Cell Migration Behavior Research Articles

Design, simulation and application of multichannel microfluidic chip for cell migration

Cell migration is defined as the directional movement of cells toward a specific chemical concentration gradient, which plays a crucial role in embryo development, wound healing and tumor metastasis. However, current research methods showed low flux and are only suitable for single-factor assessment, and it was difficult to comprehensively consider the effects of other parameters such as different concentration gradients on cell migration behavior. In this paper, a four-channel microfluidic chip was designed. Its characteristics were as follows: it relied on laminar flow and diffusion mechanisms to establish and maintain a concentration gradient; it was suitable for observation of cell migration in different concentration gradient environment under a single microscope field; four cell isolation zones (20 μm width) were integrated into the microfluidic device to calibrate the initial cell position, which ensured the accuracy of the experimental results. In particular, we used COMSOL Multiphysics software to simulate the structure of the chip, which demonstrated the necessity of designing S-shaped microchannel and horizontal pressure balance channel to maintain concentration gradient. Finally, neutrophils were incubated with advanced glycation end products (AGEs, 0, 0.2, 0.5, 1.0 μmol·L -1), which were closely related to diabetes mellitus and its complications. The migration behavior of incubated neutrophils was studied in the 100 nmol·L -1 of chemokine (N-formylmethionyl-leucyl-phenyl-alanine) concentration gradient. The results prove the reliability and practicability of the microfluidic chip.

Fibroblast Derived Skin Wound Healing Modeling on Chip under the Influence of Micro-Capillary Shear Stress.

Fibroblast cell migration plays a crucial role in the wound-healing process. Hence, its quantitative investigation is important to understand the mechanism of the wound-healing process. The dynamic nature of the wound-healing process can be easily implemented using a microfluidic-based wound-healing assay. This work presented the use of a microfluidics device to simulate traumatic wounds on fibroblast cell monolayers by utilizing trypsin flow and PDMS barrier. In this study, a microfluidic chip with a transparent silk film is reported. The placement of film provides 3D cell culture conditions that mimic a 3D extracellular matrix (ECM) like environment and allows real-time monitoring of cells. A numerical study was conducted to evaluate the influence of dynamic medium-induced shear stress on the base and wall of the microchannel. This could facilitate the optimization of the inlet flow conditions of the media in the channel. At the same time, it could help in identifying stress spots in the channel. The scaffolds were placed in those spots for evaluating the influence of shear forces on the migratory behavior of fibroblast cells. The in vitro microfluidic assembly was then evaluated for cell migration under the influence of external shear forces during the wound-healing phenomena. A faster wound healing was obtained at the end of 24 h of the creation of the wound in the presence of optimal shear stress. On increasing the shear stress beyond a threshold limit, it dissociates fibroblast cells from the surface of the substrate, thereby decelerating the wound-healing process. The above phenomena were transformed in both coplanar microfluidics surfaces (by realizing in the multichannel interlinked model) and transitional microfluidics channels (by realizing in the sandwich model).

The Oxygen Gradient in Hypoxic Conditions Enhances and Guides Dictyostelium discoideum Migration

Spatiotemporal variations of oxygen concentration affect the cell behaviors that are involved in physiological and pathological events. In our previous study with Dictyostelium discoideum (Dd) as a model of cell motility, aggregations of Dd cells exhibited long-lasting and highly stable migration in a self-generated hypoxic environment, forming a ring shape that spread toward the outer higher oxygen region. However, it is still unclear what kinds of changes in the migratory properties are responsible for the observed phenomena. Here, we investigated the migration of Dd to clarify the oxygen-dependent characteristics of aerokinesis and aerotaxis. Migratory behaviors of Dd cells were analyzed under various oxygen concentration gradients and uniform oxygen conditions generated in microfluidic devices. Under hypoxic conditions below 2% O2, corresponding to less than 25 µM O2 in the culture medium, the migration of Dd cells was enhanced (aerokinesis) and the oxygen gradient guided the cells toward the oxygen-rich region (aerotaxis). The aerotaxis was attributed to the increase in the frequency of migration associated with the direction of higher O2, the acceleration of migration velocity, and the enhancement of migration straightness. Thus, aerokinesis and aerotaxis are dependent on both the oxygen level and possibly relative gradient and are essential mechanisms for the migration of Dd.

The Effects of αvβ3 Integrin Blockage in Breast Tumor and Endothelial Cells under Hypoxia In Vitro.

Breast cancer is characterized by a hypoxic microenvironment inside the tumor mass, contributing to cell metastatic behavior. Hypoxia induces the expression of hypoxia-inducible factor (HIF-1α), a transcription factor for genes involved in angiogenesis and metastatic behavior, including the vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), and integrins. Integrin receptors play a key role in cell adhesion and migration, being considered targets for metastasis prevention. We investigated the migratory behavior of hypoxia-cultured triple-negative breast cancer cells (TNBC) and endothelial cells (HUVEC) upon αvβ3 integrin blocking with DisBa-01, an RGD disintegrin with high affinity to this integrin. Boyden chamber, HUVEC transmigration, and wound healing assays in the presence of DisBa-01 were performed in hypoxic conditions. DisBa-01 produced similar effects in the two oxygen conditions in the Boyden chamber and transmigration assays. In the wound healing assay, hypoxia abolished DisBa-01′s inhibitory effect on cell motility and decreased the MMP-9 activity of conditioned media. These results indicate that αvβ3 integrin function in cell motility depends on the assay and oxygen levels, and higher inhibitor concentrations may be necessary to achieve the same inhibitory effect as in normoxia. These versatile responses add more complexity to the role of the αvβ3 integrin during tumor progression.

Curvature dynamically regulates cell migration mode and motility

The intrinsic architecture of tissues and engineered scaffolds subject cells to geometrical cues in the form of mesoscale curvatures. While the effect of nano- and micro-scale topographical cues have been extensively studied for decades, surprisingly little is known about the cell response to non-planar geometries larger than cell size. To study the effect of curvature in a systematic and high-throughput manner, we developed a microfabricated chip containing arrays of concave and convex structures with a wide range of types and sizes (μm to mm) and investigated cell migration behavior on these structures.

Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating β‑catenin and increases irinotecan sensitivity in colorectal cancer.

With >1.85 million cases and 850,000 deaths annually, colorectal cancer (CRC) is the third most common cancer detected globally. CRC is an aggressive malignancy with metastasis and, in spite of advances in improved treatment regimen, distant disease failure rates remain disappointingly high. Mucin-like 1 (MUCL1) is a small glycoprotein highly expressed mainly in breast cancer. The involvement of the MUCL1 protein in CRC progression and the underlying mechanism have been largely unknown. The aim of the present study was to investigate the MUCL1 expression profile and its functional significance in CRC. The Cancer Genome Atlas dataset revealed that MUCL1 expression was higher in colorectal tumor compared with normal tissues. MUCL1 was also revealed to be expressed in human CRC cell lines. The results demonstrated that MUCL1 promoted cell proliferation and colony formation, confirming its oncogenic potential. Silencing MUCL1 with short interfering RNA inhibited the protein expression of Bcl2 family proteins, such as Bcl2 and BclxL. Targeting MUCL1 resulted in significant inhibition in cell invasive and migratory behavior of HT-29 and SW620 cells. In addition, the expression of E-cadherin increased whereas the expression of vimentin decreased in MUCL1-silenced cells, confirming inhibition of epithelial-mesenchymal transition (EMT) process. Thus, it was revealed that MUCL1 plays a notable role in cell invasion and migration by inhibiting EMT in CRC. Mechanistically, MUCL1 drives β-catenin activation by Ser-552 phosphorylation, nuclear accumulation and transcriptional activation. Targeting MUCL1 increases the drug sensitivity of CRC cells towards irinotecan. These findings thus demonstrated that MUCL1 acts as a modifier of other pathways that play an important role in CRC progression and MUCL1 was identified as a potential target for CRC therapeutics.

Annexin A6 and NPC1 regulate LDL-inducible cell migration and distribution of focal adhesions

Cholesterol is considered indispensable for cell motility, but how physiological cholesterol pools enable cells to move forward remains to be clarified. The majority of cells obtain cholesterol from the uptake of Low-Density lipoproteins (LDL) and here we demonstrate that LDL stimulates A431 squamous epithelial carcinoma and Chinese hamster ovary (CHO) cell migration and invasion. LDL also potentiated epidermal growth factor (EGF) -stimulated A431 cell migration as well as A431 invasion in 3-dimensional environments, using organotypic assays. Blocking cholesterol export from late endosomes (LE), using Niemann Pick Type C1 (NPC1) mutant cells, pharmacological NPC1 inhibition or overexpression of the annexin A6 (AnxA6) scaffold protein, compromised LDL-inducible migration and invasion. Nevertheless, NPC1 mutant cells established focal adhesions (FA) that contain activated focal adhesion kinase (pY397FAK, pY861FAK), vinculin and paxillin. Compared to controls, NPC1 mutants display increased FA numbers throughout the cell body, but lack LDL-inducible FA formation at cell edges. Strikingly, AnxA6 depletion in NPC1 mutant cells, which restores late endosomal cholesterol export in these cells, increases their cell motility and association of the cholesterol biosensor D4H with active FAK at cell edges, indicating that AnxA6-regulated transport routes contribute to cholesterol delivery to FA structures, thereby improving NPC1 mutant cell migratory behaviour.

Computational Investigation of Cell Migration Behavior in a Confluent Epithelial Monolayer

Cell migration plays a significant role in many biological activities, yet the physical mechanisms of cell migration are still not well understood. In this study, a continuum physics-based epithelial monolayer model including the intercellular interaction was employed to study the cell migration behavior in a confluent epithelial monolayer at constant cell density. The epithelial cell was modeled as isotropic elastic material. Through finite element simulation, the results revealed that the motile cell was subjected to higher stress than the other jammed cells during the migration process. Cell stiffness was implied to play a significant role in epithelial cell migration behavior. Higher stiffness results in smaller displacement and lower migration speed.

Overexpression of LIMA1 Indicates Poor Prognosis and Promotes Epithelial-Mesenchymal Transition in Head and Neck Squamous Cell Carcinoma.

Background:LIMA1 encodes LIM domain and actin binding 1, a cytoskeleton-associated protein whose loss has been linked to migration and invasion behavior of cancer cells. However, the roles of LIMA1 underlying the malignant behavior of tumors in head and neck squamous cell carcinoma (HNSC) are not fully understood.Methods:We conducted a multi-omics study on the role of LIMA1 in HNSC based on The Cancer Genome Atlas data. Subsequent in vitro experiments were performed to validate the results of bioinformatic analysis. We first identified the correlation between LIMA1 and tumor cell functional states according to single-cell sequencing data in HNSC. The potential downstream effects of LIMA1 were explored for gene ontology and Kyoto Encyclopedia of Genes and Genomes pathways through functional enrichment analysis of the gene sets that correlated with LIMA1 in HNSC. The prognostic role of LIMA1 was assessed using the log rank test to compare difference in survival between LIMA1High and LIMA1Low patients. Univariate Cox regression and multivariate Cox regression were further carried out to identify the prognostic value of LIMA1 in HNSC.Results:LIMA1 was identified as a prognostic biomarker and is associated with epithelial-mesenchymal transition (EMT) progress in HNSC. In vitro silencing of LIMA1 suppressed EMT and related pathways in HNSC.Conclusions:LIMA1 promotes EMT and further leads to tumor invasion and metastasis. Increased expression of LIMA1 indicates poor survival, identifying it as a prognostic biomarker in HNSC.

Oral administration of eucalyptol reduces cell migration and pain-like behavior in zymosan-induced arthritis mice

Rheumatoid arthritis (RA) is an inflammatory disease that utilizes nonbiologic and biologic drugs for appropriate disease management. However, high cost, adverse effects, reduced effectiveness, and risk of infection have stimulated the search for safer and more efficacious therapeutic strategies. In the present study, we aimed to evaluate the anti-inflammatory and analgesic properties of eucalyptol in an experimental model of arthritis. Mice were administered zymosan or saline intra-articularly. One hour before the zymosan administration, the mice were treated with oral eucalyptol (200-400 mg/kg) and vehicle. Cell influx, neutrophils, lymphocytes, and monocytes were measured in joint exudates. Joint pain was assessed using paw-pressure tests. Orally administered eucalyptol (200 and 400 mg/kg) significantly reduced cell influx, as well as neutrophils, lymphocytes, and monocytes, when compared with the control. Eucalyptol at a dose of 400 mg/kg significantly reversed joint pain and demonstrated analgesic activity (60%); however, 200 mg/kg failed to alter joint pain. These results indicate that oral eucalyptol promotes anti-inflammatory and analgesic activity in mice subjected to zymosan-induced arthritis.

Cellular nanointerface of vertical nanostructure arrays and its applications.

Vertically standing nanostructures with various morphologies have been developed with the emergence of the micro-/nanofabrication technology. When cells are cultured on them, various bio–nano interfaces between cells and vertical nanostructures would impact the cellular activities, depending on the shape, density, and height of nanostructures. Many cellular pathway activation processes involving a series of intracellular molecules (proteins, RNA, DNA, enzymes, etc.) would be triggered by the cell morphological changes induced by nanostructures, affecting the cell proliferation, apoptosis, differentiation, immune activation, cell adhesion, cell migration, and other behaviors. In addition, the highly localized cellular nanointerface enhances coupled stimulation on cells. Therefore, understanding the mechanism of the cellular nanointerface can not only provide innovative tools for regulating specific cell functions but also offers new aspects to understand the fundamental cellular activities that could facilitate the precise monitoring and treatment of diseases in the future. This review mainly describes the fabrication technology of vertical nanostructures, analyzing the formation of cellular nanointerfaces and the effects of cellular nanointerfaces on cells' fates and functions. At last, the applications of cellular nanointerfaces based on various nanostructures are summarized.

Pillar-Based Mechanical Induction of an Aggressive Tumorigenic Lung Cancer Cell Model.

Tissue microarchitecture imposes physical constraints to the migration of individual cells. Especially in cancer metastasis, three-dimensional structural barriers within the extracellular matrix are known to affect the migratory behavior of cells, regulating the pathological state of the cells. Here, we employed a culture platform with micropillar arrays of 2 μm diameter and 16 μm pitch (2.16 micropillar) as a mechanical stimulant. Using this platform, we investigated how a long-term culture of A549 human lung carcinoma cells on the (2.16) micropillar-embossed dishes would influence the pathological state of the cell. A549 cells grown on the (2.16) micropillar array with 10 μm height exhibited a significantly elongated morphology and enhanced migration even after the detachment and reattachment, as evidenced in the conventional wound-healing assay, single-cell tracking analysis, and in vivo tumor colonization assays. Moreover, the pillar-induced morphological deformation in nuclei was accompanied by cell-cycle arrest in the S phase, leading to suppressed proliferation. While these marked traits of morphology-migration-proliferation support more aggressive characteristics of metastatic cancer cells, typical indices of epithelial-mesenchymal transition were not found, but instead, remarkable traces of amoeboidal transition were confirmed. Our study also emphasizes the importance of mechanical stimuli from the microenvironment during pathogenesis and how gained traits can be passed onto subsequent generations, ultimately affecting their pathophysiological behavior. Furthermore, this study highlights the potential use of pillar-based mechanical stimuli as an in vitro cell culture strategy to induce more aggressive tumorigenic cancer cell models.

The Critical Importance of Spatial and Temporal Scales in Designing and Interpreting Immune Cell Migration Assays.

Intravital microscopy and other direct-imaging techniques have allowed for a characterisation of leukocyte migration that has revolutionised the field of immunology, resulting in an unprecedented understanding of the mechanisms of immune response and adaptive immunity. However, there is an assumption within the field that modern imaging techniques permit imaging parameters where the resulting cell track accurately captures a cell’s motion. This notion is almost entirely untested, and the relationship between what could be observed at a given scale and the underlying cell behaviour is undefined. Insufficient spatial and temporal resolutions within migration assays can result in misrepresentation of important physiologic processes or cause subtle changes in critical cell behaviour to be missed. In this review, we contextualise how scale can affect the perceived migratory behaviour of cells, summarise the limited approaches to mitigate this effect, and establish the need for a widely implemented framework to account for scale and correct observations of cell motion. We then extend the concept of scale to new approaches that seek to bridge the current “black box” between single-cell behaviour and systemic response.

Newly Synthesized Benzothiazole Derived Ligand and its Co(III) and Ru(III) Complexes as Biological Potent Molecules: Chemical Preparation, Structure, Antimicrobial, in vitro and in vivo Cytotoxicity Studies

Herein we describe the synthesis of 2-((E)-(2-(benzo[d]thiazol-2-yl)phenylimino)methyl)-5-methoxyphenol imine base (BMM) and its metal complexes such as [Co(BMM)2] C1, [Co(BMM)(ph)] C2, [Ru(BMM)2] C3 and [Ru(BMM)(ph)] C4. The ligand and its complexes were characterized by spectroscopic, TGA, Powder X-ray diffraction (XRD), and elemental analysis. The coordination number and geometry were confirmed by various experimental data. The Co(III) and Ru(III) complexes are assigned to be having octahedral geometry. Further, the experimental results revealed that complex C2 and C4 emerged as moderate DNA binding intercalators compared to standard intercalators and prominent nuclease activity compared with C1 and C3 complexes. The extent of interaction and intercalation mode was confirmed by monitoring electronic absorption studies, fluorescence quenching probe, and viscosity measurement. Furthermore, the complexes C1-C4 were investigated for their in vitro cytotoxic studies against a panel of human cancer cell lines, viz., A549, NIH3T3, MCF-7, and EAC. Also, the cell adhesion and migration behavior were carried out after administering the complex C2 and C4 to respective cancer cell lines. In mice, the toxicities and tolerabilities of the complex C2 and C4 were also investigated through in vivo studies. The resultant complexes were screened for their microbial inhibition studies through the good diffusion method. All the complexes display moderate microbial inhibition properties compared to their uncoordinated analog.

Effect of MicroRNA-150 (miR-150) on the Biological Activity of Breast Cancer Cells and Its Correlation with Notch Signal

Our study explores miR-150’s effect on the biological activity of breast cancer cells and its correlation with Notch signaling. Human breast cancer cells MCF-7 were divided into WZ group (MCF-7 cells); KZ group (transfected with miR-150-NC); and group II (transfected with miR-150inhibitor) followed by analysis of miR-150 expressio,n cell replication, apoptosis, invasion, migration ability and Notch1 and Notch3 expression by qRT-PCR, cloning, Hoechst33258 fluorescent staining, Transwell chamber, cell scratch test, dual luciferase report and Western blot. Lowest miR-150 expression in MCF-7 cells indicated a successful transfection (P < 0.05). Compared with KZ and WZ groups, Notch1 and Notch3 mRNA levels in group II were decreased (P <0.05); and the number of cell clones in group II was reduced (P <0.05) without difference between WZ and KZ group (P >0.05); miR-150 inhibitor reduced Notch1 and Notch3 expression (P <0.05). The fluorescence intensity of MCF-7 cells in group II was highest among three groups (P <0.05). The number of cell invasion and migration as well as Notch1 and Notch 3 expression in group II was reduced (P <0.05) without difference between group KZ and WZ (P >0.05). miR-150 expression is increased in MCF-7 cells. The miR-150 inhibitor can inhibit cell apoptosis, migration and other biological behaviors, which is related to target Notch signaling pathway.

Differential Collective Cell Migratory Behaviors Modulated by Phospholipid Nanocarriers.

Phospholipid nanocarriers have been widely explored for theranostic and nanomedicine applications. These amphiphilic nanocarriers possess outstanding cargo encapsulation efficiency, high water dispersibility, and excellent biocompatibility, which render them promising for drug delivery and bioimaging applications. While the biological applications of phospholipid nanocarriers have been well documented, the fundamental aspects of the phospholipid-cell interactions beyond cytotoxicity have been less investigated. In particular, the effect of phospholipid nanocarriers on collective cell behaviors has not been elucidated. Herein, we evaluate the interactions of phospholipid nanocarriers possessing different functional groups and sizes with normal and cancerous immortalized breast epithelial cell sheets with varying metastatic potential. Specifically, we examine the impact of nanocarrier treatments on the collective migratory dynamics of these cell sheets. We observe that phospholipid nanocarriers induce differential collective cell migratory behaviors, where the migration speed of normal and cancerous breast epithelial cell sheets is retarded and accelerated, respectively. To a certain extent, the nanocarriers are able to alter the migration trajectory of the cancerous breast epithelial cells. Furthermore, phospholipid nanocarriers could modulate the stiffness of the nuclei, cytoplasm, and cell-cell junctions of the breast epithelial cell sheets, remodel their actin filament arrangement, and regulate the expressions of the actin-related proteins. We anticipate that this work will further shed light on nanomaterial-cell interactions and provide guidelines for rational and safer designs and applications of phospholipid nanocarriers for cancer theranostics and nanomedicine.

Propagating acoustic waves on a culture substrate regulate the directional collective cell migration

Collective cell migration plays a critical role in physiological and pathological processes such as development, wound healing, and metastasis. Numerous studies have demonstrated how various types of chemical, mechanical, and electrical cues dictate the collective migratory behaviors of cells. Although an acoustic cue can be advantageous because of its noninvasiveness and biocompatibility, cell migration in response to acoustic stimulation remains poorly understood. In this study, we developed a device that is able to apply surface acoustic waves to a cell culture substrate and investigated the effect of propagating acoustic waves on collective cell migration. The migration distance estimated at various wave intensities revealed that unidirectional cell migration was enhanced at a critical wave intensity and that it was suppressed as the intensity was further increased. The increased migration might be attributable to cell orientation alignment along the direction of the propagating wave, as characterized by nucleus shape. Thicker actin bundles indicative of a high traction force were observed in cells subjected to propagating acoustic waves at the critical intensity. Our device and technique can be useful for regulating cellular functions associated with cell migration.

The expression, purification, and functional evaluation of the novel tumor suppressor fusion protein IL-24-CN.

Interleukin-24 (IL-24) can specifically induce apoptosis in a broad range of cancer cells without harming normal cells. The interaction of contortrostatin (CN) with integrins on angiogenic vascular endothelial and tumor cells is modulated by the RGD motifs that can significantly inhibit metastasis and angiogenesis. To achieve superior therapeutic efficacy by combining anti-metastasis with tumor-selective apoptosis activity, CN was fused at the C-terminus of IL-24 with a flexible linker (G4S)2, and the recombinant IL-24-CN was expressed in Escherichia coli as a Thioredoxin (Trx)/IL-24-CN fusion protein. The target protein was purified using nickel affinity chromatography. Furthermore, we simplified the purification process by purifying Trx-IL-24-CN and cleaving the Trx tag in one step. The final yield of IL-24-CN was 27.6mg/L based on flask fermentation. In vitro activity assay demonstrated that the recombinant IL-24-CN could more effectively suppress tumor growth and induce apoptosis of melanoma cells. Scratch and transwell assays suggested that IL-24-CN strongly reduced the migration and invasion behavior of melanoma cells. Immunofluorescence analysis and cell adhesion assay showed that CN could evidently improve the tumor inhibition capability of IL-24 by enhancing the affinity of recombinant protein toward cancer cells. In summary, a highly efficient strategy was developed for producing the bioactive IL-24-CN from prokaryotic cells, supporting IL-24-CN in melanoma therapy.Key points• Efficient heterologous production of recombinant IL-24-CN in E. coli using Trx fusion strategy.• Improved tumor growth suppression and apoptosis induction potency of IL-24-CN.• Enhanced cell adhesion ability of IL-24-CN in cancer cells.

Design and Fabrication of Microfluidic-Based 3D Microphysiological Systems for Studying Cell Migration and Invasion Behaviors

The cell migration and invasion behaviors play pivotal roles in tissue regeneration. For the skin repair process, a directed inflammatory response that regulates fibroblasts is critical for efficient wound healing. In this study, the authors present the design and fabrication of a microfluidic-based three-dimensional (3D) microphysiological system and how it impacts in controlling fibroblast migration and invasion under the induction of differently polarized macrophages. The microfluidic device had two chambers on opposite sides of a 1 mm micochannel, providing directed induction and sufficient width for long-term observation. The test cells could be seeded with or without matrix gel, cultured in a 2D or 3D microenvironment according to experiment settings. The microchannel allowed for any sorts of matrix filling and was on-demanding for continuous surveillance. Herein, our microfluidic device reserved the advantages of traditional methods using transwell chamber or scratch wound healing assay. In addition, it even came with more superiority such as retrievability, dynamic observation, and 3D environment simulation. The migration and invasion pattern of NIH3T3 modulated by RAW264.7 macrophages in different polarization status was demonstrated as an example. The results of the migration assay corresponded with that of the proliferation and gene expression experiments, verifying that our device was fully capable of restoring in vivo microenvironment and presenting cellular motility behaviors.

Photothermal Agarose Microfabrication Technology for Collective Cell Migration Analysis.

Agarose photothermal microfabrication technology is one of the micropatterning techniques that has the advantage of simple and flexible real-time fabrication even during the cultivation of cells. To examine the ability and limitation of the agarose microstructures, we investigated the collective epithelial cell migration behavior in two-dimensional agarose confined structures. Agarose microchannels from 10 to 211 micrometer width were fabricated with a spot heating of a focused 1480 nm wavelength infrared laser to the thin agarose layer coated on the cultivation dish after the cells occupied the reservoir. The collective cell migration velocity maintained constant regardless of their extension distance, whereas the width dependency of those velocities was maximized around 30 micrometer width and decreased both in the narrower and wider microchannels. The single-cell tracking revealed that the decrease of velocity in the narrower width was caused by the apparent increase of aspect ratio of cell shape (up to 8.9). In contrast, the decrease in the wider channels was mainly caused by the increase of the random walk-like behavior of component cells. The results confirmed the advantages of this method: (1) flexible fabrication without any pre-designing, (2) modification even during cultivation, and (3) the cells were confined in the agarose geometry.