutic agents in tissues, drug screening, and toxicity analysis. In this chapter, we first report on the fabrication and use of a novel self-filling microwell arrays (SFMWs) platform that is self-filling and enables the formation of organoids with uniform size distributions. Next, we will introduce a mathematical framework that predicts the organoid growth, cell death, and the therapeutic responses of the organoids to different therapeutic agents. Through systematic investigations, the computational model can identify shortcomings of in vitro assays and reduce the time and effort required to improve preclinical tumor models' design. Lastly, the mathematical model provides new testable hypotheses and encourages mathematically driven experiments.

A Self-Driven Carbon-Doped High-Density Microwell Array for Single Cell Analysis

A Self-Driven Carbon-Doped High-Density Microwell Array for Single Cell Analysis

Optimized Microwell Array Device for Preparation of Hair Follicle Germ-like Aggregates

Optimized Microwell Array Device for Preparation of Hair Follicle Germ-like Aggregates

Design and aligner-assisted fast fabrication of a microfluidic platform for quasi-3D cell studies on an elastic polymer.

While most studies of mechanical stimulation of cells are focused on two-dimensional (2D) and three-dimensional (3D) systems, it is rare to study the effects of cyclic stretching on cells under a quasi-3D microenvironment as a linkage between 2D and 3D. Herein, we report a new method to prepare an elastic membrane with topographic microstructures and integrate the membrane into a microfluidic chip. The fabrication difficulty lay not only in the preparation of microstructures but also in the alignment and bonding of the patterned membrane to other layers. To resolve the problem, we designed and assembled a fast aligner that is cost-effective and convenient to operate. To enable quasi-3D microenvironment of cells, we fabricated polydimethylsiloxane (PDMS) microwell arrays (formed by micropillars of a few microns in diameter) with the microwell diameters close to the cell sizes. An appropriate plasma treatment was found to afford a coating-free approach to enable cell adhesion on PDMS. We examined three types of cells in 2D, quasi-3D, and 3D microenvironments; the cell adhesion results showed that quasi-3D cells behaved between 2D and 3D cells. We also constructed transgenic human mesenchymal stem cells (hMSCs); under cyclic stretching, the visualizable live hMSCs in microwells were found to orientate differently from in a 3D Matrigel matrix and migrate differently from on a 2D flat plate. This study not only provides valuable tools for microfabrication of a microfluidic device for cell studies, but also inspires further studies of the topological effects of biomaterials on cells.

Grouped-seq for integrated phenotypic and transcriptomic screening of patient-derived tumor organoids.

Patient-derived tumor organoids (PDOs) have emerged as a reliable in vitro model for drug discovery. However, RNA sequencing-based analysis of PDOs treated with drugs has not been realized in a high-throughput format due to the limited quantity of organoids. Here, we translated a newly developed pooled RNA-seq methodology onto a superhydrophobic microwell array chip to realize an assay of genome-wide RNA output unified with phenotypic data (Grouped-seq). Over 10-fold reduction of sample and reagent consumption together with a new ligation-based barcode synthesis method lowers the cost to ∼$2 per RNA-seq sample. Patient-derived colorectal cancer (CRC) organoids with a number of 10 organoids per microwell were treated with four anti-CRC drugs across eight doses and analyzed by the Grouped-seq. Using a phenotype-assisted pathway enrichment analysis (PAPEA) method, the mechanism of actions of the drugs were correctly derived, illustrating the great potential of Grouped-seq for pharmacological screening with tumor organoids.

Use of liquid lithography to form in vitro intestinal crypts with varying microcurvature surrounding the stem cell niche

Objective. The role of the crypt microarchitecture and surrounding tissue curvature on intestinal stem/proliferative cell physiology is unknown. The utility of liquid lithography in creating polydimethylsiloxane (PDMS) micropillar stamps with controlled tip curvature was assessed. Using these stamps, the impact of microcurvature at the crypt base on intestinal cell and cytoskeletal behavior was studied. Approach. An SU-8 master mold as a support, polyols of varying surface energies as sacrificial liquids, and liquid PDMS as the solidifiable material were combined using liquid lithography to form PDMS micropillar arrays. Vapor phase deposition of organosilane onto the master mold was used to modify the surface energy of the master mold to shape the micropillar tips. Collagen was molded using the micropillar arrays forming a scaffold for culture of human primary colonic epithelial cells. Cell proliferation and cytoskeletal properties were assessed using fluorescent stains. Main results. Liquid lithography using low surface energy polyols (<55 dynes cm−1) generated convex-tipped PDMS micropillars, while polyols with higher surface energies (>55 dynes cm−1) yielded concave-tipped PDMS micropillars. Gradients of octyltrichlorosilane deposition across a master mold with an array of microwells yielded a PDMS micropillar array with a range of tip curvatures. Human primary colonic epithelial cells cultured on micropillar-molded collagen scaffolds demonstrated a stem/proliferative cell compartment at the crypt base. Crypts with a convex base demonstrated significantly lower cell proliferation at the crypt base than that of cells in crypts with either flat or concave bases. Crypts with a convex base also displayed higher levels of G-actin activity compared to that of crypts with flat or concave bases. Significance. Liquid lithography enabled creation of arrays of in vitro colonic crypts with programmable curvature. Primary cells at the crypt base sensed and responded to surface curvature by altering their proliferation and cytoskeletal properties.

A microfluidic platform for highly parallel bite by bite profiling of mosquito-borne pathogen transmission

Mosquito bites transmit a number of pathogens via salivary droplets deposited during blood-feeding, resulting in potentially fatal diseases. Little is known about the genomic content of these nanodroplets, including the transmission dynamics of live pathogens. Here we introduce Vectorchip, a low-cost, scalable microfluidic platform enabling high-throughput molecular interrogation of individual mosquito bites. We introduce an ultra-thin PDMS membrane which acts as a biting interface to arrays of micro-wells. Freely-behaving mosquitoes deposit saliva droplets by biting into these micro-wells. By modulating membrane thickness, we observe species-dependent differences in mosquito biting capacity, utilizable for selective sample collection. We demonstrate RT-PCR and focus-forming assays on-chip to detect mosquito DNA, Zika virus RNA, as well as quantify infectious Mayaro virus particles transmitted from single mosquito bites. The Vectorchip presents a promising approach for single-bite-resolution laboratory and field characterization of vector-pathogen communities, and could serve as a powerful early warning sentinel for mosquito-borne diseases.

Photolithographic Patterning of FluorAcryl for Biphilic Microwell-Based Digital Bioassays and Selection of Bacteria.

FluorAcryl 3298 (FA) is a UV-curable fluoroacrylate polymer commonly employed as a chemically resistant, hydrophobic, and oleophobic coating. Here, FA was used in a cleanroom-based microstructuring process to fabricate hydrophilic-in-hydrophobic (HiH) micropatterned surfaces containing femtoliter-sized well arrays. A short protocol involving direct UV photopatterning, an etching step, and final recovery of the hydrophobic properties of the polymer produced patterned substrates with micrometer resolution. Specifically, HiH microwell arrays were obtained with a well diameter of 10 μm and various well depths ranging from 300 nm to 1 μm with high reproducibility. The 300 nm deep microdroplet array (MDA) substrates were used for digital immunoassays, which presented a limit of detection in the attomolar range. This demonstrated the chemical functionality of the hydrophilic and hydrophobic surfaces. Furthermore, the 1 μm deep wells could efficiently capture particles such as bacteria, whereas the 300 nm deep substrates or other types of flat HiH molecular monolayers could not. Capturing a mixture of bacteria expressing red- and green-fluorescent proteins, respectively, served as a model for screening and selection of specific phenotypes using FA-MDAs. Here, green-fluorescent bacteria were specifically selected by overlaying a solution of gelatin methacryloyl (GelMA) mixed with a photoinitiator and using a high-magnification objective, together with custom pinholes, in a common fluorescence microscope to cross-link the hydrogel around the bacteria of interest. In conclusion, due to the straightforward processing, versatility, and low-price, FA is an advantageous alternative to more commonly used fluorinated materials, such as CYTOP or Teflon-AF, for the fabrication of HiH microwell arrays and other biphilic microstructures.

Summit: Automated Analysis of Arrayed Single-Cell Gel Electrophoresis.

New pipelines are required to automate the quantitation of emerging high-throughput electrophoretic (EP) assessment of DNA damage, or proteoform expression in single cells. EP cytometry consists of thousands of Western blots performed on a microscope slide-sized gel microwell array for single cells. Thus, EP cytometry images pose an analysis challenge that blends requirements for accurate and reproducible analysis encountered for both standard Western blots and protein microarrays. Here, we introduce the Summit algorithm to automate array segmentation, peak background subtraction, and Gaussian fitting for EP cytometry. The data structure storage of parameters allows users to perform quality control on identically processed data, yielding a ~6.5% difference in coefficient of quartile variation (CQV) of protein peak area under the curve (AUC) distributions measured by four users. Further, inspired by investigations of background subtraction methods to reduce technical variation in protein microarray measurements, we aimed to understand the trade-offs between EP cytometry analysis throughput and variation. We found an 11%-50% increase in protein peaks that passed quality control with a subtraction method similar to microarray "average on-boundary" versus an axial subtraction method. The background subtraction method only mildly influences AUC CQV, which varies between 1% and 4.5%. Finally, we determined that the narrow confidence interval for peak location and peak width parameters from Gaussian fitting yield minimal uncertainty in protein sizing. The AUC CQV differed by only ~1%-2% when summed over the peak width bounds versus the 95% peak width confidence interval. We expect Summit to be broadly applicable to other arrayed EP separations, or traditional Western blot analysis.