Fabrication Method for Shape-Controlled 3D Tissue Using High-Porosity Porous Structure.

Shape-controlled 3D tissues resemble natural living tissues in human and animal bodies and are essential materials for developing and improving technologies in regenerative medicine, drug discovery, and biological robotics. In previous studies, shape-controlled 3D tissues were fabricated using scaffold structures or 3D bioprinting techniques. However, controlling the shape of 3D tissues without leaving non-natural materials inside the 3D tissue and efficiently fabricating them remains challenging. In this paper, we propose a novel method for fabricating shape-controlled 3D tissues free of non-natural materials using a flexible high-porosity porous structure (HPPS). The HPPS consisted of a micromesh with pore sizes of 14.87 ± 1.83 µm, lattice widths of 2.24 ± 0.10 µm, thicknesses of 9.96 ± 0.92 µm, porosity of 69.06 ± 3.30%, and an I-shaped microchamber of depth 555.26 ± 11.17 µm. U-87 human glioma cells were cultured in an I-shaped HPPS microchamber for 48 h. After cultivation, the 3D tissue was released within a few seconds while maintaining its I-shape. Specific chemicals, such as proteolytic enzymes, were not used. Moreover, the viability of the released cells composed of shape-controlled 3D tissues free of non-natural materials was above 90%. Therefore, the proposed fabrication method is recommended for shape-controlled 3D tissues free of non-natural materials without applying significant stresses to the cells.

Localization of EGFR Mutations in Non-small-cell Lung Cancer Tissues Using Mutation-specific PNA-DNA Probes.

BACKGROUND/AIM: Epidermal growth factor receptor (EGFR) signaling inhibitors are potent therapeutic agents for EGFR-mutant non-small-cell lung cancer, but the effects of such inhibitors on the localization of EGFR mutations in tumor tissues remain to be elucidated. Thus, a simple and efficient technology for the detection of mutations in tumor tissue specimens needs to be developed. MATERIALS AND METHODS: Using an EGFR mutation-specific peptide nucleic acid (PNA)-DNA probe, the EGFR mutation-positive part of whole NSCLC tissues was visualized by immunofluorescence. Formalin-fixed paraffin-embedded sections obtained from A549, NCI-H1975, HCC827 and PC-9 tumors transplanted into nude mice were subjected to staining using PNA-DNA probes specific for the mRNA sequences producing the L858R, del E746-A750 and T790M mutations. RESULTS: The probes for the L858R mutation showed intense positive staining in H1975 cells, and the probe for the del E746-A750 mutation exhibited positive staining specifically in HCC827 and PC-9 tumors. On the other hand, A549 tumors without EGFR mutation did not show any significant staining for any PNA-DNA probe. In combination staining, the addition of cytokeratin stain increased the positive staining rate of each PNA-DNA probe. In addition, the positive staining rate of the probes for the L858R mutation was comparable to that of the antibody to EGFR L858R mutated protein. CONCLUSION: PNA-DNA probes specific for EGFR mutations might be useful tools to detect heterogeneous mutant EGFR expression in cancer tissues and efficiently evaluate the effect of EGFR signaling inhibitors on tissues of EGFR-mutant cancer.

FRET probe for detecting two mutations in one EGFR mRNA.

Technologies for visualizing and tracking RNA are essential in molecular biology, including in disease-related fields. In this study, we propose a novel probe set (DAt-probe and T-probe) that simultaneously detects two mutations in the same RNA using fluorescence resonance energy transfer (FRET). The DAt-probe carrying the fluorophore Atto488 and the quencher Dabcyl were used to detect a cancer mutation (exon19del), and the T-probe carrying the fluorophore Tamra was used to detect drug resistance mutations (T790M) in epidermal growth factor receptor (EGFR) mRNA. These probes were designed to induce FRET when both mutations were present in the mRNA. Gel electrophoresis confirmed that the two probes could efficiently bind to the mutant mRNA. We measured the FRET ratios using wild-type and double-mutant RNAs and found a significant difference between them. Even in living cells, the FRET probe could visualize mutant RNA. As a result, we conclude that this probe set provides a method for detecting two mutations in the single EGFR mRNA via FRET.

Single-Cell Microarray Chip with Inverse-Tapered Wells to Maintain High Ratio of Cell Trapping.

A single-cell microarray (SCM) influenced by gravitational force is expected to be one of the simple methods in various fields such as DNA analysis and antibody production. After trapping the cells in the SCM chip, it is necessary to remove the liquid from the SCM to wash away the un-trapped cells on the chip and treat the reagents for analysis. The flow generated during this liquid exchange causes the trapped cells to drop out of conventional vertical wells. In this study, we propose an inverse-tapered well to keep trapped cells from escaping from the SCM. The wells with tapered side walls have a reduced force of flow toward the opening, which prevents trapped cells from escaping. The proposed SCM chip was fabricated using 3D photolithography and polydimethylsiloxane molding techniques. In the trapping experiment using HeLa cells, the cell residual rate increased more than two-fold for the SCM chip with the inverse-tapered well with a taper angle of 30° compared to that for the conventional vertical SCM chip after multiple rounds of liquid exchanges. The proposed well structure increases the number of trapped cells and decreases the cell dropout rate to improve the efficiency of cellular analysis.

Fluorescence ratiometric DNA detection by peptide nucleic acid-pyrene binary probes.

We developed a method for detecting DNA by excimer fluorescence from two peptide nucleic acids (PNAs) modified with a pyrene (Pyr). The two PNA-Pyr probes were prepared by solid-phase peptide synthesis, and we assessed fluorescence from the mixture of probes with DNA. From the results, excimer fluorescence derived from the two PNA-Pyr probes forming hybrids with the complementary DNA was observed, and the two probes showed the maximum excimer/monomer ratio when the probes and DNA were hybridized at a 1:1:1 ratio, indicating that the PNA-Pyr probes can detect target DNA. Furthermore, we adjusted the spatial arrangement between the two PNA-Pyr hybrids formed on the DNA to promote optimal excimer formation. As a result, optimal excimer formation was achieved by spacing the two nucleobases between the formed two hybrids and further inserting a hexamethylene linker (C6) between the PNA and Pyr of the PNA-Pyr probe on one side.

Selective cell retrieval method using light-responsive gas-generating polymer-based microarrays.

Selective cell retrieval from base material is necessary for developing and improving cell analyzing technologies as well as regenerative medicine. Many conventional technologies, such as micromanipulators, are developed for selective cell retrieval. However, selective cell retrieval at the single-cell level remains challenging because it is quite difficult to retrieve adhered single cells from base material with ease, rapidity, and no damage. Here, we propose a novel selective cell retrieval method using microarrays made of a light-responsive gas-generating polymer (LGP microarray). The convex LGP microarray was fabricated by a molding process using polystyrene microarray chips. LGP microarrays generate N2 gas when exposed to a specific light used for fluorescence microscopy. A human cervical cancer cell (HeLa) suspension was spread on the LGP microarray coated by fibronectin. After these HeLa cells were adhered to the surface of the LGP microarray structure, light at a wavelength of 365 nm was used to irradiate the LGP microarray. All the target HeLa cells were selectively released from the light-irradiated surface area of the LGP microarray by the generated N2 gas. The LGP microarray system was also applied to single-cell retrieval, and we easily and rapidly retrieved 100% of the single HeLa cells from the microarrays. In addition, approximately 90% of single HeLa cells retrieved from the LGP microarray proliferated on a chamber of a 96-well plate. Therefore, the LGP microarray system enables easy and selective retrieval of adhered cell groups or single cells with only harmless light irradiation.

Novel Quick Cell Patterning Using Light-Responsive Gas-Generating Polymer and Fluorescence Microscope.

Conventional cell patterning methods are mainly based on hydrophilic/hydrophobic differences or chemical coating for cell adhesion/non-adhesion with wavering strength as it varies with the substrate surface conditions, including the cell type and the extracellular matrix components (ECMs) coating; thus, the versatility and stability of cell patterning methods must be improved. In this study, we propose a new cell patterning method using a light-responsive gas-generating polymer (LGP) and a conventional fluorescence microscope. Herein, cells and cellular tissues are easily released from the substrate surface by the nitrogen gas bubbles generated from LGP by the excitation light for fluorescence observation without harming the cells. The LGP-implanted chip was fabricated by packing LGP into a polystyrene (PS) microarray chip with a concave pattern. HeLa cells were spread on the LGP-implanted chips coated with three different ECMs (fibronectin, collagen, and poly-D-lysine), and all HeLa cells on the three LGP patterns were released. The pattern error between the LGP pattern and the remaining HeLa cells was 8.81 ± 4.24 µm, less than single-cell size. In addition, the LGP-implanted chip method can be applied to millimeter-scale patterns, with less than 30 s required for cell patterning. Therefore, the proposed method is a simple and rapid cell patterning method with high cell patterning accuracy of less than the cell size error, high scalability, versatility, and stability unaffected by the cell type or the ECM coating.

Fluorophore-PNA-Quencher/Quencher-DNA probe for miRNA detection.

Micro RNAs (miRNAs) are involved in a variety of biological functions and are attracting attention as diagnostic and prognostic markers for various diseases. Highly sensitive RNA detection methods are required to determine miRNA expression levels and intracellular localization. In this study, we designed new double-stranded peptide nucleic acid (PNA)/DNA probes consisting of a fluorophore-PNA-quencher (fPq) and a quencher-DNA (qD) for miR-221 detection. We optimized the fPq structure, PNA-DNA hybrid length, and hybrid position. The resultant fPq-2/qD-6b probe was a 6-bp hybrid probe with a 10-base fPq and a 6-base qD. The signal-to-background ratios of the probes showed that fPq-2/qD-6b had a higher target sensitivity than fPq (PNA beacon)-type and fP/qD-type probes. The results of the detection limit and target specificity indicate that the fPq/qD probe is promising for RNA detection in both cells and cell extracts as well as for miRNA diagnosis.

Editorial for the Special Issue on Micro and Nano Devices for Cell Analysis.

In recent years, miniaturized systems (micro- and nano-devices) called a lab-on-a-chip or micro-total analysis system (µ-TAS) have received attention as new systems for chemical and biochemical analyses [...].

Analysis of Single Nucleotide-Mutated Single-Cancer Cells Using the Combined Technologies of Single-Cell Microarray Chips and Peptide Nucleic Acid-DNA Probes.

Research into cancer cells that harbor gene mutations relating to anticancer drug-resistance at the single-cell level has focused on the diagnosis of, or treatment for, cancer. Several methods have been reported for detecting gene-mutated cells within a large number of non-mutated cells; however, target single nucleotide-mutated cells within a large number of cell samples, such as cancer tissue, are still difficult to analyze. In this study, a new system is developed to detect and isolate single-cancer cells expressing the T790M-mutated epidermal growth factor receptor (EGFR) mRNA from multiple non-mutated cancer cells by combining single-cell microarray chips and peptide nucleic acid (PNA)-DNA probes. The single-cell microarray chip is made of polystyrene with 62,410 microchambers (31-40 µm diameter). The T790M-mutated lung cancer cell line, NCI-H1975, and non-mutated lung cancer cell line, A549, were successfully separated into single cells in each microchambers on the chip. Only NCI-H1975 cell was stained on the chip with a fluorescein isothiocyanate (FITC)-conjugated PNA probe for specifically detecting T790M mutation. Of the NCI-H1975 cells that spiked into A549 cells, 0-20% were quantitatively analyzed within 1 h, depending on the spike concentration. Therefore, our system could be useful in analyzing cancer tissue that contains a few anticancer drug-resistant cells.

Multi-Color Enhanced Fluorescence Imaging of a Breast Cancer Cell with A Hole-Arrayed Plasmonic Chip.

Breast cancer cells of MDA-MB-231 express various types of membrane proteins in the cell membrane. In this study, two types of membrane proteins in MDA-MB-231 cells were observed using a plasmonic chip with an epifluorescence microscope. The targeted membrane proteins were epithelial cell adhesion molecules (EpCAMs) and epidermal growth factor receptor (EGFR), and Alexa®488-EGFR antibody and allophycocyanin (APC)-labeled EpCAM antibody were applied to the fluorescent detection. The plasmonic chip used in this study is composed of a two-dimensional hole-array structure, which is expected to enhance the fluorescence at different resonance wavelengths due to two kinds of grating pitches in a square side and a diagonal direction. As a result of multi-color imaging, the enhancement factor of Alexa®488-EGFR and APC-EpCAM was 13 ± 2 and 12 ± 2 times greater on the plasmonic chip, respectively. The excited wavelength or emission wavelength of each fluorescent agent is due to consistency with plasmon resonance wavelength in the hole-arrayed chip. The multi-color fluorescence images of breast cancer cells were improved by the hole-arrayed plasmonic chip.

Imaging analysis of EGFR mutated cancer cells using peptide nucleic acid (PNA)-DNA probes.

Lung cancer cells harbor various gene mutations in the mRNA sequence of the Epidermal Growth Factor Receptor (EGFR), especially the mutations of exon19del E746-A750, T790M, and L858R. This results in cancer progression and resistance to anticancer drugs (tyrosine kinase inhibitor; TKI). Therefore, the imaging analysis of EGFR mutations is required for the treatment planning for non-small cell lung cancers. This study focused on the imaging analysis of a single nucleotide substitute in EGFR mutated cancer cells. We developed three novel peptide nucleic acid (PNA)-DNA probes for recognizing and detecting the following three gene mutations in EGFR gene mutations. The PNA-DNA probes consist of fluorescein isothiocyanate (FITC) conjugated PNA as a detection probe and Dabcyl conjugated DNA as a quencher probe. The PNA-DNA probes were used to validate the feasibility for detecting three EGFR mutated sequences: exon19del E746-A750, T790M, and L858R. The three probes emitted fluorescent dose-dependent signals against three target DNA and RNA. Using the three PNA-DNA probes, we succeeded in distinguishing three kinds of lung-cancer cell lines (H1975, PC-9, and A549) which have different EGFR mutations by the fluorescence in situ hybridization (FISH) method.

Dual-Color Fluorescence Imaging of EpCAM and EGFR in Breast Cancer Cells with a Bull's Eye-Type Plasmonic Chip.

Surface plasmon field-enhanced fluorescence microscopic observation of a live breast cancer cell was performed with a plasmonic chip. Two cell lines, MDA-MB-231 and Michigan Cancer Foundation-7 (MCF-7), were selected as breast cancer cells, with two kinds of membrane protein, epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR), observed in both cells. The membrane proteins are surface markers used to differentiate and classify breast cancer cells. EGFR and EpCAM were detected with Alexa Fluor® 488-labeled anti-EGFR antibody (488-EGFR) and allophycocyanin (APC)-labeled anti-EpCAM antibody (APC-EpCAM), respectively. In MDA-MB231 cells, three-fold plus or minus one and seven-fold plus or minus two brighter fluorescence of 488-EGFR were observed on the 480-nm pitch and the 400-nm pitch compared with that on a glass slide. Results show the 400-nm pitch is useful. Dual-color fluorescence of 488-EGFR and APC-EpCAM in MDA-MB231 was clearly observed with seven-fold plus or minus two and nine-fold plus or minus three, respectively, on the 400-nm pitch pattern of a plasmonic chip. Therefore, the 400-nm pitch contributed to the dual-color fluorescence enhancement for these wavelengths. An optimal grating pitch of a plasmonic chip improved a fluorescence image of membrane proteins with the help of the surface plasmon-enhanced field.

Separation and Analysis of Adherent and Non-Adherent Cancer Cells Using a Single-Cell Microarray Chip.

A new single-cell microarray chip was designed and developed to separate and analyze single adherent and non-adherent cancer cells. The single-cell microarray chip is made of polystyrene with over 60,000 microchambers of 10 different size patterns (31-40 µm upper diameter, 11-20 µm lower diameter). A drop of suspension of adherent carcinoma (NCI-H1650) and non-adherent leukocyte (CCRF-CEM) cells was placed onto the chip, and single-cell occupancy of NCI-H1650 and CCRF-CEM was determined to be 79% and 84%, respectively. This was achieved by controlling the chip design and surface treatment. Analysis of protein expression in single NCI-H1650 and CCRF-CEM cells was performed on the single-cell microarray chip by multi-antibody staining. Additionally, with this system, we retrieved positive single cells from the microchambers by a micromanipulator. Thus, this system demonstrates the potential for easy and accurate separation and analysis of various types of single cells.

Hydrophilic-treated plastic plates for wide-range analysis of Giemsa-stained red blood cells and automated Plasmodium infection rate counting.

BACKGROUND: Malaria is a red blood cell (RBC) infection caused by Plasmodium parasites. To determine RBC infection rate, which is essential for malaria study and diagnosis, microscopic evaluation of Giemsa-stained thin blood smears on glass slides ('Giemsa microscopy') has been performed as the accepted gold standard for over 100 years. However, only a small area of the blood smear provides a monolayer of RBCs suitable for determination of infection rate, which is one of the major reasons for the low parasite detection rate by Giemsa microscopy. In addition, because Giemsa microscopy is exacting and time-consuming, automated counting of infection rates is highly desirable. RESULTS: A method that allows for microscopic examination of Giemsa-stained cells spread in a monolayer on almost the whole surface of hydrophilic-treated cyclic olefin copolymer (COC) plates was established. Because wide-range Giemsa microscopy can be performed on a hydrophilic-treated plate, the method may enable more reliable diagnosis of malaria in patients with low parasitaemia burden. Furthermore, the number of RBCs and parasites stained with a fluorescent nuclear staining dye could be counted automatically with a software tool, without Giemsa staining. As a result, researchers studying malaria may calculate the infection rate easily, rapidly, and accurately even in low parasitaemia. CONCLUSION: Because the running cost of these methods is very low and they do not involve complicated techniques, the use of hydrophilic COC plates may contribute to improved and more accurate diagnosis and research of malaria.

Sensitive Detection of Cell Surface Membrane Proteins in Living Breast Cancer Cells Using Multicolor Fluorescence Microscopy with a Plasmonic Chip.

A plasmonic chip was applied to live cancer cell imaging. The epithelial cell adhesion molecule (EpCAM) is a surface marker that can be used to classify breast cancer cell lines into distinct differentiation states. EpCAM and the nuclei of two kinds of living breast cancer cells, MDA-MB231 and MCF-7, were stained with allophycocyanin (APC)-labeled anti-EpCAM antibody and 4',6-diamidino-2-phenylindole (DAPI), respectively, and the cells were scattered on either a plasmonic chip (metal-coated wavelength-scale grating substrate) or a control glass slide. Multicolor fluorescence microscopic imaging allowed fluorescence images of APC-EpCAM to be obtained on the plasmonic chip that were more than 10 times brighter compared with those on the glass slide. In contrast, in the fluorescence images of DAPI-stained nuclei, no difference in brightness was observed between substrates. The fluorescence enhancement of APC-EpCAM in the cell membrane in contact with the plasmonic chip is thought to be due to the excitation of APC molecules localized within the surface plasmon field. Analysis of the cross section of a fluorescence image revealed a distribution of EpCAM at a higher level of fluorescence in the center of the cell image because of contact between the cell membrane and the plasmonic chip. In contrast, fluorescence images of APC-EpCAM taken on a glass slide were so dark that only the outline of the cell was characterized. The plasmonic chip thus constitutes a simple and powerful tool for analyzing the distribution and kinetics of surface marker proteins in cell membranes contacting the chip.

Prognostic Impact of Circulating Tumor Cell Detected Using a Novel Fluidic Cell Microarray Chip System in Patients with Breast Cancer.

Various types of circulating tumor cell (CTC) detection systems have recently been developed that show a high CTC detection rate. However, it is a big challenge to find a system that can provide better prognostic value than CellSearch in head-to-head comparison. We have developed a novel semi-automated CTC enumeration system (fluidic cell microarray chip system, FCMC) that captures CTC independently of tumor-specific markers or physical properties. Here, we compared the CTC detection sensitivity and the prognostic value of FCMC with CellSearch in breast cancer patients. FCMC was validated in preclinical studies using spike-in samples and in blood samples from 20 healthy donors and 22 breast cancer patients in this study. Using spike-in samples, a statistically higher detection rate (p=0.010) of MDA-MB-231 cells and an equivalent detection rate (p=0.497) of MCF-7 cells were obtained with FCMC in comparison with CellSearch. The number of CTC detected in samples from patients that was above a threshold value as determined from healthy donors was evaluated. The CTC number detected using FCMC was significantly higher than that using CellSearch (p=0.00037). CTC numbers obtained using either FCMC or CellSearch had prognostic value, as assessed by progression free survival. The hazard ratio between CTC+ and CTC- was 4.229 in CellSearch (95% CI, 1.31 to 13.66; p=0.01591); in contrast, it was 11.31 in FCMC (95% CI, 2.245 to 57.0; p=0.000244). CTC detected using FCMC, like the CTC detected using CellSearch, have the potential to be a strong prognostic factor for cancer patients.

Application of a cell microarray chip system for accurate, highly sensitive, and rapid diagnosis for malaria in Uganda.

Accurate, sensitive, rapid, and easy operative diagnosis is necessary to prevent the spread of malaria. A cell microarray chip system including a push column for the recovery of erythrocytes and a fluorescence detector was employed for malaria diagnosis in Uganda. The chip with 20,944 microchambers (105 µm width and 50 µm depth) was made of polystyrene. For the analysis, 6 µl of whole blood was employed, and leukocytes were practically removed by filtration through SiO2-nano-fibers in a column. Regular formation of an erythrocyte monolayer in each microchamber was observed following dispersion of an erythrocyte suspension in a nuclear staining dye, SYTO 21, onto the chip surface and washing. About 500,000 erythrocytes were analyzed in a total of 4675 microchambers, and malaria parasite-infected erythrocytes could be detected in 5 min by using the fluorescence detector. The percentage of infected erythrocytes in each of 41 patients was determined. Accurate and quantitative detection of the parasites could be performed. A good correlation between examinations via optical microscopy and by our chip system was demonstrated over the parasitemia range of 0.0039-2.3438% by linear regression analysis (R(2) = 0.9945). Thus, we showed the potential of this chip system for the diagnosis of malaria.

Nanoparticles speckled by ready-to-conjugate lanthanide complexes for multimodal imaging.

Multimodal and multifunctional contrast agents receive enormous attention in the biomedical imaging field. Such contrast agents are routinely prepared by the incorporation of organic molecules and inorganic nanoparticles (NPs) into host materials such as gold NPs, silica NPs, polymer NPs, and liposomes. Despite their non-cytotoxic nature, the large size of these NPs limits the in vivo distribution and clearance and inflames complex pharmacokinetics, which hinder the regulatory approval for clinical applications. Herein, we report a unique method that combines magnetic resonance imaging (MRI) and fluorescence imaging modalities together in nanoscale entities by the simple, direct and stable conjugation of novel biotinylated coordination complexes of gadolinium(III) to CdSe/ZnS quantum dots (QD) and terbium(III) to super paramagnetic iron oxide NPs (SPION) but without any host material. Subsequently, we evaluate the potentials of such lanthanide-speckled fluorescent-magnetic NPs for bioimaging at single-molecule, cell and in vivo levels. The simple preparation and small size make such fluorescent-magnetic NPs promising contrast agents for biomedical imaging.

Singlet-oxygen-sensitizing near-infrared-fluorescent multimodal nanoparticles.

Nanoprobes based on quantum clusters (QC) with near-infrared fluorescence, magnetic-resonance-imaging contrast, and singlet-oxygen-sensitized intracellular fluorescence are studied. The generation of singlet oxygen and singlet-oxygen-sensitized fluorescence uncaging by magnetic and NIR-emitting nanoparticles are exploited for multimodal bioimaging in vitro.