Cutting and Bonding Parafilm® to Fast Prototyping Flexible Hanging Drop Chips for 3D Spheroid Cultures.

A fast and low-cost fabrication process of flexible hanging drop chips for 3D spheroid cultures was proposed by cutting and bonding Parafilm®, a cohesive thermoplastic. The Parafilm® Hanging Drop Chip (PHDC) was assembled by two-layer of Parafilm® sheet with different sizes of holes. The hole on the upper layer of the Parafilm® is smaller than the hole on the bottom layer. The impact of hole size and sample volume on hanging drop formation and 3D spheroid formations in the hanging drop were investigated. The results showed that 20 µL solution on PHDC with a 3 mm hole could form stabile drop and facilitate spheroid formation. The initial cell number determinates the size of the formed spheroids. Exchanging liquid from the upper hole of the PHDC enables the co-culture of two types of cells in one spheroid and drug efficacy testing in hanging drops. The relative expression of cell adhesion and hypoxia-related genes from spheroids in hanging drop and conventional culture plate suggested the relevance of 3D spheroids and in vivo tumor tissue. The economical hanging drop chip can be fabricated without wet chemistry or expensive fabrication equipment, strengthening its application potential in conventional biological laboratories.

One-Step Dip-Coating-Fabricated Core-Shell Silk Fibroin Rice Paper Fibrous Scaffolds for 3D Tumor Spheroid Formation.

Bioscaffolds are important substrates for supporting three-dimensional (3D) cell cultures. Silk fibroin (SF) is an attractive biomaterial in tissue engineering because of its good biocompatibility and mechanical properties. Electrospinning is one of the most often used approaches to fabricate SF fibrous scaffolds; yet, this technique still faces many challenges, such as low yield, residual organic solvents, limited extensibility of fibers, and a lack of spatial control over pore size. To circumvent these limitations, a core-shell SF on rice paper (SF@RP) fibrous scaffold was fabricated using a mild one-step dip-coating method. The cellulose fiber matrix of RP is the physical basis of the 3D scaffold, whereas the SF coating on the cellulose fiber controls the adhesion/spreading of the cells. The results indicated that by tuning the secondary structure of SF on the surface of a SF@RP scaffold, the cell behavior on SF@RP could be tuned. Tumor spheroids can be formed on SF@RP scaffolds with a dominant random secondary structure, in contrast to cells adhering and spreading on SF@RP scaffolds with a higher ratio of ß-sheet secondary structures. Direct culturing of breast cancer MDA-MB-231 and MCF-7, lung cancer A549, prostate cancer DU145, and liver cancer HepG2 cells could spontaneously lead to corresponding tumor spheroids on SF@RP. In addition, the physiological characteristics of HepG2 tumor spheroids were investigated, and the results showed that compared with HepG2 monolayer cells, CYP3A4, CYP1A1, and albumin gene expression levels in HepG2 cell spheres formed on SF@RP scaffolds were significantly higher. Moreover, these spheroids showed higher drug resistance. In summary, these SF@RP scaffolds prepared by the dip-coating method are biocompatible substrates for cell culture, especially for tumor cell spheroid formation.

On-chip RT-LAMP and colorimetric detection of the prostate cancer 3 biomarker with an integrated thermal and imaging box.

To achieve low-cost, compact, and portable nucleic acid testing, an integrated device containing a three-dimensional printing fabricated reverse transcription loop-mediated isothermal amplification (RT-LAMP) chip, a thermal module, and an imaging module was developed. Samples and RT-LAMP reagents were loaded on a sponge-like polyvinyl alcohol pad on a chip, whereas the colorimetric detection zone was a dry paper pre-loaded with Calcine. The sealed chip was incubated on the integrated thermal module, and the RT-LAMP products were pressed into the Calcine pre-loaded dry paper by a stick. Colorimetric changes could be visually observed by the naked eye or imaged with a smartphone camera through the imaging module. For detection of the prostate cancer antigen 3 (PCA3) biomarker, LAMP primers were designed and verified. The specificity of Calcine pre-loaded dry paper based on colorimetric detection of positive LAMP products was investigated. The reaction conditions for on-chip RT-LAMP such as amplification time, temperature, and volume were optimized. Finally, a detection limit of 0.34 fg/µL RNA was achieved with the proposed on-chip RT-LAMP and colorimetric detection method for PCA3. Since the thermal plate is powered by a 12-V battery and the color change can be imaged with a smartphone, the integrated platform can be operated on-site, highlighting its potential in point-of-care testing applications.

Separation and Characterization of Prostate Cancer Cell Subtype according to Their Motility Using a Multi-Layer CiGiP Culture.

Cancer cell metastasis has been recognized as one hallmark of malignant tumor progression; thus, measuring the motility of cells, especially tumor cell migration, is important for evaluating the therapeutic effects of anti-tumor drugs. Here, we used a paper-based cell migration platform to separate and isolate cells according to their distinct motility. A multi-layer cells-in-gels-in-paper (CiGiP) stack was assembled. Only a small portion of DU 145 prostate cancer cells seeded in the middle layer could successfully migrate into the top and bottom layers of the stack, showing heterogeneous motility. The cells with distinct migration were isolated for further analysis. Quantitative PCR assay results demonstrated that cells with higher migration potential had increased expression of the ALDH1A1, SRY (sex-determining region Y)-box 2, NANOG, and octamer-binding transcription 4. Increased doxorubicin tolerance was also observed in cells that migrated through the CiGiP layers. In summary, the separation and characterization of prostate cancer cell subtype can be achieved by using the multi-layer CiGiP cell migration platform.

Synthesis and luminescent properties of LuAG : Ce3+ transparent ceramics by solvo-thermal method.

The precursor powders of LuAG : Ce3+ transparent ceramics were synthesized by solvo-thermal method. The crystal structure and morphology of powders were analyzed by means of Fourier transform infra-red spectroscopy, X-ray diffraction and scanning electron microscopy. The precursor powders were sintered into transparent ceramics in vacuum and then in nitrogen without any additive. The surface morphology of the transparent unpolished ceramics was characterized using scanning electron microscopy. Some factors that affect the transparency of ceramics were discussed. The UV-Vis fluorescence excitation and emission spectra of LuAG : Ce3+ transparent ceramics were measured. The vacuum ultraviolet spectra of transparent ceramics were investigated using the synchrotron radiation as the excitation source. The excitation mechanism of Ce3+ was discussed at different excitation wavelength.

[Preparation and luminescence of down-conversion material beta--NaYF4 : Tb3+, Yb3+].

NaYF4 : Tb3+, Yb3+ down-conversion (DC) phosphors were synthesized by hydrothermal method. X-ray diffraction (XRD), photoluminescence (PL) and photoluminescence excitation (PLE) spectra were used to characterize the samples. Experiment results revealed that samples of NaYF4 : Tb3+, Yb3+ crystallized in hexagonal shape without cubic shape. When the doping concentration of Tb3+ and Yb3+ was altered, the lattice structure of samples did not change, indicating that the Tb3+ and Yb3+ ions are completely dissolved in the NaYF4 host lattice by substitution for the Y3+. The emission from 5D4 --> 7F6 (489 nm), 5D4 --> 7F5 (542 nm), 5D4 --> F4 (584 nm), and 5D4 --> F3 (619 nm) of Tb3+ ions was observed, in which the dominant emission was at 542 nm. With single Tb3+ doping, no near-infrared (NIR) emission was observed under excitation of 355 nm pulsed laser. However, while with Tb3+ and Yb3+ codoping, the NIR emission at around 950 -1 100 nm from Yb3+ (2F5/2 --> 2F7/2) was observed under the same excitation. The dependence of the visible and NIR-emissions on Yb3+ doping concentration has been investigated. These results show that there is energy transfer process between Tb2+ and Yb3+. Furthermore, it is a possible DC process through cooperative energy transfer from Tb3+ to Yb3+. When the doping concentration is 1% mol Tb3+ and 6% mol Yb3+ respectively, the intensity of NIR emission reaches its strongest.

[Analysis of mannoligosaccharides by liquid chromatography-electrospray ionization mass spectrometry].

Oligosaccharide characterization has been of utmost interest in various areas such as medicine, biochemistry, and food chemistry. These biologically relevant molecules are ideally suited for mass spectrometric investigation, because of the capability of this technique in offering structure and relative molecular mass information. Therefore, liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was applied to characterize the acetolysis of mannan from Saccharomyces cerevisiae. The electrospray using Na+ as adducts proved to be superior to the LC-MS for the determination of mannoligosaccharides. LC separation was accomplished by the use of NH2 column and the elution by acetonitrile-water (70:30, volume ratio). The results showed that mannoligosaccharides side chain consisted of mannose, mannobiose, mannotriose and mannotetraose. The method developed is accurate, fast and convenient and can be used to characterize the relative molecular mass of the oligosaccharides.