Two-dimensional segmentation fusion tool: an extensible, free-to-use, user-friendly tool for combining different bidimensional segmentations.

Introduction: In several fields, the process of fusing multiple two-dimensional (2D) closed lines is an important step. For instance, this is fundamental in histology and oncology in general. The treatment of a tumor consists of numerous steps and activities. Among them, segmenting the cancer area, that is, the correct identification of its spatial location by the segmentation technique, is one of the most important and at the same time complex and delicate steps. The difficulty in deriving reliable segmentations stems from the lack of a standard for identifying the edges and surrounding tissues of the tumor area. For this reason, the entire process is affected by considerable subjectivity. Given a tumor image, different practitioners can associate different segmentations with it, and the diagnoses produced may differ. Moreover, experimental data show that the analysis of the same area by the same physician at two separate timepoints may result in different lines being produced. Accordingly, it is challenging to establish which contour line is the ground truth. Methods: Starting from multiple segmentations related to the same tumor, statistical metrics and computational procedures could be exploited to combine them for determining the most reliable contour line. In particular, numerous algorithms have been developed over time for this procedure, but none of them is validated yet. Accordingly, in this field, there is no ground truth, and research is still active. Results: In this work, we developed the Two-Dimensional Segmentation Fusion Tool (TDSFT), a user-friendly tool distributed as a free-to-use standalone application for MAC, Linux, and Windows, which offers a simple and extensible interface where numerous algorithms are proposed to "compute the mean" (i.e., the process to fuse, combine, and "average") multiple 2D lines. Conclusions: The TDSFT can support medical specialists, but it can also be used in other fields where it is required to combine 2D close lines. In addition, the TDSFT is designed to be easily extended with new algorithms thanks to a dedicated graphical interface for configuring new parameters. The TDSFT can be downloaded from the following link: https://sourceforge.net/p/tdsft.

Enhancing the conductivity of PEDOT:PSS films for biomedical applications via hydrothermal treatment.

This paper reports a new biocompatible conductivity enhancement of poly (3,4-ethylenedioxythiophene):poly (styrene sulfonate) (PEDOT:PSS) films for biomedical applications. Conductivity of PEDOT:PSS layer was reproducibly from 0.495 to 125.367 S cm-1 by hydrothermal (HT) treatment. The HT treatment employs water (relative humidity > 80%) and heat (temperature > 61 °C) instead of organic solvent doping and post-treatments, which can leave undesirable residue. The treatment can be performed using the sterilizing conditions of an autoclave. Additionally, it is possible to simultaneously reduce the electrical resistance, and sterilize the electrode for practical use. The key to conductivity enhancement was the structural rearrangement of PEDOT:PSS, which was determined using atomic force microscopy, X-ray diffraction, Raman spectroscopy, X-ray photoelectron spectroscopy, and ultraviolet-visible spectroscopy. It was found that PEDOT inter-bridging occurred as a result of the structural rearrangement. Therefore, the conductivity increased on account of the continuous conductive pathways of the PEDOT chains. To test the biocompatible enhancement technique for biomedical applications, certain demonstrations, such as the monitoring of joint movements and skin temperature, and measuring electrocardiogram signals were conducted with the hydrothermal-treated PEDOT:PSS electrode. This simple, biocompatible treatment exhibited significant potential for use in other biomedical applications as well.

Simultaneous on-chip isolation and characterization of circulating tumor cell sub-populations.

The diagnosis of tumor metastasis using circulating tumor cells (CTCs) has been considered an important developmental target for several decades but remains a formidable challenge because of the rarity and heterogeneity of CTCs. Additional downstream analysis is required after isolating CTCs on-chip for subtype verification. To solve those problems, we have developed microfluidic based integrated system which uses magnetic field gradient and immune-fluorescence differences to on-chip isolation and discrimination of CTCs simultaneously. The system presented in the present study can isolate CTCs with an efficiency of >99% by utilizing magnetic nanoparticles conjugated to CTC membranes. Furthermore, the statuses of three biomarkers can be determined on-chip simultaneously. The devised microfluidic system can differentiate eight different subtypes of heterogenic CTCs by on-chip isolation and based on the statuses of three biomarkers (HER2, ER, and PR) which are critical variables to five-year overall survivals for breast cancer patients.

On-chip isolation and enrichment of circulating cell-free DNA using microfluidic device.

Circulating cell-free DNA (cfDNA), containing cancer-specific DNAs derived from tumor cells, plays an important role in real-time monitoring of disease progression. Due to the abnormal growth of cancer and the promotion of cancer cell apoptosis by chemotherapy, the higher cfDNA concentration than healthy individuals is closely correlated with the diagnosis and treatment of cancer. Also, the mutation detection in tumor cell-derived cfDNA can be used to predict tumor progression. Human blood contains many blood cells (red blood cells, white blood cells, and platelets), proteins, extracellular vesicles, and so on. These blood components act as the inhibitors when the cfDNA is analyzed using polymerase chain reaction. So, analysis of cfDNA using whole blood directly may affect the sensitivity of the analysis or result in false-negative. The conventional methods of cfDNA isolation, such as silica absorption and polymer-mediated enrichment, are labor-intensive and time-consuming processes that can also lead to the loss of cfDNA in cumbersome procedures. Here, we designed an integrated microfluidic chip capable of on-chip cfDNA extracting to reduce sample loss and processing time. Our proposed device minimizes the number of experimental steps from 5 to 1, the total processing time from 42 to 19 min, and the required volume of washing reagents from 2 to 0.4 ml for cfDNA enrichment compared to the conventional method.

Salivary Exosome and Cell-Free DNA for Cancer Detection.

Liquid biopsies are easier to acquire patient derived samples than conventional tissue biopsies, and their use enables real-time monitoring of the disease through continuous sampling after initial diagnosis, resulting in a paradigm shift to customized treatment according to the patient's prognosis. Among the various liquid biopsy samples, saliva is easily obtained by spitting or swab sucking without needing an expert for sample collection. In addition, it is known that disease related biomarkers that exist in the blood and have undergone extensive research exist in saliva even at a lower concentration than the blood. Thus, interest in the use of saliva as a liquid biopsy has increased. In this review, we focused on the salivary exosome and cell-free DNA (cfDNA) among the various biomarkers in saliva. Since the exosome and cfDNA in saliva are present at lower concentrations than the biomarkers in blood, it is important to separate and concentrate them before conducting down-stream analyses such as exosome cargo analysis, quantitative polymerase chain reaction (qPCR), and sequencing. However, saliva is difficult to apply directly to microfluidics-based systems for separation because of its high viscosity and the presence of various foreign substances. Therefore, we reviewed the microfluidics-based saliva pretreatment method and then compared the commercially available kit and the microfluidic chip for isolation and enrichment of the exosome and cfDNA in saliva.

Progress in Circulating Tumor Cell Research Using Microfluidic Devices.

Circulating tumor cells (CTCs) are a popular topic in cancer research because they can be obtained by liquid biopsy, a minimally invasive procedure with more sample accessibility than tissue biopsy, to monitor a patient's condition. Over the past decades, CTC research has covered a wide variety of topics such as enumeration, profiling, and correlation between CTC number and patient overall survival. It is important to isolate and enrich CTCs before performing CTC analysis because CTCs in the blood stream are very rare (0⁻10 CTCs/mL of blood). Among the various approaches to separating CTCs, here, we review the research trends in the isolation and analysis of CTCs using microfluidics. Microfluidics provides many attractive advantages for CTC studies such as continuous sample processing to reduce target cell loss and easy integration of various functions into a chip, making "do-everything-on-a-chip" possible. However, tumor cells obtained from different sites within a tumor exhibit heterogenetic features. Thus, heterogeneous CTC profiling should be conducted at a single-cell level after isolation to guide the optimal therapeutic path. We describe the studies on single-CTC analysis based on microfluidic devices. Additionally, as a critical concern in CTC studies, we explain the use of CTCs in cancer research, despite their rarity and heterogeneity, compared with other currently emerging circulating biomarkers, including exosomes and cell-free DNA (cfDNA). Finally, the commercialization of products for CTC separation and analysis is discussed.

Mass fabrication of uniform sized 3D tumor spheroid using high-throughput microfluidic system.

In vivo tumors develop in a three-dimensional manner and have unique and complex characteristics. Physico-biochemical barriers on tumors cause drug resistance and limit drug delivery efficiency. Currently, 2D cancer cell monolayer platforms are frequently used to test the efficiency of new drug materials. However, the monolayer platform generally overestimates drug efficiency because of the absence of physico-biochemical barriers. Many literatures indicated that a 3D tumor spheroid model has very similar characteristics to in vivo tumor models, and studies demonstrated the accurate prediction of drug efficiency using this model. The use of a 3D tumor spheroid model in drug development process remains challenging because of the low generation yield and difficulties in size control. In this study, we developed a droplet-based microfluidic system that can generate cancer cells encapsulated by micro-droplets with very high generation yield (16-20 Hz, 1000 droplets/min). The system can control the number of encapsulated cancer cells in the droplet or diameter of the 3D spheroid model precisely between 50 and 150 µm. Moreover, the formed 3D tumor spheroid model can be cultured for >2 weeks by an additional step of droplet disruption and recollection, and can grow up to 245 µm in diameter.

Spiral shape microfluidic channel for selective isolating of heterogenic circulating tumor cells.

Detecting heterogenic tumor cells that are traveling in our body through blood stream for the tumor metastasis is one way for cancer prognosis. Due to the heterogeneity of circulating tumor cells (CTCs), further identification of tumor cell types should be accompanied with CTCs isolation from blood cells in peripheral blood sample. Both negative enrichment and recollection of isolated CTCs are required in the downstream analysis, which are time-consuming, labor-intensive, and massive equipment required. To solve these limitations, we have developed a simple and disposable spiral shape microfluidic channel that can separate all CTCs from blood cells, and at the same time, can identify the types of CTCs based on epithelial cell adhesion molecule (EpCAM) expression level. Two different types of tumor cells, MCF-7 and MDA-MB-231, both from the same origin of breast carcinoma cells, were used to demonstrate the functionality of the developed system. The spiral channel system could capture the EpCAM positive and negative CTCs with 96.3% and 81.2% purity, respectively, while both EpCAM positive and negative CTCs were differently positioned along the microfluidic channel. The average selectivity of EpCAM positive and negative CTCs is 6.1:4.8. In addition, the throughput of the system was optimized at a sample flow rate of 150µl/min. The developed system successfully demonstrated its potential to identify biomarkers, including EpCAM, for detecting the heterogenic CTCs.

Selective isolation of magnetic nanoparticle-mediated heterogeneity subpopulation of circulating tumor cells using magnetic gradient based microfluidic system.

Relocation mechanisms of the circulating tumor cells (CTCs) from the primary site to the secondary site through the blood vessel network cause tumor metastasis. Despite of the importance to diagnose the cancer metastasis by CTCs, still it is formidable challenge to use in the clinical purpose because of the rarity and the heterogeneity of CTCs in the cancer patient's peripheral blood sample. In this study we have developed magnetic force gradient based microfluidic chip (Mag-Gradient Chip) for isolating the total number of CTCs in the sample and characterizing the state of CTCs simultaneously with respect to the epithelial cell adhesion molecule (EpCAM) expression level. We have synthesized magnetic nanoparticles (MNPs) using hydrothermal method and functionalized anti-EpCAM on their surface for the specific binding with CTCs. The Mag-Gradient Chip designed to isolate and classify the CTCs by isolating at the different location in the chip using magnetic force differences depending on the EpCAM expression level. We observed 95.7% of EpCAM positive and 79.3% of EpCAM negative CTCs isolated in the Mag-Gradient Chip. At the same time, the 71.3% of isolated EpCAM positive CTCs were isolated at the first half area whereas the 76.9% of EpCAM negative CTCs were collected at the latter half area. The Mag-Gradient Chip can isolate the 3ml of heterogeneous CTCs sample in 1h with high isolating yield. The EpCAM expression level dose not means essential condition of the metastatic CTCs, but the Mag-Gradient Chip can shorten the date to diagnose the cancer metastasis in clinic.

Simulation of complex transport of nanoparticles around a tumor using tumor-microenvironment-on-chip.

Delivery of therapeutic agents selectively to tumor tissue, which is referred as "targeted delivery," is one of the most ardently pursued goals of cancer therapy. Recent advances in nanotechnology enable numerous types of nanoparticles (NPs) whose properties can be designed for targeted delivery to tumors. In spite of promising early results, the delivery and therapeutic efficacy of the majority of NPs are still quite limited. This is mainly attributed to the limitation of currently available tumor models to test these NPs and systematically study the effects of complex transport and pathophysiological barriers around the tumors. In this study, thus, we developed a new in vitro tumor model to recapitulate the tumor microenvironment determining the transport around tumors. This model, named tumor-microenvironment-on-chip (T-MOC), consists of 3-dimensional microfluidic channels where tumor cells and endothelial cells are cultured within extracellular matrix under perfusion of interstitial fluid. Using this T-MOC platform, the transport of NPs and its variation due to tumor microenvironmental parameters have been studied including cut-off pore size, interstitial fluid pressure, and tumor tissue microstructure. The results suggest that T-MOC is capable of simulating the complex transport around the tumor, and providing detailed information about NP transport behavior. This finding confirms that NPs should be designed considering their dynamic interactions with tumor microenvironment.

Development of an in vitro 3D tumor model to study therapeutic efficiency of an anticancer drug.

The importance and advantages of three-dimensional (3D) cell cultures have been well-recognized. Tumor cells cultured in a 3D culture system as multicellular tumor spheroids (MTS) can bridge the gap between in vitro and in vivo anticancer drug evaluations. An in vitro 3D tumor model capable of providing close predictions of in vivo drug efficacy will enhance our understanding, design, and development of better drug delivery systems. Here, we developed an in vitro 3D tumor model by adapting the hydrogel template strategy to culture uniformly sized spheroids in a hydrogel scaffold containing microwells. The in vitro 3D tumor model was to closely simulate an in vivo solid tumor and its microenvironment for evaluation of anticancer drug delivery systems. MTS cultured in the hydrogel scaffold are used to examine the effect of culture conditions on the drug responses. Free MTS released from the scaffold are transferred to a microfluidic channel to simulate a dynamic in vivo microenvironment. The in vitro 3D tumor model that mimics biologically relevant parameters of in vivo microenvironments such as cell-cell and cell-ECM interactions, and a dynamic environment would be a valuable device to examine efficiency of anticancer drug and targeting specificity. These models have potential to provide in vivo correlated information to improve and optimize drug delivery systems for an effective chemotherapy.