Investigation of anti-cancer effects of new pyrazino[1,2-a]benzimidazole derivatives on human glioblastoma cells through 2D in vitro model and 3D-printed microfluidic device.

AIMS: Recent studies show targeted therapy of new pyrazino[1,2-a]benzimidazole derivatives with COX-II inhibitory effects on different cancer cells. This study aimed to investigate 2D cell culture and 3D spheroid formation of glioblastoma multiforme (GBM) cells using a microfluidic device after exposure to these compounds. MAIN METHODS: After isolating astrocytes from human GBM samples, IC50 of 2,6-dimethyl pyrazino[1,2-a]benzimidazole (L1) and 3,4,5-trimethoxy pyrazino[1,2-a]benzimidazole (L2) were determined as 13 µM and 85 µM, respectively. Then, in all experiments, cells were exposed to subtoxic concentrations of L1 (6.5 µM) and L2 (42.5 µM), which were ½IC50. In the following, in two phases, cell cycle, migration, and gene expression through 2D cell culture and tumor spheroid formation ability using a 3D-printed microfluidic chip were assessed. KEY FINDINGS: The obtained results showed that both compounds have positive effects in reducing G2/M cell population and GBM cell migration. Furthermore, real-time gene expression data showed that L1 and L2 significantly impact the upregulation of P21 and P53 and down-regulation of cyclin D1, MMP2, and MMP9. On the other hand, GBM spheroids exposed to L1 and L2 become smaller with fewer live cells. SIGNIFICANCE: Our data on human isolated astrocyte cells in 2D and 3D cell culture conditions showed that L1 and L2 compounds could reduce GBM cells' invasion by controlling gene expressions associated with migration and proliferation. Moreover, designing microfluidic platform and related cell culture protocols facilitates the broad screening of 3D multicellular tumor spheroids derived from GBM tumor biopsies and provides effective drug development for brain gliomas.

A combined 3D printing/CNC micro-milling method to fabricate a large-scale microfluidic device with the small size 3D architectures: an application for tumor spheroid production.

The fabrication of a large-scale microfluidic mold with 3D microstructures for manufacturing of the conical microwell chip using a combined projection micro-stereolithography (PµSL) 3D printing/CNC micro-milling method for tumor spheroid formation is presented. The PµSL technique is known as the most promising method of manufacturing microfluidic chips due to the possibility of creating complex three-dimensional microstructures with high resolution in the range of several micrometers. The purpose of applying the proposed method is to investigate the influence of microwell depths on the formation of tumor spheroids. In the conventional methods, the construction of three-dimensional microstructures and multi-height chips is difficult, time-consuming, and is performed using a multi-step lithography process. Microwell depth is an essential parameter for microwell design since it directly affects the shear stress of the fluid flow and the diffusion of nutrients, respiratory gases, and growth factors. In this study, a chip was made with microwells of different depth varying from 100 to 500 µm. The mold of the microwell section is printed by the lab-made PµSL printer with 6 and 1 µm lateral and vertical resolutions. Other parts of the mold, such as the main chamber and micro-channels, were manufactured using the CNC micro-milling method. Finally, different parts of the master mold were assembled and used for PDMS casting. The proposed technique drastically simplifies the fabrication and rapid prototyping of large-scale microfluidic devices with high-resolution microstructures by combining 3D printing with the CNC micro-milling method.

Publisher Correction: A compact LED-based projection microstereolithography for producing 3D microstructures.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A compact LED-based projection microstereolithography for producing 3D microstructures.

Projection microstereolithography (PµSL) is a promising additive manufacturing technique due to its low cost, accuracy, speed, and also the diversity of the materials that it can use. Recently it has shown great potentials in various applications such as microfluidics, tissue engineering, micro-optics, biomedical microdevices, and so on. However, studies on PµSL are still ongoing in terms of the quality and accuracy of the construction process, which particularly affect the fabrication of complex 3D microstructures and make it attractive enough to be considered for commercial applications. In this paper, a compact LED-based PµSL 3D printer for the fabrication of 3D microstructures was developed, and the effective parameters that influence the quality of construction were thoroughly investigated and optimized. Accordingly, a customized optical system, including illumination optics and projection optics, was designed using optical engineering principles. This custom 3D printer was proposed for the PµSL process, which besides improving the quality of construction, led to the reduction of the size of the device, its cost-effectiveness, and the repeatability of its performance. To demonstrate the performance of the fabricated device, a variety of complex 3D microstructures such as porous, hollow, helical, and self-support microstructures were constructed. In addition, the repeatability of the device was assessed by fabricating microstructure arrays. The device performance showed that the lateral accuracy of printing was better than 5 µm, and the smallest thickness of the printed layer was 1 µm. Moreover, the maximum printable size of the device was 6.4 mm × 4 mm × 40 mm.

On-chip analysis of carbon dots effect on yeast replicative lifespan.

Carbon dots (CDs) are promising nanomaterials for biosensing, bioimaging, and drug delivery due to their large surface area, excellent optical properties, and thermal and chemical stability. However, biosafety of CDs is still understudied, and there is not a generally accepted standard to evaluate the toxicity of CDs. We present a gradient network generator microfluidic device for dose-dependent testing of toxicity of CDs to a unicellular eukaryotic model organism, yeast Pichia pastoris. We fully characterized the microfluidic model and compare its performance with a conventional method. The gradient generator increased the contact area between the mixing species and enabled a high-throughput testing of CDs in a wide range of concentrations in cell chambers. Real time monitoring of yeast cell proliferation in the presence of CDs showed dose-dependent growth inhibition and various budding cell shape profiles. Comparing the result of microfluidic platform and conventional method revealed statistically significant differences in the proliferation rate of the cells between the two platforms. To understand the toxicity mechanism, we studied binding of CDs to P. pastoris and found increasing interactions of CDs with the cell surface at CDs larger concentrations. This study demonstrated the utility of the gradient generator microfluidic device as a convenient tool for toxicity assessment of nanomaterials at a cellular level.

Investigation the cytotoxicity and photo-induced toxicity of carbon dot on yeast cell.

Carbon dots (CDs) as a new fluorescent material with excellent water solubility, chemical inertness, and easy surface modification are a good candidate for bioimaging and biosensing due to their low toxicity and good biocompatibility. Although carbon is not an intrinsically toxic substance, carbon nanomaterials such as CDs may cause risks to human health and the potentially hazardous effects of CDs on various living systems must be completely determined. So far, cytotoxicity studies of CDs have focused on human cells and are mainly conducted on limited cell lines. In the present study, toxicity assessment of CDs was evaluated on yeast cells Pichia pastoris as a unicellular eukaryotic model. Results revealed dose-dependent toxicity of CDs on yeast cells and less relative cell growth in 25 mg/ml of CDs as compared to the control group. CDs binding curve confirmed the interaction between CDs and surface of yeast cells. SEM images showed that the CDs caused cell shrinkage and hole formation on the surface of yeast cells and also induced slightly cell deformation. It was demonstrated that CDs could generate the ROS dose-dependently. Finally, results showed the growth inhibition and ROS generation effects of CDs were enhanced at light exposure, as an important environmental factor. These findings could have important implications for applications of CDs.