Extracellularly Detectable Electrochemical Signals of Living Cells Originate from Metabolic Reactions.

Direct detection of cellular redox signals has shown immense potential as a novel living cell analysis tool. However, the origin of such signals remains unknown, which hinders the widespread use of electrochemical methods for cellular research. In this study, the authors found that intracellular metabolic pathways that generate adenosine triphosphate (ATP) are the main contributors to extracellularly detectable electrochemical signals. This is achieved through the detection of living cells (4,706 cells/chip, linearity: 0.985) at a linear range of 7,466-48,866. Based on this discovery, the authors demonstrated that the cellular signals detected by differential pulse voltammetry (DPV) can be rapidly amplified with a developed medium containing metabolic activator cocktails (MACs). The DPV approach combined with MAC treatment shows a remarkable performance to detect the effects of the anticancer drug CPI-613 on cervical cancer both at a low drug concentration (2 µm) and an extremely short treatment time (1 hour). Furthermore, the senescence of mesenchymal stem cells could also be sensitively quantified using the DPV+MAC method even at a low passage number (P6). Collectively, their findings unveiled the origin of redox signals in living cells, which has important implications for the characterization of various cellular functions and behaviors using electrochemical approaches.

A Spheroid-Forming Hybrid Gold Nanostructure Platform That Electrochemically Detects Anticancer Effects of Curcumin in a Multicellular Brain Cancer Model.

In this study, a multifunctional platform that enables the highly efficient formation of 3D multicellular cancer spheroids and precise real-time assessments of the anticancer effects of curcumin in a brain tumor coculture model is reported. A highly conductive gold nanostructure (HCGN) is fabricated to facilitate cancer spheroid formation without using anti-cell adhesion molecules. A neuroblastoma (SH-SY5Y) and glioblastoma (U-87MG) coculture model is generated on HCGN with a specific cell-to-cell ratio (SH-SY5Y: U-87MG = 1:1), and their redox behaviors are successfully measured without destroying the distinct 3D structure of the multicellular spheroids. Using electrochemical signals as an indicator of spheroid viability, the effects of potential anticancer compounds on cocultured spheroids are further assessed. Remarkably, decreased cell viability in 3D spheroids caused by a low concentration of curcumin (30 µM) is detectable using the electrochemical method (29.4%) but not with a conventional colorimetric assay (CCK-8). The detection is repeated more than ten times for both short- (63 h) and long-term cultivation (144 h) without damaging the spheroids, enabling real-time, non-destructive pharmacokinetic analysis of various drug candidates. Therefore, it can be concluded that the hybrid platform is a highly promising, precise, and high-throughput drug screening tool based on 3D cell cultivation.

Recent Advances in Electrochemical Sensors for the Detection of Biomolecules and Whole Cells.

Electrochemical sensors are considered an auspicious tool to detect biomolecules (e.g., DNA, proteins, and lipids), which are valuable sources for the early diagnosis of diseases and disorders. Advances in electrochemical sensing platforms have enabled the development of a new type of biosensor, enabling label-free, non-destructive detection of viability, function, and the genetic signature of whole cells. Numerous studies have attempted to enhance both the sensitivity and selectivity of electrochemical sensors, which are the most critical parameters for assessing sensor performance. Various nanomaterials, including metal nanoparticles, carbon nanotubes, graphene and its derivatives, and metal oxide nanoparticles, have been used to improve the electrical conductivity and electrocatalytic properties of working electrodes, increasing sensor sensitivity. Further modifications have been implemented to advance sensor platform selectivity and biocompatibility using biomaterials such as antibodies, aptamers, extracellular matrix (ECM) proteins, and peptide composites. This paper summarizes recent electrochemical sensors designed to detect target biomolecules and animal cells (cancer cells and stem cells). We hope that this review will inspire researchers to increase their efforts to accelerate biosensor progress-enabling a prosperous future in regenerative medicine and the biomedical industry.

Vertically Coated Graphene Oxide Micro-Well Arrays for Highly Efficient Cancer Spheroid Formation and Drug Screening.

Research on the 3D culturing of cancer cells that better mimic in vivo solid tumors is important for efficient drug screening. Herein, a new platform that effectively facilitates the formation of cancer spheroids for anticancer drug screening is reported. Cytophilic graphene oxide (GO), when selectively coated on the sidewalls of micro-wells fabricated from a cell-adhesion-resistive polymer, is found to efficiently initiates distinct donut-like formation of cancer cell spheroids. Scanning electron microscopy and Raman mapping are used to analyze vertically coated GO micropatterns (vGO-MPs) of different sizes (100-250 µm) on polymer platforms, and human liver cancer cells (HepG2), as a model cancer, are seeded on these platforms. Remarkably, the 150 µm-sized platform is found to easily and rapidly generate 3D spheroids in the absence of cell-adhesion proteins. In addition, owing to the unique characteristics of GO, vGO-MPs are highly stable for long periods of time (≈1 month), even under harsh conditions (>70 °C). Moreover, the anticancer effects of two drugs (hydroxyurea and cisplatin) and the potential anticancer compound (curcumin) on HepG2 cells are demonstrated by simply measuring decreases in spheroid sizes. Hence, this new platform is highly promising as a cancer spheroid-forming material for rapid drug screening.

A fibronectin-coated gold nanostructure composite for electrochemical detection of effects of curcumin-carrying nanoliposomes on human stomach cancer cells.

Curcumin, which is produced by the medicinal herbaceous plant Curcuma longa, has been widely investigated for use as a potential anticancer drug. In this study, the potential toxicity of curcumin-carrying nanoliposomes (curcumin-NLC) toward human stomach cancer cells (MKN-28) was investigated using a new cell-based electrochemical sensing platform. To satisfy both biocompatibility and electroconductivity of the electrodes, the density of the gold nanostructure and the coating conditions of extracellular matrix proteins (fibronectin and collagen) were optimized. The developed platform enabled the successful adhesion and long-term growth of stomach cancer cells on the chip surface, allowing label-free and real-time monitoring of cell viability in a quantitative manner. According to the electrochemical results, both bare curcumin and curcumin-NLC showed toxicity toward MKN-28 cells in the concentration range of 10-100 µM, which was consistent with the results obtained from a conventional colorimetric method (CCK-8). Remarkably, at a low concentration range (<50 µM), this electrochemical platform determined the decrease in cell viability to be approximately 22.8%, 33.9% and 53.1% in the presence of 10, 30, and 50 µM of curcumin-NLC, respectively, compared with the 1.3%, 18.5%, and 28.1% determined by CCK-8, making it 1.7-2 times more sensitive than the conventional colorimetric assay. Hence, it can be concluded that the newly developed fibronectin-coated electroconductive platform is highly promising as an electrochemical detection tool for the sensitive and precise assessment of the anticancer effects of various food-derived compounds with low toxicity.

High density gold nanostructure composites for precise electrochemical detection of human embryonic stem cells in cell mixture.

Precise detection of undifferentiated human pluripotent stem cells (hPSCs) and their entire subsequent elimination are incredibly important in preventing teratoma formations after transplantation. Recently, electrochemical sensing platforms have demonstrated immense potential as a new tool to detect remaining hPSCs in label-free and non-destructive manner. Nevertheless, one of the critical huddles of this electrochemical sensing approach is its low sensitivity since even low concentrations of remaining hPSCs were reported to form teratoma once transplanted. To address this issue, in this study, we report an engineering-based approach to improve the sensitivity of electrochemical sensing platform for hPSC detection. By optimizing the density of gold nanostructure and the matrigel concentration to improve both electro-catalytic property and biocompatibility, the sensitivity of the developed platform toward hESCs detection could reach 12,500 cells/chip, which is close to the known critical concentration of hPSCs (˜10,000 cells) that induce teratoma formation in vivo. Remarkably, the electrochemical signals were not detectable from other types of stem cell-derived endothelial cells (CB-EPCs) even at high concentrations of CB-EPCs (40,000 cells/chip), proving the high selectivity of the developed platform toward hPSC detection. Hence, the developed platform could be highly useful to solve the safety issues that are related with clinical application of hPSC-derived cells.

In situ label-free monitoring of human adipose-derived mesenchymal stem cell differentiation into multiple lineages.

Precise characterizations of stem cell differentiation into specific lineages, especially in non-destructive and non-invasive manner, are extremely important for generating patient-specific cells without mass loss of differentiated cells. Here, we report a new method capable of in situ label-free quantification of stem cell differentiation into multiple lineages, even at a single cell level. The human adipose-derived mesenchymal stem cells (hADMSCs) were first differentiated into two different types of cells (osteoblasts and adipocytes) and these differentiated cells were then intensively analyzed by micro-Raman spectroscopy. Interestingly, the Raman peaks assigned to lipid droplets and hydroxyapatite were found to be highly specific to the adipocyte (fat cell) and osteoblast (bone cell) and were thus found to be useful for generating label-free single cell Raman images in combination with CH3 (2935 cm-1) peaks for visualizing cell shape. Remarkably, based on these Raman images, we found that the osteogenesis of hADMSCs could be determined and quantified after 9 days of differentiation, which is a week earlier than with the typical alizarin red staining method. In the case of adipogenesis, the increase of lipid droplets in the cytoplasm at the single cell level could be clearly visualized and detected during the entire period of adipogenesis, which is impossible using any other currently available methods such as Oil Red O and immunostaining. Hence, the new method reported in this study is highly promising as an analytical tool for precise in-situ monitoring of stem cell differentiation, and could facilitate the use of stem cell-based materials for the regenerative therapies.

Effects of two-dimensional materials on human mesenchymal stem cell behaviors.

Graphene, a typical two-dimensional (2D) material, is known to affect a variety of stem cell behaviors including adhesion, spreading, growth, and differentiation. Here, we report for the first time the effects of four different emerging 2D materials on human adipose-derived mesenchymal stem cells (hADMSCs). Graphene oxide (GO), molybdenum sulfide (MoS2), tungsten sulfide (WS2), and boron nitride (BN) were selected as model two-dimensional materials and were coated on cell-culture substrates by a drop-casting method. Acute toxicity was not observed with any of the four different 2D materials at a low concentration range (<5 µg/ml). Interestingly, the 2D material-modified substrates exhibited a higher cell adhesion, spreading, and proliferation when compared with a non-treated (NT) substrate. Remarkably, in the case of differentiation, the MoS2-, WS2-, and BN-modified substrates exhibited a better performance in terms of guiding the adipogenesis of hADMSCs when compared with both NT and GO-modified substrates, based on the mRNA expression level (qPCR) and amount of lipid droplets (ORO staining). In contrast, the osteogenesis was found to be most efficiently induced by the GO-coated substrate (50 µg/mL) among all 2D-material coated substrates. In summary, 2D materials could act as favorable sources for controlling the stem cell growth and differentiation, which might be highly advantageous in both biomedical research and therapy.