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A microfluidics-based three-dimensional skin-on-chip (SoC) model is developed in this study to enable quantitative studies of transendothelial and transepithelial migration of human T lymphocytes in mimicked skin inflammatory microenvironments and to test new drug candidates. The keys results include 1) CCL20-dependent T cell transmigration is significantly inhibited by an engineered CCL20 locked dimer (CCL20LD), supporting the potential immunotherapeutic use of CCL20LD for treating skin diseases such as psoriasis; 2) transepithelial migration of T cells in response to a CXCL12 gradient mimicking T cell egress from the skin is significantly reduced by a sphingosine-1-phosphate (S1P) background, suggesting the role of S1P for T cell retention in inflamed skin tissues; and 3) T cell transmigration is induced by inflammatory cytokine stimulated epithelial cells in the SoC model. Collectively, the developed SoC model recreates a dynamic multi-cellular micro-environment that enables quantitative studies of T cell transmigration at a single cell level in response to physiological cutaneous inflammatory mediators and potential drugs.

Apresentação

Mobile sensing based on the integration of microfluidic device and smartphone, so-called MS2 technology, has enabled many applications over recent years, and continues to stimulate growing interest in both research communities and industries. In particular, it has been envisioned that MS2 technology can be developed for various cell functional assays to enable basic research and clinical applications. Toward this direction, in this paper, we describe the development of a MS2-based cell functional assay for testing cell migration (the Mkit). The system is constructed as an integrated test kit, which includes microfluidic chips, a smartphone-based imaging platform, the phone apps for image capturing and data analysis, and a set of reagent and accessories for performing the cell migration assay. We demonstrated that the Mkit can effectively measure purified neutrophil and cancer cell chemotaxis. Furthermore, neutrophil chemotaxis can be tested from a drop of whole blood using the Mkit with red blood cell (RBC) lysis. The effects of chemoattractant dose and gradient profile on neutrophil chemotaxis were also tested using the Mkit. In addition to research applications, we demonstrated the effective use of the Mkit for on-site test at the hospital and for testing clinical samples from chronic obstructive pulmonary disease patient. Thus, this developed Mkit provides an easy and integrated experimental platform for cell migration related research and potential medical diagnostic applications.

Activin A belongs to the superfamily of transforming growth factor beta (TGFβ) and is a critical regulatory cytokine in breast cancer and inflammation. However, the role of activin A in migration of breast cancer cells and immune cells was not well characterized. Here, a microfluidic device was used to examine the effect of activin A on the migration of human breast cancer cell line MDA-MB-231 cells and human blood neutrophils as well as their migratory interaction. We found that activin A promoted the basal migration but impaired epidermal growth factor (EGF)-induced migration of breast cancer cells. By contrast, activin A reduced neutrophil chemotaxis and transendothelial migration to N-Formyl-Met-Leu-Phe (fMLP). Finally, activin A promoted neutrophil chemotaxis to the supernatant from breast cancer cell culture. Collectively, our study revealed the different roles of activin A in regulating the migration of breast cancer cells and neutrophils and their migratory interaction. These findings suggested the potential of activin A as a therapeutic target for inflammation and breast cancers.

Chemotaxis is a classic mechanism for guiding cell migration and an important topic in both fundamental cell biology and health sciences. Neutrophils are a widely used model to study eukaryotic cell migration and neutrophil chemotaxis itself can lead to protective or harmful immune actions to the body. While much has been learnt from past research about how neutrophils effectively navigate through a chemoattractant gradient, many interesting questions remain unclear. For example, while it is tempting to model neutrophil chemotaxis using the well-established biased random walk theory, the experimental proof was challenged by the cell's highly persistent migrating nature. A special experimental design is required to test the key predictions from the random walk model. Another question that has interested the cell migration community for decades concerns the existence of chemotactic memory and its underlying mechanism. Although chemotactic memory has been suggested in various studies, a clear quantitative experimental demonstration will improve our understanding of the migratory memory effect. Motivated by these questions, we developed a microfluidic cell migration assay (so-called dual-docking chip or D2-Chip) that can test both the biased random walk model and the memory effect for neutrophil chemotaxis on a single chip enabled by multi-region gradient generation and dual-region cell alignment. Our results provide experimental support for the biased random walk model and chemotactic memory for neutrophil chemotaxis. Quantitative data analyses provide new insights into neutrophil chemotaxis and memory by making connections to entropic disorder, cell morphology and oscillating migratory response.

A presente pesquisa é fruto da dissertação de mestrado que identificou a presença de lutas por posições dos pesquisadores participantes do Observatório das Violências nas escolas, reconhecendo nessa organização, uma forma estratégica de negociar o conhecimento. Dentro dessa perspectiva, o artigo propôs refletir sobre o discurso científico institucionalizado pelos pesquisadores do Ensino Superior no âmbito do Observatório da Violência nas Escolas. Por meio da análise das árvores científicas extraídas do levantamento da plataforma do curriculum lattes dos 10 pesquisadores professores de Ensino Superior foi possível identificar o uso de movimentos intelectuais e inclinações teóricas voltadas para a pesquisa maior, o da Violência Escolar. Esses deslocamentos evidenciados se configuraram numa lógica própria do campo, com estratégia inconsciente ou parcialmente controlada, que externou um “interesse desinteressado” dentro da Academia, uma forma de “jogar o jogo” que a própria ciência sugestiona. Essa constituição revelou forças internas no campo científico que se materializaram em esforços dos pesquisadores no trabalho em parceria, consubstanciados pelos discursos caracterizadores de legitimidade no campo. Sendo perceptível perante a análise do percurso científico dos pesquisadores, o uso da representação social interessada, que estabeleceu um discurso científico socializado, imprimindo novas identidades científicas aos pesquisadores.

Neutrophil migration and chemotaxis are fundamentally important biological processes and have direct relevance to various health problems. Microfluidic devices provide useful experimental tools for the quantitative analysis of neutrophil chemotaxis in controlled microenvironments. However, such experiments often require specialized research facilities and lengthy cell preparation from a large amount of blood. In this paper, we report a new, yet simple, all-on-chip method for the magnetic isolation of untouched neutrophils directly from small volumes of blood, followed by chemotaxis testing on the same microfluidic device. Furthermore, we incorporated a cell-docking structure to the microfluidic device for better control of the cells’ initial positions before the chemotaxis test and for improved data analysis. The whole experiment can be performed in less than 25 minutes. We successfully validated this method by testing neutrophil chemotaxis to both purified chemoattractant (i.e. fMLP) and clinical samples (sputum from patients with Chronic Obstructive Pulmonary Disease, COPD). Thus, the “all-on-chip” method can be a useful tool for research and clinical applications that require rapid and accurate chemotaxis testing of untouched neutrophils.

Chronic obstructive pulmonary disease (COPD) is a common lung disease characterized by narrowed airways, resulting in serious breathing difficulty. Previous studies have demonstrated that inflammatory infiltration of leukocytes in the airway is associated with the pathogenesis of COPD. In the present study, we employed a microfluidic approach to assess the effect of COPD sputum on activated human peripheral blood T cell migration and chemotaxis under well-controlled gradient conditions. Our results showed considerable basal migration of T cells derived from peripheral blood of COPD patients and healthy controls in the medium control groups. By contrast, the migration of T cells from COPD patients and healthy controls was significantly inhibited in the presence of a gradient of sputum supernatant from COPD patients. Furthermore, chemotaxis of T cells from COPD patients or healthy subjects toward an SDF-1α gradient was clearly inhibited by sputum samples from the COPD patients. The inhibition effect revealed by the microfluidic cell migration experiments provides new information about the complex involvement of T cell trafficking in COPD.

Cell-based assays including both cell identification assays and cell functional assays are broadly used in life science research and for medical applications. Traditionally, cell-based assays are performed in professional laboratories. Although such an approach is reliable, it significantly limits the accessibility of cell-based assays by many researchers without specialized cell imaging facilities and does not permit on-site use at most clinics. Over recent years, growing efforts have been made to overcome this limitation by developing various portable cell imaging platforms. Herein, we provide a timely review on the recent development of portable imaging systems and their biomedical applications. We describe the respective applications of three main classes of portable imaging technologies including miniaturized microscope, lens-free imaging device and smartphone-based microscope for cell-based assays. We conclude by discussing our views on the opportunities, problems and future directions of this emerging field.

Cardiovascular disease (CVD) and cancer are two leading causes of death worldwide. CVD and cancer share risk factors such as obesity and diabetes mellitus and have common diagnostic biomarkers such as interleukin-6 and C-reactive protein. Thus, timely and accurate diagnosis of these two correlated diseases is of high interest to both the research and healthcare communities. Most conventional methods for CVD and cancer biomarker detection such as microwell plate-based immunoassay and polymerase chain reaction often suffer from high costs, low test speeds, and complicated procedures. Recently, lab-on-a-chip (LoC)-based platforms have been increasingly developed for CVD and cancer biomarker sensing and analysis using various molecular and cell-based diagnostic biomarkers. These new platforms not only enable better sample preparation, chemical manipulation and reaction, high-throughput and portability, but also provide attractive features such as label-free detection and improved sensitivity due to the integration of various novel detection techniques. These features effectively improve the diagnostic test speed and simplify the detection procedure. In addition, microfluidic cell assays and organ-on-chip models offer new potential approaches for CVD and cancer diagnosis. Here we provide a mini-review focusing on recent development of LoC-based methods for CVD and cancer diagnostic biomarker measurements, and our perspectives of the challenges, opportunities and future directions.

Mobile sensing based on the integration of microfluidic device and smartphone, so-called MS2 technology, has enabled many applications over recent years, and continues to stimulate growing interest in both research communities and industries. In particular, it has been envisioned that MS2 technology can be developed for various cell functional assays to enable basic research and clinical applications. Toward this direction, in this paper, we describe the development of a MS2-based cell functional assay for testing cell migration (the Mkit). The system is constructed as an integrated test kit, which includes microfluidic chips, a smartphone-based imaging platform, the phone apps for image capturing and data analysis, and a set of reagent and accessories for performing the cell migration assay. We demonstrated that the Mkit can effectively measure purified neutrophil and cancer cell chemotaxis. Furthermore, neutrophil chemotaxis can be tested from a drop of whole blood using the Mkit with red blood cell (RBC) lysis. The effects of chemoattractant dose and gradient profile on neutrophil chemotaxis were also tested using the Mkit. In addition to research applications, we demonstrated the effective use of the Mkit for on-site test at the hospital and for testing clinical samples from chronic obstructive pulmonary disease patient. Thus, this developed Mkit provides an easy and integrated experimental platform for cell migration related research and potential medical diagnostic applications.

Netrin-1 is well-known for its chemoattractive and chemorepulsive properties for axon guidance. Early studies report that netrin-1 inhibits granulocyte migration. On the other hand, netrin-1 can promote cancer cell migration and invasion. The underlying mechanisms are not well understood, which requires more in-depth characterizations of netrin-1 mediated immune and cancer cell migration. The present study, for the first time, employs microfluidic devices that are recently developed to quantitatively investigate the effects of netrin-1 on the motility and chemotaxis of human blood neutrophils and human breast cancer cells under well-controlled gradient conditions. The results show that netrin-1 reduces chemokinetic motility of human neutrophils, which is accompanied with reduced cell polarization and spreading. In addition, netrin-1 reduces neutrophil chemotaxis to N-formyl-Met-Leu-Phe on fibronectin substrate but interestingly not on collagen substrate. By contrast, netrin-1 promotes the migration of human breast cancer cells. Furthermore, it is found that netrin-1 reduces neutrophil chemotaxis to the supernatant of human breast cancer cell culture. Collectively, this microfluidic cell migration study provides quantitative characterizations of the effects of netrin-1 on the motility and chemotaxis of neutrophils and breast cancer cells, and further suggests the potential role of netrin-1 in regulating neutrophil recruitment to breast cancer microenvironments.

Background: Natural Killer (NK) cells play a key role in innate immunity against viral, microbial infections and transformed cells and their migration for effector function to peripheral tissues or inflamed lymph nodes are tightly regulated. Of interest, production of Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) by cancer cells is correlated to host immune suppression and tumor metastasis, suggesting an immune evasion property of GM-CSF. Here we examined role(s) of recombinant GM-CSF in the regulation of NK-cell migratory properties in vitro. Methods: Previously published “Y” shape microfluidic platform was used to study the roles of GM-CSF gradient on NK-cell migrations. IL-2 activated human primary NK cells were used in the migration studies. Results: Our microfluidic-based migration study demonstrated a novel role of GM-CSF in regulating repulsive NK-cell migration under the stable GM-CSF gradient (at 20 ng/ml), followed by subsequent arrest in cell migration. Blocking of GM-CSF-Rα abolished the repulsive migratory behavior but not the arrest. In contrast, lower concentrations of GM-CSF induced hyper-polarization, immediate arrest of NK cells, and little/or no NK-cell migrations. Circularity measurement in controls and above experiments confirmed statistically the correlation between hyperpolarization and migration arrest. Future analyses will elucidate the mechanisms underlying the dual roles of GM-CSF in the regulation of NK-cell migratory properties.