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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]

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 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]

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]

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]

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]

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]

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]

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]

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]

High-grade gliomas are considered an incurable disease. Despite all the various therapy options available, patient survival remains low, and the tumor usually returns. Tumor resistance to conventional therapy and stimulation of the migratory activity of surviving cells are the main factors that lead to recurrent tumors. When developing new treatment approaches, the effect is most often evaluated on standard and phenotypically depleted cancer cell lines. Moreover, there is much focus on the anti-proliferative effect of such therapies without considering the possible stimulation of migratory activity. In this paper, we studied how glioma cell migration changes after exposure to bi-(AID-1-T), an anti-proliferative aptamer. We investigated the effect of this aptamer on eight human glioma cell cultures (Grades III and IV) that were derived from patients' tumor tissue; the difference between primary and recurrent tumors was taken into account. Despite its strong anti-proliferative activity, bi-(AID-1-T) was shown to induce migration of recurrent tumor cells. This result shows the importance of studying the effect of therapeutic molecules on the invasive properties of glioma tumor cells in order to reduce the likelihood of inducing tumor recurrence. [ABSTRACT FROM AUTHOR]

Space-related stressors such as microgravity are associated with cellular and molecular alterations of the immune and inflammatory homeostasis that have been linked to the disorders that astronauts suffer from during their missions. Most of the research of the past 30 years has consistently established that innate adaptive immune cells represent a target of microgravity, which leads to their defective or dysfunctional activation, as well as to an altered ability to produce soluble mediators—e.g., cytokines/chemokines and bioactive lipids—that altogether control tissue homeostasis. Bioactive lipids include a vast array of endogenous molecules of immune origin that control the induction, intensity and outcome of the inflammatory events. However, none of the papers published so far focus on a newly characterized class of lipid mediators called specialized pro-resolving mediators (SPMs), which orchestrate the "resolution of inflammation"—i.e., the active control and confinement of the inflammatory torrent mostly driven by eicosanoids. SPMs are emerging as crucial players in those processes that avoid acute inflammation to degenerate into a chronic event. Given that SPMs, along with their metabolism and signaling, are being increasingly linked to many inflammatory disorders, their study seems of the outmost importance in the research of pathological processes involved in space-related diseases, also with the perspective of developing therapeutic countermeasures. Here, we show that microgravity, simulated in the rotary cell culture system (RCCS) developed by NASA, rearranges SPM receptors both at the gene and protein level, in human monocytes but not in lymphocytes. Moreover, RCCS treatment reduces the biosynthesis of a prominent SPM like resolvin (Rv) D1. These findings strongly suggest that not only microgravity can impair the functioning of immune cells at the level of bioactive lipids directly involved in proper inflammation, but it does so in a cell-specific manner, possibly perturbing immune homeostasis with monocytes being primary targets. [ABSTRACT FROM AUTHOR]

In vitro reconstruction of highly mature engineered heart tissues (EHTs) is attempted for the selection of cardiotoxic drugs suitable for individual patients before administration. Mechanical contractile force generated in the EHTs is known to be a critical indicator for evaluating the EHT response. However, measuring contractile force requires anchoring the EHT in a tailored force‐sensing cell culture chamber, causing technical difficulties in the stable evaluation of contractile force in long‐term culture. This paper proposes a hydrogel‐sheathed human induced pluripotent stem cell (hiPSC)‐derived heart microtissue (H3M) that can provide an anchor‐free contractile force measurement platform in commonly used multi‐well plates. The contractile force associated with tissue formation and drug response is calculated by motion tracking and finite element analysis on the bending angle of the hydrogel sheath. From the experiment of the drug response, H3M is an excellent drug screening platform with high sensitivity and early testing capability compared to conventionally anchored EHT. This unique platform would be useful and versatile for regenerative therapy and drug discovery research in EHT. [ABSTRACT FROM AUTHOR]

Simple Summary: Primordial germ cells (PGCs) play a critical role in the preservation of poultry genetic resources and transgenic research, but there are significant efficiency issues with isolating PGCs from chicken embryonic blood, especially from a single embryo. In this study, we developed a simple and rapid method to isolate PGCs from single chicken embryonic blood. This method is based on the characteristics of PGCs found in the study. That is, when co-cultured with chicken embryonic fibroblasts (CEFs) of passages two to three, PGCs can migrate to the lower layer of CEF through pores smaller than their diameter, which is called cell culture inserts/CEF adhesion method. The PGCs isolated using this method retain their stem cell characteristics and migration ability, and exhibit good proliferation efficiency. These cells can be directly utilized for subsequent transgenic experiments or for the preservation of germplasm resources. Primordial germ cells (PGCs) play a crucial role in preserving poultry genetic resources and conducting transgenic research. A system for the rapid isolation of PGCs from single chicken embryonic blood was established in this paper. We found that PGCs can migrate to the lower layer of chicken embryonic fibroblasts (CEFs) through pores smaller than their diameter, while blood cells cannot, when co-cultured with CEFs of passages two to three. Based on the characteristics of PGCs, we developed a new PGC isolation method (cell culture insert/CEF adhesion method) that utilizes a 3 μm cell culture insert and CEFs of passages two to three. Using this method, approximately 700 PGCs can be isolated from the blood of a single chicken embryo at Hamburger and Hamilton (H&H) stage 17 of development. The separation rate achieved was 87.5%, with a separation purity of 95%. The separation rate of this method was 41.4% higher than the common Percoll density gradient centrifugation method and 33.6% higher than lysis with ACK buffer. PGCs isolated from embryonic blood could proliferate 37-fold within 2 weeks when cultured in a feeder-free culture system. They also continued to express the SSEA-1 and DAZL proteins and retained the ability to migrate in vivo. Overall, PGCs separated using cell culture inserts/CEF adhesion method retain their stem cell characteristics and migration ability. PGCs also exhibit good proliferation efficiency, making them suitable for subsequent transgenic experiments or genetic resource preservation. [ABSTRACT FROM AUTHOR]

We suggest a novel approach for searching natural compounds with anti-aging and rejuvenation potential using cell cultures, with a high potential for the further in vivo applications. The present paper discusses ways of defining age for cell populations with large numbers of cells and suggests a method of assessing how young or old a cell population is based on a cell age profile approach. This approach uses experimental distributions of the cells over the cell cycle stages, acquired using flow cytometry. This paper discusses how such a profile should evolve under homeostatic maintenance of cell numbers in the proliferation niches. We describe promising results from experiments on a commercial substance claiming rejuvenating and anti-aging activity acting upon the cultures of human mononuclear cells and dermal fibroblasts. The chosen substance promotes a shift towards larger proportion of cells in synthesis and proliferation stages, and increases cell culture longevity. Further, we describe promising in vivo testing results of a selected food supplement. Based on the described concept of cell age profile and available test results, a strategy to search for natural compounds with regenerative, anti-aging and rejuvenation potential is suggested and proposed for wider and thorough testing. Proposed methodology of age assessment is rather generic and can be used for quantitative assessment of the anti-aging and rejuvenation potential of different interventions. Further research aimed at the tests of the suggested strategy using more substances and different interventions, and the thorough studies of molecular mechanisms related to the action of the substance used for testing the suggested search methodology, are needed. [ABSTRACT FROM AUTHOR]

Charge variants, as an important quality attribute of mAbs, must be comprehensively characterized and monitored during development. However, due to their complex structure, the characterization of charge variants is challenging, labor-intensive, and time-consuming when using traditional approaches. This work combines on-line and off-line 2D-LC-MS to comprehensively characterize mAb charge variants and quickly offer precise instructions for process development. Six charge variant peaks of mAb 1 were identified using the developed platform. Off-line 2D-LC-MS analysis at the peptide level showed that the acidic peak P1 and the basic peaks P4 and P5 were caused by the deamidation of asparagine, the oxidation of methionine, and incomplete C-terminal K loss, respectively. On-line 2D-LC-MS at the intact protein level was used to identify the root causes, and it was found that the acidic peak P2 and the basic peak P6 were due to the glutathionylation of cysteine and succinimidation of aspartic acid, respectively, which were not found in off-line 2D-LC-MS because of the loss occurring during pre-treatment. These results suggest that process development could focus on cell culture for adjustment of glutathionylation. In this paper, we propose the concept of precision process development based on on-line 2D-LC-MS, which could quickly offer useful data with only 0.6 mg mAb within 6 h for precise instructions for process development. Overall, the combination of on-line and off-line 2D-LC-MS can characterize mAb charge variants more comprehensively, precisely, and quickly than other approaches. This is a very effective platform with routine operations that provides precise instructions for process development within hours, and will help to accelerate the development of innovative therapeutics. [ABSTRACT FROM AUTHOR]

Cell culture‐conditioned medium (CCM) is a valuable source of extracellular vesicles (EVs) for basic scientific, therapeutic and diagnostic applications. Cell culturing parameters affect the biochemical composition, release and possibly the function of CCM‐derived EVs (CCM‐EV). The CCM‐EV task force of the Rigor and Standardization Subcommittee of the International Society for Extracellular Vesicles aims to identify relevant cell culturing parameters, describe their effects based on current knowledge, recommend reporting parameters and identify outstanding questions. While some recommendations are valid for all cell types, cell‐specific recommendations may need to be established for non‐mammalian sources, such as bacteria, yeast and plant cells. Current progress towards these goals is summarized in this perspective paper, along with a checklist to facilitate transparent reporting of cell culturing parameters to improve the reproducibility of CCM‐EV research. [ABSTRACT FROM AUTHOR]

Cancers of digestive tract such as colorectal cancer (CRC) and gastric cancer (GC) are the most commonly detected types of cancer worldwide and their origin can be associated with oxidative stress conditions. Commonly known and followed antioxidants, such as vitamin C and E, are widely considered as potential anti-cancer agents. Raman spectra have great potential in the biochemical characterization of matter based on the fact that each molecule has its own unique vibrational properties. Raman spectroscopy allows to precisely characterize components (proteins, lipids, nucleic acids). The paper presents the application of the Raman spectroscopy technique for the analysis of tissue samples and cells of the human colon and stomach. The main goal of this study is to show the differences between healthy and cancerous tissues from the human digestive tract and human normal and cancer colon and gastric cell lines. The paper presents the spectroscopic characterization of normal colon cells, CCD-18 Co, in physiological and oxidative conditions and effect of oxidative injury of normal colon cells upon supplementation with vitamin C at various concentrations based on Raman spectra. The obtained results were related to the Raman spectra recorded for human colon cancer cells—CaCo-2. In addition, the effect of the antioxidant in the form of vitamin E on gastric cancer cells, HTB-135, is presented and compared with normal gastric cells—CRL-7869. All measured gastric samples were biochemically and structurally characterized by means of Raman spectroscopy and imaging. Statistically assisted analysis has shown that normal, ROS injured and cancerous human gastrointestinal cells can be distinguished based on their unique vibrational properties. ANOVA tests, PCA (Principal Component Analysis) and PLSDA (Partial Least Squares Discriminant Analysis) have confirmed the main role of nucleic acids, proteins and lipids in differentiation of human colon and stomach normal and cancer tissues and cells. The conducted research based on Raman spectra proved that antioxidants in the form of vitamin C and E exhibit anti-cancer properties. In consequence, conducted studies proved that label-free Raman spectroscopy may play an important role in clinical diagnostic differentiation of human normal and cancerous gastrointestinal tissues and may be a source of intraoperative information supporting histopathological analysis. [ABSTRACT FROM AUTHOR]

This document is a summary of a special issue of the International Journal of Molecular Sciences titled "Drug Repurposing for Cancer Therapies." The issue focuses on the use of approved or investigational drugs for cancer treatment, outside of their original clinical indication. The research papers in the issue highlight specific repurposed drugs that have shown efficacy against various types of cancer. Examples include the use of amodiaquine for breast cancer, disulfiram for thyroid carcinoma, pirfenidone for non-small-cell lung cancer, tramadol for endometrial cancer, and a combination therapy targeting polo-like kinase 1 and ATP citrate synthase for cancer patients with higher expression levels of these proteins. The issue also includes a review article on the advantages of studying repurposed drugs for personalized breast cancer treatment. Overall, the special issue provides insights into the potential of repurposed drugs for cancer therapy, both as single agents and in combination therapies. [Extracted from the article]

This paper aims to review the evidence of the cellular activity on titanium samples exposed to Plasma of Argon (PoA) treatment. A systematic review was carried out based on the PRISMA statement by searching the Cochrane Library, PubMed, Web of Science, EMBASE and Scopus, up to October 2020. Papers were selected according to PICOS format that is: Population (P): osteoblasts, fibroblasts, gingival cells; Intervention (I): PoA disinfection treatment; Comparison (C): untreated controls; Outcome (O): cell culture; Setting (S): in vitro assays. The quality assessment was performed according to the CRIS Guidelines (Checklist for Reporting In vitro Studies). A total of 661 articles were found, of which 16 were included. The quality assessment revealed an overall poor quality of the studies analyzed. In vitro studies on the potential of PoA showed a potential effect in promoting higher cell adhesion and protein adsorption in the earliest times (hours). This outcome was not so evident when later stages of cell growth on the surfaces were tested and compared to the control groups. Only one study was conducted in vivo on a human sample regarding abutment cleaning. No meta-analysis was conducted because of the variety of experimental settings, mixed methods and different cell lines studied. PoA seems to be effective in promoting cell adhesion and protein adsorption. The duration of this effect remains unclear. Further evidence is required to demonstrate the long-term efficacy of the treatment and to support the use of PoA treatment in clinical practice. [ABSTRACT FROM AUTHOR]

Simple Summary: In addition to millions of normal red and white blood cells, the blood of cancer patients contains a variety of very rare cell types associated with the tumor. A simple blood draw or 'liquid biopsy' can, thus, provide important information about the state of the disease no matter where it exists in the body. In this paper, we describe a method for imaging all rare cells in a blood draw and the means to define each cell type using a combination of genomic, proteomic, and morphological measurements to distinguish tumor cells from mesenchymal and endothelial cells that are present in the tumor microenvironment. We compare the results of this assay among multiple prostate and breast cancer patients and show that this cellular profiling method is valid across patients and cancer types and may provide important biomarkers for assessing disease in real time. Bi-directional crosstalk between the tumor and the tumor microenvironment (TME) has been shown to increase the rate of tumor evolution and to play a key role in neoplastic progression, therapeutic resistance, and a patient's overall survival. Here, we set out to use a comprehensive liquid-biopsy analysis to study cancer and specific TME cells in circulation and their association with disease status. Cytokeratin+, CD45- circulating rare cells (CRCs) from nine breast and four prostate cancer patients were characterized through morphometrics, single-cell copy number analysis, and targeted multiplexed proteomics to delineate cancer cell lineage from other rare cells originating in the TME. We show that we can detect epithelial circulating tumor cells (EPI.CTC), CTCs undergoing epithelial-to-mesenchymal transition (EMT.CTC) and circulating endothelial cells (CECs) using a universal rare event detection platform (HDSCA). Longitudinal analysis of an index patient finds that CTCs are present at the time of disease progression, while CECs are predominately present at the time of stable disease. In a small cohort of prostate and breast cancer patients, we find high inter-patient and temporal intra-patient variability in the expression of tissue specific markers such as ER, HER2, AR, PSA and PSMA and EpCAM. Our study stresses the importance of the multi-omic characterization of circulating rare cells in patients with breast and prostate carcinomas, specifically highlighting overlapping and cell type defining proteo-genomic characteristics of CTCs and CECs. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T cell therapy has emerged as a promising modality for anti-cancer treatment. Its efficacy is quite remarkable in hematological tumors. Owing to their excellent clinical results, gene- modified cell therapies, including T cells, natural killer (NK) cells, and macrophages, are being actively studied in both academia and industry. However, the protocol to make CAR immune cells is too complicated, so it is still unclear how to efficiently produce the potent CAR immune cells. To manufacture effective CAR immune cells, we need to be aware of not only how to obtain highly infective viral particles, but also how to transduce CAR genes into immune cells. In this paper, we provide detailed information on spinoculation, which is one of the best known protocols to transduce genes into immune cells, in a methodological view. Our data indicate that gene transduction is significantly dependent on speed and duration of centrifugation, concentration and number of viral particles, the concentration of polybrene, and number of infected immune cells. In addition, we investigated on the optimal polyethylene glycol (PEG) solution to concentrate the viral supernatant and the optimized DNA ratios transfected into 293T cells to produce high titer of viral particles. This study provides useful information for practical production of the gene-modified immune cells using viral vectors. [ABSTRACT FROM AUTHOR]

Cannabidiol (CBD) has previously been shown to inhibit inflammatory cytokine production in both in vitro and in vivo studies of neurodegenerative diseases. To date, the CBD treatment of these diseases by quantitative targeting directly to the brain is one of the greatest challenges. In this paper, we present a new particulate system capable of delivering CBD into the brain via the intranasal route. Intranasal administration of CBD-loaded starch nanoparticles resulted in higher levels of cannabidiol in the brain compared to an identically administered cannabidiol solution. The production and the characterization of starch-based nanoparticles was reported, as well as the evaluation of their penetration and anti-inflammatory activity in cells. Cannabidiol-loaded starch nanoparticles were prepared by crosslinking with divanillin, using the nanoprecipitation method. Evaluation of the anti-inflammatory activity in vitro was performed using the BV2 microglia cell line. The starch nanoparticles appeared under electron microscopy in clusters sized approximately 200 nm in diameter. In cultures of lipopolysaccharide-induced inflamed BV2 cells, the cannabidiol-loaded starch nanoparticles demonstrated low toxicity while effectively reducing nitric oxide production and IL-6 levels. The anti-inflammatory effect was comparable to that of a glucocorticoid. Starch-based nanoparticle formulations combined with intranasal administration may provide a suitable platform for efficacious cannabidiol delivery and activity in the central nervous system. [ABSTRACT FROM AUTHOR]

This paper presents an innovative experimental setup that employs the principles of audio technology to subject adherent cells to rhythmic vertical vibrations. We employ a novel approach that combines three-axis acceleration measurements and particle tracking velocimetry to evaluate the setup's performance. This allows us to estimate crucial parameters such as root mean square acceleration, fluid flow patterns, and shear stress generated within the cell culture wells when subjected to various vibration types. The experimental conditions consisted of four vibrational modes: No Vibration, Continuous Vibration, Regular Pulse, and Variable Pulse. To evaluate the effects on cells, we utilized fluorescence microscopy and a customized feature extraction algorithm to analyze the F-actin filament structures. Our findings indicate a consistent trend across all vibrated cell cultures, revealing a reduction in size and altered orientation (2D angle) of the filaments. Furthermore, we observed cell accumulations in the G1 cell cycle phase in cells treated with Continuous Vibration and Regular Pulse. Our results demonstrate a negative correlation between the magnitude of mechanical stimuli and the size of F-actin filaments, as well as a positive correlation with the accumulations of cells in the G1 phase of the cell cycle. By unraveling these analyses, this study paves the way for future investigations and provides a compelling framework for comprehending the intricate cellular responses to rhythmic mechanical stimulation. [ABSTRACT FROM AUTHOR]

Simple Summary: Neuroblastoma is a pediatric tumor originating from the precursors of sympathetic nerves. The disease is known for its high heterogeneity. Hence, developing adequate preclinical models reflecting the complex biology of neuroblastoma is particularly challenging. This paper describes the current status of the available neuroblastoma models with their strengths and limitations, and demonstrates the future perspectives for preclinical neuroblastoma research. Preclinical in vitro and in vivo models remain indispensable tools in cancer research. These classic models, including two- and three-dimensional cell culture techniques and animal models, are crucial for basic and translational studies. However, each model has its own limitations and typically does not fully recapitulate the course of the human disease. Therefore, there is an urgent need for the development of novel, advanced systems that can allow for efficient evaluation of the mechanisms underlying cancer development and progression, more accurately reflect the disease pathophysiology and complexity, and effectively inform therapeutic decisions for patients. Preclinical models are especially important for rare cancers, such as neuroblastoma, where the availability of patient-derived specimens that could be used for potential therapy evaluation and screening is limited. Neuroblastoma modeling is further complicated by the disease heterogeneity. In this review, we present the current status of preclinical models for neuroblastoma research, discuss their development and characteristics emphasizing strengths and limitations, and describe the necessity of the development of novel, more advanced and clinically relevant approaches. [ABSTRACT FROM AUTHOR]

Simple Summary: When prostate cancer cells spread to other parts of the body (metastasise), they frequently migrate to the bone. Here they encounter a microenvironment that differs greatly from that in the primary tumour (prostate), namely interactions with bone-specific cells, including fibroblasts. To better understand the contribution of these fibroblasts to tumour progression, it is the aim of the current paper to propagate a biologically relevant fibroblast population that mimics the cellular and molecular profile of fibroblasts as found in the metastatic bone environment in the body. Using three-dimensional (3D) cell culture models, we were able to transform normal fibroblast cells into fibroblasts that express modified protein and gene profiles similar to those found in fibroblasts in the metastatic bone environment in the body. The exploitation of these engineered 3D models could help further unravel the novel biology regulating metastatic growth and the role fibroblasts play in the colonisation process. Elucidation of these processes has the potential to aid in the development of therapies not currently available for late-stage cancers. A dynamic interplay between prostate cancer (PCa) cells and reactive bone stroma modulates the growth of metastases within the bone microenvironment. Of the stromal cells, metastasis-associated fibroblasts (MAFs) are known to contribute but are the least studied cell type in PCa tumour progression. It is the aim of the current study to establish a biologically relevant 3D in vitro model that mimics the cellular and molecular profiles of MAFs found in vivo. Using 3D in vitro cell culture models, the bone-derived fibroblast cell line, HS-5, was treated with conditioned media from metastatic-derived PCa cell lines, PC3 and MDA-PCa 2b, or mouse-derived fibroblasts 3T3. Two corresponding reactive cell lines were propagated: HS5-PC3 and HS5-MDA, and evaluated for alterations in morphology, phenotype, cellular behaviour, plus protein and genomic profiles. HS5-PC3 and HS5-MDA displayed distinct alterations in expression levels of N-Cadherin, non-functional E-Cadherin, alpha-smooth muscle actin (α-SMA), Tenascin C, and vimentin, along with transforming growth factor receptor expression (TGF β R1 and R2), consistent with subpopulations of MAFs reported in vivo. Transcriptomic analysis revealed a reversion of HS5-PC3 towards a metastatic phenotype with an upregulation in pathways known to regulate cancer invasion, proliferation, and angiogenesis. The exploitation of these engineered 3D models could help further unravel the novel biology regulating metastatic growth and the role fibroblasts play in the colonisation process. [ABSTRACT FROM AUTHOR]

In recent decades, significant progress has been made in liver tissue engineering through the use of 3D bioprinting technology. This technology offers the ability to create personalized biological structures with precise geometric design capabilities. The complex and multifaceted nature of liver diseases underscores the need for advanced technologies to accurately mimic the physiological and mechanical characteristics, as well as organ-level functions, of liver tissue in vitro. Bioprinting stands out as a superior option over traditional two-dimensional cell culture models and animal models due to its stronger biomimetic advantages. Through the use of bioprinting, it is possible to create liver tissue with a level of structural and functional complexity that more closely resembles the real organ, allowing for more accurate disease modeling and drug testing. As a result, it is a promising tool for restoring and replacing damaged tissue and organs in the field of liver tissue engineering and drug research. This article aims to present a comprehensive overview of the progress made in liver tissue engineering using bioprinting technology to provide valuable insights for researchers. The paper provides a detailed account of the history of liver tissue engineering, highlights the current 3D bioprinting methods and bioinks that are widely used, and accentuates the importance of existing in vitro liver tissue models based on 3D bioprinting and their biomedical applications. Additionally, the article explores the challenges faced by 3D bioprinting and predicts future trends in the field. The progress of 3D bioprinting technology is poised to bring new approaches to printing liver tissue in vitro, while offering powerful tools for drug development, testing, liver disease modeling, transplantation, and regeneration, which hold great academic and practical significance. [ABSTRACT FROM AUTHOR]

The purpose of this paper was to discuss the achievements of in vitro modeling in terms of the blood–brain barrier [BBB] and to create a clear overview of this research area, which is useful in research planning. The text was divided into three main parts. The first part describes the BBB as a functional structure, its constitution, cellular and noncellular components, mechanisms of functioning and importance for the central nervous system, in terms of both protection and nourishment. The second part is an overview of parameters important in terms of establishing and maintaining a barrier phenotype that allows for formulating criteria of evaluation of the BBB in vitro models. The third and last part discusses certain techniques for developing the BBB in vitro models. It describes subsequent research approaches and models, as they underwent change alongside technological advancement. On the one hand, we discuss possibilities and limitations of different research approaches: primary cultures vs. cell lines and monocultures vs. multicultures. On the other hand, we review advantages and disadvantages of specific models, such as models-on-a-chip, 3D models or microfluidic models. We not only attempt to state the usefulness of specific models in different kinds of research on the BBB but also emphasize the significance of this area of research for advancement of neuroscience and the pharmaceutical industry. [ABSTRACT FROM AUTHOR]

Breast cancer is a highly harmful malignant tumor, which poses a great threat to women's body and mind, and the mortality rate ranks second among all women's diseases. The incidence rate accounts for 7-10% of various malignant tumors in the whole body, second only to uterine cancer in women, and has become the main cause of threatening women's health. Advanced breast cancer is often considered an incurable disease. The family of heterogeneous nuclear ribonucleoprotein complexes is composed of about 20 hnRNP proteins with molecular weights ranging from 32 to 120 kDa, and they are named according to their molecular weights. Among them, hnRNPA2 and hnRNPB1 are the two most important members of the hnRNP family, both derived from the same gene on chromosome 7p15. Therefore, research to understand the molecular mechanism and process of breast cancer progression has an important role in promoting the current medical research on breast cancer treatment methods. Therefore, studying the mechanism of tumorigenesis is the key to tumor prevention and treatment. Therefore, this paper proposes that A2/B1 promotes the stability of NRF2 mRNA and inhibits ferroptosis and cell proliferation in breast cancer cells. The article mainly introduces the disease diagnosis method based on artificial neural network and its neural network algorithm. In the experimental part, the activity of hnRNP A2/B1 on cancer cells is deeply studied. The results show that the absorbance of the MTT method increases continuously with the extension of the culture time, and the maximum reaches 1.2. This fully shows that its absorption capacity is very strong, especially after 24 hours, the absorption rate rises from 0.6 to 0.9, which shows that 24 hours is the best absorption time. And it can also be found that hnRNPA2/B1 has a significant inhibitory effect on breast cancer cells; it can reduce the effect on breast cancer cell cycle and apoptosis. [ABSTRACT FROM AUTHOR]

This paper presents an analysis of the regulation activity of the partially purified preparations of cellular aconitate hydratase (AH) on the yeast Yarrowia lipolytica cultivated at extreme pH. As a result of purification, enzyme preparations were obtained from cells grown on media at pH 4.0, 5.5, and 9.0, purified by 48-, 46-, and 51-fold and having a specific activity of 0.43, 0.55 and 0.36 E/mg protein, respectively. The kinetic parameters of preparations from cells cultured at extreme pH demonstrated: (1) an increase in the affinity for citrate and isocitrate; and (2) a shift in the pH optima to the acidic and alkaline side in accordance with the modulation of the medium pH. The regulatory properties of the enzyme from cells subjected to alkaline stress showed increased sensitivity to Fe2+ ions and high peroxide resistance. Reduced glutathione (GSH) stimulated AH, while oxidized glutathione (GSSG) inhibited AH. A more pronounced effect of both GSH and GSSG was noted for the enzyme obtained from cells grown at pH 5.5. The data obtained provide new approaches to the use of Y. lipolytica as a model of eukaryotic cells demonstrating the development of a stress-induced pathology and to conducting a detailed analysis of enzymatic activity for its correction. [ABSTRACT FROM AUTHOR]

Despite recent advances in the mathematical modeling of biological processes and real-world situations raised in the day-to-day life phase, some phenomena such as immune cell populations remain poorly understood. The mathematical modeling of complex phenomena such as immune cell populations using nonlinear differential equations seems to be a quite promising and appropriate tool to model such complex and nonlinear phenomena. Fractional differential equations have recently gained a significant deal of attention and demonstrated their relevance in modeling real phenomena rather than their counterpart, classical (integer) derivative differential equations. We report in this paper a mathematical approach susceptible to answering some relevant questions regarding the side effects of ionizing radiation (IR) on DNA with a particular focus on double-strand breaks (DSBs), leading to the destruction of the cell population. A theoretical elucidation of the population memory was carried out within the framework of fractional differential equations (FODEs). Using FODEs, the mathematical approach presented herein ensures connections between fractional calculus and the nonlocal feature of the fractional order of immune cell populations by taking into account the memory trace and genetic qualities that are capable of integrating all previous actions and considering the system's long-term history. An illustration of both fractional modeling, which provides an excellent framework for the description of memory and hereditary properties of immune cell populations, is elucidated. The mathematics presented in this research hold promise for modeling real-life phenomena and paves the way for obtaining accurate model parameters resulting from the mathematical modeling. Finally, the numerical simulations are conducted for the analytical approach presented herein to elucidate the effect of various parameters that govern the influence of ionizing irradiation on DNA in immune cell populations as well as the evolution of cell population dynamics, and the results are presented using plots and contrasted with previous theoretical findings. [ABSTRACT FROM AUTHOR]

The development of electrospun nanofibers based on cellulose and its derivatives is an inalienable task of modern materials science branches related to biomedical engineering. The considerable compatibility with multiple cell lines and capability to form unaligned nanofibrous frameworks help reproduce the properties of natural extracellular matrix and ensure scaffold applications as cell carriers promoting substantial cell adhesion, growth, and proliferation. In this paper, we are focusing on the structural features of cellulose itself and electrospun cellulosic fibers, including fiber diameter, spacing, and alignment responsible for facilitated cell capture. The study emphasizes the role of the most frequently discussed cellulose derivatives (cellulose acetate, carboxymethylcellulose, hydroxypropyl cellulose, etc.) and composites in scaffolding and cell culturing. The key issues of the electrospinning technique in scaffold design and insufficient micromechanics assessment are discussed. Based on recent studies aiming at the fabrication of artificial 2D and 3D nanofiber matrices, the current research provides the applicability assessment of the scaffolds toward osteoblasts (hFOB line), fibroblastic (NIH/3T3, HDF, HFF-1, L929 lines), endothelial (HUVEC line), and several other cell types. Furthermore, a critical aspect of cell adhesion through the adsorption of proteins on the surfaces is touched upon. [ABSTRACT FROM AUTHOR]

(1) Background: Obesity, a complex metabolic disease resulting from an imbalance between food consumption and energy expenditure, leads to an increase in adipocytes and chronic inflammatory conditions. The aim of this paper was to synthesize a small series of carvacrol derivatives (CD1-3) that are able to reduce both adipogenesis and the inflammatory status often associated with the progression of the obesity disease. (2) Methods: The synthesis of CD1-3 was performed using classical procedures in a solution phase. Biological studies were performed on three cell lines: 3T3-L1, WJ-MSCs, and THP-1. The anti-adipogenic properties of CD1-3 were evaluated using western blotting and densitometric analysis by assessing the expression of obesity-related proteins, such as ChREBP. The anti-inflammatory effect was estimated by measuring the reduction in TNF-α expression in CD1-3-treated THP-1 cells. (3) Results: CD1-3—obtained through a direct linkage between the carboxylic moiety of anti-inflammatory drugs (Ibuprofen, Flurbiprofen, and Naproxen) and the hydroxyl group of carvacrol—have an inhibitory effect on the accumulation of lipids in both 3T3-L1 and WJ-MSCs cell cultures and an anti-inflammatory effect by reducing TNF- α levels in THP-1 cells. (4) Conclusions: Considering the physicochemical properties, stability, and biological data, the CD3 derivative—obtained by a direct linkage between carvacrol and naproxen—resulted in the best candidate, displaying anti-obesity and anti-inflammatory effects in vitro. [ABSTRACT FROM AUTHOR]

Cardiomyocyte alignment in myocardium tissue plays a significant role in the physiological, electrical, and mechanical functions of the myocardium. It remains, however, difficult to align cardiac cells in a 3D in vitro heart model. This paper proposes a simple method to align cells using microfabricated Polydimethylsiloxane (PDMS) grooves with large dimensions (of up to 350 µm in width), similar to the dimensions of trabeculae carneae, the smallest functional unit of the myocardium. Two cell groups were used in this work; first, H9c2 cells in combination with Nor10 cells for proof of concept, and second, neonatal cardiac cells to investigate the functionality of the 3D model. This model compared the patterned and nonpatterned 3D constructs, as well as the 2D cell cultures, with and without patterns. In addition to alignment, we assessed the functionality of our proposed 3D model by comparing beating rates between aligned and non-aligned structures. In order to assess the practicality of the model, the 3D aligned structures should be demonstrated to be detachable and alignable. This evaluation is crucial to the use of this 3D functional model in future studies related to drug screening, building blocks for tissue engineering, and as a heart-on-chip by integrating microfluidics. [ABSTRACT FROM AUTHOR]

The off-target effects of light-activated or targeted liposomes are difficult to distinguish in traditional well plate experiments. Additionally, the absence of fluid flow in traditional cell models can lead to overestimation of nanoparticle uptake. In this paper, we established a perfusion cell culture platform to study light-activated liposomes and determined the effect of flow on the liposomal cell uptake. The optimal cell culturing parameters for the A549 cells under flow conditions were determined by monitoring cell viability. To determine optimal liposome treatment times, particle uptake was measured with flow cytometry. The suitability of commercial QuasiVivo flow-chambers for near-infrared light activation was assessed with a calcein release study. The chamber material did not hinder the light activation and subsequent calcein release from the liposomes. Furthermore, our results show that the standard cell culturing techniques are not directly translatable to flow cultures. For non-coated liposomes, the uptake was hindered by flow. Interestingly, hyaluronic acid coating diminished the uptake differences between the flow and static conditions. The study demonstrates that flow affects the liposomal uptake by lung cancer cell line A549. The flow also complicates the cell attachment of A549 cells. Moreover, we show that the QuasiVivo platform is suitable for light-activation studies. [ABSTRACT FROM AUTHOR]

Chemotherapy is the most used method after surgery in the treatment of colon cancer. Cancer cells escape the recognition mechanism of immune system cells to survive and develop chemoresistance. Therefore, the use of immunotherapy in combination with chemotherapy can increase the effectiveness of the treatment. Nanoparticles have been used clinically to increase the accumulation of therapeutics in target tissues and reduce toxicity. In this paper, nanoplexes were formed via cationic cyclodextrin polymer, 5-Fluorouracil, and Interleukin-2 based on the opposite charge interaction of macromolecules without undergoing any structural changes or losing the biological activity of Interleukin-2. Anticancer activities of nanoplexes were determined in two-dimensional and three-dimensional cell culture setups. The dual drug-loaded cyclodextrin nanoplexes diffused deeper into the spheroids and accelerated apoptosis when compared with 5-FU solutions. In the colorectal tumor-bearing animal model, survival rate, antitumor activity, metastasis, and immune response parameters were assessed using a cyclodextrin derivative, which was found to be safe based on the ALT/AST levels in healthy mice. Histomorphometric analysis showed that the groups treated with the nanoplex formulation had significantly fewer initial tumors and lung foci when compared with the control. The dual drug-loaded nanoplex could be a promising drug delivery technique in the immunochemotherapy of colorectal cancer. [ABSTRACT FROM AUTHOR]

Ginger and its derivatives have been shown to be effective in the prevention and treatment of cancer. We undertook a systematic review to answer the question of whether ginger has a role in modifying the biomarkers of cancer in cell culture conditions and on colorectal cancer in randomized clinical trials. We performed a comprehensive search of the literature from Scopus, Embase, Web of Science, PubMed, Cochrane central register of controlled trials, and Cochrane database of systematic reviews. At first, all 12 papers studied the effect of ginger or its derivatives on cell culture conditions. The results of cell culture studies show that ginger has a powerful role in inducing apoptosis. In the second part, five studies of clinical trials were analyzed. By analyzing antitumor markers of clinical trials, ginger increased some anticancer markers but performed poorly in inducing some anticancer markers. This systematic review showed that the consumption of ginger extract has the potential to prevent and treat colorectal cancer but this ability is weak. [ABSTRACT FROM AUTHOR]

Simple Summary: The compounds named PROTACs are formed by two fragments, which bring together a particular protein with a ubiquitinating enzyme. This allows ubiquitination and degradation of the targeted protein. Ubiquitination-mediated protein degradation is an important regulatory process to control the expression levels of proteins and maintain the homeostatic conditions in cells. Thus, by re-directing a mechanism that is normally used for cell regulation, PROTACs allow to remove specific proteins associated to particular diseases or pathological conditions. In this paper, we report a type of PROTAC that targets for degradation the protein named p38α, whose functions have been linked to cancer and other diseases. We show that these PROTACs effectively reduce p38α protein expression not only in several cancer cell lines but also in tumors generated in mice. These compounds may provide an attractive strategy to evaluate potential therapeutic applications for targeting p38α in the clinical context. We report a series of small molecule proteolysis-targeting chimeras (PROTACs) that target the protein kinase p38α for degradation. These PROTACs are based on a ligand of the VHL E3 ubiquitin ligase, which is linked to an ATP competitive inhibitor of p38α. We provide evidence that these compounds can induce the specific degradation of p38α, but not p38β and other related kinases, at nanomolar concentrations in several mammalian cell lines. We also show that the p38α-specific PROTACs are soluble in aqueous solutions and therefore suitable for their administration to mice. Systemic administration of the PROTACs induces p38α degradation only in the liver, probably due to the PROTAC becoming inactivated in that organ, but upon local administration the PROTACs induce p38α degradation in mammary tumors. Our compounds provide an alternative to traditional chemical inhibitors for targeting p38α signaling in cultured cells and in vivo. [ABSTRACT FROM AUTHOR]

This paper reports perfusion culture of human umbilical vein endothelial cells (HUVECs) on a microporous membrane in a pressure-driven microphysiological system (PD-MPS), which we developed previously as a multi-throughput perfusion culture platform. We designed fluidic culture unit with microporous membrane to culture HUVECs under fluidic shear stress and constructed a perfusion culture model in the PD-MPS platform. Four fluidic culture units were arranged in the microplate-sized device, which enables four-throughput assay for characterization of HUVECs under flow. Medium flow was generated above and below the membrane by sequential pneumatic pressure to apply physiological shear stress to HUVECs. HUVECs exhibited aligned morphology to the direction of the flow with shear stress of 11.5–17.7 dyn/cm2 under the flow condition, while they randomly aligned under static culture condition in a 6 well plate. We also observed 3.3- and 5.0-fold increase in the expression levels of the thrombomodulin and endothelial nitric oxide synthase mRNAs, respectively, under the flow condition in the PD-MPS compared to the static culture in 6 well plate. We also observed actin filament aligned to the direction of flow in HUVECs cultured under the flow condition. [Display omitted] [ABSTRACT FROM AUTHOR]

Cancer tissue engineering using three-dimensional (3D) cell culture scaffolds has been reported to handle the limitations of two-dimensional (2D) cell culture in vitro and in vivo cancer models. In this paper, the scaffolds were produced via the freeze-drying technique and chemical cross-linking. 1-ethyl-3(3-dimethylamino propyl) carbodiimide hydrochloride (EDC) has been used as a crosslinker for conjugation between gelatin and silk fibroin (SF). The gelatin-fibroin (GF) scaffold evaluated the physicochemical characteristics such as microstructure, functional groups, swelling rate, degradability as well as some biological characteristics including in vitro cytotoxicity, for physicochemical characteristics such as microstructure, functional groups, swelling rate, degradability, and some biological characteristics including in vitro cytotoxicity and cell adhesion, cell proliferation and the spheroid forming capacity of MCF-7 cells on this scaffold. The results showed that the GF scaffold with the uniform pore size demonstrated suitable physicochemical properties that controlled swelling and degradation characteristics. The GF scaffold had no toxicity on human fibroblast cells (hF), and supported MCF-7 cells' adhesion, proliferation, and spheroid formation. In conclusion, this study successfully considered the potential of the GF scaffold as an in vitro 3D cell-culture scaffold in cancer tissue engineering in particular and tissue engineering in general. [ABSTRACT FROM AUTHOR]