Knockout cancer by nano-delivered immunotherapy using perfusion-aided scaffold-based tumor-on-a-chip.

Cancer is a multifactorial disease produced by mutations in the oncogenes and tumor suppressor genes, which result in uncontrolled cell proliferation and resistance to cell death. Cancer progresses due to the escape of altered cells from immune monitoring, which is facilitated by the tumor's mutual interaction with its microenvironment. Understanding the mechanisms involved in immune surveillance evasion and the significance of the tumor microenvironment might thus aid in developing improved therapies. Although in vivo models are commonly utilized, they could be better for time, cost, and ethical concerns. As a result, it is critical to replicate an in vivo model and recreate the cellular and tissue-level functionalities. A 3D cell culture, which gives a 3D architecture similar to that found in vivo, is an appropriate model. Furthermore, numerous cell types can be cocultured, establishing cellular interactions between TME and tumor cells. Moreover, microfluidics perfusion can provide precision flow rates, thus simulating tissue/organ function. Immunotherapy can be used with the perfused 3D cell culture technique to help develop successful therapeutics. Immunotherapy employing nano delivery can target the spot and silence the responsible genes, ensuring treatment effectiveness while minimizing adverse effects. This study focuses on the importance of 3D cell culture in understanding the pathophysiology of 3D tumors and TME, the function of TME in drug resistance, tumor progression, and the development of advanced anticancer therapies for high-throughput drug screening.

Isolation of Extracellular Vesicles from Cell Culture and Blood Through Nano-Targeted DLD Microfluidic Device.

Extracellular vesicles (EVs) are secreted by cells and found in biological fluids such as blood, with concentration correlated with oncogenic signals, making them attractive biomarkers for liquid biopsy. The current gold-standard method for EVs isolation requires an ultracentrifugation (UC) step among others. The cost and complexity of this technique are forbiddingly high for many researchers, as well as for routine use in biological laboratories and hospitals. This chapter reports on a simple microfluidic method for EVs isolation, based on a microfluidic size sorting technique named Deterministic Lateral Displacement (DLD). With the design of micrometric DLD array, we demonstrated the potential of our DLD devices for the isolation of nano-biological objects such as EVs, with main population size distribution consistent with UC technique.

Microchip for Immunomagnetic Sorting of Circulating Tumor Cells (CTCs).

Circulating tumor cells (CTCs) isolated directly from whole blood opens new perspectives for cancer monitoring and the development of personalized treatments. However, due to their rarity among the multitude of blood cells, it remains a challenge to recover them alive with high level of purity, i.e., with few remaining white blood cells, and in a time frame compatible with the clinical context. Microfluidic chips have emerged as promising tools to address these challenges. We propose a two-step workflow including a pre-enrichment step, performed by a size-based pre-enrichment system, and a purification step, performed by an immunomagnetic chip. Here, we describe the protocol for the fabrication of the immunomagnetic microchip, the preparation of the sample, and the procedure for injection into the microchip allowing the sorting of the CTCs.

On-Chip Magnetic Extraction of Circulating Cell-Free DNA from Biological Samples.

In recent years, the analysis of circulating cell-free DNA (cfDNA) containing tumor-derived DNA has emerged as a noninvasive means for cancer monitoring and personalized medicine. However, the isolation of cfDNA from peripheral blood has remained a challenge due to the low abundance and high fragmentation of these molecules. Here, we present a dynamic Magnetic ExTRactiOn (METRO) protocol using microfluidic fluidized bed technology to isolate circulating cfDNA from raw biological materials such as undiluted serum. This protocol maximizes the surface area for DNA binding within the chip in order to capture short DNA fragments. It uses only a few µL of sample and reagents. The protocol can be automated, and it is fully compatible with sensitive DNA amplification methods such as droplet-based digital PCR (ddPCR).

Selective Targeting of Tumor Cells in a Microfluidic Tumor Model with Multiple Cell Types.

Organ-on-a-chip technology allows researchers to precisely monitor drug efficacy in 3D tissue culture systems that are physiologically more relevant to humans compared to 2D cultures and that allow better control over experimental conditions as compared to animal models. Specifically, the high control over microenvironmental conditions combined with the broad range of direct measurements that can be performed in these systems makes organ-on-a-chip devices a versatile tool to investigate tumor targeting and drug delivery. Here, we describe a detailed protocol for studying the cell-selective targeting of protein drugs to tumor cells on an organ-on-a-chip system using a co-culture consisting of BT-474 cancer cells and C5120 human fibroblasts as an example.

Micropump integrated white blood cell separation platform for detection of chronic granulomatous disease.

White blood cells (WBCs) are robust defenders during antigenic challenges and prime immune cell functioning indicators. High-purity WBC separation is vital for various clinical assays and disease diagnosis. Red blood cells (RBCs) are a major hindrance in WBC separation, constituting 1000 times the WBC population. The study showcases a low-cost micropump integrated microfluidic platform to provide highly purified WBCs for point-of-care testing. An integrated user-friendly microfluidic platform was designed to separate WBCs from finger-prick blood (⁓5 µL), employing an inertial focusing technique. We achieved an efficient WBC separation with 86% WBC purity and 99.99% RBC removal rate in less than 1 min. In addition, the microdevice allows lab-on-chip colorimetric evaluation of chronic granulomatous disease (CGD), a rare genetic disorder affecting globally. The assay duration, straight from separation to disease detection, requires only 20 min. Hence, the proposed microfluidic platform can further be implemented to streamline various clinical procedures involving WBCs in healthcare industries.

Evaluation of Pm2.5 Influence on Human Lung Cancer Cells Using a Microfluidic Platform.

In this study, we developed a microfluidic device that is able to monitor cell biology under continuous PM2.5 treatment. The effects of PM2.5 on human alveolar basal epithelial cells, A549 cells, and uncovered several significant findings were investigated. The results showed that PM2.5 exposure did not lead to a notable decrease in cell viability, indicating that PM2.5 did not cause cellular injury or death. However, the study found that PM2.5 exposure increased the invasion and migration abilities of A549 cells, suggesting that PM2.5 might promote cell invasiveness. Results of RNA sequencing revealed 423 genes that displayed significant differential expression in response to PM2.5 exposure, with a particular focus on pathways associated with the generation of reactive oxygen species (ROS) and mitochondrial dysfunction. Real-time detection demonstrated an increase in ROS production in A549 cells after exposure to PM2.5. JC1 assay, which indicated a loss of mitochondrial membrane potential (ΔΨm) in A549 cells exposed to PM2.5. The disruption of mitochondrial membrane potential further supports the detrimental effects of PM2.5 on A549 cells. These findings highlight several adverse effects of PM2.5 on A549 cells, including enhanced invasion and migration capabilities, altered gene expression related to ROS pathways, increased ROS production and disruption of mitochondrial membrane potential. These findings contribute to our understanding of the potential mechanisms through which PM2.5 can impact cellular function and health.

Development of an alginate-chitosan biopolymer composite with dECM bioink additive for organ-on-a-chip articular cartilage.

In vitro use of articular cartilage on an organ-on-a-chip (OOAC) via microfluidics is challenging owing to the dense extracellular matrix (ECM) composed of numerous protein moieties and few chondrocytes, which has limited proliferation potential and microscale translation. Hence, this study proposes a novel approach for using a combination of biopolymers and decellularised ECM (dECM) as a bioink additive in the development of scalable OOAC using a microfluidic platform. The bioink was tested with native chondrocytes and mesenchymal stem cell-induced chondrocytes using biopolymers of alginate and chitosan composite hydrogels. Two-dimensional (2D) and three-dimensional (3D) biomimetic tissue construction approaches have been used to characterise the morphology and cellular marker expression (by histology and confocal laser scanning microscopy), viability (cell viability dye using flow cytometry), and genotypic expression of ECM-specific markers (by quantitative PCR). The results demonstrated that the bioink had a significant impact on the increase in phenotypic and genotypic expression, with a statistical significance level of p < 0.05 according to Student's t-test. The use of a cell-laden biopolymer as a bioink optimised the niche conditions for obtaining hyaline-type cartilage under culture conditions, paving the way for testing mechano-responsive properties and translating these findings to a cartilage-on-a-chip microfluidics system.

Microfluidic Electroporation Arrays for Investigating Electroporation-Induced Cellular Rupture Dynamics.

Electroporation is pivotal in bioelectrochemistry for cellular manipulation, with prominent applications in drug delivery and cell membrane studies. A comprehensive understanding of pore generation requires an in-depth analysis of the critical pore size and the corresponding energy barrier at the onset of cell rupture. However, many studies have been limited to basic models such as artificial membranes or theoretical simulations. Challenging this paradigm, our study pioneers using a microfluidic electroporation chip array. This tool subjects live breast cancer cell species to a diverse spectrum of alternating current electric field conditions, driving electroporation-induced cell rupture. We conclusively determined the rupture voltages across varying applied voltage loading rates, enabling an unprecedented characterization of electric cell rupture dynamics encompassing critical pore radius and energy barrier. Further bolstering our investigation, we probed cells subjected to cholesterol depletion via methyl-ß-cyclodextrin and revealed a strong correlation with electroporation. This work not only elucidates the dynamics of electric rupture in live cell membranes but also sets a robust foundation for future explorations into the mechanisms and energetics of live cell electroporation.

Drug screening on digital microfluidics for cancer precision medicine.

Drug screening based on in-vitro primary tumor cell culture has demonstrated potential in personalized cancer diagnosis. However, the limited number of tumor cells, especially from patients with early stage cancer, has hindered the widespread application of this technique. Hence, we developed a digital microfluidic system for drug screening using primary tumor cells and established a working protocol for precision medicine. Smart control logic was developed to increase the throughput of the system and decrease its footprint to parallelly screen three drugs on a 4 × 4 cm2 chip in a device measuring 23 × 16 × 3.5 cm3. We validated this method in an MDA-MB-231 breast cancer xenograft mouse model and liver cancer specimens from patients, demonstrating tumor suppression in mice/patients treated with drugs that were screened to be effective on individual primary tumor cells. Mice treated with drugs screened on-chip as ineffective exhibited similar results to those in the control groups. The effective drug identified through on-chip screening demonstrated consistency with the absence of mutations in their related genes determined via exome sequencing of individual tumors, further validating this protocol. Therefore, this technique and system may promote advances in precision medicine for cancer treatment and, eventually, for any disease.

Biosensor-Enhanced Organ-on-a-Chip Models for Investigating Glioblastoma Tumor Microenvironment Dynamics.

Glioblastoma, an aggressive primary brain tumor, poses a significant challenge owing to its dynamic and intricate tumor microenvironment. This review investigates the innovative integration of biosensor-enhanced organ-on-a-chip (OOC) models as a novel strategy for an in-depth exploration of glioblastoma tumor microenvironment dynamics. In recent years, the transformative approach of incorporating biosensors into OOC platforms has enabled real-time monitoring and analysis of cellular behaviors within a controlled microenvironment. Conventional in vitro and in vivo models exhibit inherent limitations in accurately replicating the complex nature of glioblastoma progression. This review addresses the existing research gap by pioneering the integration of biosensor-enhanced OOC models, providing a comprehensive platform for investigating glioblastoma tumor microenvironment dynamics. The applications of this combined approach in studying glioblastoma dynamics are critically scrutinized, emphasizing its potential to bridge the gap between simplistic models and the intricate in vivo conditions. Furthermore, the article discusses the implications of biosensor-enhanced OOC models in elucidating the dynamic features of the tumor microenvironment, encompassing cell migration, proliferation, and interactions. By furnishing real-time insights, these models significantly contribute to unraveling the complex biology of glioblastoma, thereby influencing the development of more accurate diagnostic and therapeutic strategies.

Assessing personalized responses to anti-PD-1 treatment using patient-derived lung tumor-on-chip.

There is a compelling need for approaches to predict the efficacy of immunotherapy drugs. Tumor-on-chip technology exploits microfluidics to generate 3D cell co-cultures embedded in hydrogels that recapitulate simplified tumor ecosystems. Here, we present the development and validation of lung tumor-on-chip platforms to quickly and precisely measure ex vivo the effects of immune checkpoint inhibitors on T cell-mediated cancer cell death by exploiting the power of live imaging and advanced image analysis algorithms. The integration of autologous immunosuppressive FAP+ cancer-associated fibroblasts impaired the response to anti-PD-1, indicating that tumors-on-chips are capable of recapitulating stroma-dependent mechanisms of immunotherapy resistance. For a small cohort of non-small cell lung cancer patients, we generated personalized tumors-on-chips with their autologous primary cells isolated from fresh tumor samples, and we measured the responses to anti-PD-1 treatment. These results support the power of tumor-on-chip technology in immuno-oncology research and open a path to future clinical validations.

Establishment of a novel microfluidic co-culture system for simultaneous analysis of multiple indicators of gefitinib sensitivity in colorectal cancer cells.

The therapeutic effect of gefitinib on colorectal cancer (CRC) is unclear, but it has been reported that stromal cells in the tumor microenvironment may have an impact on drug sensitivity. Herein, we established a microfluidic co-culture system and explored the sensitivity of CRC cells co-cultured with cancer-associated fibroblasts (CAFs) to gefitinib. The system consisted of a multichannel chip and a Petri dish. The chambers in the chip and dish were designed to continuously supply nutrients for long-term cell survival and create chemokine gradients for driving cell invasion without any external equipment. Using this system, the proliferation and invasiveness of cells were simultaneously evaluated by quantifying the area of cells and the migration distance of cells. In addition, the system combined with live cell workstation could evaluate the dynamic drug response of co-cultured cells and track individual cell trajectories in real-time. When CRC cells were co-cultured with CAFs, CAFs promoted CRC cell proliferation and invasion and reduced the sensitivity of cells to gefitinib through the exosomes secreted by CAFs. Furthermore, the cells that migrated out of the chip were collected, and EMT-related markers were determined by immunofluorescent and western blot assays. The results demonstrated that CAFs affected the response of CRC cells to gefitinib by inducing EMT, providing new ideas for further research on the resistance mechanism of gefitinib. This suggests that targeting CAFs or exosomes might be a new approach to enhance CRC sensitivity to gefitinib, and our system could be a novel platform for investigating the crosstalk between tumor cells and CAFs and understanding multiple biological changes of the tumor cells in the tumor microenvironment.

Multiplexed Opto-Microfluidic Biosensing: Advanced Platform for Prostate Cancer Detection.

Cancer stands as a prominent global cause of mortality, necessitating early detection to augment survival rates and alleviate economic burdens on healthcare systems. In particular, prostate cancer (PCa), impacting 1.41 million men globally in 2020, accentuates the demand for sensitive and cost-effective detection methods beyond traditional prostate-specific antigen (PSA) testing. While clinical techniques exhibit limitations, biosensors emerge as compact, user-friendly alternatives to traditional laboratory approaches. However, existing biosensors predominantly concentrate on PSA detection, prompting the necessity for advancing toward multiplex sensing platforms. This study introduces a compact opto-microfluidic sensor featuring a substrate of gold nanospikes, fabricated via electrodeposition, for enhanced sensitivity. Embedded within a microfluidic chip, this nanomaterial enables the precise and concurrent measurement of PSA, alongside two complementary PCa biomarkers, matrix metalloproteinase-2 (MMP-2) and anti-α-methylacyl-CoA racemase (anti-AMACR) in diluted human plasma, offering a comprehensive approach to PSA analysis. Taking advantage of the localized surface plasmon resonance principle, this biosensor offers robustness and sensitivity in real sample analysis without the need for labeling agents. With the limit of detection at 0.22, 0.37, and 0.18 ng/mL for PSA, MMP-2, and anti-AMACR, respectively, this biosensing platform holds promise for point-of-care analysis, underscoring its potential impact on medical diagnostics.

Noninvasive and Continuous Monitoring of On-Chip Stem Cell Osteogenesis Using a Reusable Electrochemical Immunobiosensor.

Noninvasive monitoring of biofabricated tissues during the biomanufacturing process is needed to obtain reproducible, healthy, and functional tissues. Measuring the levels of biomarkers secreted from tissues is a promising strategy to understand the status of tissues during biofabrication. Continuous and real-time information from cultivated tissues enables users to achieve scalable manufacturing. Label-free biosensors are promising candidates for detecting cell secretomes since they can be noninvasive and do not require labor-intensive processes such as cell lysing. Moreover, most conventional monitoring techniques are single-use, conducted at the end of the fabrication process, and, challengingly, are not permissive to in-line and continual detection. To address these challenges, we developed a noninvasive and continual monitoring platform to evaluate the status of cells during the biofabrication process, with a particular focus on monitoring the transient processes that stem cells go through during in vitro differentiation over extended periods. We designed and evaluated a reusable electrochemical immunosensor with the capacity for detecting trace amounts of secreted osteogenic markers, such as osteopontin (OPN). The sensor has a low limit of detection (LOD), high sensitivity, and outstanding selectivity in complex biological media. We used this OPN immunosensor to continuously monitor on-chip osteogenesis of human mesenchymal stem cells (hMSCs) cultured 2D and 3D hydrogel constructs inside a microfluidic bioreactor for more than a month and were able to observe changing levels of OPN secretion during culture. The proposed platform can potentially be adopted for monitoring a variety of biological applications and further developed into a fully automated system for applications in advanced cellular biomanufacturing.

Spatially controlled diffusion range of tumor-associated angiogenic factors to develop a tumor model using a microfluidic resistive circuit.

Developing a tumor model with vessels has been a challenge in microfluidics. This difficulty is because cancer cells can overgrow in a co-culture system. The up-regulation of anti-angiogenic factors during the initial tumor development can hinder neovascularization. The standard method is to develop a quiescent vessel network before loading a tumor construct in an adjacent chamber, which simulates the interaction between a tumor and its surrounding vessels. Here, we present a new method that allows a vessel network and a tumor to develop simultaneously in two linked chambers. The physiological environment of these two chambers is controlled by a microfluidic resistive circuit using two symmetric long microchannels. Applying the resistive circuit, a diffusion-dominated environment with a small 2-D pressure gradient is created across the two chambers with velocity <10.9 nm s-1 and Péclet number <6.3 × 10-5. This 2-D pressure gradient creates a V-shaped velocity clamp to confine the tumor-associated angiogenic factors at pores between the two chambers, and it has two functions. At the early stage, vasculogenesis is stimulated to grow a vessel network in the vessel chamber with minimal influence from the tumor that is still developed in the adjacent chamber. At the post-tumor-development stage, the induced steep concentration gradient at pores mimics vessel-tumor interactions to stimulate angiogenesis to grow vessels toward the tumor. Applying this method, we demonstrate that vasculogenic vessels can grow first, followed by stimulating angiogenesis. Angiogenic vessels can grow into stroma tissue up to 1.3 mm long, and vessels can also grow into or wrap around a 625 µm tumor spheroid or a tumor tissue developed from a cell suspension. In summary, our study suggests that the interactions between a developing vasculature and a growing tumor must be controlled differently throughout the tissue development process, including at the early stage when vessels are still forming and at the later stage when the tumor needs to interact with the vessels.

Drug testing of monodisperse arrays of live microdissected tumors using a valved multiwell microfluidic platform.

Cancer drug testing in animals is an extremely poor predictor of the drug's safety and efficacy observed in humans. Hence there is a pressing need for functional testing platforms that better predict traditional and immunotherapy responses in human, live tumor tissue or tissue constructs, and at the same time are compatible with the use of mouse tumor tissue to facilitate building more accurate disease models. Since many cancer drug actions rely on mechanisms that depend on the tumor microenvironment (TME), such platforms should also retain as much of the native TME as possible. Additionally, platforms based on miniaturization technologies are desirable to reduce animal use and sensitivity to human tissue scarcity. Present high-throughput testing platforms that have some of these features, e.g. based on patient-derived tumor organoids, require a growth step that alters the TME. On the other hand, microdissected tumors (µDTs) or "spheroids" that retain an intact TME have shown promising responses to immunomodulators acting on native immune cells. However, difficult tissue handling after microdissection has reduced the throughput of drug testing on µDTs, thereby constraining the inherent advantages of producing numerous TME-preserving units of tissue for drug testing. Here we demonstrate a microfluidic 96-well platform designed for drug treatment of hundreds of similarly-sized, cuboidal µDTs ("cuboids") produced from a single tumor sample. The platform organizes a monodisperse array of four cuboids per well in 384 hydrodynamic traps. The microfluidic device, entirely fabricated in thermoplastics, features 96 microvalves that fluidically isolate each well after the cuboid loading step for straightforward multi-drug testing. Since our platform makes the most of scarce tumor tissue, it can potentially be applied to human biopsies that preserve the human TME while minimizing animal testing.

Investigation of the in vitro toxic effects induced by real-time aerosol of electronic cigarette solvents using microfluidic chips.

The safety of propylene glycol (PG) and vegetable glycerin (VG) as solvents in electronic cigarette liquid has received increasing attention and discussion. However, the conclusions derived from toxicity assessments conducted through animal experiments and traditional in vitro methodologies have consistently been contentious. This study constructed an original real-time aerosol exposure system, centered around a self-designed microfluidic bionic-lung chip, to assess the biological effects following exposure to aerosols from different solvents (PG, PG/VG mixture alone and PG/VG mixture in combination with nicotine) on BEAS-2B cells. The study aimed to investigate the impact of aerosols from different solvents on gene expression profiles, intracellular biomarkers (i.e., reactive oxygen species content, nitric oxide content, and caspase-3/7 activity), and extracellular biomarkers (i.e., IL-6, IL-8, TNF-α, and malondialdehyde) of BEAS-2B cells on-chip. Transcriptome analyses suggest that ribosomal function could serve as a potential target for the impact of aerosols derived from various solvents on the biological responses of BEAS-2B cells on-chip. And the results showed that aerosols of PG/VG mixtures had significantly less effect on intracellular and extracellular biomarkers in BEAS-2B cells than aerosols of PG, whereas increasing nicotine levels might elevate these effects of aerosol from PG/VG mixture.

Magnetic Nanoparticle-Based Microfluidic Platform for Automated Enrichment of High-Purity Extracellular Vesicles.

Extracellular vesicles (EVs) are available in various biological fluids and have highly heterogeneous sizes, origins, contents, and functions. Rapid enrichment of high-purity EVs remains crucial for enhancing research on EVs in tumors. In this work, we present a magnetic nanoparticle-based microfluidic platform (ExoCPR) for on-chip isolation, purification, and mild recovery of EVs from cell culture supernatant and plasma within 29 min. The ExoCPR chip integrates bubble-driven micromixers and immiscible filtration assisted by surface tension (IFAST) technology. The bubble-driven micromixer enhances the mixing between immunomagnetic beads and EVs, eliminating the need for manual pipetting or off-chip oscillatory incubation. The high-purity EVs were obtained after passing through the immiscible phase interface where hydrophilic or hydrophobic impurities nonspecifically bound to SIMI were removed. The ExoCPR chip had a capture efficiency of 75.8% and a release efficiency of 62.7% for model EVs. We also demonstrated the powerful performance of the ExoCPR in isolating EVs from biological samples (>90% purity). This chip was further employed in clinical plasma samples and showed that the number of GPC3-positive EVs isolated from hepatocellular carcinoma patients was significantly higher than that of healthy individuals. This ExoCPR chip may provide a promising tool for EV-based liquid biopsy and other fundamental research.

Time-encoded electrical detection of trace RNA biomarker by integrating programmable molecular amplifier on chip.

One of the serious challenges facing modern point-of-care (PoC) molecular diagnostic platforms relate to reliable detection of low concentration biomarkers such as nucleic acids or proteins in biological samples. Non-specific analyte-receptor interactions due to competitive binding in the presence of abundant molecules, inefficient mass transport and very low number of analyte molecules in sample volume, in general pose critical hurdles for successful implementation of such PoC platforms for clinical use. Focusing on these specific challenges, this work reports a unique PoC biosensor that combines the advantages of nanoscale biologically-sensitive field-effect transistor arrays (BioFET-arrays) realized in a wafer-scale top-down nanofabrication as high sensitivity electrical transducers with that of sophisticated molecular programs (MPs) customized for selective recognition of analyte miRNAs and amplification resulting in an overall augmentation of signal transduction strategy. The MPs realize a programmable universal molecular amplifier (PUMA) in fluidic matrix on chip and provide a biomarker-triggered exponential release of small nucleic acid sequences easily detected by receptor-modified BioFETs. A common miRNA biomarker LET7a was selected for successful demonstration of this novel biosensor, achieving limit of detection (LoD) down to 10 fM and wide dynamic ranges (10 pM-10 nM) in complex physiological solutions. As the determination of biomarker concentration is implemented by following the electrical signal related to analyte-triggered PUMA in time-domain instead of measuring the threshold shifts of BioFETs, and circumvents direct hybridization of biomarkers at transducer surface, this new strategy also allows for multiple usage (>3 times) of the biosensor platform suggesting exceptional cost-effectiveness for practical use.