3D tumor spheroid microarray for high-throughput, high-content natural killer cell-mediated cytotoxicity.

Immunotherapy has emerged as a promising approach to treating several forms of cancer. Use of immune cells, such as natural killer (NK) cells, along with small molecule drugs and antibodies through antibody dependent cell-mediated cytotoxicity (ADCC) has been investigated as a potential combination therapy for some difficult to treat solid tumors. Nevertheless, there remains a need to develop tools that support co-culture of target cancer cells and effector immune cells in a contextually relevant three-dimensional (3D) environment to provide a rapid means to screen for and optimize ADCC-drug combinations. To that end, here we have developed a high throughput 330 micropillar-microwell sandwich platform that enables 3D co-culture of NK92-CD16 cells with pancreatic (MiaPaCa-2) and breast cancer cell lines (MCF-7 and MDA-MB-231). The platform successfully mimicked hypoxic conditions found in a tumor microenvironment and was used to demonstrate NK-cell mediated cell cytotoxicity in combination with two monoclonal antibodies; Trastuzumab and Atezolizumab. The platform was also used to show dose response behavior of target cancer cells with reduced EC50 values for paclitaxel (an anti-cancer chemotherapeutic) when treated with both NK cells and antibody. Such a platform may be used to develop more personalized cancer therapies using patient-derived cancer cells.

Impact of tumor cellularity on the HER2 amplification assay by OncoScan™ in breast cancer.

HER2 amplification is seen in 20-25% of primary breast cancer cases, and HER2 detection is performed routinely in primary operable, as well as metastatic breast cancer patients. Currently, HER2 is the only gene of which amplification is routinely assayed by fluorescent in situ hybridization (FISH) and/or immunohistochemistry (IHC). However, histochemical assay (FISH/IHC) of multiple target genes is laborious and time-consuming, and simultaneous amplification by microarray is preferred. OncoScan™ is a microarray-based assay capable of whole-genome copy number analysis using DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues. In the current study, we aimed to investigate the impact of tumor cellularity on the accuracy of OncoScan™ in the determination of HER2 amplification. Our results demonstrated that HER2 amplification by OncoScan™ is accurate, and has a high concordance rate of 93.3% with FISH. However, the concordance rate is poor (66.7%) in cases with a tumor cellularity < 20%. Nevertheless, the addition of FISH to breast tumors with a tumor cellularity < 20% and a HER2 copy number of 4 appears to be useful to minimize false-negative results by OncoScan™.

Functional nanoarrays for investigating stem cell fate and function.

Stem cells show excellent potential in the field of tissue engineering and regenerative medicine based on their excellent capability to not only self-renew but also differentiate into a specialized cell type of interest. However, the lack of a non-destructive monitoring system makes it challenging to identify and characterize differentiated cells before their transplantation without compromising cell viability. Thus, the development of a non-destructive monitoring method for analyzing cell function is highly desired and can significantly benefit stem cell-based therapies. Recently, nanomaterial-based scaffolds (e.g., nanoarrays) have made possible considerable advances in controlling the differentiation of stem cells and characterization of the differentiation status sensitively in real time. This review provides a selective overview of the recent progress in the synthesis methods of nanoarrays and their applications in controlling stem cell fate and monitoring live cell functions electrochemically. We believe that the topics discussed in this review can provide brief and concise guidelines for the development of novel nanoarrays and promote the interest in live cell study applications. A method which can not only control but also monitor stem cell fate and function will be a promising technology that can accelerate stem cell therapies.

Characterizing the Tumor Immune Microenvironment with Tyramide-Based Multiplex Immunofluorescence.

Multiplex immunofluorescence (mIF) allows simultaneous antibody-based detection of multiple markers with a nuclear counterstain on a single tissue section. Recent studies have demonstrated that mIF is becoming an important tool for immune profiling the tumor microenvironment, further advancing our understanding of the interplay between cancer and the immune system, and identifying predictive biomarkers of response to immunotherapy. Expediting mIF discoveries is leading to improved diagnostic panels, whereas it is important that mIF protocols be standardized to facilitate their transition into clinical use. Manual processing of sections for mIF is time consuming and a potential source of variability across numerous samples. To increase reproducibility and throughput we demonstrate the use of an automated slide stainer for mIF incorporating tyramide signal amplification (TSA). We describe two panels aimed at characterizing the tumor immune microenvironment. Panel 1 included CD3, CD20, CD117, FOXP3, Ki67, pancytokeratins (CK), and DAPI, and Panel 2 included CD3, CD8, CD68, PD-1, PD-L1, CK, and DAPI. Primary antibodies were first tested by standard immunohistochemistry and single-plex IF, then multiplex panels were developed and images were obtained using a Vectra 3.0 multispectral imaging system. Various methods for image analysis (identifying cell types, determining cell densities, characterizing cell-cell associations) are outlined. These mIF protocols will be invaluable tools for immune profiling the tumor microenvironment.

A simple microsphere-based mold to rapidly fabricate microwell arrays for multisize 3D tumor culture.

Three-dimensional (3D) tumor has been considered as the best in vitro model for cancer research. In recent years, various methods have been developed to controllable prepare multisize 3D tumors. Nonetheless, reported technologies are still problematic and difficult to produce 3D tumors with highly uniform size and cell content. Here, a novel and simple microsphere-based mold approach is proposed to rapidly fabricate spherical microwell arrays for multisize 3D tumors formation, culture, and recovery. Larger amounts of HepG2 3D tumors with excellent quality and uniformity can be efficiently generated using this method. In addition, the tumor size can also be simply controlled by adjusting the diameter of the microwell arrays. All experimental results indicated that the proposed method offers a promising platform to generate and recover highly controlled multisize 3D tumors for various cell-based biomedical research.

Intestine-on-chip device increases ECM remodeling inducing faster epithelial cell differentiation.

An intestine-on-chip has been developed to study intestinal physiology and pathophysiology as well as intestinal transport absorption and toxicity studies in a controlled and human similar environment. Here, we report that dynamic culture of an intestine-on-chip enhances extracellular matrix (ECM) remodeling of the stroma, basement membrane production and speeds up epithelial differentiation. We developed a three-dimensional human intestinal stromal equivalent composed of human intestinal subepithelial myofibroblasts embedded in their own ECM. Then, we cultured human colon carcinoma-derived cells in both static and dynamic conditions in the opportunely designed microfluidic system until the formation of a well-oriented epithelium. This low cost and handy microfluidic device allows to qualitatively and quantitatively detect epithelial polarization and mucus production as well as monitor barrier function and ECM remodeling after nutraceutical treatment.

An introduction of an easy-operating and economical technique for tissue microarray preparation.

AIM: Tissue microarray (TMA) is a powerful and effective tool for in situ tissue analysis. However, manual TMA construction methods showed varied qualities. This study aimed to raise a standardised TMA preparation technique that can be easily operated and is economical. METHODS: A sampling needle was used to punch the tissue rods from the donor block and holes in the recipient block. To indicate the dots' positions and ensure vertical punching, a novel auxiliary device made using commercial three-dimensional printing technology was attached. The TMA block was made up of tissue rods and a recipient block. RESULTS: A 77-rod (7×11) TMA block was constructed. The rows and columns were fixed in straight lines. There was no specimen loss during the process of embedding. CONCLUSIONS: An alternative method for the construction of TMA blocks that met the basic requirement of many laboratories and can be effortlessly performed was presented.

Sample preparation strategies for high-throughput mass spectrometry imaging of primary tumor organoids.

Patient-derived 3D organoids show great promise for understanding patient heterogeneity and chemotherapy response in human-derived tissue. The combination of organoid culture techniques with mass spectrometry imaging provides a label-free methodology for characterizing drug penetration, patient-specific response, and drug biotransformation. However, current methods used to grow tumor organoids employ extracellular matrices that can produce small molecule background signal during mass spectrometry imaging analysis. Here, we develop a method to isolate 3D human tumor organoids out of a Matrigel extracellular matrix into gelatin mass spectrometry compatible microarrays for high-throughput mass spectrometry imaging analysis. The alignment of multiple organoids in the same z-axis is essential for sectioning organoids together and for maintaining reproducible sample preparation on a single glass slide for up to hundreds of organoids. This method successfully removes organoids from extracellular matrix interference and provides an organized array for high-throughput imaging analysis to easily identify organoids by eye for area selection and further analysis. With this method, mass spectrometry imaging can be readily applied to organoid systems for preclinical drug development and personalized medicine research initiatives.

Investigation of uniform sized multicellular spheroids raised by microwell arrays after the combined treatment of electric field and anti-cancer drug.

Nowadays, cancer disease is continuously identified as the leading cause of mortality worldwide. Cancer chemotherapeutic agents have been continuously developing to achieve high curative effectiveness and low side effects. However, solid tumors present the properties of low drug penetration and resistance of quiescent cells. Radiation therapy is concurrently given in some cases; but it induces different levels of adverse effects. In the current work, uniform sized multicellular spheroids were raised by microwell arrays to mimic the architecture of solid tumors. Investigation of the response of the spheroids was conducted after the treatment of alternating electric field. The result showed that the electric field could induce early apoptosis by disturbing cell membrane. Moreover, combined treatment of electric field and anti-cancer drug was applied to the spheroids. The electric field synergistically enhanced the treatment efficacy because the anti-cancer drug could permeate through the disrupted cell membrane. Significant improvement of late apoptosis was shown by the combined treatment. Because the electric field treatment induces limited side effect to the patient, lower dosage of anti-cancer drug may be applied to the patients for achieving curative effectiveness.

Single cell arrays of hematological cancer cells for assessment of lymphocyte cytotoxicity dynamics, serial killing, and extracellular molecules.

Cytotoxicity exerted by cytotoxic lymphocytes against cancer cells is an essential cellular function for successful cancer immunotherapy. Standard cytotoxicity assays mostly provide population level information, whereas live cell imaging-based cytotoxicity assays can assess single cell level heterogeneity. However, long term tracking of individual cytotoxic lymphocyte-hematological cancer cell interactions is technically challenging because both cells can float around and form multi-cellular aggregates. To overcome this limitation, single hematological cancer cell arrays with immobilized hematological cancer cells are fabricated using microwell arrays. Using this new platform, single cell level natural killer (NK) cell cytotoxicity against leukemic cells is quantitatively assessed. Depending on microwell surface adhesiveness and inter-microwell distances, distinct modes of NK-leukemic cell interactions that result in different NK cell cytotoxicity are observed. For microwell arrays coated with bovine serum albumin, which prevents cell adhesion, NK cells stably contacted cancer cells with rounded morphologies, whereas for microwell arrays coated with fibronectin (FN), which triggers integrin signals, NK cells contacting cancer cells exhibited dynamic behaviors with elongated morphologies and constantly explored extracellular spaces by membrane extension. In addition, FN on extracellular spaces facilitate NK cell detachment from leukemic cells after killing by providing anchorage for force transmission, and promote cytotoxicity and serial killing. Single hematologic cell arrays are not only an efficient method for lymphocyte cytotoxicity analysis but also a useful tool to study the role of signaling molecules in extracellular spaces on lymphocyte cytotoxicity.

Liquid biopsy using the nanotube-CTC-chip: capture of invasive CTCs with high purity using preferential adherence in breast cancer patients.

In this paper, we report the development of the nanotube-CTC-chip for isolation of tumor-derived epithelial cells (circulating tumor cells, CTCs) from peripheral blood, with high purity, by exploiting the physical mechanisms of preferential adherence of CTCs on a nanotube surface. The nanotube-CTC-chip is a new 76-element microarray technology that combines carbon nanotube surfaces with microarray batch manufacturing techniques for the capture and isolation of tumor-derived epithelial cells. Using a combination of red blood cell (RBC) lysis and preferential adherence, we demonstrate the capture and enrichment of CTCs with a 5-log reduction of contaminating WBCs. EpCAM negative MDA-MB-231/luciferase-2A-green fluorescent protein (GFP) cells were spiked in the blood of wild mice and enriched using an RBC lysis protocol. The enriched samples were then processed using the nanotube-CTC-chip for preferential CTC adherence on the nanosurface and counting the GFP cells yielded anywhere from 89% to 100% capture from the droplets. Electron microscopy (EM) studies showed focal adhesion with filaments from the cell body to the nanotube surface. We compared the nanotube preferential adherence to collagen adhesion matrix (CAM) scaffolding, reported as a viable strategy for CTC capture in patients. The CAM scaffolding on the device surface yielded 50% adherence with 100% tracking of cancer cells (adhered vs. non-adhered) versus carbon nanotubes with >90% adherence and 100% tracking for the same protocol. The nanotube-CTC-chip successfully captured CTCs in the peripheral blood of breast cancer patients (stage 1-4) with a range of 4-238 CTCs per 8.5 ml blood or 0.5-28 CTCs per ml. CTCs (based on CK8/18, Her2, EGFR) were successfully identified in 7/7 breast cancer patients, and no CTCs were captured in healthy controls (n = 2). CTC enumeration based on multiple markers using the nanotube-CTC-chip enables dynamic views of metastatic progression and could potentially have predictive capabilities for diagnosis and treatment response.

Controlled Nanoscale Topographies for Osteogenic Differentiation of Mesenchymal Stem Cells.

Nanotopography with length scales of the order of extracellular matrix elements offers the possibility of regulating cell behavior. Investigation of the impact of nanotopography on cell response has been limited by the inability to precisely control geometries, especially at high spatial resolutions and across practically large areas. In this paper, we demonstrate well-controlled and periodic nanopillar arrays of silicon and investigate their impact on osteogenic differentiation of human mesenchymal stem cells (hMSCs). Silicon nanopillar arrays with critical dimensions in the range of 40-200 nm, exhibiting standard deviations below 15% across full wafers, were realized using the self-assembly of block copolymer colloids. Immunofluorescence and quantitative polymerase chain reaction measurements reveal clear dependence of osteogenic differentiation of hMSCs on the diameter and periodicity of the arrays. Further, the differentiation of hMSCs was found to be dependent on the age of the donor. While osteoblastic differentiation was found to be promoted by the pillars with larger diameters and heights independent of donor age, they were found to be different for different spacings. Pillar arrays with smaller pitch promoted differentiation from a young donor, while a larger spacing promoted those of an old donor. These findings can contribute for the development of personalized treatments of bone diseases, namely, novel implant nanostructuring depending on patient age.

A Novel Controllable Cell Array Printing Technique on Microfluidic Chips.

GOAL: The construction of single-cell array is known as the challenging technology to manipulate cell position and number and accomplish cell analysis in biomedical engineering. METHODS: We put forward a novel controllable cell printing technique for rapid, precise, convenient, high cell viability, multicellular, and high-throughput printing. We also proposed a novel microfluidic device to verify the effectiveness of the printing and study the migration ability and anti-cancer drug responses of cancer cell as important applications. RESULTS: This technique offered a minimum process time of 5 min, a maximum positional accuracy of 10 µm, 0.1 nL liquid volume level per droplet, above 87% cell viability after seven days and the ability to print different multicellular arrays. We found that the cell compared to cell culture in petri dish after 48 h. In addition, there was a significant different inhibition on cancer cells migration ability and cell drug activities with different concentrations of paclitaxel. CONCLUSION: This novel controllable cell array printing technique on the microfluidic platforms provides a useful method with high-quality printing and cell viability for the applications of single-cell analysis and high-throughput drug screening. SIGNIFICANCE: The controllable cell printing technique could apply in many biological processes and biomedical engineering applications, such as cell analysis, cancer development, and drug screening and metabolism. Combined with the microfluidic chips, tissue engineering, and sensors, this technique will be widely used for the construction and analysis of biological and biomedical model.

Personalised organs-on-chips: functional testing for precision medicine.

Organs-on-chips are microfluidic systems with controlled, dynamic microenvironments in which cultured cells exhibit functions that emulate organ-level physiology. They can in principle be 'personalised' to reflect individual physiology, for example by including blood samples, primary human tissue, and cells derived from induced pluripotent stem cell-derived cells, as well as by tuning key physico-chemical parameters of the cell culture microenvironment based on personal health data. The personalised nature of such systems, combined with physiologically relevant read-outs, provides new opportunities for person-specific assessment of drug efficacy and safety, as well as personalised strategies for disease prevention and treatment; together, this is known as 'precision medicine'. There are multiple reports of how to personalise organs-on-chips, with examples including airway-on-a-chip systems containing primary patient alveolar epithelial cells, vessels-on-chips with shapes based on personal biomedical imaging data and lung-on-a-chip systems that can be exposed to various regimes of cigarette smoking. In addition, multi-organ chip systems even allow the systematic and dynamic integration of more complex combinations of personalised cell culture parameters. Current personalised organs-on-chips have not yet been used for precision medicine as such. The major challenges that affect the implementation of personalised organs-on-chips in precision medicine are related to obtaining access to personal samples and corresponding health data, as well as to obtaining data on patient outcomes that can confirm the predictive value of personalised organs-on-chips. We argue here that involving all biomedical stakeholders from clinicians and patients to pharmaceutical companies will be integral to transition personalised organs-on-chips to precision medicine.

A lung/liver-on-a-chip platform for acute and chronic toxicity studies.

The merging of three-dimensional in vitro models with multi-organ-on-a-chip (MOC) technology has taken in vitro assessment of chemicals to an unprecedented level. By connecting multiple organotypic models, MOC allows for the crosstalk between different organs to be studied to evaluate a compound's safety and efficacy better than with single cultures. The technology could also improve the toxicological assessment of aerosols that have been implicated in the development of chronic obstructive pulmonary disease, asthma, or lung cancer. Here we report the development of a lung/liver-on-a-chip, connecting in a single circuit, normal human bronchial epithelial (NHBE) cells cultured at the air-liquid interface (ALI), and HepaRG™ liver spheroids. Maintenance of the individual tissues in the chip increased NHBE ALI tissue transepithelial electrical resistance and decreased HepaRG™ spheroid adenosine triphosphate content as well as cytochrome P450 (CYP) 1A1/1B1 inducibility. CYP inducibility was partly restored when HepaRG™ spheroids were cocultured with NHBE ALI tissues. Both tissues remained viable and functional for 28 days when cocultured in the chip. The capacity of the HepaRG™ spheroids to metabolize compounds present in the medium and to modulate their toxicity was proven using aflatoxin B1 (AFB1). AFB1 toxicity in NHBE ALI tissues decreased when HepaRG™ spheroids were present in the same chip circuit, proving that the HepaRG™-mediated detoxification is protecting/decreasing from AFB1-mediated cytotoxicity. The lung/liver-on-a-chip platform presented here offers new opportunities to study the toxicity of inhaled aerosols or to demonstrate the safety and efficacy of new drug candidates targeting the human lung.

On-chip photodynamic therapy - monitoring cell metabolism using electrochemical microsensors.

We introduce a new system which combines metabolic monitoring using electrochemical microsensors with photodynamic therapy on-chip for the first time. Oxygen consumption of T-47D breast cancer cells was measured during therapy with protoporphyrin IX. We determined the efficacy of the therapy and revealed its recovery effects, which underlines the high relevance of continuous monitoring.

Self-filling microwell arrays (SFMAs) for tumor spheroid formation.

Tumor spheroid formation in microwell arrays is a promising approach for high-throughput screening of chemotherapeutic agents. This method offers the advantage of better mimicking the complexities of tumors as compared to conventional monolayer culture systems. However, using these technologies to their full potential is hindered by the inability to seed the cells within the wells uniformly and with high yield and reproducibility. Moreover, standard manufacturing approaches for fabrication of microwell arrays rely on lithography and etching techniques, which are costly, labor-intensive, and time-consuming. Herein, we report on the development of self-filling microwell arrays (SFMAs) in which cells are directed from a loading chamber to microwells using inclined guiding channels. The SFMAs are fabricated by replica molding of three-dimensionally (3D) printed molds in agarose. We characterize the fabrication process, demonstrate the ability to culture breast adenocarcinoma MCF-7 and glioma U87 in SFMAs and perform drug toxicity studies. We envision that the proposed innovative approach opens avenues of opportunities for high-throughput three-dimensional cell culture for drug screening and disease modeling.

Medium throughput breathing human primary cell alveolus-on-chip model.

Organs-on-chips have the potential to improve drug development efficiency and decrease the need for animal testing. For the successful integration of these devices in research and industry, they must reproduce in vivo contexts as closely as possible and be easy to use. Here, we describe a 'breathing' lung-on-chip array equipped with a passive medium exchange mechanism that provide an in vivo-like environment to primary human lung alveolar cells (hAEpCs) and primary lung endothelial cells. This configuration allows the preservation of the phenotype and the function of hAEpCs for several days, the conservation of the epithelial barrier functionality, while enabling simple sampling of the supernatant from the basal chamber. In addition, the chip design increases experimental throughput and enables trans-epithelial electrical resistance measurements using standard equipment. Biological validation revealed that human primary alveolar type I (ATI) and type II-like (ATII) epithelial cells could be successfully cultured on the chip over multiple days. Moreover, the effect of the physiological cyclic strain showed that the epithelial barrier permeability was significantly affected. Long-term co-culture of primary human lung epithelial and endothelial cells demonstrated the potential of the lung-on-chip array for reproducible cell culture under physiological conditions. Thus, this breathing lung-on-chip array, in combination with patients' primary ATI, ATII, and lung endothelial cells, has the potential to become a valuable tool for lung research, drug discovery and precision medicine.

A 96-well microplate bioreactor platform supporting individual dual perfusion and high-throughput assessment of simple or biofabricated 3D tissue models.

Traditional 2D monolayer cell cultures and submillimeter 3D tissue construct cultures used widely in tissue engineering are limited in their ability to extrapolate experimental data to predict in vivo responses due to their simplistic organization and lack of stimuli. The rise of biofabrication and bioreactor technologies has sought to address this through the development of techniques to spatially organize components of a tissue construct, and devices to supply these tissue constructs with an increasingly in vivo-like environment. Current bioreactors supporting both parenchymal and barrier tissue constructs in interconnected systems for body-on-a-chip platforms have chosen to emphasize study throughput or system/tissue complexity. Here, we report a platform to address this disparity in throughput and both system complexity (by supporting multiple in situ assessment methods) and tissue complexity (by adopting a construct-agnostic format). We introduce an ANSI/SLAS-compliant microplate and docking station fabricated via stereolithography (SLA), or precision machining, to provide up to 96 samples (Ø6 × 10 mm) with two individually-addressable fluid circuits (192 total), loading access, and inspection window for imaging during perfusion. Biofabricated ovarian cancer models were developed to demonstrate the in situ assessment capabilities via microscopy and a perfused resazurin-based metabolic activity assay. In situ microscopy highlighted flexibility of the sample housing to accommodate a range of sample geometries. Utility for drug screening was demonstrated by exposing the ovarian cancer models to an anticancer drug (doxorubicin) and generating the dose-response curve in situ, while achieving an assay quality similar to static wellplate culture. The potential for quantitative analysis of temporal tissue development and screening studies was confirmed by imaging soft- (gelatin) and hard-tissue (calcium chloride) analogs inside the bioreactor via spectral computed tomography (CT) scanning. As a proof-of-concept for particle tracing studies, flowing microparticles were visualized to inform the design of hydrogel constructs. Finally, the ability for mechanistic yet high-throughput screening was demonstrated in a vascular coculture model adopting endothelial and mesenchymal stem cells (HUVEC-MSC), encapsulated in gelatin-norbornene (gel-NOR) hydrogel cast into SLA-printed well inserts. This study illustrates the potential of a scalable dual perfusion bioreactor platform for parenchymal and barrier tissue constructs to support a broad range of multi-organ-on-a-chip applications.

An acoustofluidic trap and transfer approach for organizing a high density single cell array.

We demonstrate a hybrid microfluidic system that combines fluidic trapping and acoustic switching to organize an array of single cells at high density. The fluidic trapping step is achieved by balancing the hydrodynamic resistances of three parallel channel segments forming a microfluidic trifurcation, the purpose of which was to capture single cells in a high-density array. Next, the cells were transferred into adjacent larger compartments by generating an array of streaming micro-vortices to move the cells to the desired streamlines in a massively parallel format. This approach can compartmentalize single cells with efficiencies of ≈67% in compartments that have diameters on the order of ∼100 um, which is an appropriate size for single cell proliferation studies and other single cell biochemical measurements.