Search

The utilization of 3D cell culture for spheroid formation holds significant implications in cancer research, contributing to a fundamental understanding of the disease and aiding drug development. Conventional methods such as the hanging drop technique and other alternatives encounter limitations due to smaller drop volumes, leading to nutrient starvation and restricted culture duration. In this study, we present a straightforward approach to creating superhydrophobic paper cones capable of accommodating large volumes of culture media drops. These paper cones have sterility, autoclavability, and bacterial repellent properties. Leveraging these attributes, we successfully generate large spheroids of ovarian cancer cells and, as a proof of concept, conduct drug screening to assess the impact of carboplatin. Thus, our method enables the preparation of flexible superhydrophobic surfaces for laboratory applications in an expeditious manner, exemplified here through spheroid formation and drug screening demonstrations. [ABSTRACT FROM AUTHOR]

This paper reports a spin-disc paper-based device with 10 individual detection units containing electromagnetic modules controlling the sample incubation time before chemiluminescence (CL) signal detection. After the sample was added to the top paper chip and incubated with the enzyme, the electromagnet was turned off to allow contact between the top and bottom paper. The H2O2 generated by the sample flowed vertically to the bottom paper and initiated the oxidase of the luminol to generate the CL signal. After one detection the disc was automatically rotated to the next position to repeat the above detection. The advantage of using the device over the lateral flow and the in situ detection was firstly proved using the detection of H2O2 and the glucose/lactate sample with 5 minute incubation. The CL intensity was increased 300 times/1000 times as the glucose/lactate was incubated for 5 minutes compared to the non-incubated samples. Afterward, the device was employed to separately detect glucose and lactate diluted in PBS, artificial sweat, artificial saliva, and fresh cell culture media. Finally, the device was employed to detect the glucose and lactate in the media collected over the 24 hour culture of PC3 cells. The uptake and production rates of glucose and lactate were correspondingly determined as 0.328 ± 0.015 pmol h−1 per cell and 1.254 ± 0.053 pmol h−1 per cell, respectively. The reported device has wide application potential due to its capabilities in automatic detection of multiple samples with very high sensitivity and small sample volume (down to 0.5 μL). [ABSTRACT FROM AUTHOR]

Cellular viability measurements quantify decreased proliferation or increased cytotoxicity caused by drug candidates or potential environmental toxins. Direct viability measures count each cell to provide an accurate readout. This approach can prove analytically challenging and time-consuming when cells are maintained in 3D structures akin to tissues or solid tumors. While less labor-intensive, indirect viability measures can be less accurate due to the heterogeneous structural and chemical microenvironment that arises when cells are maintained in tissue-like architectures and in contact with extracellular matrices. Here we determine the analytical figures of merit of five indirect viability assays in the paper-based cell culture platform we continue to develop in our laboratory: calcein-AM staining, the CellTiter-Glo assay, imaging fluorescent protein expression, propidium iodide staining, and the resazurin assay. We also determined the compatibility of each indirect assay with hypoxic conditions, intra-experimental repeatability, inter-experimental reproducibility, and ability to predict a potency value for a known antineoplastic drug. Our results show that each assay has benefits and drawbacks to consider when choosing the appropriate readout to answer a particular research question. We also highlight that only one indirect readout is unaffected by hypoxia, a commonly overlooked variable in cell culture that likely yields inaccurate viability measures. [ABSTRACT FROM AUTHOR]

Exosomes are vesicles released by healthy and cancer cells into the extracellular matrix and bodily fluid. Cancer cell-derived exosomes have attracted much attention in early-stage detection and prognostication of treatment response. Thus, detecting exosomes is of great interest to biology and medicine. However, many conventional detection methods require high-cost equipment and centralized laboratory facilities, making diagnostics inaccessible in limited-resource settings. This study reports a proof-of-concept low-cost electrochemical paper-based analytical device to quantify both the total bulk and cancer cell-derived exosomes in cell culture media. The device employs a sandwich immune assay design, where exosomes are initially captured using the electrode-bound generic antibodies (i.e. CD9) and subsequently detected via ovarian cancer-specific CA125 antibodies. Our proposed device quantifies the total bulk exosome concentration with a detection limit of 9.3 × 107 exosomes per mL and ovarian cancer cell-derived exosomes with a detection limit of 7.1 × 108 exosomes per mL, with a relative standard deviation of <10% (n = 3). We suggest that this low-cost and simple electrochemical paper-based device could be an alternative tool for detecting disease-specific exosomes in biological samples with the potential to be further developed for point-of-care diagnosis. [ABSTRACT FROM AUTHOR]

The blood–brain barrier (BBB) protects the central nervous system from toxins and pathogens in the blood by regulating permeation of molecules through the barrier interface. In vitro BBB models described to date reproduce some aspects of BBB functionality, but also suffer from incomplete phenotypic expression of brain endothelial traits, difficulty in reproducibility and fabrication, or overall cost. To address these limitations, a 3D BBB model based on a hybrid paper/nanofiber scaffold is described. The cell culture platform utilizes lens paper as a framework to accommodate 3D culture of astrocytes. An electrospun nanofiber layer is coated onto one face of the paper to mimic the basement membrane and support growth of an organized 2D layer of endothelial cells (ECs). Human induced pluripotent stem cell‐derived ECs and astrocytes are co‐cultured to develop a human BBB model. Morphological and spatial organization of model are validated with confocal microscopy. Measurements of transendothelial resistance and permeability demonstrate the BBB model develops a high‐quality barrier and responds to hyperosmolar treatments. RNA‐sequencing shows introduction of astrocytes both regulates EC tight junction proteins and improves endothelial phenotypes related to vasculogenesis. This model shows promise as a model platform for future in vitro studies of the BBB. [ABSTRACT FROM AUTHOR]

The study was focused on assessment of potential health risks of paper-based food contact materials (FCMs) in a step-wise approach using three toxicological bioassays in vitro and chemical analyses of migrating contaminants. 3T3 NRU cytotoxicity test showed high sensitivity to detect basal toxicity of FCMs extracts and served as a first-line test for selection of samples for further testing. The reconstructed human intestine model EpiIntestinal showed more realistic tissue response than cell culture monolayer and higher resistance despite prolonged exposure to the selected 6 samples, i.e. negligible decrease of viability and intestinal penetration, nevertheless an increase of IL-8 after exposure to black printed sample extract. Yeast based assays identified weak agonistic/antagonostic activity to human androgen receptor of the black printed sample. In accordance with the biological effects, the targeted LC and GC analytical methods confirmed the presence of high amounts of phthalates, photoinitiators and PAHs that could justify the hazard of the black printed sample. Heavily printed uncoated FCMs are recognized not to be suitable for direct contact with food. The selected bioassays and chemical analyses might be useful tools to detect targeted biological effects of xenobiotics suspected to contribute to human exposure from food. • An effect-directed approach combining bioassays and chemical analyses was proved as useful to assess possible impact of FCMs on human health. • bioassays included cytotoxicity, toxicity and penetration using reconstructed human intestinal model and endocrine disruption screen. • Extensively printed napkin samples exhibited highest toxicological risks and released phthalates, photoinitiators and PAHs. • The black printed paper extract caused IL-8 increase and agonistic activity to human androgen receptor. [ABSTRACT FROM AUTHOR]

Advances in synthetic biology have led to an arsenal of proof-of-principle bacterial circuits that can be leveraged for applications ranging from therapeutics to bioproduction. A unifying challenge for most applications is the presence of selective pressures that lead to high mutation rates for engineered bacteria. A common strategy is to develop cloning technologies aimed at increasing the fixation time for deleterious mutations in single cells. We adopt a complementary approach that is guided by ecological interactions, whereby cyclical population control is engineered to stabilize the functionality of intracellular gene circuits. Three strains of Escherichia coli were designed such that each strain could kill or be killed by one of the other two strains. The resulting “rock-paper-scissors” dynamic demonstrates rapid cycling of strains in microfluidic devices and leads to an increase in the stability of gene circuit functionality in cell culture. [ABSTRACT FROM AUTHOR]

Recently, paper has gained traction in the biotechnology research field due to its ability to be a substrate for 3D cell culture. In this work, we demonstrate the application of paper-based 3D cell culture for rapid and easy screening of the effect of natural compounds on melanin production. Whatman No. 1 filter paper was used as the substrate for B16F10 melanoma cell culture. The use of paper is beneficial for supporting the 3D structure of cells, which makes the result more reliable due to the similarity to in vivo conditions. Furthermore, paper is beneficial for melanin observation due to melanin's black color, which is easily in situ visualized after it is cultured on white paper. Matrigel was used to encapsulate cells before being pipetted onto the paper to prevent the passing of cells through paper pores. The intensity of melanin can then be observed with the naked eye and analyzed by scanning the paper. The analysis process took only 20 minutes, which is faster than that of the conventional absorbance spectroscopy, owing to the elimination of centrifugation, melanin solubilization, and the absorbance measurement step. The color intensity on the paper showed a direct proportion with increased α-MSH concentrations, confirming that the color on the paper was melanin. The 3D structure of cells was confirmed by using a scanning electron microscope. To demonstrate the application of the paper-based scaffold, paper-based 3D cell culture was used for screening the effects of Kojic acid and Arbutin on melanin production, which showed increased anti-melanogenesis effects with increased concentrations of natural compounds. High cell viability was observed over 120 hours. In conclusion, the developed paper-based scaffold can be used for screening the effect of natural compounds on melanin production, as a rapid and simple method with low cost. [ABSTRACT FROM AUTHOR]

Bouteloua gracilis (blue grama grass) is one of the most drought and grazing tolerant plants in the short-grass ecosystem. To obtain information on their photosystem activities, we measured the fast (< 1 s) chlorophyll a fluorescence transient (the OJIP curve) from their leaves, and isolated cells grown photoautotrophically in suspension in a culture medium, or with added sucrose. One of our goals was to study the effect of different sucrose concentrations (0, 0.15, 0.3, and 3%) on PSII activity in isolated cells. Our results on cells suspended in culture medium, using the JIP-test, showed a decrease in PSII activity at increasing sucrose concentrations, while the photoautotrophic cells showed an optimal PSII activity, close to that of the leaves. Further, our data on cells grown in 0, 0.15, and 0.3% sucrose, but with added CO2, and measured while the cells were deposited on filter paper, indicate that supplementary CO2 can increase the PSII activity in the presence of sucrose, although further research is necessary to understand these results. [ABSTRACT FROM AUTHOR]

Andrographis paniculata is one of the most important “Southern Medicines” in China. It is used for clearing heat and detoxifying, cooling blood and reducing swelling. Its main active ingredient andrographolide has functions in anti-cancer, anti-HIV, anti-inflammation and liver protection. Andrographolide is difficult to be synthesized artificially, mainly relying on extraction from cultivated plant materials. However, the quality of cultivated medicinal materials is affected by various factors such as soil, climate, water and fertilizer managements, and A. paniculata has a long growth cycle, occupying land resources. The technology of plant in vitro culture has significant advantages in rapid propagation of seedling and accumulation of active ingredients, which is one of the important ways to achieve production of active ingredients rapidly and efficiently in A. paniculata. The in vitro regeneration technology system of A. paniculata is becoming increasingly perfect, and the in vitro regeneration technology from explants to complete plants is becoming more and more mature, and it has been applied in seedling propagation and ploidy breeding. At the same time, during callus culture, cell suspension culture, adventitious root culture and hairy root culture of A. paniculata, the accumulation of andrographolide and other active ingredients in the culture could be greatly increased by optimizing the culture conditions and using appropriate inducers. This paper comprehensively and systematically reviewed the research advances on the in vitro culture technology of A. paniculata and production of andrographolide from the aspects of tissue, cell, adventitious root, and hairy root cultures. This paper aimed to provide reference for promoting the development and application of in vitro culture technology of A. paniculata, as well as for the study of in vitro production of andrographolide. It also put forward three aspects that need be focused on in future research on in vitro culture technology of A. paniculata and the production of andrographolides by this technology: (1)To mature and improve the tissues in vitro regeneration technology system of A. paniculata, and to establish a comprehensive and systematic evaluation system; (2)To further increase the yield of andrographolide and other important active ingredients by optimizing the culture conditions and its combination with efficient inducers; (3)To carry out researches in bioreactor culture of the production of andrographolide by cell suspension culture technology. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, offer groundbreaking therapeutic potential for degenerative diseases and cellular repair. Despite their significance, a comprehensive bibliometric analysis in this field, particularly in relation to age-related macular degeneration (AMD), is yet to be conducted. This study aims to map the foundational and emerging areas in stem cell and AMD research through bibliometric analysis. This study analyzed articles and reviews on stem cells and AMD from 2000 to 2022, sourced from the Web of Science Core Collection. We used VOSviewer and CiteSpace for analysis and visualization of data pertaining to countries, institutions, authors, journals, references and key words. Statistical analyses were conducted using R language and Microsoft Excel 365. In total, 539 publications were included, indicating an increase in global literature on stem cells and AMD from 2000 to 2022. The USA was the leading contributor, with 239 papers and the highest H-index, also the USA had the highest average citation rate per article (59.82). Notably, 50% of the top 10 institutions were from the USA, with the University of California system being the most productive. Key authors included Masayo Takahashi, Michiko Mandai, Dennis Clegg, Pete J. Coffey, Boris Stanzel, and Budd A. Tucker. Investigative Ophthalmology & Visual Science published the majority of relevant papers (n = 27). Key words like "clinical trial," "stem cell therapy," "retinal organoid," and "retinal progenitor cells" were predominant. Research on stem cells and AMD has grown significantly, highlighting the need for increased global cooperation. Current research prioritizes the relationship between "ipsc," "induced pluripotent stem cell," "cell culture," and "human embryonic stem cell." As stem cell culture and safety have advanced, focus has shifted to prognosis and complications post-transplantation, signifying the movement of stem cell research from labs to clinical settings. [ABSTRACT FROM AUTHOR]

Nanocomposite (NC) papers using bifunctional nanoparticles are prepared for 3D cell growth experiments. Cells show better affinity to NC papers than to paper itself and differentiate the chemical group on the external surface of the nanoparticles. Cells possess spherical shaped morphology and form agglomerates onto the (NC) papers like as observed by cell growth in 3D materials. Furthermore, the preparation of “Janus” paper and the differentiation of its two distinct faces by cells is presented. [ABSTRACT FROM AUTHOR]

In order to achieve high predictive value of cell chemosensitivity test for clinical efficacy, cancer cells were suggested to be encapsulated and cultured in hydrogel to mimic the natural microenvironment of tumors. However, handling 3D cells/hydrogel culture construct is tedious and cellular response is difficult to be quantitatively analyzed. In the current study, a novel platform for conducting 3D cell culture and analyzing cell viability has been developed towards a high throughput drug screening. Cells encapsulated in the hydrogel were cultured in the microwells of a paper substrate. The substrate was then immersed in the culture medium containing drug for 2 days. At 24 and 48 h during the culture course, the paper substrate was placed on the measurement electrodes for conducting the impedance measurement in order to quantify the cell viability in the hydrogel. Cell viability of two human hepatoma cell lines (Huh7 and Hep-G2) was quantitatively investigated under the treatment of two drugs (doxorubicin and etoposide). The results represented by IC 50 values revealed that Huh7 cells had a higher drug resistance than Hep-G2 cells and doxorubicin had a higher efficacy than etoposide for treating hepatocellular carcinoma. The current work has demonstrated a high throughput, convenient, and quantitative platform for the investigation of chemosensitivity of cells cultured in the 3D environment. [ABSTRACT FROM AUTHOR]

Abstract The feasibility of using nanocellulose-based mille-feuille filter paper for upstream applications in serum-free growth media filtration, i.e. Dulbecco's modified Eagle's medium (DMEM) and Luria-Bertani medium (LBM), was tested. The filter performance with respect to ΦΧ174 bacteriophage (28 nm) removal as a model small-size virus was characterized for filters of varying thicknesses, i.e. 11 and 33 µm, at varying operating pressures, i.e. 1 and 3 bar. The filters demonstrated generally good model small-size virus removal properties with LRV ≥ 5, especially for 33 µm filters. The 33 µm filters were more robust and exhibited better virus removal and throughput properties than 11 µm filters, although their flux was generally lower. The performance of the 33 and 11 µm nanocellulose-based filter papers was further verified for upscaled bioprocessing by 10-fold increase in the loading volume. The results of the present work show that the 33 µm nanocellulose-based filter paper could be an interesting alternative for large volume cell culture medium filtration during upstream bioprocessing. Graphical abstract fx1 Highlights • Feasibility of using virus removal filter paper for upstream bioprocessing studied. • High virus removal capacity of nanocellulose-based filter paper shown. • Higher pressure significantly increases virus retention. • Filter thickness positively correlates with virus removal efficiency. [ABSTRACT FROM AUTHOR]

Paper-based cultures are an emerging platform for preparing 3D tissue-like structures. Chemical gradients can be imposed upon these cultures, generating microenvironments similar to those found in poorly vascularized tumors. There is increasing evidence that the tumor microenvironment is responsible for promoting drug resistance and increased invasiveness. Acidosis, or the acidification of the extracellular space, is particularly important in promoting these aggressive cancer phenotypes. To better understand how cells respond to acidosis there is a need for 3D culture platforms that not only model relevant disease states but also contain sensors capable of quantifying small molecules in the extracellular environment. In this work, we describe pH-sensing optodes that are capable of generating high spatial and temporal resolution maps of pH gradients in paper-based cultures. This sensor was fabricated by suspending microparticles containing pH-sensitive (fluorescein) and pH-insensitive (diphenylanthracene) dyes in a polyurethane hydrogel, which was then coated onto a transparent film. The pH-sensing films have a fast response time, are reversible, stable in long-term culture environments, have minimal photobleaching, and are not cytotoxic. These films have a pK[sub a] of 7.61 ± 0.04 and are sensitive in the pH range corresponding to normal and tumorigenic tissues. With these optodes, we measured the spatiotemporal evolution of pH gradients in paper-based tumor models. [ABSTRACT FROM AUTHOR]

Paper has recently attracted increasing attention as a substrate for various biomedical applications. By modifying its physical and chemical properties, paper can be used as an alternative to conventional cell culture substrates. Because it can be stacked into a three-dimensional (3D) structure, which can better mimic the in vivo cell microenvironment. Paper has shown great potential as a 3D cell culture platform for developing normal and diseased models. This platform gives precise control over extracellular matrix (ECM) composition as well as cell distribution and precise analysis of the interactions between cells. Paper-based platforms have been applied for pathophysiological studies and therapeutic intervention studies. In this paper, we first discuss the modifications of the physical and chemical properties of paper to develop various 2D and 3D cell culture platforms. We then review the applications of paper-based cell culture platforms for the construction of in vitro disease models, drug screening, and cell cryopreservation applications. Because of its advantages such as biocompatibility, eco-friendliness, cost efficiency, and ease of large-scale production, we believe that paper-based cell culture platforms would play an important role in the fields of biomedicine. [ABSTRACT FROM AUTHOR]

Culturing cells at the air–liquid interface (ALI) is essential for creating functional in vitro models of lung tissues. We present the use of direct-patterned laser-treated hydrophobic paper as an effective semi-permeable membrane, ideal for ALI cell culture. The surface properties of the paper are modified through a selective CO2 laser-assisted treatment to create a unique porous substrate with hydrophilic regions that regulate fluid diffusion and cell attachment. To select the appropriate model, four promising hydrophobic films were compared with each other in terms of gas permeability and long-term strength in an aqueous environment (wet-strength). Among the investigated substrates, parchment paper showed the fastest rate of oxygen permeability (3 times more than conventional transwell cell culture membranes), with the least variation in its dry and wet tensile strengths (124 MPa and 58 MPa, remaining unchanged after 7 days of submersion in PBS).The final paper-based platform provides an ideal, robust, and inexpensive device for generating monolayers of lung epithelial cells on-chip in a high-throughput fashion for disease modelling and in vitro drug testing. [ABSTRACT FROM AUTHOR]

[Display omitted] • An overview of bacterial and viral detection using paper-based devices is highlighted. • Fabrication techniques, fluid control of the paper-based devices are summarized. • Recently updated approaches to pathogen detection are introduced. • The benefits and limitations of each method are discussed. The early detection of pathogens plays a significant role in reducing fatalities and improving prognosis. Laboratory tests such as the cell culture, polymerase chain reaction, and immunoassays are performed for the identification of pathogens; however, these are time-consuming and require trained personnel, limiting their prompt use at the point of need. Several microfluidic devices have been developed for point-of-care testing (POCT), including microfluidic paper-based analytical devices (µPADs). Paper, as a material, holds great promise for POCT owing to its advantages such as low cost, easy fabrication, easy disposal, and equipment-free operation by capillary action, which coincides with the requirements for an ideal rapid test. Considering this, we summarize major µPAD fabrication techniques as well as the fluid controlling strategies published to date. The development of various fabrication techniques is important for finding appropriate mass-production methods, and fluid control determines the complexity of the analysis. We also emphasize on recent bacterial and viral detection strategies from recent publications. Finally, this review discusses the challenges of developing an integrated device that can provide quantitative results to further improve the performance of bacterial and viral detection. [ABSTRACT FROM AUTHOR]

We developed a simple and low-cost cell culture monitoring system utilizing a paper-based analytical device (PAD) and a smartphone. The PAD simultaneously analyses glucose and lactate concentrations in the cell culture medium. Focusing on the fact that animal cells consume glucose and produce lactate under anaerobic conditions, oxidase- and horseradish peroxidase (HRP) enzyme-mediated colorimetric assays were integrated into the PAD. The PAD was designed to have three laminated layers. By using a double-sided adhesive tape as the middle layer and wax coating, a bifurcated fluidic channel was prepared to manipulate sample flow. At the inlet and the outlets of the channel, a sample drop zone and two detection zones for glucose and lactate, respectively, were positioned. When sample solution is loaded onto the drop zone, it flows to the detection zone through the hydrophilic fluidic channel via capillary force. Upon reaching the detection zone, the sample reacts with glucose and lactate oxidases (GOx and LOx) and HRP, immobilized on the detection zone along with colorless chromophores. By the Trinder’s reaction, the colorless chromophore is converted to a blue-colored product, generating concentration-dependent signal. With a gadget designed to aid the image acquisition, the PAD was positioned to the smartphone-embedded camera. Images of the detection zones were acquired using a mobile application and the color intensities were quantified as sensor signals. For the glucose assay using GOx/HRP format, we obtained the limit of detection (LOD ∼0.3 mM) and the limit of quantification (LOQ ∼0.9 mM) values in the dynamic detection range from 0.3 to 8.0 mM of glucose. For lactate assay using LOx/HRP, the LOD (0.02 mM) and the LOQ (0.06 mM) values were registered in the dynamic detection range from 0.02 to 0.50 mM of lactate. With the device, simultaneous analyses of glucose and lactate in cell culture media were conducted, exhibiting highly accurate and reproducible results. Based on the results, we propose that the optical sensing system developed is feasible for practical monitoring of animal cell culture. [ABSTRACT FROM AUTHOR]

Paper-based scaffolds are an attractive material for generating 3D tissue-like cultures because paper is readily available and does not require specialized equipment to pattern, cut, or use. By controlling the exchange of fresh culture medium with the paper-based scaffolds, we can engineer diffusion-dominated environments similar to those found in spheroids or solid tumors. Oxygen tension directly regulates cellular phenotype and invasiveness through hypoxia-inducible transcription factors and also has chemotactic properties. To date, gradients of oxygen generated in the paper-based cultures have relied on cellular response-based readouts. In this work, we prepared a luminescent thin film capable of quantifying oxygen tensions in apposed cell-containing paper-based scaffolds. The oxygen sensors, which are polystyrene films containing a Pd(II) tetrakis(pentafluorophenyl)porphyrin dye, are photostable, stable in culture conditions, and not cytotoxic. They have a linear response for oxygen tensions ranging from 0 to 160 mmHg O, and a Stern-Volmer constant ( K) of 0.239 ± 0.003 mmHg O. We used these oxygen-sensing films to measure the spatial and temporal changes in oxygen tension for paper-based cultures containing a breast cancer line that was engineered to constitutively express a fluorescent protein. By acquiring images of the oxygen-sensing film and the fluorescently labeled cells, we were able to approximate the oxygen consumption rates of the cells in our cultures. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is the most important opportunistic pathogen leading to serious and life-threatening infections, especially in immunocompromised patients. Because of its remarkable capacity to resist antibiotics, the selection of the right antibiotics with the exact dose for the appropriate duration is critical to effectively treat the infections and prevent antibiotic resistance. Although conventional genotypic and phenotypic antibiotic susceptibility testing (AST) methods have been dramatically advanced, they have suffered from many technical and operational issues as a generalized antibiotic stewardship program. Furthermore, given that most microbial infections are caused by their biofilms, the existing AST methods do not provide evidence-based antibiotic prescribing guidance for biofilm-based infections because the results are based on individual bacteria traditionally grown in their planktonic form. In this work, we create an innovative susceptibility testing technique for P. aeruginosa that offers clinically relevant guidelines and widely adaptable stewardship to effectively treat the infections and minimize antibiotic resistance. Our approach evaluates the antibiotic efficacy by continuously monitoring the accumulated electrical outputs from the bacterial extracellular electron transfer (EET) process in the presence of antibiotics. Our innovative paper-based culturing 3-D scaffold promotes surface-associated growth of bacterial colonies and biofilms. The platform replicates a natural habitat for P. aeruginosa where it can grow similarly to sites it infects. Our technique enables an all-electrical, real-time, easy-to-use, portable AST that can be easily translatable to clinical settings. The entire procedure takes 96 min to provide evidence-based antimicrobial prescribing guidance for biofilm-based infections. [Display omitted] [ABSTRACT FROM AUTHOR]

Recently, 3D cell culture technique was proposed to provide a more physiologically-meaningful environment for cell-based assays. With the development of microfluidics technology, cellular response can be quantified by impedance measurement technique in a real-time and non-invasive manner. However, handling of these microfluidic systems requires a trained engineering personnel and the operation is not compatible to traditional biological research laboratories. In this work, we incorporated the impedance measurement technique to paper-based 3D cell culture model and demonstrated non-invasive quantification of cells encapsulated in hydrogel during the culture course. A cellulose filter paper was patterned with an array of circular microchambers. Cells were encapsulated in hydrogel and loaded to the microchambers for culturing cells in 3D environment. At the preset schedule during the culture course, the paper was placed on a glass substrate with measurement electrodes for the impedance measurement. Cells in each microchamber was represented by impedance magnitude and cell proliferation could be studied over time. Also, conventional bio-assay was performed to further confirm the feasibility of the impedimetric quantification of cells encapsulated in hydrogel cultured in the paper-based microchamber. This technique provides a convenient, fast, and non-invasive approach to monitor cells cultured in 3D environment. It has potential to be developed for routine 3D cell culture protocol in biological research laboratories. [ABSTRACT FROM AUTHOR]

Background Evidence about the clinical impact of rapid diagnostic tests (RDTs) for the diagnosis of bloodstream infections is limited, and whether RDT are superior to conventional blood cultures (B Cs) embedded within antimicrobial stewardship programs (ASPs) is unknown. Methods We performed network meta-analyses using results from studies of patients with bloodstream infection with the aim of comparing the clinical impact of RDT (applied on positive BC broth or whole blood) to conventional BC, both assessed with and without ASP with respect to mortality, length of stay (LOS), and time to optimal therapy. Results Eighty-eight papers were selected, including 25 682 patient encounters. There was an appreciable amount of statistical heterogeneity within each meta-analysis. The network meta-analyses showed a significant reduction in mortality associated with the use of RDT + ASP versus BC alone (odds ratio [OR], 0.72; 95% confidence interval [CI],.59–.87) and with the use of RDT + ASP versus BC + ASP (OR, 0.78; 95% CI,.63–.96). No benefit in survival was found associated with the use of RDT alone nor with BC + ASP compared to BC alone. A reduction in LOS was associated with RDT + ASP versus BC alone (OR, 0.91; 95% CI,.84–.98) whereas no difference in LOS was shown between any other groups. A reduced time to optimal therapy was shown when RDT + ASP was compared to BC alone (−29 hours; 95% CI, −35 to −23), BC + ASP (−18 hours; 95% CI, −27 to −10), and to RDT alone (−12 hours; 95% CI, −20 to −3). Conclusions The use of RDT + ASP may lead to a survival benefit even when introduced in settings already adopting effective ASP in association with conventional BC. [ABSTRACT FROM AUTHOR]

Stereolithography 3D printing, although an increasingly used fabrication method for microfluidic chips, has the main disadvantage of producing monolithic chips in a single material. We propose to incorporate during printing various objects using a "print-pause-print" strategy. Here, we demonstrate that this novel approach can be used to incorporate glass slides, hydrosoluble films, paper pads, steel balls, elastic or nanoporous membranes and silicon-based microdevices, in order to add microfluidic functionalities as diverse as valves, fluidic diodes, shallow chambers, imaging windows for bacteria tracking, storage of reagents, blue energy harvesting or filters for cell capture and culture. [ABSTRACT FROM AUTHOR]

Bioimpedance spectrum (BIS) measurements have a great future in in vitro experiments, meeting all the requirements for non-destructive and label-free methods. Nevertheless, a real basic research can provide the necessary milestones to achieve the success of the method. In this paper a self-developed technology-based approach for in vitro assays is proposed. Authors invented a special graphene-based measuring plate in order to assess the high sensitivity and reproducibility of introduced technique. The design of the self-produced BIS plates maximizes the detection capacity of qualitative changes in cell culture and it is robust against physical effects and artifacts. The plates do not influence the viability and proliferation, however the results are robust, stable and reproducible regardless of when and where the experiments are carried out. In this study, physiological saline concentrations, two cancer and stem cell lines were utilized. All the results were statistically tested and confirmed. The findings of the assays show, that the introduced BIS technology is appropriate to be used in vitro experiments with high efficacy. The experimental results demonstrate high correlation values across the replicates, and the model parameters suggested that the characteristic differences among the various cell lines can be detected using appropriate hypothesis tests. [ABSTRACT FROM AUTHOR]

Wild bananas and their relatives are potentially utilized for pre-breeding due to their genetic diversity, disease resistance, and tolerance to abiotic stress, and other desirable traits. The embryonic suspension culture of wild bananas provides a means to harness this genetic diversity for banana genetic improvement. This paper elucidates the response of different subspecies M. acuminata (ssp. malaccensis, microcarpa, sumatrana, and breviformis) to the induction, growth, and behavior of suspension cultures and their regeneration into plantlets. Different subspecies exhibit varied responses starting from the embryogenic culture induction stage, culture proliferation, to plantlet formation. The highest competence for plant regeneration through somatic embryogenesis was found in ssp. malaccensis, followed by microcarpa, sumatrana, and breviformis. The wild banana embryogenic culture consists of somatic embryos, somatic embryo masses, proembryos, and proembryonic masses, and it proliferates through somatic embryo budding and proembryo proliferation. Maintenance and proliferation of suspension cultures were achieved through subculturing medium-sized cell aggregates (300 to 1000 µm). With an inoculum density of 0.3 g per 30 mL medium, the culture's proliferation rate reached seven times within 25 d. Embryogenic cultures from the suspensions of ssp. malaccensis and microcarpa were capable of forming somatic embryos upon transfer to a semi-solid somatic embryo development medium and later developed shoots on a semi-solid plant regeneration medium, with conversion efficiencies of 35% and 17%, respectively. [ABSTRACT FROM AUTHOR]

In the process of tissue engineering, several types of stresses can influence the outcome of tissue regeneration. This outcome can be understood by designing hydrogels that mimic this process and studying how such hydrogel scaffolds and cells behave under a set of stresses. Here, a hydrogel formulation is proposed to create biomimetic scaffolds suitable for fibroblast cell culture. Subsequently, we examine the impact of external stresses on fibroblast cells cultured on both solid and porous hydrogels. These stresses included mechanical tension and altered-gravity conditions experienced during the 83rd parabolic flight campaign conducted by the European Space Agency. This study shows distinct cellular responses characterized by cell aggregation and redistribution in regions of intensified stress concentration. This paper presents a new biomimetic hydrogel that fulfills tissue-engineering requirements in terms of biocompatibility and mechanical stability. Moreover, it contributes to our comprehension of cellular biomechanics under diverse gravitational conditions, shedding light on the dynamic cellular adaptations versus varying stress environments. [ABSTRACT FROM AUTHOR]

Biological cells replicate their genomes in a well-planned manner. The DNA replication program of an organism determines the timing at which different genomic regions are replicated, with fundamental consequences for cell homeostasis and genome stability. In a growing cell culture, genomic regions that are replicated early should be more abundant than regions that are replicated late. This abundance pattern can be experimentally measured using deep sequencing. However, a general quantitative theory linking this pattern to the replication program is still lacking. In this paper, we predict the abundance of DNA fragments in asynchronously growing cultures from any given stochastic model of the DNA replication program. As key examples, we present stochastic models of the DNA replication programs in budding yeast and Escherichia coli. In both cases, our model results are in excellent agreement with experimental data and permit to infer key information about the replication program. In particular, our method is able to infer the locations of known replication origins in budding yeast with high accuracy. These examples demonstrate that our method can provide insight into a broad range of organisms, from bacteria to eukaryotes. Author summary: Biological cells replicate their genome in a planned manner. One way of obtaining experimental information about this plan is by deep sequencing a growing culture of cells. The idea underlying these experiments is that genomic regions that are replicated earlier would be present in higher abundances than regions replicated at a later stage. In this paper, we make use of this idea to obtain precise quantitative information on replication programs from sequencing. As a main application, we infer the locations of origins of replication in budding yeast from sequencing data. Our inference is consistent with direct experimental evidences on the locations of these origins. Our method can be in principle be applied to any organism that can be cultured and sequenced, and has therefore the potential to shed light on the replication program of a broad class of organisms. [ABSTRACT FROM AUTHOR]

Simple Summary: The field of cell culture technology involves the cultivation of cells in a controlled environment, mimicking the natural conditions found in living organisms. This process is performed on a large scale, utilizing a specially designed culture medium. The goal is to create either undifferentiated individual cells or multicellular aggregates with minimal differentiation. The present paper offers a comprehensive overview of the advancements and applications of primary cell culture techniques in fish. It places a particular focus on revealing the physiological roles played by fish cells during their in vitro cultivation. Maintaining the functional characteristics of fish cells is of paramount importance, as it is essential for gaining a deeper understanding of the underlying mechanisms governing various physiological processes. To facilitate the use of fish cells in research and practical applications, it is crucial to standardize the separation techniques for these cells and optimize the conditions for their culture. This not only contributes to our knowledge of fish health, disease development, and drug discovery but also drives progress in the aquaculture industry. Fish primary cell culture has emerged as a valuable tool for investigating the physiological roles and responses of various cell types found in fish species. This review aims to provide an overview of the advancements and applications of fish primary cell culture techniques, focusing on the profiling of physiological roles exhibited by fish cells in vitro. Fish primary cell culture involves the isolation and cultivation of cells directly derived from fish tissues, maintaining their functional characteristics and enabling researchers to study their behavior and responses under controlled conditions. Over the years, significant progress has been made in optimizing the culture conditions, establishing standardized protocols, and improving the characterization techniques for fish primary cell cultures. The review highlights the diverse cell types that have been successfully cultured from different fish species, including gonad cells, pituitary cells, muscle cells, hepatocytes, kidney and immune cells, adipocyte cells and myeloid cells, brain cells, primary fin cells, gill cells, and other cells. Each cell type exhibits distinct physiological functions, contributing to vital processes such as metabolism, tissue regeneration, immune response, and toxin metabolism. Furthermore, this paper explores the pivotal role of fish primary cell culture in elucidating the mechanisms underlying various physiological processes. Researchers have utilized fish primary cell cultures to study the effects of environmental factors, toxins, pathogens, and pharmaceutical compounds on cellular functions, providing valuable insights into fish health, disease pathogenesis, and drug development. The paper also discusses the application of fish primary cell cultures in aquaculture research, particularly in investigating fish growth, nutrition, reproduction, and stress responses. By mimicking the in vivo conditions in vitro, primary cell culture has proven instrumental in identifying key factors influencing fish health and performance, thereby contributing to the development of sustainable aquaculture practices. [ABSTRACT FROM AUTHOR]

Low cost and user-friendly paper microfluidic devices, combined with DNA-based biosensors with binding capacities for specific molecules, have been proposed for the developing of novel platforms that ease and speed-up the process of cell secretion monitoring. In this work, we present the first cellulose microfluidic paper-based analytical device for the single-step detection of cell secreted Vascular Endothelial Growth Factor through a self-reporting Structure Switching Signaling Aptamer. A three-part Structure Switching Signaling Aptamer was designed with an aptameric sequence specific for VEGF, which provides a quantifiable fluorescent signal through the displacement of a quencher upon VEGF recognition. The VEGF biosensor was integrated in cellulose paper, enabling the homogenous distribution of the sensor in the paper substrate and the detection of as low as 0.34 ng of VEGF in 30 min through fluorescence intensity analysis. As a proof-of-concept, the biosensor was incorporated in a microfluidic paper-based analytical device format containing a VEGF detection zone and a control zone, which was applied for the detection of cell secreted VEGF in the supernatant of mesenchymal stem cells culture plates, demonstrating its potential use in cell biology research. [Display omitted] • A Structure Switching Signaling Aptamer (SSSA) was developed for the single-step detection of VEGF through fluorescence analysis. • The SSSA was incorporated into a simple-to-use cellulose paper device, enabling the detection of 0.34 ng of the growth factor in 30 minutes. • The paper microfluidic device was successfully applied for the quantification of VEGF secreted from a Mesenchymal Stromal Cell culture. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the toxicity of extracts from disposable chopsticks, toothpicks, and paper cups on L-929 cells. Methods: We followed national standards to prepare the extracts from disposable chopsticks, toothpicks, and paper cups used for the cell culture media, and the morphology of L-929 cells was observed with an optical microscope. The loss rate for adherent cells was evaluated with the trypan blue exclusion method, and cell proliferation was determined using the WST-1 assay. Results: Compared with the control group, the cells cultured in media containing the extracts showed signs of apoptosis and necrosis after culturing for 4 or 7 days, and the loss rate for adherent cells was significantly increased ( P < 0.05). An obvious decrease in cell viability was also observed ( P < 0.05). Conclusion: The extracts from disposable chopsticks, toothpicks, and paper cups can affect the growth and proliferation of L-929 cells and are potentially toxic to humans. [ABSTRACT FROM AUTHOR]

Abstract: We report the development of a new type of flexible electrochemical biosensors based on graphene paper loaded with closely-packed Au@Pt core–shell nanoparticles as a freestanding cell culture substrate for real-time monitoring cell secretion of nitric oxide. The hybrid electrode was fabricated through a modular approach in which 2D-assembly of nanoparticles formed at the oil–water interface was transferred onto graphene paper by dip-coating. We have shown that the independently optimized metal nanostructures and graphene paper were integrated into functional electrodes with high electrocatalytic activity. When used for the detection of nitric oxide, the flexible electrodes have demonstrated high sensitivity, a wide linear range, and a low detection limit, which, in combination with its biocompatibility, offer unique opportunities for the real-time monitoring of nitric oxide secretion by human endothelial vein cells grown on the electrode. These interesting findings collectively demonstrate the potential of our modular approach for designing high-performance flexible electrodes with tailored surface properties. [Copyright &y& Elsevier]

Conference papers originating from the 27th International Symposium on Amyotrophic Lateral Sclerosis (ALS) and Motor Neurone Diseases with a focus on in vitro experimental modeling are presented. The conference papers cover a range of topics which include a cellular model of ALS with SQSTM1 mutations and the oligomerization and subcellular localization of missense/in-frame deletion ALS2 mutants in cultured cells. The development of a biomolecular fluorescence complementation assay is mentioned.

There is a growing interest in realizing the benefits of continuous processing in biologics manufacturing, which is reflected by the significant number of industrial and academic researchers who are actively involved in the development of continuous bioprocessing systems. These efforts are further encouraged by guidance expressed in recent US FDA conference presentations. The advantages of continuous manufacturing include sustained operation with consistent product quality, reduced equipment size, high-volumetric productivity, streamlined process flow, low-process cycle times, and reduced capital and operating cost. This technology, however, poses challenges, which need to be addressed before routine implementation is considered. This paper, which is based on the available literature and input from a large number of reviewers, is intended to provide a consensus of the opportunities, technical needs, and strategic directions for continuous bioprocessing. The discussion is supported by several examples illustrating various architectures of continuous bioprocessing systems. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:813-820, 2015 [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) cell culture system, as an alternative approach for traditional cell culture, attracts great attention because of physiological relevance and great microenvironment similarity to human conditions. Herein, a facile paper-polylactic (PLA) platform that was fabricated by wax printing and 3D printing, coupled with electrochemical sensor, was designed for the construction and intervention of 3D cell damage model. Pheochromocytoma cells (PC12) and bone marrow mesenchymal stem cells (BMSCs) were seeded on the paper-PLA 3D platforms and displayed the features of uniform distribution, good adhesion and perfect proliferation, as well as decreased circularity when compared to those grown on the two-dimensional (2D) interfaces. The electrochemical sensors revealed cell viability by monitoring dopamine released by cell models, ascertaining the applicability of the paper-PLA platform to a long-term 3D cell culture and drug assessment. Additionally, the results revealed that donepezil and BMSCs-secreted active molecules exhibited stronger cytoprotective effect against amyloid-beta oligomers-induced cell damage on the paper-PLA 3D printed platforms, indicating the cell damage model and the cell intervention model were achieved successfully in the simulated in vivo physiological microenvironment. Thus, the proposed paper-PLA platform may serve as a promising candidate for efficient drug screening and toxicity evaluation due to its simple structure, low cost, and convenient integration of 3D cell culture and activity evaluation. [Display omitted] • The PC12 cells and MSCs encapsulated in agarose gel were proliferated on the facile paper-PLA 3D printed platform. • Cell viability of different cell models was evaluated by paper-PLA 3D printed device combined with electrochemical sensor. • The type of paper may affect the activity of key small molecules secreted by BMSCs. • The facile paper-PLA 3D printed platform offers an ideal 3D cell culture product for drug screening and toxicity evaluation. [ABSTRACT FROM AUTHOR]

We examine the use of time series data, derived from Electric Cell-substrate Impedance Sensing (ECIS), to differentiate between standard mammalian cell cultures and those infected with a mycoplasma organism. With the goal of easy visualization and interpretation, we perform low-dimensional feature-based classification, extracting application-relevant features from the ECIS time courses. We can achieve very high classification accuracy using only two features, which depend on the cell line under examination. Initial results also show the existence of experimental variation between plates and suggest types of features that may prove more robust to such variation. Our paper is the first to perform a broad examination of ECIS time course features in the context of detecting contamination; to combine different types of features to achieve classification accuracy while preserving interpretability; and to describe and suggest possibilities for ameliorating plate-to-plate variation. [ABSTRACT FROM AUTHOR]

siRNA is an important tool for modulating gene expression in current biomedical research. It would be highly desirable for siRNA to respond to an external stimulus. In this paper, we report a convenient, photolabile caging agent to regulate siRNA functions. 2-bromo-4'-hydroxyacetophenone (BHAP) can readily modify phosphorothioate backbones and inhibit siRNAs. Mild UV irradiation will cleave the modifying moiety to generate natural nucleic acid backbones, thus activating siRNA functions. Such modification is conveniently conducted in an aqueous solution with high efficiency and is cost-effective and scalable. This approach provides a convenient tool for the controlled regulation of gene expression by deploying minimal usage of complex organic synthesis for site-specific installation of the caging group to siRNA unlike previous reported works that required a series of intricate organic synthesis and cumbersome purification techniques to achieve similar aims. This study will open new doors for optochemical regulation of a variety of genes by pHP caging group in mammalian cell culture. [ABSTRACT FROM AUTHOR]

During the development of the nervous system, neuronal cells extend axons and dendrites that form complex neuronal networks, which are essential for transmitting and processing information. Understanding the physical processes that underlie the formation of neuronal networks is essential for gaining a deeper insight into higher-order brain functions such as sensory processing, learning, and memory. In the process of creating networks, axons travel towards other recipient neurons, directed by a combination of internal and external cues that include genetic instructions, biochemical signals, as well as external mechanical and geometrical stimuli. Although there have been significant recent advances, the basic principles governing axonal growth, collective dynamics, and the development of neuronal networks remain poorly understood. In this paper, we present a detailed analysis of nonlinear dynamics for axonal growth on surfaces with periodic geometrical patterns. We show that axonal growth on these surfaces is described by nonlinear Langevin equations with speed-dependent deterministic terms and gaussian stochastic noise. This theoretical model yields a comprehensive description of axonal growth at both intermediate and long time scales (tens of hours after cell plating), and predicts key dynamical parameters, such as speed and angular correlation functions, axonal mean squared lengths, and diffusion (cell motility) coefficients. We use this model to perform simulations of axonal trajectories on the growth surfaces, in turn demonstrating very good agreement between simulated growth and the experimental results. These results provide important insights into the current understanding of the dynamical behavior of neurons, the self-wiring of the nervous system, as well as for designing innovative biomimetic neural network models. [ABSTRACT FROM AUTHOR]

Biomimetic scaffolds imitate native tissue and can take a multidimensional form. They are biocompatible and can influence cellular metabolism, making them attractive bioengineering platforms. The use of biomimetic scaffolds adds complexity to traditional cell cultivation methods. The most commonly used technique involves cultivating cells on a flat surface in a two-dimensional format due to its simplicity. A three-dimensional (3D) format can provide a microenvironment for surrounding cells. There are two main techniques for obtaining 3D structures based on the presence of scaffolding. Scaffold-free techniques consist of spheroid technologies. Meanwhile, scaffold techniques contain organoids and all constructs that use various types of scaffolds, ranging from decellularized extracellular matrix (dECM) through hydrogels that are one of the most extensively studied forms of potential scaffolds for 3D culture up to 4D bioprinted biomaterials. 3D bioprinting is one of the most important techniques used to create biomimetic scaffolds. The versatility of this technique allows the use of many different types of inks, mainly hydrogels, as well as cells and inorganic substances. Increasing amounts of data provide evidence of vast potential of biomimetic scaffolds usage in tissue engineering and personalized medicine, with the main area of potential application being the regeneration of skin and musculoskeletal systems. Recent papers also indicate increasing amounts of in vivo tests of products based on biomimetic scaffolds, which further strengthen the importance of this branch of tissue engineering and emphasize the need for extensive research to provide safe for humansbiomimetic tissues and organs. In this review article, we provide a review of the recent advancements in the field of biomimetic scaffolds preceded by an overview of cell culture technologies that led to the development of biomimetic scaffold techniques as the most complex type of cell culture. [ABSTRACT FROM AUTHOR]

[Display omitted] • We reviewed the literature on isolation of Toxoplasma gondii directly in cell culture. • Four experiments were reported to isolate T. gondii from different animals in Vero and HFF cell cultures. • We recommend an in vitro protocol to isolate T. gondii. • This method is simpler, more cost-effective, ethically more acceptable, and less time-sensitive. Toxoplasma gondii is an apicomplexan protozoan parasite that can infect mammals and birds. The infection can cause acute toxoplasmosis and death in susceptible hosts. Bioassay using cats and mice has been the standard for the isolation of T. gondii from infected hosts for the past several decades. However, bioassay is labor-intensive, expensive, and involves using laboratory animals. To search alternative approaches and o work towards replacement of animal experiments, we summarized the key literature and conducted four experiments to isolate T. gondii in vitro by cell culture. A few heart tissue samples from animals with the highest antibody titers in a given collection were used for T. gondii isolation. These experiments included samples from five out of 51 wild ducks, four of 46 wild turkeys, six of 24 white-tailed deer, as well as from six kangaroos that had died with acute toxoplasmosis in a zoo. These experiments resulted in three isolates from five chronically infected wild ducks (60%), four isolates from four chronically infected wild turkeys (100%), one isolate from six chronically infected white-tailed deer (17%), and four isolates from six kangaroos with acute toxoplasmosis (67%). In addition, five isolates from the five chronically infected wild ducks were obtained by bioassay in mice, showing a 100% success rate, which is higher than the 60% rate by direct cell culture. These T. gondii isolates were successfully propagated in human foreskin fibroblast (HFF) or Vero cells, and genotyped by multilocus PCR-RFLP markers. The results showed that it is practical to isolate T. gondii directly in cell culture. Although the cell culture approach may not be as sensitive as the bioassay, it does provide an alternative that is simple, cost-effective, ethically more acceptable, and less time-sensitive to isolate T. gondii. In this paper we propose a procedure that may be applied and further optimized for isolation of T. gondii. [ABSTRACT FROM AUTHOR]

Deer antlers are unique because they are cast and regenerate each year. They are the fastest-growing structure, reaching an astonishing growth rate of up to 2.75 cm/day in length and more than 20 cm2/day of skin. Surprisingly, no study so far has assessed the metabolic rate of the antler. High metabolic rate needs highly efficient (or large) mitochondria, and it involves a high creation or reactive oxygen species (ROS), origin of oxidative stress. The speed of creation of ROS and the oxidative stress are inversely related to ageing and many diseases such as cancer or age-related diseases. However, antler must have the most efficient anti-oxidant system, as it rarely shows any departure from a perfect growth. This paper examines recent studies showing surprising applications in medicine of growing-antler extracts, or the information regarding its physiology. A recent study (Wang et al. (2019) , Science 364, eaav6335) has shown that antlers have evolved a speed of growth faster than cancer, based on high expression of proto-oncogenes. As a result, deer has evolved tumour-suppression genes to control the high risk of developing cancer. This may explain why several studies have found in vitro and in vivo anti-cancer effects of deer velvet-antler extract in human tumours, such as cell cultures and animal models of cancers such as brain cancer (glioblastoma), prostate cancer, and others. We will also discuss findings in the study of the cyclic osteoporosis of the deer, with unexpected similarities in their proteomics and gene expression with that of the human pathological osteoporosis. Last, we will examine potential applications based on having the highest metabolic rate. If the future studies establish the antler as the tissue having the fastest metabolism and the best antioxidant system, this may have implications for understanding how to fight oxidative stress, which, in turn, will have direct implications for aging and age-related diseases (and others, from cancer to osteoporosis and Alzheimer's for example). It may also show that velvet-antler extract is a general anti-cancer compound, and this may show the path to find an anti-cancer medicine that has no secondary toxic effects in healthy cells. Antler is the only mammal structure regenerating each year, and it is the fastest-growing tissue (much faster-growing than cancer). For this reason, several interesting medical applications are reviewed in this paper, such as anti-cancer activity of growing-antler extract. Studying growing antler may help develop powerful and general anti-cancer treatments without secondary effects, and the high metabolic rate and antioxidant system of the antler may also have other potential medical applications, such as delaying aging- or fight-associated diseases. [ABSTRACT FROM AUTHOR]

The yellowing of paper on aging causes major aesthetic damages of cultural heritage. It is due to cellulose oxidation, a complex process with many possible products still to be clarified. By comparing ultraviolet-visible reflectance spectra of ancient and artificially aged modern papers with ab initio time-dependent density functional theory calculations, we identify and estimate the abundance of oxidized functional groups acting as chromophores and responsible of paper yellowing. This knowledge can be used to set up strategies and selective chemical treatments preventing paper yellowing. [ABSTRACT FROM AUTHOR]

In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc). The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously ("cells-in-gels-in-paper" or CiGiP), this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM) gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers) containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, "sections" all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures. [ABSTRACT FROM AUTHOR]

Fundamental investigations of human biology, and the development of therapeutics, commonly rely on 2D cell-culture systems that do not accurately recapitulate the structure, function, or physiology of living tissues. Systems for 3D cultures exist but do not replicate the spatial distributions of oxygen, metabolites, and signaling molecules found in tissues. Microfabrication can create architecturally complex scaffolds for 3D cell cultures that circumvent some of these limitations; unfortunately, these approaches require instrumentation not commonly available in biology laboratories. Here we report that stacking and destacking layers of paper impregnated with suspensions of cells in extracellular matrix hydrogel makes it possible to control oxygen and nutrient gradients in 3D and to analyze molecular and genetic responses. Stacking assembles the "tissue", whereas destacking disassembles it, and allows its analysis. Breast cancer cells cultured within stacks of layered paper recapitulate behaviors observed both in 3D tumor spheroids in vitro and in tumors in vivo: Proliferating cells in the stacks localize in an outer layer a few hundreds of microns thick, and growth-arrested, apoptotic, and necrotic cells concentrate in the hypoxic core where hypoxia-sensitive genes are overex- pressed. Altering gas permeability at the ends of stacks controlled the gradient in the concentration of the 02 and was sufficient by itself to determine the distribution of viable cells in 3D. Cell cultures in stacked, paper-supported gels offer a uniquely flexible approach to study cell responses to 3D molecular gradients and to mimic tissue- and organ-level functions. [ABSTRACT FROM AUTHOR]

Abstract: This article presents the WHO recommendations on the use of rabies vaccines excerpted from the recently published Rabies vaccines: WHO position paper. This document replaces the WHO position paper entitled Rabies vaccines WHO position paper published in the Weekly Epidemiological Record in December 2007. Footnotes to this paper provide a limited number of core references; their abstracts as well as a more comprehensive list of references may be found at http://www.who.int/immunization/documents/positionpapers/en/index.html. Grading tables which assess the quality of scientific evidence for key conclusions are also available through this link and are referenced in the position paper. In accordance with its mandate to provide guidance to Member States on health policy matters, WHO issues a series of regularly updated position papers on vaccines and combinations of vaccines against diseases that have an international public health impact. These papers are concerned primarily with the use of vaccines in large-scale immunization programmes; they summarize essential background information on diseases and vaccines, and conclude with WHO''s current position on the use of vaccines in the global context. This updated paper reflects the recent recommendations of WHO''s Strategic Advisory Group of Experts on immunization, or SAGE. [Copyright &y& Elsevier]

Background & Aims: Cancer chemotherapy is facing many problems such as patients' intolerance or resistance of cancer cells to the used compounds and drugs. In this regard, researchers are continuously trying to design new anti-cancer compounds and use them. Test to find a combination that can overcome these problems. Considering that structures containing Indole rings and toxic Carbazone and Thiazoles have already shown different biological effects. In this article, hybrids of these chemical structures will be designed and their effects will be investigated, maybe new compounds can be obtained to overcome some of the problems in the way of chemotherapy. The purpose of this paper is to synthesize and investigate the anticancer properties of new hybrid compounds of Indole attached to Triazole and thiosemicarbazone pharmacophores using the click method. Derivatives of indole and 3,2,1-triazoles and thiosemicarbazone are of special importance due to various biological properties such as anti-inflammatory, antibacterial, anti-HIV, anti-fungal, and anti-cancer. In this research, new derivatives of 3, 2,1-triazoles attached to 2-aryl-1H-indole-3-substituted system by click reaction between 2-aryl-1-prop-2-ynyl-1H-indole-3-substituted and azide Aromatics were synthesized in the presence of copper(II) acetate and sodium ascorbate catalyst in ethanol solvent. Also, a series of new derivatives of 2-aryl-1H-indole-3-substituted hybridized with thiosemicarbazone and triazole were synthesized in the presence of Paratoluenesulfonic acid catalyst in ethanol solvent, and the cytotoxic activity of these compounds on Hela and MCF cell lines. Compounds with heterocyclic structures, because they exist in many natural products and exhibit different physicochemical properties, also have different biological effects, which has diversified their use in the treatment of various diseases. Cancer chemotherapy is faced with many problems such as patient intolerance or resistance of cancer cells to the used compounds and drugs. In this regard, researchers are constantly trying to design new anti-cancer compounds and test them to find a combination. achieve that can overcome these problems. They are considering that structures containing indole rings and toxic carbazone and thiazoles have already shown different biological effects. In this article, hybrids of these chemical structures will be designed and their effects will be investigated, maybe new compounds can be obtained to overcome some of the problems in chemotherapy. Derivatives of indole and 3,2,1-triazoles and thiosemicarbazone are of special importance due to their various biological properties, including anti-inflammatory, antibacterial, anti-HIV, anti-fungal, and anti-cancer. In this research, new derivatives of 3, 2,1-triazoles attached to 2- aryl-1H-indole-3-substituted system by click reaction between 2-aryl-1-prop-2-ynyl-1Hindole-3-substituted and azide Aromatics were synthesized in the presence of copper(II) acetate and sodium ascorbate catalyst in ethanol solvent. Also, a series of new derivatives of 2-aryl-1H-indole-3-substituted hybridized with thiosemicarbazone and triazole were synthesized in the presence of para toluene sulfonic acid catalyst in ethanol solvent, and the cytotoxic activity of these compounds on Hela and MCF cell lines. 7 was investigated. To solve the problem of cancer from compounds with different pharmacophores such as indole, triazole (due to the presence in many cytotoxic and antimicrobial compounds and the effectiveness of this skeleton), and thiosemicarbazone and connecting these three pharmacophores to find new compounds that have possible cytotoxicity were used. Methods: Derivatives of Indole and 3,2,1-triazoles and thiosemicarbazone are of special importance due to various biological properties such as anti-inflammatory, antibacterial, antiHIV, anti-fungal, and anti-cancer. In this paper, the click reaction was used for the synthesis of compounds. The click reactions were performed under mild conditions in the presence of cheap solvent and in a short time, and the product with high efficiency and purity (lowest amount of side products) is obtained. Click reaction of new derivatives of 3,2,1-triazoles attached to the 2-aryl-1H-indole-3-substituted system between 2-aryl-1-prop-2-ynyl-1Hindole-3-substituted Decompound Aromatic Azides were synthesized in the presence of copper(II) acetate and sodium ascorbate catalyst in ethanol solvent. Also, a series of new derivatives of 2-aryl-1H-indole-3-substituted hybridized with thiosemicarbazone and Triazole were also synthesized in the presence of Paratoluenesulfonic acid catalyst in ethanol solvent, and the structure of all the synthesized compounds was analyzed by IR, H-NMR1 and 13 CNMR were confirmed. Results: In order to solve the problem of cancer from compounds with different Pharmacophores such as Indole, Triazole (due to the presence in many cytotoxic and antimicrobial compounds and the effectiveness of this skeleton), and thiosemicarbazone and connecting these three pharmacophores to each other It was used to find new compounds that have possible cytotoxicity. The cytotoxic activity of these compounds on Hela and MCF-7 cell lines was evaluated by the MTT method. Conclusion: The results showed that the investigated compounds had a significant cytotoxic effect on the Hela cell line only at the highest concentration (1000 µM) and had no effect at other concentrations and in some cases, including the effect on the MCF line. It seems that the compounds even increased cell growth. This increase in growth is likely to be due to the structural similarity of some compounds with phenolic structures, which has been shown in previous studies that phenolic groups act similar to estrogenic compounds and therefore can mimic growth. According to the results, these compounds can be considered as non-toxic agents for other therapeutic applications. [ABSTRACT FROM AUTHOR]

In this study we obtained Fourier transform infrared (FTIR) spectra of fixed prostate cell lines of differing types as well as the primary epithelial cells from benign prostatic hyperplasia (BPH). Results showed that by using multivariate chemometric analysis it was possible to discriminate and classify these cell lines, which gave rise to sensitivity and specificity values of >94% and >98%, respectively. Following on from these results the possible influences of different factors on the discrimination and classification of the prostate cell lines were examined. Firstly, the effect of using different growth media during cell culturing was investigated, with results indicating that this did not influence chemometric discrimination. Secondly, differences in the nucleus-to-cytoplasm (N/C) ratio were examined, and it was concluded that this factor was not the main reason for the discrimination and classification of the prostate cancer (CaP) cell lines. In conclusion, given the fact that neither growth media nor N/C ratio could totally explain the classification it is likely that actual biochemical differences between the cell lines is the major contributing factor. [ABSTRACT FROM AUTHOR]

In this study the suitability of Raman spectroscopy for the determination of carbon nanotube mediated toxicity on human alveolar carcinoma epithelial cells (A549) is explored. The exposure of this cell line represents the primary pathway of exposure in humans, that of inhalation. Peak ratio analysis demonstrates a dose-dependent response which correlates to previous toxicological studies. Principal component analysis is employed to further classify cellular response as a function of dose and to examine differences between spectra as a function of exposed concentration. To further illustrate the potential of Raman spectroscopy in this field, Partial Least Squares (PLS) regression and genetic algorithm feature selection have been utilised to demonstrate that clonogenic endpoints, and therefore toxic response, can be potentially predicted from spectra of cells exposed to un-determined doses, removing the need for costly and time consuming biochemical assays. This preliminary study demonstrates the potential of Raman spectroscopy as a probe of cytotoxicity to nanoparticle exposure. [ABSTRACT FROM AUTHOR]

The human body is made up of approximately 40 trillion cells in close contact, with the cellular density of individual tissues varying from 1 million to 1 billion cells per cubic centimetre. Interactions between different cell types (termed heterotypic) are thus common in vivo. Communication between cells can take the form of direct cell–cell contact mediated by plasma membrane proteins or through paracrine signalling mediated through the release, diffusion, and receipt of soluble factors. There is currently no systematic method to investigate the relative contributions of these mechanisms to cell behaviour. In this paper, we detail the conception, development and validation of a microfluidic device that allows cell–cell contact and paracrine signalling in defined areas and over a variety of biologically relevant length scales, referred to as the interactome-device or 'I-device'. Importantly, by intrinsic device design features, cells in different regions in the device are exposed to four different interaction types, including a) no heterotypic cell interaction, b) only paracrine signalling, c) only cell–cell direct contact, or d) both forms of interaction (paracrine and cell–cell direct contact) together. The device design was validated by both mathematical modelling and experiments. Perfused stem cell culture over the medium term and the formation of direct contact between cells in the culture chambers was confirmed. The I-device offers significant flexibility, being able to be applied to any combination of adherent cells to determine the relative contributions of different communication mechanisms to cellular outcomes. [ABSTRACT FROM AUTHOR]

Primary cell lines are invaluable for exploring cancer biology and investigating novel treatments. Despite their numerous advantages, primary cultures are laborious to obtain and maintain in culture. Hence, established cell lines are still more common. This study aimed to evaluate a range of techniques for isolating primary breast cancer cultures, employing distinct enzymatic compositions, incubation durations, and mechanical approaches, including filtration. Out of several protocols, we opted for a highly effective method (Method 5) that gave rise to a primary cell culture (BC160). This method combines mechanical disaggregation and enzymatic digestion with hyaluronidase and collagenase. Moreover, the paper addresses common issues in isolating primary cultures, shedding light on the struggle against fibroblasts overgrowing cancer cell populations. To make primary cell lines a preferred model, it is essential to elaborate and categorise isolation methods, develop approaches to separate heterogeneous cultures and investigate factors influencing the establishment of primary cell lines. [ABSTRACT FROM AUTHOR]