The use of microfluidic sperm sorting (MFSS) systems in infertility treatment is increasing due to their practicality and ease of use. While often presented as highly effective, their efficacy in patients with varying sperm analysis results remains uncertain. In this study, we evaluated the effectiveness of MFSS compared with the swim-up (SU) technique in terms of oxygen radical levels and spermiogram parameters. Samples from each patient were processed using both methods, followed by assessments of sperm concentration, motility, morphology, DNA integrity, acrosomal status, and mitochondrial membrane potential. Participants were selected based on sperm analysis and categorized as normozoospermic (n = 40) or non-normozoospermic (n = 28). An analysis of separation techniques revealed no significant differences, except for a lower percentage of DNA-fragmented sperm in the MFSS group compared with SU within the non-normozoospermic cohort (SU: 10.0% vs. MFSS: 5.69%, p = 0.027). No differences were observed between SU and MFSS in normozoospermic men. The MFSS method is a simple technique, frequently used in laboratories, that yields good results but does not offer a substantial advantage over SU. The primary benefit of MFSS appears to be a significant reduction in the proportion of sperm with DNA fragmentation compared with SU in patients with abnormal sperm analysis results.
- MeSH
- Semen Analysis methods MeSH
- Adult MeSH
- DNA Fragmentation MeSH
- Sperm Injections, Intracytoplasmic * methods MeSH
- Humans MeSH
- Membrane Potential, Mitochondrial MeSH
- Microfluidics * methods MeSH
- Sperm Motility * MeSH
- Infertility, Male therapy MeSH
- Cell Separation * methods MeSH
- Spermatozoa * cytology metabolism MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Male MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
Proteomics provides an understanding of biological systems by enabling the detailed study of protein expression profiles, which is crucial for early disease diagnosis. Microfluidic-based proteomics enhances this field by integrating complex proteome analysis into compact and efficient systems. This review focuses on developing microfluidic chip structures for proteomics, covering on-chip sample pretreatment, protein extraction, purification, and identification in recent years. Furthermore, our work aims to inspire researchers to select proper methodologies in designing novel, efficient assays for proteomics applications by analyzing trends and innovations in this field.
- MeSH
- Biosensing Techniques instrumentation methods MeSH
- Equipment Design MeSH
- Lab-On-A-Chip Devices * MeSH
- Humans MeSH
- Microfluidics methods MeSH
- Microfluidic Analytical Techniques instrumentation MeSH
- Proteins analysis isolation & purification MeSH
- Proteome analysis isolation & purification chemistry MeSH
- Proteomics * methods MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
The review focuses on the design of detection cells, the use of microcontrollers for processing and evaluation of the detection signal, and the development of multi-detection systems for electromigration, liquid chromatography, flow-through and microfluidic techniques. A separate section is the introduction of modern 3D printing techniques and the use of new printing materials for the design of multidetection systems. In addition to traditional utilisation in separation techniques, new versions of contactless conductivity detectors are finding applications in FIA, SIA, portable and paper based analytical systems or as independent sensors. Applicationwise, C4Ds find new use in gas detection, segmented flow monitoring, as part of point of care devices, and in many other biomedical, environmental, agricultural and industrial applications.
- Publication type
- Journal Article MeSH
- Review MeSH
As organoids and organ-on-chip (OoC) systems move toward preclinical and clinical applications, there is an increased need for method validation. Using a liquid chromatography-mass spectrometry (LC-MS)-based approach, we developed a method for measuring small-molecule drugs and metabolites in the cell medium directly sampled from liver organoids/OoC systems. The LC-MS setup was coupled to an automatic filtration and filter flush system with online solid-phase extraction (SPE), allowing for robust and automated sample cleanup/analysis. For the matrix, rich in, e.g., protein, salts, and amino acids, no preinjection sample preparation steps (protein precipitation, SPE, etc.) were necessary. The approach was demonstrated with tolbutamide and its liver metabolite, 4-hydroxytolbutamide (4HT). The method was validated for analysis of cell media of human stem cell-derived liver organoids cultured in static conditions and on a microfluidic platform according to Food and Drug Administration (FDA) guidelines with regards to selectivity, matrix effects, accuracy, precision, etc. The system allows for hundreds of injections without replacing chromatography hardware. In summary, drug/metabolite analysis of organoids/OoCs can be performed robustly with minimal sample preparation.
- MeSH
- Chromatography, Liquid methods MeSH
- Solid Phase Extraction MeSH
- Mass Spectrometry methods MeSH
- Liver * metabolism MeSH
- Liquid Chromatography-Mass Spectrometry MeSH
- Small Molecule Libraries analysis metabolism chemistry MeSH
- Lab-On-A-Chip Devices MeSH
- Pharmaceutical Preparations metabolism analysis MeSH
- Humans MeSH
- Organoids * metabolism cytology MeSH
- Tolbutamide metabolism analysis MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Validation Study MeSH
Persistent infection with high-risk types of human papillomaviruses (HPV) is a major cause of cervical cancer, and an important factor in other malignancies, for example, head and neck cancer. Despite recent progress in screening and vaccination, the incidence and mortality are still relatively high, especially in low-income countries. The mortality and financial burden associated with the treatment could be decreased if a simple, rapid, and inexpensive technology for HPV testing becomes available, targeting individuals for further monitoring with increased risk of developing cancer. Commercial HPV tests available in the market are often relatively expensive, time-consuming, and require sophisticated instrumentation, which limits their more widespread utilization. To address these challenges, novel technologies are being implemented also for HPV diagnostics that include for example, isothermal amplification techniques, lateral flow assays, CRISPR-Cas-based systems, as well as microfluidics, paperfluidics and lab-on-a-chip devices, ideal for point-of-care testing in decentralized settings. In this review, we first evaluate current commercial HPV tests, followed by a description of advanced technologies, explanation of their principles, critical evaluation of their strengths and weaknesses, and suggestions for their possible implementation into medical diagnostics.
- MeSH
- Papillomavirus Infections * complications MeSH
- Humans MeSH
- Human Papillomavirus Viruses MeSH
- Uterine Cervical Neoplasms * MeSH
- Papillomaviridae genetics MeSH
- Technology MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
The application of microfluidic devices as next-generation cell and tissue culture systems has increased impressively in the last decades. With that, a plethora of materials as well as fabrication methods for these devices have emerged. Here, we describe the rapid prototyping of microfluidic devices, using micromilling and vapour-assisted thermal bonding of polymethyl methacrylate (PMMA), to create a spheroid-on-a-chip culture system. Surface roughness of the micromilled structures was assessed using scanning electron microscopy (SEM) and atomic force microscopy (AFM), showing that the fabrication procedure can impact the surface quality of micromilled substrates with milling tracks that can be readily observed in micromilled channels. A roughness of approximately 153 nm was created. Chloroform vapour-assisted bonding was used for simultaneous surface smoothing and bonding. A 30-s treatment with chloroform-vapour was able to reduce the surface roughness and smooth it to approximately 39 nm roughness. Subsequent bonding of multilayer PMMA-based microfluidic chips created a durable assembly, as shown by tensile testing. MDA-MB-231 breast cancer cells were cultured as multicellular tumour spheroids in the device and their characteristics evaluated using immunofluorescence staining. Spheroids could be successfully maintained for at least three weeks. They consisted of a characteristic hypoxic core, along with expression of the quiescence marker, p27kip1. This core was surrounded by a ring of Ki67-positive, proliferative cells. Overall, the method described represents a versatile approach to generate microfluidic devices compatible with biological applications.
Three-dimensional (3D) cell cultures are to date the gold standard in biomedical research fields due to their enhanced biological functions compared to conventional two-dimensional (2D) cultures. 3D cell spheroids, as well as organoids, are better suited to replicate tissue functions, which enables their use both as in vitro models for basic research and toxicology, as well as building blocks used in tissue/organ biofabrication approaches. Culturing 3D spheroids from bone-derived cells is an emerging technology for both disease modelling and drug screening applications. Bone tissue models are mainly limited by the implementation of sophisticated devices and procedures that can foster a tissue-specific 3D cell microenvironment along with a dynamic cultivation regime. In this study, we consequently developed, optimized and characterized an advanced perfused microfluidic platform to improve the reliability of 3D bone cell cultivation and to enhance aspects of bone tissue maturation in vitro. Moreover, biomechanical stimulation generated by fluid flow inside the arrayed chamber, was used to mimic a more dynamic cell environment emulating a highly vascularized bone we expected to improve the osteogenic 3D microenvironment in the developed multifunctional spheroid-array platform. The optimized 3D cell culture protocols in our murine bone-on-a-chip spheroid model exhibited increased mineralization and viability compared to static conditions. As a proof-of-concept, we successfully confirmed on the beneficial effects of a dynamic culture environment on osteogenesis and used our platform for analysis of bone-derived spheroids produced from primary human pre-osteoblasts. To conclude, the newly developed system represents a powerful tool for studying human bone patho/physiology in vitro under more relevant and dynamic culture conditions converging the advantages of microfluidic platforms with multi-spheroid array technologies.
- Publication type
- Journal Article MeSH
Live cells act as biological lenses and can be employed as real-world optical components in bio-hybrid systems. Imaging at nanoscale, optical tweezers, lithography and also photonic waveguiding are some of the already proven functionalities, boosted by the advantage that cells are fully biocompatible for intra-body applications. So far, various cell types have been studied for this purpose, such as red blood cells, bacterial cells, stem cells and yeast cells. White Blood Cells (WBCs) play a very important role in the regulation of the human body activities and are usually monitored for assessing its health. WBCs can be considered bio-lenses but, to the best of our knowledge, characterization of their optical properties have not been investigated yet. Here, we report for the first time an accurate study of two model classes of WBCs (i.e., monocytes and lymphocytes) by means of a digital holographic microscope coupled with a microfluidic system, assuming WBCs bio-lens characteristics. Thus, quantitative phase maps for many WBCs have been retrieved in flow-cytometry (FC) by achieving a significant statistical analysis to prove the enhancement in differentiation among sphere-like bio-lenses according to their sizes (i.e., diameter d) exploiting intensity parameters of the modulated light in proximity of the cell optical axis. We show that the measure of the low intensity area (S: Iz
- MeSH
- Holography * methods MeSH
- Humans MeSH
- Lymphocytes MeSH
- Microscopy * methods MeSH
- Monocytes MeSH
- Optics and Photonics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Animal testing has made a significant and unequalled contribution to important discoveries and advancements in the fields of research, medicine, vaccine development, and drug discovery. Each year, millions of animals are sacrificed for various experiments, and this is an ongoing process. However, the debate on the ethical and sensible usage of animals in in vivo experimentation is equally important. The need to explore and adopt newer alternatives to animals so as to comply with the goal of reduce, refine, and replace needs attention. Besides the ever-increasing debate on ethical issues, animal research has additional drawbacks (need of trained labour, requirement of breeding area, lengthy protocols, high expenses, transport barriers, difficulty to extrapolate data from animals to humans, etc.). With this scenario, the present review has been framed to give a comprehensive insight into the possible alternative options worth exploring in this direction especially targeting replacements for animal models of bacterial infections. There have been some excellent reviews discussing on the alternate methods for replacing and reducing animals in drug research. However, reviews that discuss the replacements in the field of medical bacteriology with emphasis on animal bacterial infection models are purely limited. The present review discusses on the use of (a) non-mammalian models and (b) alternative systems such as microfluidic chip-based models and microdosing aiming to give a detailed insight into the prospects of these alternative platforms to reduce the number of animals being used in infection studies. This would enlighten the scientific community working in this direction to be well acquainted with the available new approaches and alternatives so that the 3R strategy can be successfully implemented in the field of antibacterial drug research and testing.
- MeSH
- Bacterial Infections * prevention & control veterinary MeSH
- Animal Experimentation * MeSH
- Humans MeSH
- Models, Animal MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) show potent efficacy in several ALK-driven tumors, but the development of resistance limits their long-term clinical impact. Although resistance mechanisms have been studied extensively in ALK-driven non-small cell lung cancer, they are poorly understood in ALK-driven anaplastic large cell lymphoma (ALCL). Here, we identify a survival pathway supported by the tumor microenvironment that activates phosphatidylinositol 3-kinase γ (PI3K-γ) signaling through the C-C motif chemokine receptor 7 (CCR7). We found increased PI3K signaling in patients and ALCL cell lines resistant to ALK TKIs. PI3Kγ expression was predictive of a lack of response to ALK TKI in patients with ALCL. Expression of CCR7, PI3Kγ, and PI3Kδ were up-regulated during ALK or STAT3 inhibition or degradation and a constitutively active PI3Kγ isoform cooperated with oncogenic ALK to accelerate lymphomagenesis in mice. In a three-dimensional microfluidic chip, endothelial cells that produce the CCR7 ligands CCL19/CCL21 protected ALCL cells from apoptosis induced by crizotinib. The PI3Kγ/δ inhibitor duvelisib potentiated crizotinib activity against ALCL lines and patient-derived xenografts. Furthermore, genetic deletion of CCR7 blocked the central nervous system dissemination and perivascular growth of ALCL in mice treated with crizotinib. Thus, blockade of PI3Kγ or CCR7 signaling together with ALK TKI treatment reduces primary resistance and the survival of persister lymphoma cells in ALCL.
- MeSH
- Anaplastic Lymphoma Kinase MeSH
- Lymphoma, Large-Cell, Anaplastic * drug therapy genetics pathology MeSH
- Endothelial Cells metabolism MeSH
- Phosphatidylinositol 3-Kinases MeSH
- Protein Kinase Inhibitors pharmacology therapeutic use MeSH
- Tyrosine Kinase Inhibitors MeSH
- Crizotinib pharmacology therapeutic use MeSH
- Humans MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Tumor Microenvironment MeSH
- Lung Neoplasms * drug therapy MeSH
- Carcinoma, Non-Small-Cell Lung * drug therapy MeSH
- Receptors, CCR7 genetics MeSH
- Receptor Protein-Tyrosine Kinases metabolism MeSH
- Protein-Tyrosine Kinases MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
