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Abstract The vasculature of the brain consists of specialized endothelial cells that form a blood-brain barrier (BBB). This barrier, in conjunction with supporting cell types, forms the neurovascular unit (NVU). The NVU restricts the passage of certain substances from the bloodstream while selectively permitting essential nutrients and molecules to enter the brain. This protective role is crucial for optimal brain function, but presents a significant obstacle in treating neurological conditions, necessitating chemical modifications or advanced drug delivery methods for most drugs to cross the NVU. A deeper understanding of NVU in health and disease will aid in the identification of new therapeutic targets and drug delivery strategies for improved treatment of neurological disorders. To achieve this goal, we need models that reflect the human BBB and NVU in health and disease. Although animal models of the brain’s vasculature have proven valuable, they are often of limited translational relevance due to interspecies differences or inability to faithfully mimic human disease conditions. For this reason, human in vitro models are essential to improve our understanding of the brain’s vasculature under healthy and diseased conditions. This review delves into the advancements in in vitro modeling of the BBB and NVU, with a particular focus on microfluidic models. After providing a historical overview of the field, we shift our focus to recent developments, offering insights into the latest achievements and their associated constraints. We briefly examine the importance of chip materials and methods to facilitate fluid flow, emphasizing their critical roles in achieving the necessary throughput for the integration of microfluidic models into routine experimentation. Subsequently, we highlight the recent strides made in enhancing the biological complexity of microfluidic NVU models and propose recommendations for elevating the biological relevance of future iterations. Importantly, the NVU is an intricate structure and it is improbable that any model will fully encompass all its aspects. Fit-for-purpose models offer a valuable compromise between physiological relevance and ease-of-use and hold the future of NVU modeling: as simple as possible, as complex as needed.

Abstract Background Fragmentomics, the investigation of fragmentation patterns of cell-free DNA (cfDNA), has emerged as a promising strategy for the early detection of multiple cancers in the field of liquid biopsy. However, the clinical application of this approach has been hindered by a limited understanding of cfDNA biology. Furthermore, the prevalence of hematopoietic cell-derived cfDNA in plasma complicates the in vivo investigation of tissue-specific cfDNA other than that of hematopoietic origin. While conventional two-dimensional cell lines have contributed to research on cfDNA biology, their limited representation of in vivo tissue contexts underscores the need for more robust models. In this study, we propose three-dimensional organoids as a novel in vitro model for studying cfDNA biology, focusing on multifaceted fragmentomic analyses. Results We established nine patient-derived organoid lines from normal lung airway, normal gastric, and gastric cancer tissues. We then extracted cfDNA from the culture medium of these organoids in both proliferative and apoptotic states. Using whole-genome sequencing data from cfDNA, we analyzed various fragmentomic features, including fragment size, footprints, end motifs, and repeat types at the end. The distribution of cfDNA fragment sizes in organoids, especially in apoptosis samples, was similar to that found in plasma, implying occupancy by mononucleosomes. The footprints determined by sequencing depth exhibited distinct patterns depending on fragment sizes, reflecting occupancy by a variety of DNA-binding proteins. Notably, we discovered that short fragments (

BackgroundType 1 diabetes (T1D) is an autoimmune disease characterized by impaired immune tolerance to β-cell antigens and progressive destruction of insulin-producing β-cells. Animal models have provided valuable insights for understanding the etiology and pathogenesis of this disease, but they fall short of reflecting the extensive heterogeneity of the disease in humans, which is contributed by various combinations of risk gene alleles and unique environmental factors. Collectively, these factors have been used to define subgroups of patients, termed endotypes, with distinct predominating disease characteristics.Scope of reviewHere, we review the gaps filled by these models in understanding the intricate involvement and regulation of the immune system in human T1D pathogenesis. We describe the various models developed so far and the scientific questions that have been addressed using them. Finally, we discuss the limitations of these models, primarily ascribed to hosting a human immune system (HIS) in a xenogeneic recipient, and what remains to be done to improve their physiological relevance.Major conclusionsTo understand the role of genetic and environmental factors or evaluate immune-modifying therapies in humans, it is critical to develop and apply models in which human cells can be manipulated and their functions studied under conditions that recapitulate as closely as possible the physiological conditions of the human body. While microphysiological systems and living tissue slices provide some of these conditions, HIS mice enable more extensive analyses using in vivo systems.

Peripheral Nerve Injuries (PNI) frequently occur secondary to traumatic injuries. Recovery from these injuries can be expectedly poor, especially in proximal injuries. In order to study and improve peripheral nerve regeneration, scientists rely on peripheral nerve models to identify and test therapeutic interventions. In this review, we discuss the best described and most commonly used peripheral nerve models that scientists have and continue to use to study peripheral nerve physiology and function.

Among all of the materials used in tissue engineering in order to develop bioequivalents, collagen shows to be the most promising due to its superb biocompatibility and biodegradability, thus becoming one of the most widely used materials for scaffold production. However, current imaging techniques of the cells within collagen scaffolds have several limitations, which lead to an urgent need for novel methods of visualization. In this work, we have obtained groups of collagen scaffolds and selected the contrasting agents in order to study pores and patterns of cell growth in a non-disruptive manner via X-ray computed microtomography (micro-CT). After the comparison of multiple contrast agents, a 3% aqueous phosphotungstic acid solution in distilled water was identified as the most effective amongst the media, requiring 24 h of incubation. The differences in intensity values between collagen fibers, pores, and masses of cells allow for the accurate segmentation needed for further analysis. Moreover, the presented protocol allows visualization of porous collagen scaffolds under aqueous conditions, which is crucial for the multimodal study of the native structure of samples.

Nephrotoxicity stands as one of the most limiting effects in the development and validation of new drugs. The kidney, among the organs evaluated in toxicity assessments, has a higher susceptibility, with nephrotoxic potential frequently evading detection until late in clinical trials. Traditional cell culture, which has been widely used for decades, does not recapitulate the structure and complexity of the native tissue, which can affect cell function, and the response to cytotoxins does not resemble what occurs in the kidney. In the current study, we aimed to address these challenges by creating in vitro kidney models that faithfully biomimic the dynamics of the renal proximal tubule, using the well-established RPTEC/TERT1 cell line. For doing so, two models were developed, one recreating tubule-like structures (2.5D model) and the other using microfluidic technology (kidney-on-a-chip). The 2.5D model allowed tubular structures to be generated in the absence of hydrogels, and the kidney-on-a-chip model allowed shear stress to be applied to the cell culture, which is a physiological stimulus in the renal tissue. After characterization of both models, different nephrotoxic compounds such as cisplatin, tacrolimus, and daunorubicin were used to study cell responses after treatment. The developed models in our study could be a valuable tool for pre-clinical nephrotoxic testing of drugs and new compounds.

Cancer-associated cachexia is a multifactorial syndrome characterised by systemic inflammation and hypermetabolism that affects different tissues and organs. Is characterised by progressive and irreversible weight loss, mainly due to skeletal muscle wasting and often accompanied by loss of fat mass. Due to its complexity, and lack of effective treatment, this syndrome is a sign of poor prognosis in cancer patients. Cellular models constitute a valuable and powerful tool offering insights into the molecular pathways and cellular responses associated with cancer cachexia. Currently, there are robust and widely used cell lines used to establish models to study the pathophysiology of muscle wasting and adipose tissue loss. Various methods can be used to induce the cachectic phenotype in the cells, utilising genetic engineering or different inducing agents such as hormones, inflammatory factors and chemotherapeutic drugs. The available experimental data on their metabolic properties and transcriptional and proteomic profiles allows the selection of the most suitable research model to replicate the relevant aspects of cachexia. In this review, we make an overview of the in vitro models used to study biological aspects of cancer-associated cachexia and analyse their strengths and limitations in replicating the complex physiological environment and pathological processes of the syndrome. Herein, we also briefly approach the difficulty of modelling the contribution of different organs and crosstalk between different tissues.

Pediatric high-grade gliomas (pHGG) are malignant and usually fatal central nervous system (CNS) WHO Grade 4 tumors. The majority of pHGG consist of diffuse midline gliomas (DMG), H3.3 or H3.1 K27 altered, or diffuse hemispheric gliomas (DHG) (H3.3 G34-mutant). Due to diffuse tumor infiltration of eloquent brain areas, especially for DMG, surgery has often been limited and chemotherapy has not been effective, leaving fractionated radiation to the involved field as the current standard of care. pHGG has only been classified as molecularly distinct from adult HGG since 2012 through Next-Generation sequencing approaches, which have shown pHGG to be epigenetically regulated and specific tumor sub-types to be representative of dysregulated differentiating cells. To translate discovery research into novel therapies, improved pre-clinical models that more adequately represent the tumor biology of pHGG are required. This review will summarize the molecular characteristics of different pHGG sub-types, with a specific focus on histone K27M mutations and the dysregulated gene expression profiles arising from these mutations. Current and emerging pre-clinical models for pHGG will be discussed, including commonly used patient-derived cell lines and in vivo modeling techniques, encompassing patient-derived xenograft murine models and genetically engineered mouse models (GEMMs). Lastly, emerging techniques to model CNS tumors within a human brain environment using brain organoids through co-culture will be explored. As models that more reliably represent pHGG continue to be developed, targetable biological and genetic vulnerabilities in the disease will be more rapidly identified, leading to better treatments and improved clinical outcomes.

Traumatic brain injury (TBI), which is characterized by damage to the brain resulting from a sudden traumatic event, is a major cause of death and disability worldwide. It has short- and long-term effects, including neuroinflammation, cognitive deficits, and depression. TBI consists of multiple steps that may sometimes have opposing effects or mechanisms, making it challenging to investigate and translate new knowledge into effective therapies. In order to better understand and address the underlying mechanisms of TBI, we have developed an in vitro platform that allows dynamic simulation of TBI conditions by applying external magnetic forces to induce acceleration and deceleration injury, which is often observed in human TBI. Endothelial and neuron-like cells were successfully grown on magnetic gels and applied to the platform. Both cell types showed an instant response to the TBI model, but the endothelial cells were able to recover quickly?in contrast to the neuron-like cells. In conclusion, the presented in vitro model mimics the mechanical processes of acceleration/deceleration injury involved in TBI and will be a valuable resource for further research on brain injury.

Abstract The blood–brain barrier (BBB) is a sophisticated structure whose full functionality is required for maintaining the executive functions of the central nervous system (CNS). Tight control of transport across the barrier means that most drugs, particularly large size, which includes powerful biologicals, cannot reach their targets in the brain. Notwithstanding the remarkable advances in characterizing the cellular nature of the BBB and consequences of BBB dysfunction in pathology (brain metastasis, neurological diseases), it remains challenging to deliver drugs to the CNS. Herein, we outline the basic architecture and key molecular constituents of the BBB. In addition, we review the current status of approaches that are being explored to temporarily open the BBB in order to allow accumulation of therapeutics in the CNS. Undoubtedly, the major concern in field is whether it is possible to open the BBB in a meaningful way without causing negative consequences. In this context, we have also listed few other important key considerations that can improve our understanding about the dynamics of the BBB.

Katharina Weßollek,1 Yvonne Marquardt,1 Sylvia Wagner-Schiffler,2 Jens Malte Baron,1,* Sebastian Huth1,* 1Department of Dermatology and Allergology, University Hospital RWTH Aachen, Aachen, Germany; 2Praxis für Dermatologie, Aachen, Germany*These authors contributed equally to this workCorrespondence: Katharina Weßollek, Department of Dermatology and Allergology, University Hospital RWTH Aachen, Aachen, Germany, Tel +49 241 7501 8240, Fax +49 241 7501 8290, Email kwessollek@ukaachen.dePurpose: In vitro study on the molecular effects of post-treatment after micro-needling applications with a dexpanthenol-containing ointment (DCO) using 3D skin models.Patients and Methods: In this in vitro study, full-thickness human 3D skin models were treated with a micro-needling device according to its clinical application. For post-treatment, some of the models were additionally treated with a dexpanthenol-containing ointment (DCO). Histological samples were taken at 0, 24 and 48 hours. Gene expression analysis was performed after 24 hours.Results: Histological examination showed that DCO post-treated 3D skin models revealed a completed wound closure 24 hours after the micro-needling procedure. In contrast, DCO-untreated models still clearly exhibited the micro-needling lesions after the same period of time. After 48 hours, all models revealed a completed wound healing. In skin models that received micro-needling but no post-treatment with DCO, microarray analysis identified an upregulation of proinflammatory cytokines and chemokines and a downregulation of skin barrier and differentiation markers. In contrast, post-treatment with DCO leads to accelerated wound healing without affecting the initial inflammatory response caused by micro-needling, which leads to the subsequent collagen expression. This data was supported by qRT-PCR analyses.Conclusion: Post-treatment with DCO accelerates epidermal wound healing after micro-needling of 3D skin models without impairing the immunostimulatory properties of micro-needling. These findings can help to optimise the aftercare routine after micro-needling procedures and to shorten the downtime for the patient after treatment.Keywords: in vitro models, cosmetic procedures, aftercare, skin needling, molecular effects, human organotypic skin equivalents

At present, personalised cellular immunotherapy is considered a promising approach to the treatment of malignant neoplasms. The effectiveness of these cellular immunotherapy methods is evaluated in the context of clinical and biological tumour characteristics and the state of the immune system of a particular patient. One of the immunotherapy options for cancer is the development of autologous dendritic cell vaccines.The aim of this study was to analyse current methodological approaches to the evaluation of the quality, efficacy, and safety of dendritic cell cancer vaccines.This review describes the functional role of dendritic cells in immune response regulation. The paper presents the results of literature analysis covering current approaches to obtaining dendritic cell vaccines with specific characteristics, quality assessment, studies of the anti-tumour efficacy of cell therapy products, and the experience of conducting non-clinical and clinical studies. The review highlights specific aspects of international experience in the registration and clinical use of cell therapy products. The authors discuss methodological approaches to non-clinical studies of dendritic cell vaccines, which should aim to obtain information to select the dose, route, and mode of administration and to identify immunological markers correlating to the clinical efficacy of cell therapy products. The paper covers international experience in conducting clinical trials of dendritic cell vaccines for various malignant neoplasms. The authors propose a list of quality attributes of human somatic cell-based medicinal products for further clinical use.

New drug discovery is under growing pressure to satisfy the demand from a wide range of domains, especially from the pharmaceutical industry and healthcare services. Assessment of drug efficacy and safety prior to human clinical trials is a crucial part of drug development, which deserves greater emphasis to reduce the cost and time in drug discovery. Recent advances in microfabrication and tissue engineering have given rise to organ-on-a-chip, an in vitro model capable of recapitulating human organ functions in vivo and providing insight into disease pathophysiology, which offers a potential alternative to animal models for more efficient pre-clinical screening of drug candidates. In this review, we first give a snapshot of general considerations for organ-on-a-chip device design. Then, we comprehensively review the recent advances in organ-on-a-chip for drug screening. Finally, we summarize some key challenges of the progress in this field and discuss future prospects of organ-on-a-chip development. Overall, this review highlights the new avenue that organ-on-a-chip opens for drug development, therapeutic innovation, and precision medicine.

Abstract Infectious diseases are increasingly recognized as a major threat worldwide due to the rise of antimicrobial resistance and the emergence of novel pathogens. In vitro models that can adequately mimic in vivo gastrointestinal physiology are in high demand to elucidate mechanisms behind pathogen infectivity, and to aid the design of effective preventive and therapeutic interventions. There exists a trade‐off between simple and high throughput models and those that are more complex and physiologically relevant. The complexity of the model used shall be guided by the biological question to be addressed. This review provides an overview of the structure and function of the intestine and the models that are developed to emulate this. Conventional models are discussed in addition to emerging models which employ engineering principles to equip them with necessary advanced monitoring capabilities for intestinal host‐pathogen interrogation. Limitations of current models and future perspectives on the field are presented.

Glioblastoma multiforme (GBM) is the most common primary brain tumor, characterized by a remarkable inner complexity and inter-tumor variability. Moreover, it is very aggressive and resistant to conventional treatments, so that it rapidly relapse. Therefore, there is an immediate need for experimental strategies to enhance our comprehension of GBM, aiming to mitigate its economic and social impact. Here, we described different in vivo and in vitro strategies currently used for the study of GBM. First, we gave a brief and general overview of the classical in vivo models, including xenograft mouse and zebrafish models and canine models, offering a wide range of advantages but also presenting a series of strong limitations. Thus, we described in vitro models, starting from more traditional 2D culture models, comparing different approaches and critically exposing the advantages and disadvantages of using one or the other methods. We also briefly described GBM 2D culture systems that allow recreating multiple cell-cell and cell-extracellular matrix contacts but still do not reflect the complexity of in vivo tumors. We finally described the intricacies of the more novel 3D in vitro models, e.g., spheroids and organoids. These sophisticated models have demonstrated exceptional suitability across a wide spectrum of applications in cancer research, ranging from fundamental scientific inquiries to applications in translational research. Their adaptability and three-dimensional architecture render them invaluable tools, offering new insights and paving the way for advancements in both basic and applied research.

In vitro cellular models denote a crucial part of drug discovery programs as they aid in identifying successful drug candidates based on their initial efficacy and potency. While tremendous headway has been achieved in improving 2D and 3D culture techniques, there is still a need for physiologically relevant systems that can mimic or alter cellular responses without the addition of external biochemical stimuli. A way forward to alter cellular responses is using physical cues, like 3D topographical inorganic substrates, to differentiate macrophage-like cells. Herein, protein secretion and gene expression markers for various macrophage subsets cultivated on a 3D topographical substrate are investigated. The results show that macrophages differentiate into anti-inflammatory M2-type macrophages, secreting increased IL-10 levels compared to the controls. Remarkably, these macrophage cells are differentiated into the M2d subset, making up the main component of tumour-associated macrophages (TAMs), as measured by upregulated Il-10 and Vegf mRNA. M2d subset differentiation is attributed to the topographical substrates with 3D fractal-like geometries arrayed over the surface, else primarily achieved by tumour-associated factors in vivo. From a broad perspective, this work paves the way for implementing 3D topographical inorganic surfaces for drug discovery programs, harnessing the advantages of in vitro assays without external stimulation and allowing the rapid characterisation of therapeutic modalities in physiologically relevant environments.

BackgroundThe latent HIV reservoir represents the major barrier to a cure. One curative strategy is targeting diseased cells for elimination based on biomarkers that uniquely define these cells. Single-cell RNA sequencing (scRNA-seq) has enabled the identification of gene expression profiles associated with disease at the single-cell level. Because HIV provirus in many cells during latency is not entirely silent, it became possible to determine gene expression patterns in a subset of cells latently infected with HIV.ObjectiveThe primary objective of this study was the identification of the gene expression profiles of single latently infected CD4+ T cells using scRNA-seq. Different conditions of latency establishment were considered. The identified profiles were then explored to prioritize the identified genes for future experimental validation.MethodsTo facilitate gene prioritization, three approaches were used. First, we characterized and compared the gene expression profiles of HIV latency established in different environments: in cells that encountered an activation stimulus and then returned to quiescence, and in resting cells that were infected directly via cell-to-cell viral transmission from autologous activated, productively infected cells. Second, we characterized and compared the gene expression profiles of HIV latency established with viruses of different tropisms, using an isogenic pair of CXCR4- and CCR5-tropic viruses. Lastly, we used proviral expression patterns in cells from people with HIV to more accurately define the latently infected cells in vitro.ResultsOur analyses demonstrated that a subset of genes is expressed differentially between latently infected and uninfected cells consistently under most conditions tested, including cells from people with HIV. Our second important observation was the presence of latency signatures, associated with variable conditions when latency was established, including cellular exposure and responsiveness to a T cell receptor stimulus and the tropism of the infecting virus.ConclusionCommon signatures, specifically genes that encode proteins localized to the cell surface, should be prioritized for further testing at the protein level as biomarkers for the ability to enrich or target latently infected cells. Cell- and tropism-dependent biomarkers may need to be considered in developing targeting strategies to ensure that all the different reservoir subsets are eliminated.

The vasculature of the brain consists of specialized endothelial cells that form a blood-brain barrier (BBB). This barrier, in conjunction with supporting cell types, forms the neurovascular unit (NVU). The NVU restricts the passage of certain substances from the bloodstream while selectively permitting essential nutrients and molecules to enter the brain. This protective role is crucial for optimal brain function, but presents a significant obstacle in treating neurological conditions, necessitating chemical modifications or advanced drug delivery methods for most drugs to cross the NVU. A deeper understanding of NVU in health and disease will aid in the identification of new therapeutic targets and drug delivery strategies for improved treatment of neurological disorders. To achieve this goal, we need models that reflect the human BBB and NVU in health and disease. Although animal models of the brain's vasculature have proven valuable, they are often of limited translational relevance due to interspecies differences or inability to faithfully mimic human disease conditions. For this reason, human in vitro models are essential to improve our understanding of the brain's vasculature under healthy and diseased conditions. This review delves into the advancements in in vitro modeling of the BBB and NVU, with a particular focus on microfluidic models. After providing a historical overview of the field, we shift our focus to recent developments, offering insights into the latest achievements and their associated constraints. We briefly examine the importance of chip materials and methods to facilitate fluid flow, emphasizing their critical roles in achieving the necessary throughput for the integration of microfluidic models into routine experimentation. Subsequently, we highlight the recent strides made in enhancing the biological complexity of microfluidic NVU models and propose recommendations for elevating the biological relevance of future iterations. Importantly, the NVU is an intricate structure and it is improbable that any model will fully encompass all its aspects. Fit-for-purpose models offer a valuable compromise between physiological relevance and ease-of-use and hold the future of NVU modeling: as simple as possible, as complex as needed. [ABSTRACT FROM AUTHOR]

Lung cancer is the leading cause of cancer death in the United States and worldwide, and a major source of cancer health disparities. Lung cancer cell lines provide key in vitro models for molecular studies of lung cancer development and progression, and for pre-clinical drug testing. To ensure health equity, it is imperative that cell lines representing different lung cancer histological types, carrying different cancer driver genes, and representing different genders, races, and ethnicities should be available. This is particularly relevant for cell lines from Black men, who experience the highest lung cancer mortality in the United States. Here, we undertook a review of the available lung cancer cell lines and their racial and ethnic origin. We noted a marked imbalance in the availability of cell lines from different races and ethnicities. Cell lines from Black patients were strongly underrepresented, and we identified no cell lines from Hispanic/Latin(x) (H/L), American Indian/American Native (AI/AN), or Native Hawaiian or other Pacific Islander (NHOPI) patients. The majority of cell lines were derived from White and Asian patients. Also missing are cell lines representing the cells-oforigin of the major lung cancer histological types, which can be used to model lung cancer development and to study the effects of environmental exposures on lung tissues. To our knowledge, the few available immortalized alveolar epithelial cell lines are all derived from White subjects, and the race and ethnicity of a handful of cell lines derived from bronchial epithelial cells are unknown. The lack of an appropriately diverse collection of lung cancer cell lines and lung cancer cell-of-origin lines severely limits racially and ethnically inclusive lung cancer research. It impedes the ability to develop inclusive models, screen comprehensively for effective compounds, pre-clinically test new drugs, and optimize precision medicine. It thereby hinders the development of therapies that can increase the survival of minority and underserved patients. The noted lack of cell lines from underrepresented groups should constitute a call to action to establish additional cell lines and ensure adequate representation of all population groups in this critical pre-clinical research resource. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease causes a major burden to patients and healthcare systems, raising the need to develop effective therapies. Technological advances in cell culture, allied with ethical issues, have propelled in vitro models as essential tools to study disease aetiology, its progression, and possible therapies. Several cell‐based in vitro models of intestinal inflammation have been used, varying in their complexity and methodology to induce inflammation. Immortalized cell lines are extensively used due to their long‐term survival, in contrast to primary cultures that are short‐lived but patient‐specific. Recently, organoids and organ‐chips have demonstrated great potential by being physiologically more relevant. This review aims to shed light on the intricate nature of intestinal inflammation and cover recent works that report cell‐based in vitro models of human intestinal inflammation, encompassing diverse approaches and outcomes. [ABSTRACT FROM AUTHOR]

Microphysiological systems are an emerging area of in vitro drug development, and their independent evaluation is important for wide adoption and use. The primary goal of this study was to test reproducibility and robustness of a renal proximal tubule microphysiological system, OrganoPlate 3-lane 40, as an in vitro model for drug transport and toxicity studies. This microfluidic model was compared with static multiwell cultures and tested using several human renal proximal tubule epithelial cell (RPTEC) types. The model was characterized in terms of the functional transport for various tubule-specific proteins, epithelial permeability of small molecules (cisplatin, tenofovir, and perfluorooctanoic acid) versus large molecules (fluorescent dextrans, 60–150 kDa), and gene expression response to a nephrotoxic xenobiotic. The advantages offered by OrganoPlate 3-lane 40 as compared with multiwell cultures are the presence of media flow, albeit intermittent, and increased throughput compared with other microfluidic models. However, OrganoPlate 3-lane 40 model appeared to offer only limited (eg, MRP-mediated transport) advantages in terms of either gene expression or functional transport when compared with the multiwell plate culture conditions. Although OrganoPlate 3-lane 40 can be used to study cellular uptake and direct toxic effects of small molecules, it may have limited utility for drug transport studies. Overall, this study offers refined experimental protocols and comprehensive comparative data on the function of RPETCs in traditional multiwell culture and microfluidic OrganoPlate 3-lane 40, information that will be invaluable for the prospective end-users of in vitro models of the human proximal tubule. [ABSTRACT FROM AUTHOR]

For several decades now, researchers have been trying to answer the demand of clinical oncologists to create an ideal preclinical model of head and neck squamous cell carcinoma (HNSCC) that is accessible, reproducible, and relevant. Over the past years, the development of cellular technologies has naturally allowed us to move from primitive short-lived primary 2D cell cultures to complex patient-derived 3D models that reproduce the cellular composition, architecture, mutational, or viral load of native tumor tissue. Depending on the tasks and capabilities, a scientific laboratory can choose from several types of models: primary cell cultures, immortalized cell lines, spheroids or heterospheroids, tissue engineering models, bioprinted models, organoids, tumor explants, and histocultures. HNSCC in vitro models make it possible to screen agents with potential antitumor activity, study the contribution of the tumor microenvironment to its progression and metastasis, determine the prognostic significance of individual biomarkers (including using genetic engineering methods), study the effect of viral infection on the pathogenesis of the disease, and adjust treatment tactics for a specific patient or groups of patients. Promising experimental results have created a scientific basis for the registration of several clinical studies using HNSCC in vitro models. [ABSTRACT FROM AUTHOR]

Ageing is an irreversible and inevitable biological process and a significant risk factor for the development of various diseases, also affecting the musculoskeletal system, resulting from the accumulation of cell senescence. The aim of this systematic review was to collect the in vitro studies conducted over the past decade in which cell senescence was induced through various methods, with the purpose of evaluating the molecular and cellular mechanisms underlying senescence and to identify treatments capable of delaying senescence. Through three electronic databases, 22 in vitro studies were identified and included in this systematic review. Disc, cartilage, or muscle cells or tissues and mesenchymal stem cells were employed to set-up in vitro models of senescence. The most common technique used to induce cell senescence was the addition to the culture medium of tumor necrosis factor (TNF)α and/or interleukin (IL)1β, followed by irradiation, compression, hydrogen peroxide (H2O2), microgravity, in vitro expansion up to passage 10, and cells harvested from damaged areas of explants. Few studies evaluated possible treatments to anti-senescence effects. The included studies used in vitro models of senescence in musculoskeletal tissues, providing powerful tools to evaluate age-related changes and pathologies, also contributing to the development of new therapeutic approaches. [ABSTRACT FROM AUTHOR]

Background: Fragmentomics, the investigation of fragmentation patterns of cell-free DNA (cfDNA), has emerged as a promising strategy for the early detection of multiple cancers in the field of liquid biopsy. However, the clinical application of this approach has been hindered by a limited understanding of cfDNA biology. Furthermore, the prevalence of hematopoietic cell-derived cfDNA in plasma complicates the in vivo investigation of tissue-specific cfDNA other than that of hematopoietic origin. While conventional two-dimensional cell lines have contributed to research on cfDNA biology, their limited representation of in vivo tissue contexts underscores the need for more robust models. In this study, we propose three-dimensional organoids as a novel in vitro model for studying cfDNA biology, focusing on multifaceted fragmentomic analyses. Results: We established nine patient-derived organoid lines from normal lung airway, normal gastric, and gastric cancer tissues. We then extracted cfDNA from the culture medium of these organoids in both proliferative and apoptotic states. Using whole-genome sequencing data from cfDNA, we analyzed various fragmentomic features, including fragment size, footprints, end motifs, and repeat types at the end. The distribution of cfDNA fragment sizes in organoids, especially in apoptosis samples, was similar to that found in plasma, implying occupancy by mononucleosomes. The footprints determined by sequencing depth exhibited distinct patterns depending on fragment sizes, reflecting occupancy by a variety of DNA-binding proteins. Notably, we discovered that short fragments (< 118 bp) were exclusively enriched in the proliferative state and exhibited distinct fragmentomic profiles, characterized by 3 bp palindromic end motifs and specific repeats. Conclusions: In conclusion, our results highlight the utility of in vitro organoid models as a valuable tool for studying cfDNA biology and its associated fragmentation patterns. This, in turn, will pave the way for further enhancements in noninvasive cancer detection methodologies based on fragmentomics. [ABSTRACT FROM AUTHOR]

Over the past 20 years, the use of pluripotent stem cells to mimic the complexities of the human neuromuscular junction has received much attention. Deciphering the key mechanisms underlying the establishment and maturation of this complex synapse has been driven by the dual goals of addressing developmental questions and gaining insight into neuromuscular disorders. This review aims to summarise the evolution and sophistication of in vitro neuromuscular junction models developed from the first differentiation of human embryonic stem cells into motor neurons to recent neuromuscular organoids. We also discuss the potential offered by these models to decipher different neuromuscular diseases characterised by defects in the presynaptic compartment, the neuromuscular junction, and the postsynaptic compartment. Finally, we discuss the emerging field that considers the use of these techniques in drug screening assay and the challenges they will face in the future. [ABSTRACT FROM AUTHOR]

Neurodegenerative diseases (NDs) are characterized by uncontrolled loss of neuronal cells leading to a progressive deterioration of brain functions. The transition rate of numerous neuroprotective drugs against Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease, leading to FDA approval, is only 8–14% in the last two decades. Thus, in spite of encouraging preclinical results, these drugs have failed in human clinical trials, demonstrating that traditional cell cultures and animal models cannot accurately replicate human pathophysiology. Hence, in vitro three-dimensional (3D) models have been developed to bridge the gap between human and animal studies. Such technological advancements in 3D culture systems, such as human-induced pluripotent stem cell (iPSC)-derived cells/organoids, organ-on-a-chip technique, and 3D bioprinting, have aided our understanding of the pathophysiology and underlying mechanisms of human NDs. Despite these recent advances, we still lack a 3D model that recapitulates all the key aspects of NDs, thus making it difficult to study the ND's etiology in-depth. Hence in this review, we propose developing a combinatorial approach that allows the integration of patient-derived iPSCs/organoids with 3D bioprinting and organ-on-a-chip technique as it would encompass the neuronal cells along with their niche. Such a 3D combinatorial approach would characterize pathological processes thoroughly, making them better suited for high-throughput drug screening and developing effective novel therapeutics targeting NDs. [ABSTRACT FROM AUTHOR]

Purpose: Neurothrombectomy catheters can disrupt or injure the vessel wall. This potential injury is often studied in animal or cadaver models, but prior work suggests that endothelialized silicone models may be an option for early in vitro assessment. The purpose of this work was to create a complex, clinically-relevant endothelialized neurovascular silicone model, and to determine the utility of the model for evaluating vessel injury due to catheter simulated use. Methods: Models of the ICA and MCA were fabricated out of silicone, sterilized, coated with fibronectin, placed in bioreactors, and endothelialized with HUVECs. These silicone vessels were maintained under flow for 3 and 7 days, and cellular linings were assessed. Subsequently, 24 silicone vessels were created and treated with neurovascular catheters. Vessels were accessed with a guidewire, microcatheter, and/or aspiration catheter, either once (1-pass) or three times (3-pass). Vessels were then fixed, and injury was evaluated through quantitative image analysis and a visual scoring system. Results: Complex silicone models were successfully endothelialized and maintained with consistent cell linings. The transparent silicone permitted catheter simulated use without fluoroscopy, and injury to the vessel wall was observed and successfully imaged and characterized. Vessels subjected to 3-passes exhibited more injury than 1-pass, and injury increased with the number and size of devices. These results illustrated expected trends and support use of these models for early assessment of vessel injury. Conclusion: Complex silicone neurovascular models can be endothelialized and used in vitro to assess and compare injury due to the use of neurovascular catheters. [ABSTRACT FROM AUTHOR]

Host–microbiota interactions can influence biological functions in animals, but direct evidence of their impact on animal evolution is limited. Gut-on-a-chip systems emulate biological properties of the natural intestine, including establishment of simplified microbial communities in the epithelial barrier. Development of chips derived from multiple animal hosts can enable evolutionary aspects of animal–microbiota interactions to be studied with unparalleled resolution. Whether and how microorganisms have shaped the evolution of their animal hosts is a major question in biology. Although many animal evolutionary processes appear to correlate with changes in their associated microbial communities, the mechanistic processes leading to these patterns and their causal relationships are still far from being resolved. Gut-on-a-chip models provide an innovative approach that expands beyond the potential of conventional microbiome profiling to study how different animals sense and react to microbes by comparing responses of animal intestinal tissue models to different microbial stimuli. This complementary knowledge can contribute to our understanding of how host genetic features facilitate or prevent different microbiomes from being assembled, and in doing so elucidate the role of host–microbiota interactions in animal evolution. [ABSTRACT FROM AUTHOR]

Despite increasing use of in vitro models that closely resemble in vivo human biology, their application in understanding downstream effects of airway toxicity, such as inflammation, are at an early stage. In this study, we used various assays to examine the inflammatory response induced in MucilAir™ tissues and A549 cells exposed to three products known to induce toxicity. Reduced barrier integrity was observed in tissues following exposure to each product, with reduced viability and increased cytotoxicity also shown. Similar changes in viability were also observed in A549 cells. Furthermore, whole cigarette smoke (CS) induced downstream phenotypic THP-1 changes and endothelial cell adhesion, an early marker of atherosclerosis. In contrast, exposure to next-generation delivery product (NGP) aerosol did not induce this response. Cytokine, histological and RNA analysis highlighted increased biomarkers linked to inflammatory pathways and immune cell differentiation following exposure to whole cigarette smoke, including GM-CSF, IL-1β, cleaved caspase-3 and cytochrome P450 enzymes. As a result of similar observations in human airway inflammation, we propose that our exposure platform could act as a representative model for studying such events in vitro. Furthermore, this model could be used to test the inflammatory or anti-inflammatory impact posed by inhaled compounds delivered to the lung. • Exposure of bronchial and alveolar cells to known toxicants at the air liquid interface induced varying levels of toxicity and inflammation. • Recovery media from exposed cells induced downstream phenotypic changes in THP-1 monocytes. • Phenotypically altered THP-1 monocytes demonstrated increased adhesion to endothelial cells - an early marker of atherosclerosis. [ABSTRACT FROM AUTHOR]

The female reproductive tract (FRT) is home to diverse microbial communities that play a pivotal role in reproductive health and disorders such as infertility, endometriosis, and cervical cancer. To understand the complex host–microbiota interactions within the FRT, models that authentically replicate the FRT's environment, including the interplay between the microbiota, mucus layer, immune system, and hormonal cycle, are key. Recent strides in organoid and microfluidic technologies are propelling research in this domain, offering insights into FRT–microbiota interactions and potential therapeutic avenues. This review delves into the current state of FRT organoid models and microbe integration techniques, evaluating their merits and challenges for specific research objectives. Emphasis is placed on innovative approaches and applications, including integrating organoids with microfluidics, and using patient-derived biobanks, as this offers potential for deeper mechanistic insights and personalized therapeutic strategies. Modeling various FRT properties in organoids is explored, from encompassing age-related epithelial features, oxygen levels, and hormonal effects to mucus layers, immune responses, and microbial interactions, highlighting their potential to transform reproductive health research and predict possible outcomes. [ABSTRACT FROM AUTHOR]

Caco-2 cell monolayers are widely employed as an in vitro model of the intestinal barrier, capable of accurately predicting the absorption of conventional small-molecule drugs. However, this model may not be applicable to all drugs, and the accuracy of absorption prediction is typically poor for high molecular weight drugs. Recently, human induced pluripotent stem (iPS) cell-derived small intestinal epithelial cells (hiPSC-SIECs), exhibiting properties similar to those of the small intestine when compared with Caco-2 cells, have been developed and are considered a novel candidate model for in vitro evaluation of intestinal drug permeability. Therefore, we evaluated the utility of human hiPSC-SIECs as a new in vitro model to predict the intestinal absorption of middle-molecular weight drugs and peptide drugs. Firstly, we showed that the hiPSC-SIEC monolayer allowed faster transport of peptide drugs (insulin and glucagon-like peptide-1) than the Caco- 2 cell monolayer. Second, we revealed that hiPSC-SIECs require divalent cations (Mg2+ and Ca2+) to maintain barrier integrity. Third, we demonstrated that experimental conditions established for Caco-2 cells are not persistently applicable to hiPSC-SICEs when analyzing absorption enhancers. Comprehensively clarifying the features of hiPSC-SICEs is essential to establish a new in vitro evaluation model. [ABSTRACT FROM AUTHOR]

Organ-on-a-chip technology is attracting growing interest across various domains as a crucial platform for drug screening and testing and is set to play a significant role in precision medicine research. Lymph nodes, being intricately structured organs essential for the body’s adaptive immune responses to antigens and foreign particles, are pivotal in assessing the immunotoxicity of novel pharmaceuticals. Significant progress has been made in research on the structure and function of the lymphatic system. However, there is still an urgent need to develop prospective tools and techniques to delve deeper into its role in various diseases’ pathological and physiological processes and to develop corresponding immunotherapeutic therapies. Organ chips can accurately reproduce the specific functional areas in lymph nodes to better simulate the complex microstructure of lymph nodes and the interactions between different immune cells, which is convenient for studying specific biological processes. This paper reviews existing lymph node chips and their design approaches. It discusses the applications of the above systems in modeling immune cell motility, cell–cell interactions, vaccine responses, drug testing, and cancer research. Finally, we summarize the challenges that current research faces in terms of structure, cell source, and extracellular matrix simulation of lymph nodes, and we provide an outlook on the future direction of integrated immune system chips.

The treatment of Parkinson’s disease has been moving into the focus of pharmaceutical development. Yet, the necessity for reliable model systems in the development phase has made research challenging and in vivo models necessary. We have established reliable, reproducible in vitro model systems to evaluate the binding and transport of dopamine-loaded PLGA nanoparticles for the treatment of Parkinson’s disease and put the results in context with comparable in vivo results. The in vitro models have provided similar results concerning the usability of the investigated nanoparticles as the previously used in vivo models and thus provide a good alternative in line with the 3R principles in pharmaceutical research.

Among the many lysosomal storage disorders (LSDs) that would benefit from the establishment of novel cell models, either patient-derived or genetically engineered, is mucopolysaccharidosis type II (MPS II). Here, we present our results on the establishment and characterization of two MPS II patient-derived stem cell line(s) from deciduous baby teeth. To the best of our knowledge, this is the first time a stem cell population has been isolated from LSD patient samples obtained from the dental pulp. Taking into account our results on the molecular and biochemical characterization of those cells and the fact that they exhibit visible and measurable disease phenotypes, we consider these cells may qualify as a valuable disease model, which may be useful for both pathophysiological assessments and in vitro screenings. Ultimately, we believe that patient-derived dental pulp stem cells (DPSCs), particularly those isolated from human exfoliated deciduous teeth (SHEDs), may represent a feasible alternative to induced pluripotent stem cells (iPSCs) in many labs with standard cell culture conditions and limited (human and economic) resources.

Abstract Inflammatory bowel disease causes a major burden to patients and healthcare systems, raising the need to develop effective therapies. Technological advances in cell culture, allied with ethical issues, have propelled in vitro models as essential tools to study disease aetiology, its progression, and possible therapies. Several cell‐based in vitro models of intestinal inflammation have been used, varying in their complexity and methodology to induce inflammation. Immortalized cell lines are extensively used due to their long‐term survival, in contrast to primary cultures that are short‐lived but patient‐specific. Recently, organoids and organ‐chips have demonstrated great potential by being physiologically more relevant. This review aims to shed light on the intricate nature of intestinal inflammation and cover recent works that report cell‐based in vitro models of human intestinal inflammation, encompassing diverse approaches and outcomes.

The placenta, being a temporary organ, plays a crucial role in facilitating the exchange of nutrients and gases between the mother and the fetus during pregnancy. Any abnormalities in the development of this vital organ not only lead to various pregnancy-related disorders that can result in fetal injury or death, but also have long-term effects on maternal health. In vitro models have been employed to study the physiological features and molecular regulatory mechanisms of placental development, aiming to gain a detailed understanding of the pathogenesis of pregnancy-related diseases. Among these models, trophoblast stem cell culture and organoids show great promise. In this review, we provide a comprehensive overview of the current mature trophoblast stem cell models and emerging organoid models, while also discussing other models in a systematic manner. We believe that this knowledge will be valuable in guiding further exploration of the complex maternal-fetal interface.

Cardiometabolic diseases and cancer are among the most common diseases worldwide and are a serious concern to the healthcare system. These conditions, apparently distant, share common molecular and cellular determinants, that can represent targets for preventive and therapeutic approaches. The bone marrow plays an important role in this context as it is the main source of cells involved in cardiovascular regeneration, and one of the main sites of liquid and solid tumor metastasis, both characterized by the cellular trafficking across the bone marrow vasculature. The bone marrow vasculature has been widely studied in animal models, however, it is clear the need for human-specific in vitro models, that resemble the bone vasculature lined by endothelial cells to study the molecular mechanisms governing cell trafficking. In this review, we summarized the current knowledge on in vitro models of bone marrow vasculature developed for cardiovascular and cancer research.

Objective: The optimal degree of proximal thoracic endograft oversizing when aiming for durable sealing in prosthetic grafts is unknown. The aim of the present study was to create an in vitro model for testing different oversized thoracic endografts in a reproducible and standardized manner and, subsequently, determine the optimal oversizing range when planning procedures with a proximal landing in prosthetic zones in the descending thoracic aorta or aortic arch. Methods: An in vitro model consisting of a fixed 24-mm polyethylene terephthalate (Dacron; DuPont) graft sutured proximally and distally to two specifically designed 40-mm rings, with four force sensing resistors attached at four equally distant positions and a USB camera attached proximally for photographic and video documentation was used for deployment of Zenith TX2 (Cook Medical Inc) dissection platform endografts with diameters between 24 and 36 mm. After deployment, ballooning with a 32-mm compliant balloon was performed to simulate real-life conditions. The assessment of oversizing included visual inspection, calculation of the valley areas created between the prosthetic wall and the stent graft fabric, distance between the stent graft peaks, the radial force exerted by the proximal sealing stent, and the pull-out force necessary for endograft extraction. Results: A total of 70 endografts were deployed with the oversizing ranging from 0% to 50%: 10 × 24 mm, 10 × 26 mm, 10 × 28 mm, 10 × 30 mm, 10 × 32 mm, 10 × 34 mm, and 10 × 36 mm. Two cases of infolding occurred with 50% oversizing. The valley areas increased from 8.79 ± 0.23 mm2 with 16.7% oversizing to 14.26 ± 0.45 mm2 with 50% oversizing (P < .001). A significant difference was found in the pull-out force required for endografts with 15%. The mean radial force of the proximal sealing stent was greater after remodeling with a compliant balloon (0.55 ± 0.02 N vs 0.60 ± 0.02 N after ballooning; P < .001). However, greater oversizing did not lead to an increase in the radial force exerted by the proximal sealing stent. Conclusions: The findings from the present study offer additional insight into the mechanics of oversized stent grafts in surgical grafts. In endografts with the Zenith stent design (TX2), oversizing of 50% was associated with major infolding in 20% of cases. Long-term in vitro and in vivo testing is required to understand how these mechanical properties affect the clinical outcomes of oversizing. : Clinical Relevance: A paucity of data is available regarding the optimal thoracic endograft oversizing that should be used when landing proximally in prosthetic grafts located in the descending thoracic aortic or aortic arch. The present study is one of the first to attempt to establish a definition of optimal oversizing for in vitro studies. It is also one of the few studies to specifically consider this subcohort of patients with complex aortic disease. Evidence from the present study suggests using oversizing of 20% to 30% for proximal sealing in prosthetic grafts. The results showed low pull-out force requirements for endografts with

The blood–brain barrier, which is formed by tightly interconnected microvascular endothelial cells, separates the brain from the peripheral circulation. Together with other central nervous system-resident cell types, including pericytes and astrocytes, the blood–brain barrier forms the neurovascular unit. Upon neuroinflammation, this barrier becomes leaky, allowing molecules and cells to enter the brain and to potentially harm the tissue of the central nervous system. Despite the significance of animal models in research, they may not always adequately reflect human pathophysiology. Therefore, human models are needed. This review will provide an overview of the blood–brain barrier in terms of both health and disease. It will describe all key elements of the in vitro models and will explore how different compositions can be utilized to effectively model a variety of neuroinflammatory conditions. Furthermore, it will explore the existing types of models that are used in basic research to study the respective pathologies thus far. [ABSTRACT FROM AUTHOR]

Aim: To investigate the mechanism of liver fibrosis in chronic hepatitis C virus (HCV) infection we developed an in vitro model. Methods: Huh7 cells were transfected with HCV (JFH1 or Jc1) or sub-genomic replicons and expression of pro-fibrotic cytokines determined by qPCR. Media from infected cells was transferred onto LX2 cells or primary rat stellate cells and expression of pro-fibrotic genes measured by qPCR. Results: Replication competent HCV, but not sub-genomic replicons, induced expression of TGF-β1 and CTGF. Supernatant from infected cells induced α-SMA and COL1A1 expression and stellate cell migration, confirming functional activation. Conclusion: Infected cells produce pro-fibrogenic cytokines TGF-β1 and CTGF and activate stellate cells. This mechanism could be targeted to prevent or treat HCV-induced fibrosis. We developed a new laboratory model to study why people with hepatitis C develop liver damage. We plan to use this model to develop new treatments. We found that cells infected with whole hepatitis C virus release chemicals that cause inflammation in the liver, which can lead to liver damage and scarring. We confirmed that these chemicals act on other cells in the liver ('stellate cells'), causing them to become active and to move around. Active stellate cells are known to cause liver damage and scarring, explaining why hepatitis C causes liver damage. Our results provide new ideas to develop drugs that can prevent liver damage in people with hepatitis C. [ABSTRACT FROM AUTHOR]