Enrichment of T-lymphocytes from leukemic blood using inertial microfluidics toward improved chimeric antigen receptor-T cell manufacturing.

Chimeric antigen receptor cell therapy is a successful immunotherapy for the treatment of blood cancers. However, hurdles in their manufacturing remain including efficient isolation and purification of the T-cell starting material. Herein, we describe a one-step separation based on inertial spiral microfluidics for efficient enrichment of T-cells in B-cell acute lymphoblastic leukemia (ALL) and B-cell chronic lymphocytic leukemia patient's samples. In healthy donors used to optimize the process, the lymphocyte purity was enriched from 65% (SD ± 0.2) to 91% (SD ± 0.06) and T-cell purity was enriched from 45% (SD ± 0.1) to 73% (SD ± 0.02). Leukemic samples had higher starting B-cells compared to the healthy donor samples. Efficient enrichment and recovery of lymphocytes and T-cells were achieved in ALL samples with B-cells, monocytes and leukemic blasts depleted by 80% (SD ± 0.09), 89% (SD ± 0.1) and 74% (SD ± 0.09), respectively, and a 70% (SD ± 0.1) T-cell recovery. Chronic lymphocytic leukemia samples had lower T-cell numbers, and the separation process was less efficient compared to the ALL. This study demonstrates the use of inertial microfluidics for T-cell enrichment and depletion of B-cell blasts in ALL, suggesting its potential to address a key bottleneck of the chimeric antigen receptor-T manufacturing workflow.

Advanced models for respiratory disease and drug studies.

The global burden of respiratory diseases is enormous, with many millions of people suffering and dying prematurely every year. The global COVID-19 pandemic witnessed recently, along with increased air pollution and wildfire events, increases the urgency of identifying the most effective therapeutic measures to combat these diseases even further. Despite increasing expenditure and extensive collaborative efforts to identify and develop the most effective and safe treatments, the failure rates of drugs evaluated in human clinical trials are high. To reverse these trends and minimize the cost of drug development, ineffective drug candidates must be eliminated as early as possible by employing new, efficient, and accurate preclinical screening approaches. Animal models have been the mainstay of pulmonary research as they recapitulate the complex physiological processes, Multiorgan interplay, disease phenotypes of disease, and the pharmacokinetic behavior of drugs. Recently, the use of advanced culture technologies such as organoids and lung-on-a-chip models has gained increasing attention because of their potential to reproduce human diseased states and physiology, with clinically relevant responses to drugs and toxins. This review provides an overview of different animal models for studying respiratory diseases and evaluating drugs. We also highlight recent progress in cell culture technologies to advance integrated models and discuss current challenges and present future perspectives.

Thiolate DNAzymes on Gold Nanoparticles for Isothermal Amplification and Detection of Mesothelioma-derived Exosomal PD-L1 mRNA.

Catalytic DNAzymes have been used for isothermal amplification and rapid detection of nucleic acids, holding the potential for point-of-care testing applications. However, when Subzymes (universal substrate and DNAzyme) are tethered to the polystyrene magnetic microparticles via biotin-streptavidin bonds, the residual free Subzymes are often detached from the microparticle surface, which causes a significant degree of false positives. Here, we attached dithiol-modified Subzyme to gold nanoparticle and improved the limit of detection (LoD) by 200 times compared to that using magnetic microparticles. As a proof of concept, we applied our new method for the detection of exosomal programed cell-death ligand 1 (PD-L1) RNA. As the classical immune checkpoint, molecule PD-L1, found in small extracellular vesicles (sEVs, traditionally called exosomes), can reflect the antitumor immune response for predicting immunotherapy response. We achieved the LoD as low as 50 fM in detecting both the RNA homologous to the PD-L1 gene and exosomal PD-L1 RNAs extracted from epithelioid and nonepithelioid subtypes of mesothelioma cell lines, which only takes 8 min of reaction time. As the first application of isothermal DNAzymes for detecting exosomal PD-L1 RNA, this work suggests new point-of-care testing potentials toward clinical translations.

Machine learning reveals mesenchymal breast carcinoma cell adaptation in response to matrix stiffness.

Epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition (MET), are believed to play key roles in facilitating the metastatic cascade. Metastatic lesions often exhibit a similar epithelial-like state to that of the primary tumour, in particular, by forming carcinoma cell clusters via E-cadherin-mediated junctional complexes. However, the factors enabling mesenchymal-like micrometastatic cells to resume growth and reacquire an epithelial phenotype in the target organ microenvironment remain elusive. In this study, we developed a workflow using image-based cell profiling and machine learning to examine morphological, contextual and molecular states of individual breast carcinoma cells (MDA-MB-231). MDA-MB-231 heterogeneous response to the host organ microenvironment was modelled by substrates with controllable stiffness varying from 0.2kPa (soft tissues) to 64kPa (bone tissues). We identified 3 distinct morphological cell types (morphs) varying from compact round-shaped to flattened irregular-shaped cells with lamellipodia, predominantly populating 2-kPa and >16kPa substrates, respectively. These observations were accompanied by significant changes in E-cadherin and vimentin expression. Furthermore, we demonstrate that the bone-mimicking substrate (64kPa) induced multicellular cluster formation accompanied by E-cadherin cell surface localisation. MDA-MB-231 cells responded to different substrate stiffness by morphological adaptation, changes in proliferation rate and cytoskeleton markers, and cluster formation on bone-mimicking substrate. Our results suggest that the stiffest microenvironment can induce MET.

Simple-to-Operate Approach for Single Cell Analysis Using a Hydrophobic Surface and Nanosized Droplets.

Microfluidics-based technologies for single-cell analysis are becoming increasingly important tools in biological studies. With the increasing sophistication of microfluidics, cellular barcoding techniques, and next-generation sequencing, a more detailed picture of cellular subtype is emerging. Unfortunately, the majority of the methods developed for single-cell analysis are high-throughput and not suitable for rare cell analysis as they require a high input cell number. Here, we report a low-cost and reproducible method for rare single-cell analysis using a highly hydrophobic surface and nanosized static droplets. Our method allows rapid and efficient on-chip single-cell lysis and subsequent collection of genetic materials in nanoliter droplets using a micromanipulator or a laboratory pipette before subsequent genetic analysis. We show precise isolation of single cancer cells with high purity using two different strategies (i- cytospin and ii- static droplet array) for subsequent RNA analysis using droplet digital polymerase chain reaction (PCR) and real-time PCR. Our highly controlled isolation method opens a new avenue for the study of subcellular functional mechanisms, enabling the identification of rare cells of potential functional or pathogenic consequence.

An easy-to-operate method for single-cell isolation and retrieval using a microfluidic static droplet array.

In-depth study of cellular heterogeneity of rare cells (e.g. circulating tumour cells (CTCs) and circulating foetal cells (CFCs)) is greatly needed in disease management but has never been completely explored due to the current technological limitations. We have developed a retrieval method for single-cell detection using a static droplet array (SDA) device through liquid segmentation with almost no sample loss. We explored the potential of using SDA for low sample input and retrieving the cells of interest using everyday laboratory equipment for downstream molecular analysis. This single-cell isolation and retrieval method is low-cost, rapid and provides a solution to the remaining challenge for single rare cell detection. The entire process takes less than 15 min, is easy to fabricate and allows for on-chip analysis of cells in nanolitre droplets and retrieval of desired droplets. To validate the applicability of our device and method, we mimicked detection of single CTCs by isolating and retrieving single cells and perform real-time PCR on their mRNA contents.

Affibody Functionalized Beads for the Highly Sensitive Detection of Cancer Cell-Derived Exosomes.

Exosomes belong to the class of extracellular vesicles of endocytic origin, which are regarded as a promising source of cancer biomarkers in liquid biopsy. As a result, an accurate, sensitive, and specific quantification of these nano-sized particles is of significant importance. Affinity-based approaches are recognized as the most valuable technique for exosome isolation and characterization. Indeed, Affibody biomolecules are a type of protein scaffold engineered with small size and enjoy the features of high thermal stability, affinity, and specificity. While the utilization of antibodies, aptamers, and other biologically active substances for exosome detection has been reported widely, there are no reports describing Affibody molecules' usage for exosome detection. In this study, for the first time, we have proposed a novel strategy of using Affibody functionalized microbeads (AffiBeads) for exosome detection with a high degree of efficiency. As a proof-of-concept, anti-EGFR-AffiBeads were fabricated and applied to capture and detect human lung A549 cancer cell-derived EGFR-positive exosomes using flow cytometry and fluorescent microscopy. Moreover, the capture efficiency of the AffiBeads were compared with its counterpart antibody. Our results showed that the Affibody probe had a detection limit of 15.6 ng exosomes per mL (~12 exosomes per AffiBead). The approach proposed in the current study can be used for sensitive detection of low expression level markers on tumor-derived exosomes, providing a basis for early-stage cancer diagnosis.

A Novel Microfluidic Device-Based Neurite Outgrowth Inhibition Assay Reveals the Neurite Outgrowth-Promoting Activity of Tropomyosin Tpm3.1 in Hippocampal Neurons.

Overcoming neurite inhibition is integral for restoring neuronal connectivity after CNS injury. Actin dynamics are critical for neurite growth cone formation and extension. The tropomyosin family of proteins is a regarded as master regulator of actin dynamics. This study investigates tropomyosin isoform 3.1 (Tpm3.1) as a potential candidate for overcoming an inhibitory substrate, as it is known to influence neurite branching and outgrowth. We designed a microfluidic device that enables neurons to be grown adjacent to an inhibitory substrate, Nogo-66. Results show that neurons, overexpressing hTpm3.1, have an increased propensity to overcome Nogo-66 inhibition. We propose Tpm3.1 as a potential target for promoting neurite growth in an inhibitory environment in the central nervous system.

Single-cell profiling approaches to probing tumor heterogeneity.

Tumor heterogeneity is a major hindrance in cancer classification, diagnosis and treatment. Recent technological advances have begun to reveal the true extent of its heterogeneity. Single-cell analysis (SCA) is emerging as an important approach to detect variations in morphology, genetic or proteomic expression. In this review, we revisit the issue of inter- and intra-tumor heterogeneity, and list various modes of SCA techniques (cell-based, nucleic acid-based, protein-based, metabolite-based and lipid-based) presently used for cancer characterization. We further discuss the advantages of SCA over pooled cell analysis, as well as the limitations of conventional techniques. Emerging trends, such as high-throughput sequencing, are also mentioned as improved means for cancer profiling. Collectively, these applications have the potential for breakthroughs in cancer treatment.

Large-Volume Microfluidic Cell Sorting for Biomedical Applications.

Microfluidic cell-separation technologies have been studied for almost two decades, but the limited throughput has restricted their impact and range of application. Recent advances in microfluidics enable high-throughput cell sorting and separation, and this has led to various novel diagnostic and therapeutic applications that previously had been impossible to implement using microfluidics technologies. In this review, we focus on recent progress made in engineering large-volume microfluidic cell-sorting methods and the new applications enabled by them.

Numerical and experimental study of capillary-driven flow of PCR solution in hybrid hydrophobic microfluidic networks.

Capillary-driven microfluidics is essential for development of point-of-care diagnostic micro-devices. Polymerase chain reaction (PCR)-based micro-devices are widely developed and used in such point-of-care settings. It is imperative to characterize the fluid parameters of PCR solution for designing efficient capillary-driven microfluidic networks. Generally, for numeric modelling, the fluid parameters of PCR solution are approximated to that of water. This procedure leads to inaccurate results, which are discrepant to experimental data. This paper describes mathematical modeling and experimental validation of capillary-driven flow inside Poly-(dimethyl) siloxane (PDMS)-glass hybrid micro-channels. Using experimentally measured PCR fluid parameters, the capillary meniscus displacement in PDMS-glass microfluidic ladder network is simulated using computational fluid dynamic (CFD), and experimentally verified to match with the simulated data.

An ultra-high-throughput spiral microfluidic biochip for the enrichment of circulating tumor cells.

The detection and characterization of rare circulating tumor cells (CTCs) from the blood of cancer patients can potentially provide critical insights into tumor biology and hold great promise for cancer management. The ability to collect a large number of viable CTCs for various downstream assays such as quantitative measurements of specific biomarkers or targeted somatic mutation analysis is increasingly important in medical oncology. Here, we present a simple yet reliable microfluidic device for the ultra-high-throughput, label-free, size-based isolation of CTCs from clinically relevant blood volumes. The fast processing time of the technique (7.5 mL blood in less than 10 min) and the ability to collect more CTCs from larger blood volumes lends itself to a broad range of potential genomic and transcriptomic applications. A critical advantage of this protocol is the ability to return all fractions of blood (i.e., plasma (centrifugation), CTCs and white blood cells (WBCs) (size-based sorting)) that can be utilized for diverse biomarker studies or time-sensitive molecular assays such as RT-PCR. The clinical use of this biochip was demonstrated by detecting CTCs from 100% (10/10) of blood samples collected from patients with advanced-stage metastatic breast and lung cancers. The CTC recovery rate ranged from 20 to 135 CTCs mL(-1) and obtained under high purity (of 1 CTC out of every 30-100 WBCs which gives ∼4 log depletion of WBCs). They were identified with immunofluorescence assays (pan-cytokeratin+/CD45-) and molecular probes such as HER2/neu.

Spatial proteomic profiling of tumor and stromal compartments in non-small-cell lung cancer identifies signatures associated with overall survival.

Objectives: Non-small-cell lung carcinoma (NSCLC) is the most prevalent and lethal form of lung cancer. The need for biomarker-informed stratification of targeted therapies has underpinned the need to uncover the underlying properties of the tumor microenvironment (TME) through high-plex quantitative assays. Methods: In this study, we profiled resected NSCLC tissues from 102 patients by targeted spatial proteomics of 78 proteins across tumor, immune activation, immune cell typing, immune-oncology, drug targets, cell death and PI3K/AKT modules to identify the tumor and stromal signatures associated with overall survival (OS). Results: Survival analysis revealed that stromal CD56 (HR = 0.384, P = 0.06) and tumoral TIM3 (HR = 0.703, P = 0.05) were associated with better survival in univariate Cox models. In contrast, after adjusting for stage, BCLXL (HR = 2.093, P = 0.02) and cleaved caspase 9 (HR = 1.575, P = 0.1) negatively influenced survival. Delta testing indicated the protective effect of TIM-3 (HR = 0.614, P = 0.04) on OS. In multivariate analysis, CD56 (HR = 0.172, P = 0.001) was associated with better survival in the stroma, while B7.H3 (HR = 1.72, P = 0.008) was linked to poorer survival in the tumor. Conclusions: Deciphering the TME using high-plex spatially resolved methods is giving us new insights into compartmentalised tumor and stromal protein signatures associated with clinical endpoints in NSCLC.

Zerumbone-incorporated liquid crystalline nanoparticles inhibit proliferation and migration of non-small-cell lung cancer in vitro.

Lung cancer is the second most prevalent type of cancer and is responsible for the highest number of cancer-related deaths worldwide. Non-small-cell lung cancer (NSCLC) makes up the majority of lung cancer cases. Zerumbone (ZER) is natural compound commonly found in the roots of Zingiber zerumbet which has recently demonstrated anti-cancer activity in both in vitro and in vivo studies. Despite their medical benefits, ZER has low aqueous solubility, poor GI absorption and oral bioavailability that hinders its effectiveness. Liquid crystalline nanoparticles (LCNs) are novel drug delivery carrier that have tuneable characteristics to enhance and ease the delivery of bioactive compounds. This study aimed to formulate ZER-loaded LCNs and investigate their effectiveness against NSCLC in vitro using A549 lung cancer cells. ZER-LCNs, prepared in the study, inhibited the proliferation and migration of A549 cells. These inhibitory effects were superior to the effects of ZER alone at a concentration 10 times lower than that of free ZER, demonstrating a potent anti-cancer activity of ZER-LCNs. The underlying mechanisms of the anti-cancer effects by ZER-LCNs were associated with the transcriptional regulation of tumor suppressor genes P53 and PTEN, and metastasis-associated gene KRT18. The protein array data showed downregulation of several proliferation associated proteins such as AXL, HER1, PGRN, and BIRC5 and metastasis-associated proteins such as DKK1, CAPG, CTSS, CTSB, CTSD, and PLAU. This study provides evidence of potential for increasing the potency and effectiveness of ZER with LCN formulation and developing ZER-LCNs as a treatment strategy for mitigation and treatment of NSCLC.

Zerumbone liquid crystalline nanoparticles protect against oxidative stress, inflammation and senescence induced by cigarette smoke extract in vitro.

The purpose of this study was to evaluate the potential of zerumbone-loaded liquid crystalline nanoparticles (ZER-LCNs) in the protection of broncho-epithelial cells and alveolar macrophages against oxidative stress, inflammation and senescence induced by cigarette smoke extract in vitro. The effect of the treatment of ZER-LCNs on in vitro cell models of cigarette smoke extract (CSE)-treated mouse RAW264.7 and human BCi-NS1.1 basal epithelial cell lines was evaluated for their anti-inflammatory, antioxidant and anti-senescence activities using colorimetric and fluorescence-based assays, fluorescence imaging, RT-qPCR and proteome profiler kit. The ZER-LCNs successfully reduced the expression of pro-inflammatory markers including Il-6, Il-1ß and Tnf-α, as well as the production of nitric oxide in RAW 264.7 cells. Additionally, ZER-LCNs successfully inhibited oxidative stress through reduction of reactive oxygen species (ROS) levels and regulation of genes, namely GPX2 and GCLC in BCi-NS1.1 cells. Anti-senescence activity of ZER-LCNs was also observed in BCi-NS1.1 cells, with significant reductions in the expression of SIRT1, CDKN1A and CDKN2A. This study demonstrates strong in vitro anti-inflammatory, antioxidative and anti-senescence activities of ZER-LCNs paving the path for this formulation to be translated into a promising therapeutic agent for chronic respiratory inflammatory conditions including COPD and asthma.

A Microfluidic Approach for Enrichment and Single-Cell Characterization of Circulating Tumor Cells from Peripheral Blood.

The emergence of enabling technologies for the analysis of circulating tumor cells has been shedding new lights into cancer management in the recent years. However, majority of the technologies developed suffer from excessive cost, time-consuming workflows, and reliance on specialized equipment and operators. Herein, we propose a simple workflow for the isolation and characterization of single circulating tumor cells using microfluidic devices. The entire process can be operated by a laboratory technician without relying on any microfluidic expertise and can be completed within few hours of sample collection.

A Method for Rapid, Quantitative Evaluation of Particle Sorting in Microfluidics Using Basic Cytometry Equipment.

This paper describes, in detail, a method that uses flow cytometry to quantitatively characterise the performance of continuous-flow microfluidic devices designed to separate particles. Whilst simple, this approach overcomes many of the issues with the current commonly utilised methods (high-speed fluorescent imaging, or cell counting via either a hemocytometer or a cell counter), as it can accurately assess device performance even in complex, high concentration mixtures in a way that was previously not possible. Uniquely, this approach takes advantage of pulse processing in flow cytometry to allow quantitation of cell separation efficiencies and resulting sample purities on both single cells as well as cell clusters (such as circulating tumour cell (CTC) clusters). Furthermore, it can readily be combined with cell surface phenotyping to measure separation efficiencies and purities in complex cell mixtures. This method will facilitate the rapid development of a raft of continuous flow microfluidic devices, will be helpful in testing novel separation devices for biologically relevant clusters of cells such as CTC clusters, and will provide a quantitative assessment of device performance in complex samples, which was previously impossible.

Scaling up stem cell production: harnessing the potential of microfluidic devices.

Stem cells are specialised cells characterised by their unique ability to both self-renew and transform into a wide array of specialised cell types. The widespread interest in stem cells for regenerative medicine and cultivated meat has led to a significant demand for these cells in both research and practical applications. Despite the growing need for stem cell manufacturing, the industry faces significant obstacles, including high costs for equipment and maintenance, complicated operation, and low product quality and yield. Microfluidic technology presents a promising solution to the abovementioned challenges. As an innovative approach for manipulating liquids and cells within microchannels, microfluidics offers a plethora of advantages at an industrial scale. These benefits encompass low setup costs, ease of operation and multiplexing, minimal energy consumption, and the added advantage of being labour-free. This review presents a thorough examination of the prominent microfluidic technologies employed in stem cell research and explores their promising applications in the burgeoning stem cell industry. It thoroughly examines how microfluidics can enhance cell harvesting from tissue samples, facilitate mixing and cryopreservation, streamline microcarrier production, and efficiently conduct cell separation, purification, washing, and final cell formulation post-culture.

Sperm quality metrics were improved by a biomimetic microfluidic selection platform compared to swim-up methods.

Sperm selection is an essential component of all assisted reproductive treatments (ARTs) and is by far the most neglected step in the ART workflow in regard to technological innovation. Conventional sperm selection methodologies typically produce a higher total number of sperm with variable motilities, morphologies, and levels of DNA integrity. Gold-standard techniques, including density gradient centrifugation (DGC) and swim-up (SU), have been shown to induce DNA fragmentation through introducing reactive oxygen species (ROS) during centrifugation. Here, we demonstrate a 3D printed, biologically inspired microfluidic sperm selection device (MSSP) that utilizes multiple methods to simulate a sperms journey toward selection. Sperm are first selected based on their motility and boundary-following behavior and then on their expression of apoptotic markers, yielding over 68% more motile sperm than that of previously reported methods with a lower incidence of DNA fragmentation and apoptosis. Sperm from the MSSP also demonstrated higher motile sperm recovery after cryopreservation than that of SU or neat semen. Experiments were conducted side-by-side against conventional SU methods using human semen (n = 33) and showed over an 85% improvement in DNA integrity with an average 90% reduction in sperm apoptosis. These results that the platform is easy-to-use for sperm selection and mimics the biological function of the female reproductive tract during conception.