Microfluidic-based technologies for diagnosis, prevention, and treatment of COVID-19: recent advances and future directions.

The COVID-19 pandemic has posed significant challenges to existing healthcare systems around the world. The urgent need for the development of diagnostic and therapeutic strategies for COVID-19 has boomed the demand for new technologies that can improve current healthcare approaches, moving towards more advanced, digitalized, personalized, and patient-oriented systems. Microfluidic-based technologies involve the miniaturization of large-scale devices and laboratory-based procedures, enabling complex chemical and biological operations that are conventionally performed at the macro-scale to be carried out on the microscale or less. The advantages microfluidic systems offer such as rapid, low-cost, accurate, and on-site solutions make these tools extremely useful and effective in the fight against COVID-19. In particular, microfluidic-assisted systems are of great interest in different COVID-19-related domains, varying from direct and indirect detection of COVID-19 infections to drug and vaccine discovery and their targeted delivery. Here, we review recent advances in the use of microfluidic platforms to diagnose, treat or prevent COVID-19. We start by summarizing recent microfluidic-based diagnostic solutions applicable to COVID-19. We then highlight the key roles microfluidics play in developing COVID-19 vaccines and testing how vaccine candidates perform, with a focus on RNA-delivery technologies and nano-carriers. Next, microfluidic-based efforts devoted to assessing the efficacy of potential COVID-19 drugs, either repurposed or new, and their targeted delivery to infected sites are summarized. We conclude by providing future perspectives and research directions that are critical to effectively prevent or respond to future pandemics.

Connexin 32 overexpression increases proliferation, reduces gap junctional intercellular communication, motility and epithelial-to-mesenchymal transition in Hs578T breast cancer cells.

Connexins (Cx) are primary components of gap junctions that selectively allow molecules to be exchanged between adjacent cells, regulating multiple cellular functions. Along with their channel forming functions, connexins play a variety of roles in different stages of tumorigenesis and their roles in tumor initiation and progression is isoform- and tissue-specific. While Cx26 and Cx43 were downregulated during breast tumorigenesis, Cx32 was accumulated in the cytoplasm of the cells in lymph node metastasis of breast cancers and Cx32 was further upregulated in metastasis. Cx32's effect on cell proliferation, gap junctional communication, hemichannel activity, cellular motility and epithelial-to-mesenchymal transition (EMT) were investigated by overexpressing Cx32 in Hs578T and MCF7 breast cancer cells. Additionally, the expression and localization of Cx26 and Cx43 upon Cx32 overexpression were examined by Western blot and immunostaining experiments, respectively. We observed that MCF7 cells had endogenous Cx32 while Hs578T cells did not and when Cx32 was overexpressed in these cells, it caused a significant increase in the percentages of Hs578T cells at the S phase in addition to increasing their proliferation. Further, while Cx32 overexpression did not induce hemichannel activity in either cell, it decreased gap junctional communication between Hs578T cells. Additionally, Cx32 was mainly observed in the cytoplasm in both cells, where it did not form gap junction plaques but Cx32 overexpression reduced Cx43 levels without affecting Cx26. Moreover, migration and invasion potentials of Hs578T and migration in MCF7 were reduced upon Cx32 overexpression. Finally, the protein level of mesenchymal marker N-cadherin decreased while epithelial marker ZO-1 and E-cadherin increased in Hs578T cells. We observed that Cx32 overexpression altered cell proliferation, communication, migration and EMT in Hs578T, suggesting a tumor suppressor role in these cells while it had minor effects on MCF7 cells.

Frequency-specific sensitivity of 3T3-L1 preadipocytes to low-intensity vibratory stimulus during adipogenesis.

Adipocyte accumulation in the bone marrow is a severe complication leading to bone defects and reduced regenerative capacity. Application of external mechanical signals to bone marrow cellular niche is a non-invasive and non-pharmaceutical methodology to improve osteogenesis and suppress adipogenesis. However, in the literature, the specific parameters related to the nature of low-intensity vibratory (LIV) signals appear to be arbitrarily selected for amplitude, bouts, and applied frequency. In this study, we performed a LIV frequency sweep ranging from 30 to 120 Hz with increments of 15 Hz applied onto preadipocytes during adipogenesis for 10 d. We addressed the effect of LIV with different frequencies on single-cell density, adipogenic gene expression, lipid morphology, and triglycerides content. Results showed that LIV signals with 75-Hz frequency had the most significant suppressive effect during adipogenesis. Our results support the premise that mechanical-based interventions for suppressing adipogenesis may benefit from optimizing input parameters.

SEMA6D Differentially Regulates Proliferation, Migration, and Invasion of Breast Cell Lines.

Semaphorin 6D (SEMA6D), a member of the class 6 semaphorin family, is a membrane-associated protein that plays a key role in the development of cardiac and neural tissues. A growing body of evidence suggests that SEMA6D is also involved in tumorigenesis. In breast cancer, high SEMA6D levels are correlated with better survival rates. However, very little is known about the functional significance of SEMA6D in breast tumorigenesis. In the present study, we aimed to investigate the effects of SEMA6D expression on the normal breast cell line MCF10A and the breast cancer cell lines MCF7 and MDA MB 231. We demonstrated that SEMA6D expression increases the proliferation of MCF10A cells, whereas the opposite effect was observed in MCF7 cells. SEMA6D expression induced anchorage-independent growth in both cancer cell lines. Furthermore, migration of MCF10A and MCF7 cells and invasion of MDA MB 231 cells were elevated in response to SEMA6D overexpression. Accordingly, the genes related to epithelial-mesenchymal transition (EMT) were altered by SEMA6D expression in MCF10A and MCF7 cell lines. Finally, we provided evidence that SEMA6D levels were associated with the expression of the cell cycle, EMT, and Notch signaling pathway-related genes in breast cancer patients' data. We showed for the first time that SEMA6D overexpression has cell-specific effects on the proliferation, migration, and invasion of normal and cancer breast cell lines, which agrees with the gene expression data of clinical samples. This study lays the groundwork for future research into understanding the functional importance of SEMA6D in breast cancer.

The role of connexins in breast cancer: from misregulated cell communication to aberrant intracellular signaling.

In spite of clinical advancements and improved diagnostic techniques, breast cancers are the leading cause of cancer-associated deaths in women worldwide. Although 70% of early breast cancers can be cured, there are no efficient therapies against metastatic breast cancers. Several factors including connexins and gap junctions play roles in breast tumorigenesis. Connexins are critical for cellular processes as a linkage between connexin mutations and hereditary disorders demonstrated their importance for tissue homeostasis. Further, alterations in their expression, localization and channel activities were observed in many cancers including breast cancer. Both channel-dependent and independent functions of connexins were reported in initiation and progression of cancers. Unlike initial reports suggesting tumor suppressor functions, connexins and gap junctions have stage, context and isoform dependent effects in breast cancers similar to other cancers. In this review, we tried to describe the current understanding of connexins in tumorigenesis specifically in breast cancers.

Magnetic levitation assisted biofabrication, culture, and manipulation of 3D cellular structures using a ring magnet based setup.

Diamagnetic levitation is an emerging technology for remote manipulation of cells in cell and tissue level applications. Low-cost magnetic levitation configurations using permanent magnets are commonly composed of a culture chamber physically sandwiched between two block magnets that limit working volume and applicability. This work describes a single ring magnet-based magnetic levitation system to eliminate physical limitations for biofabrication. Developed configuration utilizes sample culture volume for construct size manipulation and long-term maintenance. Furthermore, our configuration enables convenient transfer of liquid or solid phases during the levitation. Before biofabrication, we first calibrated/ the platform for levitation with polymeric beads, considering the single cell density range of viable cells. By taking advantage of magnetic focusing and cellular self-assembly, millimeter-sized 3D structures were formed and maintained in the system allowing easy and on-site intervention in cell culture with an open operational space. We demonstrated that the levitation protocol could be adapted for levitation of various cell types (i.e., stem cell, adipocyte and cancer cell) representing cells of different densities by modifying the paramagnetic ion concentration that could be also reduced by manipulating the density of the medium. This technique allowed the manipulation and merging of separately formed 3D biological units, as well as the hybrid biofabrication with biopolymers. In conclusion, we believe that this platform will serve as an important tool in broad fields such as bottom-up tissue engineering, drug discovery and developmental biology.

Lamin A/C Is Dispensable to Mechanical Repression of Adipogenesis.

Mesenchymal stem cells (MSCs) maintain the musculoskeletal system by differentiating into multiple lineages, including osteoblasts and adipocytes. Mechanical signals, including strain and low-intensity vibration (LIV), are important regulators of MSC differentiation via control exerted through the cell structure. Lamin A/C is a protein vital to the nuclear architecture that supports chromatin organization and differentiation and contributes to the mechanical integrity of the nucleus. We investigated whether lamin A/C and mechanoresponsiveness are functionally coupled during adipogenesis in MSCs. siRNA depletion of lamin A/C increased the nuclear area, height, and volume and decreased the circularity and stiffness. Lamin A/C depletion significantly decreased markers of adipogenesis (adiponectin, cellular lipid content) as did LIV treatment despite depletion of lamin A/C. Phosphorylation of focal adhesions in response to mechanical challenge was also preserved during loss of lamin A/C. RNA-seq showed no major adipogenic transcriptome changes resulting from LIV treatment, suggesting that LIV regulation of adipogenesis may not occur at the transcriptional level. We observed that during both lamin A/C depletion and LIV, interferon signaling was downregulated, suggesting potentially shared regulatory mechanism elements that could regulate protein translation. We conclude that the mechanoregulation of adipogenesis and the mechanical activation of focal adhesions function independently from those of lamin A/C.

HologLev: A Hybrid Magnetic Levitation Platform Integrated with Lensless Holographic Microscopy for Density-Based Cell Analysis.

In clinical practice, a variety of diagnostic applications require the identification of target cells. Density has been used as a physical marker to distinguish cell populations since metabolic activities could alter the cell densities. Magnetic levitation offers great promise for separating cells at the single cell level within heterogeneous populations with respect to cell densities. Traditional magnetic levitation platforms need bulky and precise optical microscopes to visualize levitated cells. Moreover, the evaluation process of cell densities is cumbersome, which also requires trained personnel for operation. In this work, we introduce a device (HologLev) as a fusion of the magnetic levitation principle and lensless digital inline holographic microscopy (LDIHM). LDIHM provides ease of use by getting rid of bulky and expensive optics. By placing an imaging sensor just beneath the microcapillary channel without any lenses, recorded holograms are processed for determining cell densities through a fully automated digital image processing scheme. The device costs less than $100 and has a compact design that can fit into a pocket. We perform viability tests on the device by levitating three different cell lines (MDA-MB-231, U937, D1 ORL UVA) and comparing them against their dead correspondents. We also tested the differentiation of mouse osteoblastic (7F2) cells by monitoring characteristic variations in their density. Last, the response of MDA-MB-231 cancer cells to a chemotherapy drug was demonstrated in our platform. HologLev provides cost-effective, label-free, fully automated cell analysis in a compact design that could be highly desirable for laboratory and point-of-care testing applications.

Microfluidic-based virus detection methods for respiratory diseases.

With the recent SARS-CoV-2 outbreak, the importance of rapid and direct detection of respiratory disease viruses has been well recognized. The detection of these viruses with novel technologies is vital in timely prevention and treatment strategies for epidemics and pandemics. Respiratory viruses can be detected from saliva, swab samples, nasal fluid, and blood, and collected samples can be analyzed by various techniques. Conventional methods for virus detection are based on techniques relying on cell culture, antigen-antibody interactions, and nucleic acids. However, these methods require trained personnel as well as expensive equipment. Microfluidic technologies, on the other hand, are one of the most accurate and specific methods to directly detect respiratory tract viruses. During viral infections, the production of detectable amounts of relevant antibodies takes a few days to weeks, hampering the aim of prevention. Alternatively, nucleic acid-based methods can directly detect the virus-specific RNA or DNA region, even before the immune response. There are numerous methods to detect respiratory viruses, but direct detection techniques have higher specificity and sensitivity than other techniques. This review aims to summarize the methods and technologies developed for microfluidic-based direct detection of viruses that cause respiratory infection using different detection techniques. Microfluidics enables the use of minimal sample volumes and thereby leading to a time, cost, and labor effective operation. Microfluidic-based detection technologies provide affordable, portable, rapid, and sensitive analysis of intact virus or virus genetic material, which is very important in pandemic and epidemic events to control outbreaks with an effective diagnosis.

Scaffold-free biofabrication of adipocyte structures with magnetic levitation.

Tissue engineering research aims to repair the form and/or function of impaired tissues. Tissue engineering studies mostly rely on scaffold-based techniques. However, these techniques have certain challenges, such as the selection of proper scaffold material, including mechanical properties, sterilization, and fabrication processes. As an alternative, we propose a novel scaffold-free adipose tissue biofabrication technique based on magnetic levitation. In this study, a label-free magnetic levitation technique was used to form three-dimensional (3D) scaffold-free adipocyte structures with various fabrication strategies in a microcapillary-based setup. Adipogenic-differentiated 7F2 cells and growth D1 ORL UVA stem cells were used as model cells. The morphological properties of the 3D structures of single and cocultured cells were analyzed. The developed procedure leads to the formation of different patterns of single and cocultured adipocytes without a scaffold. Our results indicated that adipocytes formed loose structures while growth cells were tightly packed during 3D culture in the magnetic levitation platform. This system has potential for ex vivo modeling of adipose tissue for drug testing and transplantation applications for cell therapy in soft tissue damage. Also, it will be possible to extend this technique to other cell and tissue types.

Connexin 32 induces pro-tumorigenic features in MCF10A normal breast cells and MDA-MB-231 metastatic breast cancer cells.

Connexins (Cx), the basic subunit of gap junctions, play important roles in cell homeostasis, and their abnormal expression and function are associated with human hereditary diseases and cancers. In tumorigenesis, connexins were observed to have both anti-tumorigenic and pro-tumorigenic roles in a context- and stage-dependent manner. Initially, Cx26 and Cx43 were thought to be the only connexins involved in normal breast homeostasis and breast cancer. Later on, association of Cx32 expression with lymph node metastasis of breast cancer and subsequent demonstration of its expression in normal breast tissue suggested that Cx32 contributes to breast tissue homeostasis. Here, we aimed to determine the effects of Cx32 on normal breast cells, MCF10A, and on breast cancer cells, MDA-MB-231. Cx32 overexpression had profound effects on MCF10A cells, decreasing cell proliferation by increasing the doubling time of MCF10A. Furthermore, MCF10A cells acquired mesenchymal-like appearance upon Cx32 expression and had increased migration capacity and expression of both E-cadherin and vimentin. In contrast, Cx32 overexpression altered the EMT markers of MDA-MB-231 by increasing the expression of mesenchymal markers, such as slug and vimentin, and decreasing E-cadherin expression without affecting their proliferation and morphology. Our results indicate, for the first time in the literature, that Cx32 has tumor-promoting roles in MCF10A and MDA-MB-231 cells.

Low magnitude high frequency vibrations expedite the osteogenesis of bone marrow stem cells on paper based 3D scaffolds.

Anabolic effects of low magnitude high frequency (LMHF) vibrations on bone tissue were consistently shown in the literature in vivo, however in vitro efforts to elucidate underlying mechanisms are generally limited to 2D cell culture studies. Three dimensional cell culture platforms better mimic the natural microenvironment and biological processes usually differ in 3D compared to 2D culture. In this study, we used laboratory grade filter paper as a scaffold material for studying the effects of LHMF vibrations on osteogenesis of bone marrow mesenchymal stem cells in a 3D system. LMHF vibrations were applied 15 min/day at 0.1 g acceleration and 90 Hz frequency for 21 days to residing cells under quiescent and osteogenic conditions. mRNA expression analysis was performed for alkaline phosphatase (ALP) and osteocalcin (OCN) genes, Alizarin red S staining was performed for mineral nodule formation and infrared spectroscopy was performed for determination of extracellular matrix composition. The highest osteocalcin expression, mineral nodule formation and the phosphate bands arising from the inorganic phase was observed for the cells incubated in osteogenic induction medium with vibration. Our results showed that filter paper can be used as a model scaffold system for studying the effects of mechanical loads on cells, and LMHF vibrations induced the osteogenic differentiation of stem cells.

Stem Cell Culture Under Simulated Microgravity.

Challenging environment of space causes several pivotal alterations in living systems, especially due to microgravity. The possibility of simulating microgravity by ground-based systems provides research opportunities that may lead to the understanding of in vitro biological effects of microgravity by eliminating the challenges inherent to spaceflight experiments. Stem cells are one of the most prominent cell types, due to their self-renewal and differentiation capabilities. Research on stem cells under simulated microgravity has generated many important findings, enlightening the impact of microgravity on molecular and cellular processes of stem cells with varying potencies. Simulation techniques including clinostat, random positioning machine, rotating wall vessel and magnetic levitation-based systems have improved our knowledge on the effects of microgravity on morphology, migration, proliferation and differentiation of stem cells. Clarification of the mechanisms underlying such changes offers exciting potential for various applications such as identification of putative therapeutic targets to modulate stem cell function and stem cell based regenerative medicine.

Single Cell Densitometry and Weightlessness Culture of Mesenchymal Stem Cells Using Magnetic Levitation.

Magnetic levitation methodology enables density-based separation of microparticles/cells and sustains cell culture in different media. Levitation process can be accomplished via negative magnetophoresis (diamagnetophoresis), where the applied magnetic force compensates gravitational acceleration and the density of the diamagnetic object (e.g., cell) determines its levitation height. Here we describe a portable, sensitive, and cost-effective technology that uses the principles of magnetic levitation to measure single cell density and cell culture under desired conditions.

Applicability of Low-intensity Vibrations as a Regulatory Factor on Stem and Progenitor Cell Populations.

Persistent and transient mechanical loads can act as biological signals on all levels of an organism. It is therefore not surprising that most cell types can sense and respond to mechanical loads, similar to their interaction with biochemical and electrical signals. The presence or absence of mechanical forces can be an important determinant of form, function and health of many tissue types. Along with naturally occurring mechanical loads, it is possible to manipulate and apply external physical loads on tissues in biomedical sciences, either for prevention or treatment of catabolism related to many factors, including aging, paralysis, sedentary lifestyles and spaceflight. Mechanical loads consist of many components in their applied signal form such as magnitude, frequency, duration and intervals. Even though high magnitude mechanical loads with low frequencies (e.g. running or weight lifting) induce anabolism in musculoskeletal tissues, their applicability as anabolic agents is limited because of the required compliance and physical health of the target population. On the other hand, it is possible to use low magnitude and high frequency (e.g. in a vibratory form) mechanical loads for anabolism as well. Cells, including stem cells of the musculoskeletal tissue, are sensitive to high frequency, lowintensity mechanical signals. This sensitivity can be utilized not only for the targeted treatment of tissues, but also for stem cell expansion, differentiation and biomaterial interaction in tissue engineering applications. In this review, we reported recent advances in the application of low-intensity vibrations on stem and progenitor cell populations. Modulation of cellular behavior with low-intensity vibrations as an alternative or complementary factor to biochemical and scaffold induced signals may represent an increase of capabilities in studies related to tissue engineering.

Label-free density-based detection of adipocytes of bone marrow origin using magnetic levitation.

Adipocyte hypertrophy and hyperplasia are important parameters in describing abnormalities in adipogenesis that are concomitant to diseases such as obesity, diabetes, anorexia nervosa and osteoporosis. Therefore, technical developments in the detection of adipocytes become an important driving factor in adipogenesis research. Current techniques such as optical microscopy and flow cytometry are available in detection and examination of adipocytes, driving cell- and molecular-based research of adipogenesis. Even though microscopy techniques are common and straightforward, they are restricted in terms of manipulation and separation of the cells. Flow cytometry is an alternative, but mature adipocytes are fragile and cannot withstand the flow process. Other separation methods usually require labeling of the cells or usage of microfluidic platforms that utilize fluids with different densities. Magnetic levitation is a novel label-free technology with the principle of movement of cells towards the lower magnetic field in a paramagnetic medium depending on their individual densities. In this study, we used a magnetic levitation device for density-based single cell detection of differentiated adipogenic cells in heterogeneous populations. Results showed that the magnetic levitation platform was sensitive to changes in the lipid content of mesenchymal stem cells committed to adipogenesis and it could be successfully used to detect the adipogenic differentiation of the cells.

Cytotoxic Tolerance of Healthy and Cancerous Bone Cells to Anti-microbial Phenolic Compounds Depend on Culture Conditions.

Carnosol and carnosic acid are polyphenolic compounds found in rosemary and sage with known anti-oxidant, anti-inflammatory, and anti-microbial properties. Here, we addressed the potential use of carnosol and carnosic acid for in vitro bone tissue engineering applications, specifically depending on their cytotoxic effects on bone marrow stromal and stem cells, and osteosarcoma cells in monolayer and 3D cultures. Carnosol and carnosic acid displayed a bacteriostatic effect on Gram-positive bacteria, especially on S. aureus. The viability results indicated that bone marrow stromal cells and bone marrow stem cells were more tolerant to the presence of carnosol compared to osteosarcoma cells. 3D culture conditions increased this tolerance further for healthy cells, while not affecting the cytotoxic potential of carnosol for osteosarcoma cells. Carnosic acid was found to be more cytotoxic for all cell types used in the study. Results suggest that phenolic compounds might have potential use as anti-microbial and anti-carcinogenic agents for bone tissue engineering with further optimization for controlled release.

Biofabrication of in situ Self Assembled 3D Cell Cultures in a Weightlessness Environment Generated using Magnetic Levitation.

Magnetic levitation though negative magnetophoresis is a novel technology to simulate weightlessness and has recently found applications in material and biological sciences. Yet little is known about the ability of the magnetic levitation system to facilitate biofabrication of in situ three dimensional (3D) cellular structures. Here, we optimized a magnetic levitation though negative magnetophoresis protocol appropriate for long term levitated cell culture and developed an in situ 3D cellular assembly model with controlled cluster size and cellular pattern under simulated weightlessness. The developed strategy outlines a potential basis for the study of weightlessness on 3D living structures and with the opportunity for real-time imaging that is not possible with current ground-based simulated weightlessness techniques. The low-cost technique presented here may offer a wide range of biomedical applications in several research fields, including mechanobiology, drug discovery and developmental biology.

Magnetic Force-Based Microfluidic Techniques for Cellular and Tissue Bioengineering.

Live cell manipulation is an important biotechnological tool for cellular and tissue level bioengineering applications due to its capacity for guiding cells for separation, isolation, concentration, and patterning. Magnetic force-based cell manipulation methods offer several advantages, such as low adverse effects on cell viability and low interference with the cellular environment. Furthermore, magnetic-based operations can be readily combined with microfluidic principles by precisely allowing control over the spatiotemporal distribution of physical and chemical factors for cell manipulation. In this review, we present recent applications of magnetic force-based cell manipulation in cellular and tissue bioengineering with an emphasis on applications with microfluidic components. Following an introduction of the theoretical background of magnetic manipulation, components of magnetic force-based cell manipulation systems are described. Thereafter, different applications, including separation of certain cell fractions, enrichment of rare cells, and guidance of cells into specific macro- or micro-arrangements to mimic natural cell organization and function, are explained. Finally, we discuss the current challenges and limitations of magnetic cell manipulation technologies in microfluidic devices with an outlook on future developments in the field.