Effective isolation and in-depth analysis of Circulating Tumour Cells (CTCs) are greatly needed in diagnosis, prognosis and monitoring of the therapeutic response of cancer patients but have not been completely fulfilled by conventional approaches. The rarity of CTCs and the lack of reliable biomarkers to distinguish them from pe-ripheral blood cells have remained outstanding challenges for their clinical implementation. Herein, we devel-oped a high throughput Static Droplet Microfluidic (SDM) device with 38,400 chambers, capable of isolating and classifying the number of metabolically active CTCs in peripheral blood at single-cell resolution. Owing to the miniaturisation and compartmentalisation capability of our device, we first demonstrated the ability to precisely measure the lactate production of different types of cancer cells inside 125 pL droplets at single-cell resolution. Furthermore, we compared the metabolomic activity of leukocytes from healthy donors to cancer cells and showed the ability to differentiate them. To further prove the clinical relevance, we spiked cancer cell lines in human healthy blood and showed the possibility to detect the cancer cells from leukocytes. Lastly, we tested the workflow on 8 preclinical mammary mouse models including syngeneic 67NR (non-metastatic) and 4T1.2 (metastatic) models with Triple-Negative Breast Cancer (TNBC) as well as transgenic mouses (12-week-old MMTV-PyMT). The results have shown the ability to precisely distinguish metabolically active CTCs from the blood using the proposed SDM device. The workflow is simple and robust which can eliminate the need for specialised equipment and expertise required for single-cell analysis of CTCs and facilitate on-site metabolic screening of cancer cells.

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.

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 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.

Harvesting adherent cells from microcarriers has become one of the major challenges of the downstream bioprocessing at large scale the current method has high maintenance and operation cost, which are the results of frequent clogging, due to cell lysing effect and microcarrier cake formation on membrane-based technology. These problems hugely impede the adaptation of microcarriers technologies in large-scale cell culture and hampered the supply of cells to the clinical need. Here, we describe two 3D printing-based methods to fabricate inertial microfluidic devices for separating adherent cells from microcarriers which overcome the above-mentioned limitations. The spiral devices are employed to separate mesenchymal stem cells from the microcarriers with 99% microcarrier removal rate and 77% cell recovery rate in one round of separation.

Microbial populations play a crucial role in human health and the development of many diseases. These diseases often arise from the explosive proliferation of opportunistic bacteria, such as those in the nasal cavity. Recently, there have been increases in the prevalence of these opportunistic pathogens displaying antibiotic resistance. Thus, the study of the nasal microbiota and its bacterial diversity is critical in understanding pathogenesis and developing microbial-based therapies for well-known and emerging diseases. However, the isolation and analysis of these populations for clinical study complicates the already challenging task of identifying and profiling potentially harmful bacteria. Existing methods are limited by low sample throughput, expensive labeling, and low recovery of bacteria with ineffective removal of cells and debris. In this study, we propose a novel microfluidic channel with a zigzag configuration for enhanced isolation and detection of bacteria from human clinical nasal swabs. This microfluidic zigzag channel separates the bacteria from epithelial cells and debris by size differential focusing. As such, pure bacterial cell fractions devoid of large contaminating debris or epithelial cells are obtained. DNA sequencing performed on the separated bacteria defines the diversity and species present. This novel method of bacterial separation is simple, robust, rapid, and cost-effective and has the potential to be used for the rapid identification of bacterial cell populations from clinical samples.

Giardia is one of the most common waterborne pathogens causing around 200 × 106 diarrheal infections annually. It is of great interest to microbiological research as it is among the oldest known eukaryotic cells. Purifying Giardia from fecal samples for both research and diagnostic purposes presents one of the most difficult challenges. Traditional purification methods rely on density gradient centrifugation, membrane-based filtration, and sedimentation methods, which suffer from low recovery rates, high costs, and poor efficiency. Here, we report on the use of microfluidics to purify Giardia cysts from mouse feces. We propose a rigid spiral microfluidic device with a trapezoidal cross section to effectively separate Giardia from surrounding debris. Our characterizations reveal that the recovery rate is concentration-dependent, and our proposed device can achieve recovery rates as high as 75% with 0.75 ml/min throughput. Moreover, this device can purify Giardia from extremely turbid samples to a level where cysts are visually distinguishable with just one round of purification. This highly scalable and versatile 3D printed microfluidic device is then capable of further purifying or enhancing the recovery rate of the samples by recirculation. This device also has the potential to purify other gastrointestinal pathogens of similar size, and throughput can be significantly increased by parallelization.

Abstract Sperm Selection is an essential component of all Assisted Reproductive Treatments (ART), and is by far and large the most neglected step in the ART workflow when it comes 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 Density Gradient Centrifugation (DGC) and Swim Up (SU) have been proven to induce DNA fragmentation through the introduction of 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 towards selection. Sperm are first selected based on their motility and boundary following behavior, then on their expression of apoptotic markers, yielding over 68% more motile sperm than previously reported methods within a lower incidence of DNA fragmentation and apoptosis. Sperm from the MSSP also demonstrated higher motile sperm recovery after cryopreservation than 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 demonstrate an easy-to-use platform for sperm selection mimicking the biological function of the female reproductive tract during conception.

Cryopreservation is the final step of stem cell production before the cryostorage of the product. Conventional methods of adding cryoprotecting agents (CPA) into the cells can be manual or automated with robotic arms. However, challenging issues with these methods at industrial-scale production are the insufficient mixing of cells and CPA, leading to damage of cells, discontinuous feeding, the batch-to-batch difference in products, and, occasionally, cross-contamination. Therefore, the current study proposes an alternative way to overcome the abovementioned challenges; a highly efficient micromixer for low-cost, continuous, labour-free, and automated mixing of stem cells with CPA solutions. Our results show that our micromixer provides a more homogenous mixing of cells and CPA compared to the manual mixing method, while the cell properties, including surface markers, differentiation potential, proliferation, morphology, and therapeutic potential, are well preserved.

Microfluidic devices have shown promising applications in the bioprocessing industry. However, the lack of modularity and high cost of testing and error limit their implementation in the industry. Advances in 3D printing technologies have facilitated the conversion of microfluidic devices from research output to applicable industrial systems. Here, for the first time, we presented a 3D printed modular microfluidic system consisting of two micromixers, one spiral microfluidic separator, and one microfluidic concentrator. We showed that this system can detach and separate mesenchymal stem cells (MSCs) from microcarriers (MCs) in a short time while maintaining the cell's viability and functionality. The system can be multiplexed and scaled up to process large volumes of the industry. Importantly, this system is a closed system with no human intervention and is promising for current good manufacturing practices.

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.

The isolation of sperm cells from background cell populations and debris is an essential step in all assisted reproductive technologies. Conventional techniques for sperm recovery from testicular sperm extractions stagnate at the sample processing stage, where it can take several hours to identify viable sperm from a background of collateral cells such as white bloods cells (WBCs), red blood cells (RBCs), epithelial cells (ECs) and in some cases cancer cells. Manual identification of sperm from contaminating cells and debris is a tedious and time-consuming operation that can be suitably addressed through inertial microfluidics. Microfluidics has proven an effective technology for high-quality sperm selection based on motility. However, motility-based selection methods cannot cater for viable, non-motile sperm often present in testicular or epididymal sperm extractions and aspirations. This study demonstrates the use of a 3D printed inertial microfluidic device for the separation of sperm cells from a mixed suspension of WBCs, RBCs, ECs, and leukemic cancer cells. This technology presents a 36-fold time improvement for the recovery of sperm cells (> 96%) by separating sperm, RBCS, WBCs, ECs and cancer cells into tight bands in less than 5 min. Furthermore, microfluidic processing of sperm has no impact on sperm parameters; vitality, motility, morphology, or DNA fragmentation of sperm. Applying inertial microfluidics for non-motile sperm recovery can greatly improve the current processing procedure of testicular sperm extractions, simplifying the fertility outcomes for severe forms of male infertility that warrant the surgery.

Elasto-inertial migration in non-Newtonian fluids is a rapidly growing field with tremendous potentials for manipulating micron to submicron particles. Previous research attempts were mainly carried out in straight channels due to the complexity of particle migration, solution tuning, and data analysis in elasto-inertial microfluidics. Consequently, the combined effects of Dean drag force and solution rheology on coupled Dean drag elasto-inertial focusing phenomena have not been carefully analyzed. This study delved thoroughly into the combined effects of solution rheology and Dean drag force on elasto-inertial focusing of particles and cells within a spiral microchannel. Polyethylene oxide (PEO) of 1MDa, 2MDa, and 4MDa molecular weights were used to prepare 250, 500, and 1000 ppm non-Newtonian solutions to investigate the focusing behavior of particles and cells over a wide range of flow rates and solution rheologies. Dean coupled elasto-inertial effects were systematically investigated to demonstrate its potentials for position-adjustable and size-tunable particle and cell focusing phenomenon. Various cells and microbeads with diameters ranging from 1 to 17 μm were employed to carefully study the equilibrium position, focusing band, and migration behavior under different elastic, inertial, and Dean conditions. Following the focusing, cell viability, morphology, and growth rate were evaluated which showed cells remained undamaged from viscosity, shear rate, and chemical properties of PEO solutions. We are of the opinion that the current study can provide scientists with a better understanding of focusing phenomena in viscoelastic fluids within spiral microfluidic channels.

Telomerase activity has been demonstrated in a wide variety of most solid tumors and considered as a well-known cancer biomarker. The commonly utilized method for its detection is polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP). However, the TRAP technique suffers from false-negative results caused by the failure of PCR step. Moreover, it requires advanced equipment with a tedious and time-consuming procedure. Herein, we presented a portable nitrocellulose paper-based nanobiosensing platform for ultrafast and equipment-free detection of telomerase activity based on a simple colorimetric assay that enabled naked-eye visualization of the color change in response to enzyme activity. In this platform, hybridization was initially performed between telomere complementary oligonucleotide immobilized on gold nanoparticles (GNPs) and telomerase elongated biotinylated probe. Thereafter, the assembly was attached on activated paper strip via avidin-biotin interaction. The signal amplification was carried out by enlargement of the attached GNPs on the paper strip, forming tightly compact rod-shaped submicron structures of gold representing a visual color formation. Thanks to significant sensitivity enhancement, the color change was occurred for down to 6 cells, which can be easily observed by the naked eye. Due to the desired aspects of the developed assay including PCR-free, low cost, simple, and high sensitivity, it can be used for evaluation of telomerase activity in cell extracts for future clinical applications. Furthermore, this design has the ability to be easily integrated into lab-on-chip devices for point-of-care telomerase sensing.

During the last decade, isolation of circulating tumour cells via blood liquid biopsy of prostate cancer (PCa) has attracted significant attention as an alternative, or substitute, to conventional diagnostic tests. However, it was previously determined that localised forms of PCa shed a small number of cancer cells into the bloodstream, and a large volume of blood is required just for a single test, which is impractical. To address this issue, urine has been used as an alternative to blood for liquid biopsy as a truly non-invasive, patient-friendly test. To this end, we developed a spiral microfluidic chip capable of isolating PCa cells from the urine of PCa patients. Potential clinical utility of the chip was demonstrated using anti-Glypican-1 (GPC-1) antibody as a model of the primary antibody in immunofluorescent assay for identification and detection of the collected tumour cells. The microchannel device was first evaluated using DU-145 cells in a diluted Dulbecco’s phosphate-buffered saline sample, where it demonstrated >85 (±6) % efficiency. The microchannel proved to be functional in at least 79% of cases for capturing GPC1+ putative tumour cells from the urine of patients with localised PCa. More importantly, a correlation was found between the amount of the captured GPC1+ cells and crucial diagnostic and prognostic parameter of localised PCa—Gleason score. Thus, the technique demonstrated promise for further assessment of its diagnostic value in PCa detection, diagnosis, and prognosis.

Organ-on-a-chip is a microfluidic cell culture model that replicates key organ-specific microarchitecture and pathophysiology in vitro. The current methods to fabricate these devices rely on softlithography, which is usually tedious, laborious, and requires adroit users as well as cleanroom facilities. Recently, the use of 3D-printing technologies for the rapid fabrication of molds for polydimethylsiloxane (PDMS) casting is on the rise. However, most of the 3D-printed materials are unsuitable for PDMS casting. To address this issue, we have improved the existing techniques and introduced a modified protocol for the surface treatment of 3D-printed molds, making them ideal for repeated long-term PDMS casting. Using this protocol, we have fabricated a simple open well lung-on-a-chip model to simulate the in vivo environment of airway at air-liquid interface under dynamic condition. To validate the functionality of the developed chip, Calu-3 cells were cultured in the chip and maintained at an air-liquid interface. The model demonstrated that the cultured cells replicated the 3D culture-specific-morphology, maintained excellent barrier integrity, secreted mucus, and expressed cell surface functional P-glycoprotein; all indicative of a promising in vitro model for permeability assays, toxicological tests, and pulmonary drug delivery studies. To validate the suitability of this lung-on-a-chip in vitro model, the effects of cigarette smoke extract (CSE) on Interleukin-6 (IL-6) and Interleukin-8 (IL-8) release from cultured Calu-3 cells were examined. CSE treated cells showed significantly higher secretion of IL-6 and IL-8 over 24 h compared to the cells treated with both CSE and Budesonide, an anti-inflammatory drug. Moreover, our results illustrated that CSE reduced the expression of E-cadherin as an adherent junctional protein. In conclusion, the proposed protocol demonstrated an easy and low-cost fabrication technique which will allow a biologist with minimal technical skills to rapidly prototype molds for different/versatile organ-on-a-chip models.