Acoustofluidic Separation of Proteins Using Aptamer-Functionalized Microparticles.

We propose an acoustofluidic method for the triseparation of proteins conjugated with aptamer-coated microparticles inside a microchannel. Traveling surface acoustic waves (TSAWs) produced from a slanted-finger interdigital transducer (SFIT) are used to separate the protein-loaded microparticles of different sizes via the TSAW-driven acoustic radiation force (ARF). The acoustofluidic device consists of an SFIT deposited onto a piezoelectric lithium niobate substrate and a polydimethylsiloxane (PDMS) microfluidic channel on top of the substrate. The TSAWs propagating on the substrate penetrate into the sample fluid flow, where the human protein-conjugated microparticles are suspended, inside the PDMS microchannel. The microparticles are subjected to the TSAW-driven ARF with varying magnitude depending on their size and thus flow along different streamlines, leading to triseparation of the proteins. In this work, we used two different-sized streptavidin-functionalized polystyrene (PS) microparticles to capture two kinds of aptamers (apt15 and aptD17.4), which were labeled with a respective biotin molecule at one end. The biotin ends of the aptamers were attached to the microparticles through streptavidin-biotin linkage, whereas the free ends of the aptamers were used to capture their target proteins of thrombin (th) and immunoglobulin E (IgE). The resultant PS-apt15-th and PS-aptD17.4-IgE complexes, as well as mCardinal2, were used for experimental demonstration of acoustofluidic triseparation of the human proteins. We achieved simultaneous separation of proteins of three kinds (th, IgE, and mCardinal2) for the first time via the TSAW-driven ARF in the proposed acoustofluidic device.

Surface tethering of stromal cell-derived factor-1α carriers to stem cells enhances cell homing to ischemic muscle.

Mesenchymal stem cells are promising medicine for treating diseases and tissue defects because of their innate ability to secrete therapeutic factors. Intravenous delivery of stem cells, although favored for its minimal invasiveness, is often plagued by low cellular engraftment in the target tissue. To this end, this study hypothesizes that in situ activation of cellular expression of CXC chemokine 4 (CXCR4) would significantly improve cellular migration to injured tissue. This hypothesis was examined by tethering the surface of stem cells with poly(D,L-lactide-co-glycolide)-block-hyaluronic acid (HA) particles containing stromal cell-derived factor-1α, a model chemokine to sensitize CXCR4. The HA blocks in the particles enhanced the association rate constant to stem cells by 3.3-fold, and in turn, increased the number of cells expressing CXCR4 receptors. Consequently, these cells displayed 1.2-fold higher transendothelial migration in vitro and 1.7-fold greater trafficking to the ischemic hindlimb of a mouse than that of the untethered cells.

Efficient Capture and Raman Analysis of Circulating Tumor Cells by Nano-Undulated AgNPs-rGO Composite SERS Substrates.

The analysis of circulating tumor cells (CTCs) in the peripheral blood of cancer patients is critical in clinical research for further investigation of tumor progression and metastasis. In this study, we present a novel surface-enhanced Raman scattering (SERS) substrate for the efficient capture and characterization of cancer cells using silver nanoparticles-reduced graphene oxide (AgNPs-rGO) composites. A pulsed laser reduction of silver nanowire-graphene oxide (AgNW-GO) mixture films induces hot-spot formations among AgNPs and artificial biointerfaces consisting of rGOs. We also use in situ electric field-assisted fabrication methods to enhance the roughness of the SERS substrate. The AgNW-GO mixture films, well suited for the proposed process due to its inherent electrophoretic motion, is adjusted between indium tin oxide (ITO) transparent electrodes and the nano-undulated surface is generated by applying direct-current (DC) electric fields during the laser process. As a result, MCF7 breast cancer cells are efficiently captured on the AgNPs-rGO substrates, about four times higher than the AgNWs-GO films, and the captured living cells are successfully analyzed by SERS spectroscopy. Our newly designed bifunctional substrate can be applied as an effective system for the capture and characterization of CTCs.

Tracking adiponectin biodistribution via fluorescence molecular tomography indicates increased vascular permeability after streptozotocin-induced diabetes.

Adiponectin, a highly abundant polypeptide hormone in plasma, plays an important role in the regulation of energy metabolism in a wide variety of tissues, as well as providing important beneficial effects in diabetes, inflammation, and cardiovascular disease. To act on target tissues, adiponectin must move from the circulation to the interstitial space, suggesting that vascular permeability plays an important role in regulating adiponectin action. To test this hypothesis, fluorescently labeled adiponectin was used to monitor its biodistribution in mice with streptozotocin-induced diabetes (STZD). Adiponectin was, indeed, found to have increased sequestration in the highly fenestrated liver and other tissues within 90 min in STZD mice. In addition, increased myocardial adiponectin was detected and confirmed using computed tomography (CT) coregistration. This provided support of adiponectin delivery to affected cardiac tissue as a cardioprotective mechanism. Higher adiponectin content in the STZD heart tissues was further examined by ex vivo fluorescence molecular tomography (FMT) imaging, immunohistochemistry, and Western blot analysis. In vitro mechanistic studies using an endothelial monolayer on inserts and three-dimensional microvascular networks on microfluidic chips further confirmed that adiponectin flux was increased by high glucose. However, in the in vitro model and mouse heart tissue, high glucose levels did not change adiponectin receptor levels. An examination of the tight junction (TJ) complex revealed a decrease in the TJ protein claudin (CLDN)-7 in high glucose-treated endothelial cells, and the functional significance of this change was underscored by increased endothelium permeability upon siRNA-mediated knockdown of CLDN-7. Our data support the idea that glucose-induced effects on permeability of the vascular endothelium contribute to the actions of adiponectin by regulating its transendothelial movement from blood to the interstitial space. These observations are physiologically significant and critical when considering ways to harness the therapeutic potential of adiponectin for diabetes.

Chemotaxis Model for Breast Cancer Cells Based on Signal/Noise Ratio.

Chemotaxis, a biased migration of cells under a chemical gradient, plays a significant role in diverse biological phenomena including cancer metastasis. Stromal cells release signaling proteins to induce chemotaxis, which leads to organ-specific metastasis. Epidermal growth factor (EGF) is an example of the chemical attractants, and its gradient stimulates metastasis of breast cancer cells. Hence, the interactions between EGF and breast cancer cells have long been a subject of interest for oncologists and clinicians. However, most current approaches do not systematically separate the effects of gradient and absolute concentration of EGF on chemotaxis of breast cancer cells. In this work, we develop a theoretical model based on signal/noise ratio to represent stochastic properties and report our microfluidic experiments to verify the analytical predictions from the model. The results demonstrate that even under the same EGF concentration gradients (0-50 or 0-150 ng/mL), breast cancer cells reveal a more evident chemotaxis pattern when the absolute EGF concentrations are low. Moreover, we found that reducing the number of EGF receptors (EGFRs) with addition of EGFR antibody (1 ng/mL) can promote chemotaxis at an EGF gradient of 0-1 ng/mL as shown by chemotaxis index (0.121 ± 0.037, reduced EGFRs vs. 0.003 ± 0.041, control). This counterintuitive finding suggests that EGFR-targeted therapy may stimulate metastasis of breast cancer because the partial suppression of the receptors makes the number of receptors close to the optimal one for chemotaxis. This analysis should be considered in anticancer drug design.

Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation.

A key aspect of cancer metastases is the tendency for specific cancer cells to home to defined subsets of secondary organs. Despite these known tendencies, the underlying mechanisms remain poorly understood. Here we develop a microfluidic 3D in vitro model to analyze organ-specific human breast cancer cell extravasation into bone- and muscle-mimicking microenvironments through a microvascular network concentrically wrapped with mural cells. Extravasation rates and microvasculature permeabilities were significantly different in the bone-mimicking microenvironment compared with unconditioned or myoblast containing matrices. Blocking breast cancer cell A3 adenosine receptors resulted in higher extravasation rates of cancer cells into the myoblast-containing matrices compared with untreated cells, suggesting a role for adenosine in reducing extravasation. These results demonstrate the efficacy of our model as a drug screening platform and a promising tool to investigate specific molecular pathways involved in cancer biology, with potential applications to personalized medicine.

Visual Estimation of Bacterial Growth Level in Microfluidic Culture Systems.

Microfluidic devices are an emerging platform for a variety of experiments involving bacterial cell culture, and has advantages including cost and convenience. One inevitable step during bacterial cell culture is the measurement of cell concentration in the channel. The optical density measurement technique is generally used for bacterial growth estimation, but it is not applicable to microfluidic devices due to the small sample volumes in microfluidics. Alternately, cell counting or colony-forming unit methods may be applied, but these do not work in situ; nor do these methods show measurement results immediately. To this end, we present a new vision-based method to estimate the growth level of the bacteria in microfluidic channels. We use Fast Fourier transform (FFT) to detect the frequency level change of the microscopic image, focusing on the fact that the microscopic image becomes rough as the number of cells in the field of view increases, adding high frequencies to the spectrum of the image. Two types of microfluidic devices are used to culture bacteria in liquid and agar gel medium, and time-lapsed images are captured. The images obtained are analyzed using FFT, resulting in an increase in high-frequency noise proportional to the time passed. Furthermore, we apply the developed method in the microfluidic antibiotics susceptibility test by recognizing the regional concentration change of the bacteria that are cultured in the antibiotics gradient. Finally, a deep learning-based data regression is performed on the data obtained by the proposed vision-based method for robust reporting of data.

MineLoC: A Rapid Production of Lab-on-a-Chip Biosensors Using 3D Printer and the Sandbox Game, Minecraft.

Here, MineLoC is described as a pipeline developed to generate 3D printable models of master templates for Lab-on-a-Chip (LoC) by using a popular multi-player sandbox game “Minecraft”. The user can draw a simple diagram describing the channels and chambers of the Lab-on-a-Chip devices with pre-registered color codes which indicate the height of the generated structure. MineLoC converts the diagram into large chunks of blocks (equal sized cube units composing every object in the game) in the game world. The user and co-workers can simultaneously access the game and edit, modify, or review, which is a feature not generally supported by conventional design software. Once the review is complete, the resultant structure can be exported into a stereolithography (STL) file which can be used in additive manufacturing. Then, the Lab-on-a-Chip device can be fabricated by the standard protocol to produce a Lab-on-a-Chip. The simple polydimethylsiloxane (PDMS) device for the bacterial growth measurement used in the previous research was copied by the proposed method. The error calculation by a 3D model comparison showed an accuracy of 86%. It is anticipated that this work will facilitate more use of 3D printer-based Lab-on-a-Chip fabrication, which greatly lowers the entry barrier in the field of Lab-on-a-Chip research.

Light Emitting Marker for Robust Vision-Based On-The-Spot Bacterial Growth Detection.

Simple methods using the striped pattern paper marker and FFT (fast Fourier transformation) have been proposed as alternatives to measuring the optical density for determining the level of bacterial growth. The marker-based method can be easily automated, but due to image-processing-base of the method, the presence of light or the color of the culture broth can disturb the detection process. This paper proposes a modified version of marker-FFT-based growth detection that uses a light emitting diode (LED) array as a marker. Since the marker itself can emit the light, the measurements can be performed even when there is no light source or the bacteria are cultured in a large volume of darkly colored broth. In addition, an LED marker can function as a region of interest (ROI) indicator in the image. We expect that the proposed LED-based marker system will allow more robust growth detection compared to conventional methods.

Vision Marker-Based In Situ Examination of Bacterial Growth in Liquid Culture Media.

The detection of bacterial growth in liquid media is an essential process in determining antibiotic susceptibility or the level of bacterial presence for clinical or research purposes. We have developed a system, which enables simplified and automated detection using a camera and a striped pattern marker. The quantification of bacterial growth is possible as the bacterial growth in the culturing vessel blurs the marker image, which is placed on the back of the vessel, and the blurring results in a decrease in the high-frequency spectrum region of the marker image. The experiment results show that the FFT (fast Fourier transform)-based growth detection method is robust to the variations in the type of bacterial carrier and vessels ranging from the culture tubes to the microfluidic devices. Moreover, the automated incubator and image acquisition system are developed to be used as a comprehensive in situ detection system. We expect that this result can be applied in the automation of biological experiments, such as the Antibiotics Susceptibility Test or toxicity measurement. Furthermore, the simple framework of the proposed growth measurement method may be further utilized as an effective and convenient method for building point-of-care devices for developing countries.

Investigation of the Dysfunction Caused by High Glucose, Advanced Glycation End Products, and Interleukin-1 Beta and the Effects of Therapeutic Agents on the Microphysiological Artery Model.

Diabetes mellitus (DM) has substantial global implications and contributes to vascular inflammation and the onset of atherosclerotic cardiovascular diseases. However, translating the findings from animal models to humans has inherent limitations, necessitating a novel platform. Therefore, herein, an arterial model is established using a microphysiological system. This model successfully replicates the stratified characteristics of human arteries by integrating collagen, endothelial cells (ECs), and vascular smooth muscle cells (VSMCs). Perfusion via a peristaltic pump shows dynamic characteristics distinct from those of static culture models. High glucose, advanced glycation end products (AGEs), and interleukin-1 beta are employed to stimulate diabetic conditions, resulting in notable cellular changes and different levels of cytokines and nitric oxide. Additionally, the interactions between the disease models and oxidized low-density lipoproteins (LDL) are examined. Finally, the potential therapeutic effects of metformin, atorvastatin, and diphenyleneiodonium are investigated. Metformin and diphenyleneiodonium mitigate high-glucose- and AGE-associated pathological changes, whereas atorvastatin affects only the morphology of ECs. Altogether, the arterial model represents a pivotal advancement, offering a robust and insightful platform for investigating cardiovascular diseases and their corresponding drug development.

Hydrophobic surface induced pro-metastatic cancer cells for in vitro extravasation models.

In vitro vascularized cancer models utilizing microfluidics have emerged as a promising tool for mechanism study and drug screening. However, the lack of consideration and preparation methods for cancer cellular sources that are capable of adequately replicating the metastatic features of circulating tumor cells contributed to low relevancy with in vivo experimental results. Here, we show that the properties of cancer cellular sources have a considerable impact on the validity of the in vitro metastasis model. Notably, with a hydrophobic surface, we can create highly metastatic spheroids equipped with aggressive invasion, endothelium adhesion capabilities, and activated metabolic features. Combining these metastatic spheroids with the well-constructed microfluidic-based extravasation model, we validate that these metastatic spheroids exhibited a distinct extravasation response to epidermal growth factor (EGF) and normal human lung fibroblasts compared to the 2D cultured cancer cells, which is consistent with the previously reported results of in vivo experiments. Furthermore, the applicability of the developed model as a therapeutic screening platform for cancer extravasation is validated through profiling and inhibition of cytokines. We believe this model incorporating hydrophobic surface-cultured 3D cancer cells provides reliable experimental data in a clear and concise manner, bridging the gap between the conventional in vitro models and in vivo experiments.

Analyzing the effects of helical flow in blood vessels using acoustofluidic-based dynamic flow generator.

The effects of helical flow in a blood vessel are investigated in a dynamic flow generator using surface acoustic wave (SAW) in the microfluidic device. The SAW, generated by an interdigital transducer (IDT), induces acoustic streaming, resulting in a stable and consistent helical flow pattern in microscale channels. This approach allows rapid development of helical flow within the channel without directly contacting the medium. The precise design of the window enables the creation of distinct unidirectional vortices, which can be controlled by adjusting the amplitude of the SAW. Within this device, optimal operational parameters of the dynamic flow generator to preserve the integrity of endothelial cells are found, and in such settings, the actin filaments within the cells are aligned to the desired state. Our findings reveal that intracellular Ca2+ concentrations vary in response to flow conditions. Specifically, comparable maximum intensity and graphical patterns were observed between low-flow rate helical flow and high-flow rate Hagen-Poiseuille flow. These suggest that the cells respond to the helical flow through mechanosensitive ion channels. Finally, adherence of monocytes is effectively reduced under helical flow conditions in an inflammatory environment, highlighting the atheroprotective role of helical flow. STATEMENT OF SIGNIFICANCE: Helical flow in blood vessels is well known to prevent atherosclerosis. However, despite efforts to replicate helical flow in microscale channels, there is still a lack of in vitro models which can generate helical flow for analyzing its effects on the vascular system. In this study, we developed a method for generating steady and constant helical flow in microfluidic channel using acoustofluidic techniques. By utilizing this dynamic flow generator, we were able to observe the atheroprotective aspects of helical flow in vitro, including the enhancement of calcium ion flux and reduction of monocyte adhesion. This study paves the way for an in vitro model of dynamic cell culture and offers advanced investigation into helical flow in our circulatory system.

Generation of a 3D Outer Blood-Retinal Barrier with Advanced Choriocapillaris and Its Application in Diabetic Retinopathy in a Microphysiological System.

The outer blood-retinal barrier (oBRB) provides an optimal environment for the function of the photoreceptor by regulating the exchange of molecules between subretinal space and the choriocapillaris, and its dysfunction could impair the photoreceptor's function and vision. The existing in vitro models have limitations in reproducing the barrier function or physiological characteristics of oBRB and choriocapillaris. Here, we engineered a microphysiological system-based oBRB-choriocapillaris model that simultaneously incorporates the desired physiological characteristics and is simple to fabricate. First, we generated microvascular networks to mimic choriocapillaris and investigated the role of fibroblasts in vasculogenesis. By adding retinal pigment epithelial cells to one side of blood vessels formed with endothelial cells and fibroblasts and optimizing their culture medium conditions, we established an oBRB-choriocapillaris model. To verify the physiological similarity of our oBRB-choriocapillaris model, we identified the polarization and expression of the tight junction of the retinal pigment epithelium, Bruch's membrane, and the fenestral diaphragm of choriocapillaris. Finally, we tried to recapitulate the diabetes mellitus environment in our model with hyperglycemia and diabetes-related cytokines. This induced a decrease in tight junction integrity, loss of barrier function, and shrinkage of blood vessels, similar to the in vivo pathological changes observed in the oBRB and choriocapillaris. The oBRB-choriocapillaris model developed using a microphysiological system is expected to offer a valuable in vitro platform for retinal and choroidal vascular diseases in preclinical applications.

3D vascularized microphysiological system for investigation of tumor-endothelial crosstalk in anti-cancer drug resistance.

Despite the advantages of microfluidic system in drug screening, vascular systems responsible for the transport of drugs and nutrients have been hardly considered in the microfluidic-based chemotherapeutic screening. Considering the physiological characteristics of highly vascularized urinary tumors, we here investigated the chemotherapeutic response of bladder tumor cells using a vascularized tumor on a chip. The microfluidic chip was designed to have open-top region for tumor sample introduction and hydrophilic rail for spontaneous hydrogel patterning, which contributed to the construction of tumor-hydrogel-endothelium interfaces in a spatiotemporal on-demand manner. Utilizing the chip where intravascularly injected cisplatin diffuse across the endothelium and transport into tumor samples, chemotherapeutic responses of cisplatin-resistant or -susceptible bladder tumor cells were evaluated, showing the preservation of cellular drug resistance even within the chip. The open-top structure also enabled the direct harvest of tumor samples and post analysis in terms of secretome and gene expressions. Comparing the cisplatin efficacy of the cisplatin-resistant tumor cells in the presence or absence of endothelium, we found that the proliferation rates of tumor cells were increased in the vasculature-incorporated chip. These have suggested that our vascularized tumor chip allows the establishment of vascular-gel-tumor interfaces in spatiotemporal manners and further enables investigations of chemotherapeutic screening.

Bioprinting Methods for Fabricating In Vitro Tubular Blood Vessel Models.

Dysfunctional blood vessels are implicated in various diseases, including cardiovascular diseases, neurodegenerative diseases, and cancer. Several studies have attempted to prevent and treat vascular diseases and understand interactions between these diseases and blood vessels across different organs and tissues. Initial studies were conducted using 2-dimensional (2D) in vitro and animal models. However, these models have difficulties in mimicking the 3D microenvironment in human, simulating kinetics related to cell activities, and replicating human pathophysiology; in addition, 3D models involve remarkably high costs. Thus, in vitro bioengineered models (BMs) have recently gained attention. BMs created through biofabrication based on tissue engineering and regenerative medicine are breakthrough models that can overcome limitations of 2D and animal models. They can also simulate the natural microenvironment in a patient- and target-specific manner. In this review, we will introduce 3D bioprinting methods for fabricating bioengineered blood vessel models, which can serve as the basis for treating and preventing various vascular diseases. Additionally, we will describe possible advancements from tubular to vascular models. Last, we will discuss specific applications, limitations, and future perspectives of fabricated BMs.

Acoustofluidic lysis of cancer cells and Raman spectrum profiling.

The lysis of cancer cells inside a sessile droplet was performed using traveling surface acoustic waves (SAWs) without any chemical reagents. Raman spectrum profiling was then carried out to explore detailed cell-derived data. The Rayleigh waves formed by an interdigital transducer were made to propagate along the surface of an LiNbO3 substrate. Polystyrene microparticles (PSMPs) were used to establish mechanical cell lysis effectively, and gold nanoparticles (AuNPs) were added to enhance the Raman signals from the lysed cells by SAWs. The lysis efficiency was evaluated according to the size and concentration of the PSMPs in experiments where the frequency was varied. Lysis occurred mainly by mechanical collision using PSMPs in a high-frequency domain, and the lysis efficiency was improved by increasing the application time and the energy density of the SAWs. Raman signals from the lysed cells were greatly enhanced by nanogaps formed by the AuNPs, which were evenly distributed irrespective of the SAWs through the frequency-independent behavior of the AuNPs. Finally, detailed Raman spectra of MDA-MB-231, malignant breast cancer cells, were acquired, and various organic matter-derived peaks were observed. The 95% confidence region for cells subjected to lysis was more widely distributed than that of cells not subjected to lysis. The proposed SAW platform is expected to facilitate the detection of small quantities and to be applied in biomedical applications.

Microfluidic outer blood-retinal barrier model for inducing wet age-related macular degeneration by hypoxic stress.

Wet age-related macular degeneration (AMD) is a severe ophthalmic disease that develops in the outer blood-retinal barrier (oBRB), involving two types of cells, the retinal pigment epithelium (RPE) and the choriocapillaris endothelium (CCE). Unfortunately, the pathogenesis of AMD is unclear, and the risk of the only effective therapy (Anti-VEGF injection) has been consistently argued. Also, since oBRB is hard to observe in vivo, an in vitro model for the pathological study is necessary. Here, we propose an advanced oBRB model, enhanced in two major ways: fully vascularized CCE and the in vivo analogous distance between RPE and CCE. Our model consists of an RPE (ARPE-19) monolayer with adjacent CCE (HUVEC) embedded fibrin gel in the microfluidic chip and required four days to construct an oBRB. Notably, the intercellular distance was tuned to the in vivo scale (<100 µm) without any extraneous scaffold in between. Thus, the two cell layers can interact freely through the extracellular matrix (ECM) in vivo. This is significant as wet AMD is mainly developed through broken intercellular interaction. Thanks to this in vivo similarity, the model incubated under hypoxic conditions, similar to an oxygen-induced retinopathy animal model, showed upregulated vascularization comparable to the AMD condition. We envisage that our model can be used to assist the investigation of AMD.

Enabling perfusion through multicellular tumor spheroids promoting lumenization in a vascularized cancer model.

A tumor is composed of heterogeneous cell population, which is known as tumor stroma. In particular, blood vessels have an indispensable role in the tumor microenvironment acting as a key player in anti-cancer drug delivery. Recently, efforts have been made to accurately recapitulate the microenvironment by employing distinct cell types, however, the proper formation of perfusable tumor tissue is challenging. Here, perfusable tumor tissue is engineered by implanting multicellular tumor spheroids inside the microfluidic devices. Blood perfusion, spheroid growth, and vascular dynamics were monitored according to the spheroid composition and the contribution of internal and external vascular cells to spheroid perfusion was analyzed. Most notably, the increased penetration depth of fluorescence conjugated anti-cancer drug was observed in tri-culture spheroids. The implementation of tumor microenvironment reconstruction developed in this study not only creates a perfusable tumor vascular model but can also be utilized as a novel drug screening platform with patient-derived samples.

Microfluidic vascular models of tumor cell extravasation.

Emerging microfluidic disease models have amply demonstrated their value in many fields of cancer research. These in vitro technologies recapitulate key aspects of metastatic cancer, including the process of tumor cell arrest and extravasation at the site of the metastatic tumor. To date, extensive efforts have been made to capture key features of the microvasculature to reconstitute the pre-metastatic niche and investigate dynamic extravasation behaviors using microfluidic systems. In this mini-review, we highlight recent microfluidic vascular models of tumor cell extravasation and explore how this approach contributes to development of in vitro disease models to enhance understanding of metastasis in vivo.