Primary Cell Types Research Articles

Mapping of β3-Adrenergic Receptor in Living Cells with a Ligand-Guided Fluorescent Probe.

Tracking the dynamic distribution of native proteins in living cells or tissues in real time is essential for understanding the functional mechanisms involved in their physiological or pathological processes. The β3-adrenergic receptor (β3-AR) has important biological functions. It is expected to be a diagnostic indicator for aging, yet current probes for β-ARs are unable to dynamically monitor β3-AR in real time, which impedes the progress of β3-AR research. Here, we developed a ligand-directed anchoring probe, β3-ARP, that precisely covalently anchors with native β3-AR in living cells, for the diagnosis of senescence. The ligand-directed anchoring probe selectively recognizes and traces β3-AR and can achieve dynamic observation and monitoring of β3-AR in living cells, including the interaction between lipid droplets and mitochondria. Moreover, we were able to directly probe the distribution of β3-AR in various organs of aging mice in situ and track the location of native β3-AR in different types of primary neuronal cells using β3-ARP and two-photon imaging, which revealed the aberrant accumulation of lipid droplets and the distribution of β3-AR in neurological diseases. This research has resulted in a new covalently anchoring fluorescent probe, β3-ARP, which could serve as a powerful tool to explore the link between β3-AR and age-related diseases.

A Morphological Study of HLA-DR-Immunopositive Cells in Multiple Sclerosis Lesions and Their Implications for Pathogenesis

Background: Multiple sclerosis (MS) is characterized by white matter demyelinating plaques, which can be classified as active, chronic active, or chronic inactive based on the extent of demyelination, cellularity, and inflammation. Microglia and macrophages play a central role in these processes. This study aimed to investigate the morphological characteristics of HLA-DR-immunopositive cells in these plaques to improve our understanding of the roles of these cells in MS plaques. Methods: This study is a retrospective post-mortem histopathological study. We analyzed 90 plaques from 6 MS cases. Of the plaques studied, 77 were grouped into three categories: 28 active, 34 chronic active, and 15 chronic inactive. Additionally, five vacuolated white matter lesions, two axonal degeneration lesions, and six lesions with mixed histological features were included. Six control cases were also examined to assess HLA-DR-immunopositive cell expression across various age groups. The cells were classified based on their morphology into two types: round cells without processes (macrophages) and cells with varying processes and shapes (ramified microglia). Results: Both macrophages and ramified microglia were present in all lesion types, with a focus on identifying the predominant cell type. Of the 28 active plaques, macrophages were the primary cell type in 25 plaques, while ramified microglia predominated in 3. In the center of 49 chronic plaques, scattered ramified microglia were observed in 46, with three plaques showing a predominance of macrophages. Among the 34 chronic active lesions, ramified microglia were the main cell type in the periphery of 32 plaques, with the remaining two predominantly exhibiting macrophages. Conclusions: The predominance of macrophages in active lesions and the presence of scattered ramified microglia in the center of chronic plaques are consistent with the phagocytic role of macrophages. Meanwhile, the prevalence of ramified microglia at the periphery of chronic active lesions suggests a potential protective function in maintaining lesion stability.

Increasing intracellular dNTP levels improves prime editing efficiency.

In primary cell types, intracellular deoxynucleotide triphosphate (dNTP) levels are tightly regulated in a cell cycle-dependent manner. We report that prime editing efficiency is increased by mutations that improve the enzymatic properties of Moloney murine leukemia virus reverse transcriptase and treatments that increase intracellular dNTP levels. In combination, these modifications produce substantial increases in precise editing rates.

Cell swelling enhances ligand-driven β-adrenergic signaling

G protein-coupled receptors’ conformational landscape can be affected by their local, microscopic interactions within the cell plasma membrane. We employ here a pleiotropic stimulus, namely osmotic swelling, to alter the cortical environment within intact cells and monitor the response in terms of receptor function and downstream signaling. We observe that in osmotically swollen cells the β2-adrenergic receptor, a prototypical GPCR, favors an active conformation, resulting in cAMP transient responses to adrenergic stimulation that have increased amplitude. The results are validated in primary cell types such as adult cardiomyocytes, a model system where swelling occurs upon ischemia-reperfusion injury. Our results suggest that receptors’ function is finely modulated by their biophysical context, and specifically that osmotic swelling acts as a potentiator of downstream signaling, not only for the β2-adrenergic receptor, but also for other receptors, hinting at a more general regulatory mechanism.

An Evaluation of the Replacement of Animal-derived Biomaterials in Human Primary Cell Culture.

The likelihood that potential new drugs will successfully navigate the current translational pipeline is poor, with fewer than 10% of drug candidates making this transition successfully, even after their entry into clinical trials. Prior to this stage, candidate drugs are typically evaluated by using models of increasing complexity, beginning with basic in vitro cell culture studies and progressing through to animal studies, where many of these candidates are lost due to lack of efficacy or toxicology concerns. There are many reasons for this poor translation, but interspecies differences in functional and physiological parameters undoubtedly contribute to the problem. Improving the human-relevance of early preclinical in vitro models may help translatability, especially when targeting more nuanced species-specific cell processes. The aim of the current study was to define a set of guidelines for the effective transition of human primary cells of multiple lineages to more physiologically relevant, translatable, animal-free in vitro culture conditions. Animal-derived biomaterials (ADBs) were systematically replaced with non-animal-derived alternatives in the in vitro cell culture systems, and the impact of the substitutions subsequently assessed by comparing the kinetics and phenotypes of the cultured cells. ADBs were successfully eliminated from primary human dermal fibroblast, uterine fibroblast, pulmonary fibroblast, retinal endothelial cell and peripheral blood mononuclear cell culture systems, and the individual requirements of each cell subtype were defined to ensure the successful transition toward growth under animal-free culture conditions. We demonstrate that it is possible to transition ('humanise') a diverse set of human primary cell types by following a set of simple overarching principles that inform the selection, and guide the evaluation of new, improved, human-relevant in vitro culture conditions.

Fast and reliable quantification of aldosterone, cortisol and cortisone via LC-MS/MS to study 11β-hydroxysteroid dehydrogenase activities in primary cell cultures

Cell culture experiments can support characterization of enzymatic activities in healthy and tumorous human tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) enables simultaneous measurement of several steroids from a single sample, facilitating analysis of molecular pathways involved in steroid biosynthesis. We developed a reliable but fast method for quantification of cortisol, cortisone and aldosterone in cell culture supernatant. Validation, including investigation of matrix-matched calibration, was performed for two different cell types. Utility of the method was demonstrated in the study of 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) activity under conditions of glucocorticoid and mineralocorticoid excess in different cell types. Aldosterone, cortisol and cortisone were extracted by liquid-liquid extraction (LLE) with methyl tert-butyl ether from 1 mL of cell culture supernatant. Steroids were separated on a Kinetex biphenyl column (50 ×2.1 mm, 2.6 µm) with gradient elution of water and methanol containing 2 mM ammonium format and analysed in multiple reaction monitoring mode after positive electrospray ionization. Application of the method included cell culture experiments with two different primary cell types, human coronary artery smooth muscle cells (HCSMC) and human coronary artery endothelial cells (EC). Cells were treated with different concentrations of cortisol, aldosterone and mifepristone, a glucocorticoid receptor antagonist and quantitative PCR was performed. The method exhibits high precision (CV ≤ 6 %) and accuracy (deviation from nominal concentration ≤ 6 %) for concentrations above the limit of quantification (LoQ) which is 0.11, 0.56 and 0.69 nmol/L for aldosterone, cortisone and cortisol, respectively. Calibration curves did not differ when prepared in media or solvent. The method enabled us to confirm activity of HSD11B2 and concentration dependent conversion of cortisol to cortisone in HCSMC (median conversion ratio at 140 nM cortisol = 1.46 %). In contrast we did not observe any HSD11B2 activity in EC. Neither addition of high aldosterone, nor addition of 1 µM mifepristone had impact on glucocorticoid concentrations. Quantitative PCR revealed expression of HSD11B1 and HSD11B2 in HCSMC but not in EC. We present a fast and reliable method for quantification of cortisol, cortisone and aldosterone in cell culture supernatants. The method enabled us to study HSD11B2 activity in two different cell types and will support future experiments investigating mechanisms of target organ damage in conditions of glucocorticoid and mineralocorticoid excess.

Cold mediates maize root hair developmental plasticity via epidermis-specific transcriptomic responses.

Cold stress during early development limits maize (Zea mays L.) production in temperate zones. Low temperatures restrict root growth and reprogram gene expression. Here, we provide a systematic transcriptomic landscape of maize primary roots, their tissues, and cell types in response to cold stress. The epidermis exhibited a unique transcriptomic cold response, and genes involved in root hair formation were dynamically regulated in this cell type by cold. Consequently, activation of genes involved in root hair tip growth contributed to root hair recovery under moderate cold conditions. The maize root hair defective mutants roothair defective 5 (rth5) and roothair defective 6 (rth6) displayed enhanced cold tolerance with respect to primary root elongation. Furthermore, dehydration response element-binding protein 2.1 (dreb2.1) was the only member of the dreb subfamily of AP2/EREB transcription factor genes upregulated in primary root tissues and cell types but exclusively downregulated in root hairs upon cold stress. Plants overexpressing dreb2.1 significantly suppressed root hair elongation after moderate cold stress. Finally, the expression of rth3 was regulated by dreb2.1 under cold conditions, while rth6 transcription was regulated by dreb2.1 irrespective of the temperature regime. We demonstrated that dreb2.1 negatively regulates root hair plasticity at low temperatures by coordinating the expression of root hair defective genes in maize.

AiSEGcell: User-friendly deep learning-based segmentation of nuclei in transmitted light images.

Segmentation is required to quantify cellular structures in microscopic images. This typically requires their fluorescent labeling. Convolutional neural networks (CNNs) can detect these structures also in only transmitted light images. This eliminates the need for transgenic or dye fluorescent labeling, frees up imaging channels, reduces phototoxicity and speeds up imaging. However, this approach currently requires optimized experimental conditions and computational specialists. Here, we introduce "aiSEGcell" a user-friendly CNN-based software to segment nuclei and cells in bright field images. We extensively evaluated it for nucleus segmentation in different primary cell types in 2D cultures from different imaging modalities in hand-curated published and novel imaging data sets. We provide this curated ground-truth data with 1.1 million nuclei in 20,000 images. aiSEGcell accurately segments nuclei from even challenging bright field images, very similar to manual segmentation. It retains biologically relevant information, e.g. for demanding quantification of noisy biosensors reporting signaling pathway activity dynamics. aiSEGcell is readily adaptable to new use cases with only 32 images required for retraining. aiSEGcell is accessible through both a command line, and a napari graphical user interface. It is agnostic to computational environments and does not require user expert coding experience.

Autonomous Oscillatory Mitochondrial Respiratory Activity: Results of a Systematic Analysis Show Heterogeneity in Different In Vitro-Synchronized Cancer Cells.

Circadian oscillations of several physiological and behavioral processes are an established process in all the organisms anticipating the geophysical changes recurring during the day. The time-keeping mechanism is controlled by a transcription translation feedback loop involving a set of well-characterized transcription factors. The synchronization of cells, controlled at the organismal level by a brain central clock, can be mimicked in vitro, pointing to the notion that all the cells are endowed with an autonomous time-keeping system. Metabolism undergoes circadian control, including the mitochondrial terminal catabolic pathways, culminating under aerobic conditions in the electron transfer to oxygen through the respiratory chain coupled to the ATP synthesis according to the oxidative phosphorylation chemiosmotic mechanism. In this study, we expanded upon previous isolated observations by utilizing multiple cell types, employing various synchronization protocols and different methodologies to measure mitochondrial oxygen consumption rates under conditions simulating various metabolic stressors. The results obtained clearly demonstrate that mitochondrial respiratory activity undergoes rhythmic oscillations in all tested cell types, regardless of their individual respiratory proficiency, indicating a phenomenon that can be generalized. However, notably, while primary cell types exhibited similar rhythmic respiratory profiles, cancer-derived cell lines displayed highly heterogeneous rhythmic changes. This observation confirms on the one hand the dysregulation of the circadian control of the oxidative metabolism observed in cancer, likely contributing to its development, and on the other hand underscores the necessity of personalized chronotherapy, which necessitates a detailed characterization of the cancer chronotype.

Initiator cell death event induced by SARS-CoV-2 in the human airway epithelium.

Virus-induced cell death is a key contributor to COVID-19 pathology. Cell death induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is well studied in myeloid cells but less in its primary host cell type, angiotensin-converting enzyme 2 (ACE2)-expressing human airway epithelia (HAE). SARS-CoV-2 induces apoptosis, necroptosis, and pyroptosis in HAE organotypic cultures. Single-cell and limiting-dilution analysis revealed that necroptosis is the primary cell death event in infected cells, whereas uninfected bystanders undergo apoptosis, and pyroptosis occurs later during infection. Mechanistically, necroptosis is induced by viral Z-RNA binding to Z-DNA-binding protein 1 (ZBP1) in HAE and lung tissues from patients with COVID-19. The Delta (B.1.617.2) variant, which causes more severe disease than Omicron (B1.1.529) in humans, is associated with orders of magnitude-greater Z-RNA/ZBP1 interactions, necroptosis, and disease severity in animal models. Thus, Delta induces robust ZBP1-mediated necroptosis and more disease severity.

Mitochondrial Quality Control in Ovarian Function: From Mechanisms to Therapeutic Strategies.

Mitochondrial quality control plays a critical role in cytogenetic development by regulating various cell-death pathways and modulating the release of reactive oxygen species (ROS). Dysregulated mitochondrial quality control can lead to a broad spectrum of diseases, including reproductive disorders, particularly female infertility. Ovarian insufficiency is a significant contributor to female infertility, given its high prevalence, complex pathogenesis, and profound impact on women's health. Understanding the pathogenesis of ovarian insufficiency and devising treatment strategies based on this understanding are crucial. Oocytes and granulosa cells (GCs) are the primary ovarian cell types, with GCs regulated by oocytes, fulfilling their specific energy requirements prior to ovulation. Dysregulation of mitochondrial quality control through gene knockout or external stimuli can precipitate apoptosis, inflammatory responses, or ferroptosis in both oocytes and GCs, exacerbating ovarian insufficiency. This review aimed to delineate the regulatory mechanisms of mitochondrial quality control in GCs and oocytes during ovarian development. This study highlights the adverse consequences of dysregulated mitochondrial quality control on GCs and oocyte development and proposes therapeutic interventions for ovarian insufficiency based on mitochondrial quality control. These insights provide a foundation for future clinical approaches for treating ovarian insufficiency.

Single-Nucleus Transcriptomic Atlas of Human Pericoronary Epicardial Adipose Tissue in Normal and Pathological Conditions.

Pericoronary epicardial adipose tissue (EAT) is a unique visceral fat depot that surrounds the adventitia of the coronary arteries without any anatomic barrier. Clinical studies have demonstrated the association between EAT volume and increased risks for coronary artery disease (CAD). However, the cellular and molecular mechanisms underlying the association remain elusive. We performed single-nucleus RNA sequencing on pericoronary EAT samples collected from 3 groups of subjects: patients undergoing coronary bypass surgery for severe CAD (n=8), patients with CAD with concomitant type 2 diabetes (n=8), and patients with valvular diseases but without concomitant CAD and type 2 diabetes as the control group (n=8). Comparative analyses were performed among groups, including cellular compositional analysis, cell type-resolved transcriptomic changes, gene coexpression network analysis, and intercellular communication analysis. Immunofluorescence staining was performed to confirm the presence of CAD-associated subclusters. Unsupervised clustering of 73 386 nuclei identified 15 clusters, encompassing all known cell types in the adipose tissue. Distinct subpopulations were identified within primary cell types, including adipocytes, adipose stem and progenitor cells, and macrophages. CD83high macrophages and FOSBhigh adipocytes were significantly expanded in CAD. In comparison to normal controls, both disease groups exhibited dysregulated pathways and altered secretome in the primary cell types. Nevertheless, minimal differences were noted between the disease groups in terms of cellular composition and transcriptome. In addition, our data highlight a potential interplay between dysregulated circadian clock and altered physiological functions in adipocytes of pericoronary EAT. ANXA1 (annexin A1) and SEMA3B (semaphorin 3B) were identified as important adipokines potentially involved in functional changes of pericoronary EAT and CAD pathogenesis. We built a complete single-nucleus transcriptomic atlas of human pericoronary EAT in normal and diseased conditions of CAD. Our study lays the foundation for developing novel therapeutic strategies for treating CAD by targeting and modifying pericoronary EAT functions.

An efficient method for immortalizing mouse embryonic fibroblasts.

Mouse embryonic fibroblasts (MEFs) derived from genetically modified mice are a valuable resource for studying gene function and regulation. The MEF system can also be combined with rescue studies to characterize the function of mutant genes/proteins, such as disease-causing variants. However, primary MEFs undergo senescence soon after isolation and passaging, making long-term genetic manipulations difficult. Previously described methods for MEF immortalization are often inefficient or alter the physiological properties of the cells. Here, we describe an optimized protocol for immortalizing MEFs via CRISPR-mediated deletion of the Tp53 gene. This method is highly efficient and consistently generates immortalized MEFs, or iMEFs, within 14 days. Importantly, iMEFs closely resemble the parent cell populations, and individual iMEFs can be cloned and expanded for subsequent genetic manipulation and characterization. We envision that this protocol can be adopted to immortalize other mouse primary cell types.

Regulation of intracellular cAMP levels in osteocytes by mechano-sensitive focal adhesion kinase via PDE8A.

Osteocytes are the primary mechano-sensitive cell type in bone. Mechanical loading is sensed across the dendritic projections of osteocytes leading to transient reductions in focal adhesion kinase (FAK) activity. Knowledge regarding the signaling pathways downstream of FAK in osteocytes is incomplete. We performed tyrosine-focused phospho-proteomic profiling in osteocyte-like Ocy454 cells to identify FAK substrates. Gsα, parathyroid hormone receptor (PTH1R), and phosphodiesterase 8A (PDE8A), all proteins associated with cAMP signaling, were found as potential FAK targets based on their reduced tyrosine phosphorylation in both FAK- deficient or FAK inhibitor treated cells. Real time monitoring of intracellular cAMP levels revealed that FAK pharmacologic inhibition or gene deletion increased basal and GPCR ligand-stimulated cAMP levels and downstream phosphorylation of protein kinase A substrates. Mutating FAK phospho-acceptor sites in Gsα and PTH1R had no effect on PTH- or FAK inhibitor-stimulated cAMP levels. Since FAK inhibitor treatment augmented cAMP levels even in the presence of forskolin, we focused on potential FAK substrates downstream of cAMP generation. Indeed, PDE8A inhibition mimicked FAK inhibition at the level of increased cAMP, PKA activity, and expression of cAMP-regulated target genes. In vitro kinase assay showed that PDE8A is directly phosphorylated by FAK while immunoprecipitation assays revealed intracellular association between FAK and PDE8A. Thus, FAK inhibition in osteocytes acts synergistically with signals that activate adenylate cyclase to increase intracellular cAMP. Mechanically-regulated FAK can modulate intracellular cAMP levels via effects on PDE8A. These data suggest a novel signal transduction mechanism that mediates crosstalk between mechanical and cAMP-linked hormonal signaling in osteocytes.

Visualizing lipid nanoparticle trafficking for mRNA vaccine delivery in non-human primates.

mRNA delivered using lipid nanoparticles (LNPs) has become an important subunit vaccine modality, but mechanisms of action for mRNA vaccines remain incompletely understood. Here, we synthesized a metal chelator-lipid conjugate enabling positron emission tomography (PET) tracer labeling of LNP/mRNA vaccines for quantitative visualization of vaccine trafficking in live non-human primates (NHPs). Following i.m. injection, we observed LNPs distributing through injected muscle tissue, simultaneous with rapid trafficking to draining lymph nodes (dLNs). Deltoid injection of LNPs mimicking human vaccine administration led to stochastic LNP delivery to 3 different sets of dLNs. LNP uptake in dLNs was confirmed by histology, and cellular analysis of tissues via flow cytometry identified antigen-presenting cells as the primary cell type responsible for early LNP uptake and mRNA translation. These results provide insights into the biodistribution of mRNA vaccines administered at clinically relevant doses, injection volumes, and injection sites in an important large animal model for vaccine development.

Integration of transcriptomics and spatial biology analyses reveals Galactomyces ferment filtrate promotes epidermal interconnectivity via induction of keratinocyte differentiation, proliferation and cellular bioenergetics.

Human skin is the first line of defence from environmental factors such as solar radiation and is susceptible to premature ageing, including a disruption in epidermal differentiation and homeostasis. We evaluated the impact of a Galactomyces Ferment Filtrate (GFF) on epidermal differentiation and response to oxidative stress. We used transcriptomics, both spatial and traditional, to assess the impact of GFF on epidermal biology and homeostasis in keratinocytes (primary or immortalized) and in exvivo skin explant tissue. The effect of GFF on cell adhesion rates, cellular ATP levels and proliferation rates were quantitated. Oxidative phosphorylation and glycolytic rates were measured under normal and stress-induced conditions. Transcriptomics from keratinocytes and exvivo skin explants from multiple donors show GFF induces keratinocyte differentiation, skin barrier development and cell adhesion while simultaneously repressing cellular stress and inflammatory related processes. Spatial transcriptomics profiling of exvivo skin indicated basal keratinocytes at the epidermal-dermal junction and cornifying keratinocytes in the top layer of the epidermis as the primary cell types influenced by GFF treatment. Additionally, GFF significantly increases crosstalk between suprabasal and basal keratinocytes. To support these findings, we show that GFF can significantly increase cell adhesion and proliferation in keratinocytes. GFF also protected overall cellular bioenergetics under metabolic or oxidative stress conditions. Our findings provide novel insights into cellular differences and epidermal spatial localization in response to GFF, supporting previous findings that this filtrate has a significant impact on epidermal biology and homeostasis, particularly on spatially defined crosstalk. We propose that GFF can help maintain epidermal health by enhancing keratinocyte crosstalk and differentiation/proliferation balance as well as promoting an enhanced response to stress.

Image Analysis Using the Fluorescence Imaging of Nuclear Staining (FINS) Algorithm.

Finding appropriate image analysis techniques for a particular purpose can be difficult. In the context of the analysis of immunocytochemistry images, where the key information lies in the number of nuclei containing co-localised fluorescent signals from a marker of interest, researchers often opt to use manual counting techniques because of the paucity of available tools. Here, we present the development and validation of the Fluorescence Imaging of Nuclear Staining (FINS) algorithm for the quantification of fluorescent signals from immunocytochemically stained cells. The FINS algorithm is based on a variational segmentation of the nuclear stain channel and an iterative thresholding procedure to count co-localised fluorescent signals from nuclear proteins in other channels. We present experimental results comparing the FINS algorithm to the manual counts of seven researchers across a dataset of three human primary cell types which are immunocytochemically stained for a nuclear marker (DAPI), a biomarker of cellular proliferation (Ki67), and a biomarker of DNA damage (γH2AX). The quantitative performance of the algorithm is analysed in terms of consistency with the manual count data and acquisition time. The FINS algorithm produces data consistent with that achieved by manual counting but improves the process by reducing subjectivity and time. The algorithm is simple to use, based on software that is omnipresent in academia, and allows data review with its simple, intuitive user interface. We hope that, as the FINS tool is open-source and is custom-built for this specific application, it will streamline the analysis of immunocytochemical images.

Mechanisms modulating foam cell formation in the arterial intima: exploring new therapeutic opportunities in atherosclerosis.

In recent years, the role of macrophages as the primary cell type contributing to foam cell formation and atheroma plaque development has been widely acknowledged. However, it has been long recognized that diffuse intimal thickening (DIM), which precedes the formation of early fatty streaks in humans, primarily consists of lipid-loaded smooth muscle cells (SMCs) and their secreted proteoglycans. Recent studies have further supported the notion that SMCs constitute the majority of foam cells in advanced atherosclerotic plaques. Given that SMCs are a major component of the vascular wall, they serve as a significant source of microvesicles and exosomes, which have the potential to regulate the physiology of other vascular cells. Notably, more than half of the foam cells present in atherosclerotic lesions are of SMC origin. In this review, we describe several mechanisms underlying the formation of intimal foam-like cells in atherosclerotic plaques. Based on these mechanisms, we discuss novel therapeutic approaches that have been developed to regulate the generation of intimal foam-like cells. These innovative strategies hold promise for improving the management of atherosclerosis in the near future.

Establishment of a Four‐Cell In Vitro Blood‐Brain Barrier Model With Human Primary Brain Cells

AbstractThe blood‐brain barrier (BBB) constitutes a crucial protective anatomical layer with a microenvironment that tightly controls material transit. Constructing an in vitro BBB model to replicate in vivo features requires the sequential layering of constituent cell types. Maintaining heightened integrity in the observed tight junctions during both the establishment and post‐experiment phases is crucial to the success of these models. We have developed an in vitro BBB model that replicates the cellular composition and spatial orientation of in vivo BBB observed in humans. The experiment includes comprehensive procedures and steps aimed at enhancing the integration of the four‐cell model. Departing from conventional in vitro BBB models, our methodology eliminates the necessity for pre‐coated plates to facilitate cell adhesion, thereby improving cell visualization throughout the procedure. An in‐house coating strategy and a simple yet effective approach significantly reduce costs and provides superior imaging of cells and corresponding tight junction protein expression. Also, our BBB model includes all four primary cell types that are structural parts of the human BBB. With its innovative and user‐friendly features, our in‐house optimized in vitro four‐cell‐based BBB model showcases novel methodology and provides a promising experimental platform for drug screening processes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Coating and culture systemBasic Protocol 2: Cell seeding and Transwell insert handlingBasic Protocol 3: Assessment of model functionality

#2485 CellMatchR—RNA-seq based matching of kidney cell lines to kidney reference cell types

Abstract Background and Aims Cultured kidney cell lines are broadly used to model the physiology and pathophysiology of the kidney. Due to immortalization, passaging and culture conditions, a cell line's gene expression profile might differ greatly from the primary cells it was originally derived from. This might be further enhanced by experimental treatments with compounds or genetic modifications. It is therefore a relevant question if a given cell culture system is still the appropriate model for a certain disease or scientific investigations. Reasoning that RNA sequencing (RNA-seq) based transcriptomes are generated as part of many experimental setups and hence may be used to address this question, we developed and tested an RNA-seq based approach, CellMatchR. This approach matches kidney cell lines, primary cells and even tissue specimen to known kidney reference cell types to determine which primary kidney cell type is the most similar and to what degree global gene expression or the expression of selected marker genes differs. Method CellMatchR uses published murine and human kidney single cell- and tubule-level transcriptomic datasets from healthy mice and human donors as references. Single cell datasets were further processed to pseudobulk references for each of the contained cell types. Then RNA-seq data from cell lines or tissues of interest (test data) was compared to the reference (pseudo)bulk data with Spearman correlations of gene counts-per-million values (CPM) or Euclidean distance using log-transformed CPM values. Both approaches were systematically tested for various combinations of test and reference datasets across different species and using global gene expression (i.e. all genes measured in reference and test datasets) and a set of 315 manually curated, kidney cell type marker genes. Results We sequentially used different kidney tissue types, primary cells and cell lines as positive controls and matched them to our reference datasets. Spearman correlations of gene expression rankings showed the highest correlation coefficients with biologically correct reference cell types (examples in Fig. 1A-C). We found Spearman correlations to be superior to Euclidean distances as method of comparison. Analyses based on global gene expression compared to our curated set of 315 kidney cell type marker genes yielded similar results. Notably, correlation coefficients were higher and showed less variation between reference cell types when using the global gene set. Matchings across species, i.e. using murine test and human reference data or vice versa still yielded mostly correct results. However, correlation coefficients were generally lower (i.e. rho = 0.9 vs. 0.6) and varied more across reference datasets if comparisons were performed across species. Using published RNA-seq data for different cell lines of the proximal tubule (e.g. human HK-2 and opossum OKH cells) we observed low similarities with proximal tubule cells (Fig. 1D), whereas two tested cell lines of the collecting duct (mIMCD-3 and mpkCCD cells) plausibly showed the highest similarity to medullary cell types like collecting duct and Loop of Henle cells. The most similar reference cell types did not change in mIMCD-3 cells when kept in 2-dimensional vs. 3-dimensional culture conditions, and in mpkCCD cells when the osmolality of the culture medium was changed from 300 to 600 mosmol/kg. Also, a Pkd1 knockout in mIMCD-3 cells did not change the most similar reference cell type in our analyses (Fig. 1E and F). Conclusion Our CellMatchR approach uses publicly available kidney single cell and bulk RNA-seq datasets and combines these with simple, computationally fast yet effective statistical methods to determine similarities of kidney cell lines and tissue samples to reference cell types of interest. It can easily be implemented by trained users or integrated into online resources for usage with a visual interface. It relies on RNA-seq data from the cell lines of interest and hence presents a feasible complementary method to check the general similarity of cell culture models to kidney cell types and assess their stability across experimental conditions.