Apical Marker Research Articles

Uncovering the role of GPR39 in the kidney

G protein-coupled receptors (GPCRs) constitute the largest class of proteins in the mammalian genome. Upon activation by a specific ligand, these receptors initiate downstream cellular responses that regulate various physiological processes. One such receptor under investigation in our lab is the ghrelin family receptor GPR39. Unlike its relatives, GPR39 does not respond to peptide hormones or neuropeptides. Initial studies suggested zinc as the endogenous ligand, but recent research indicates that zinc functions as an allosteric potentiator for another unidentified endogenous ligand. To date, synthetic agonists for GPR39 have revealed functions for GPR39 in the heart, bone, skin, pancreas, and gastrointestinal tract. However, GPR39’s role in renal physiology is currently unknown, despite relatively high kidney expression. To address this gap in knowledge, we first worked to localize GPR39 within the kidney. As a reliable antibody for GPR39 is not available, we utilized a combination of RNAScope (Gpr39) and immunofluorescence (AQP2). We find that GPR39 is expressed in the renal collecting duct, with the highest expression in AQP2-positive cells (principal cells) in the inner medullary collecting duct, and lesser expression in the cortical collecting duct. To determine if GPR39 is expressed apically or basolaterally, we then cloned GPR39 with a C-terminal EGFP tag and observed basolateral targeting of GPR39 in polarized MDCK cells grown on filters. In order to query the function of GPR39 in principal cells, we used murine principal kidney cortical collecting duct cells (mpkCCD). As others have shown, we find that mpkCCD express and traffc AQP2 to the apical plasma membrane only in the presence of the vasopressin analog dDAVP. Furthermore, we observe that treatment of mpkCCD with the GPR39-specific agonist cpd1324 induces internalization of AQP2 into cytoplasmic vesicles, even in the continued presence of dDAVP. Under vehicle conditions (dDAVP + vehicle), 80.9±7.7% (mean±SD) of AQP2 stain colocalized with an apical membrane marker (WGA staining). In contrast, when mpkCCD cells were co-treated ddAVP+cpd1324, only 58.3±6.02% of AQP2 stain colocalized with the apical membrane (p < 0.001, t-test). These results indicate that GPR39 activation may act to antagonize vasopressin-induced AQP2 traffcking in mpkCCD cells. NHLBIT32HL007534 (MK) and American Heart Association Established Investigator Award (JLP). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Biomechanical Characterization of Retinal Pigment Epitheliums Derived from hPSCs Using Atomic Force Microscopy

The retinal pigment epithelium (RPE), a multifunctional cell monolayer located at the back of the eye, plays a crucial role in the survival and homeostasis of photoreceptors. Dysfunction or death of RPE cells leads to retinal degeneration and subsequent vision loss, such as in Age-related macular degeneration and some forms of Retinitis Pigmentosa. Therefore, regenerative medicine that aims to replace RPE cells by new cells obtained from the differentiation of human pluripotent stem cells, is the focus of intensive research. However, despite their critical interest in therapy, there is a lack of biomechanical RPE surface description. Such biomechanical properties are tightly related to their functions. Herein, we used atomic force microscopy (AFM) to analyze both the structural and mechanical properties of RPEs obtained from four cell lines and at different stages of epithelial formation. To characterize epitheliums, we used apical markers in immunofluorescence and showed the increase of transepithelial resistance, as well as the ability to secrete cytokines with an apico-basal polarity. Then, we used AFM to scan the apical surface of living or fixed RPE cells. We show that RPE monolayers underwent softening of apical cell center as well as stiffening of cell borders over epithelial formation. We also observed apical protrusions that depend on actin network, suggesting the formation of microvilli at the surface of RPE epitheliums. These RPE cell characteristics are essential for their functions into the retina and AFM studies may improve the characterization of the RPE epithelium suitable for cell therapy.Graphical

Steroid hormone signaling synchronizes cell migration machinery, adhesion and polarity to direct collective movement.

Migratory cells - either individually or in cohesive groups - are critical for spatiotemporally regulated processes such as embryonic development and wound healing. Their dysregulation is the underlying cause of formidable health problems such as congenital abnormalities and metastatic cancers. Border cell behavior during Drosophila oogenesis provides an effective model to study temporally regulated, collective cell migration in vivo. Developmental timing in flies is primarily controlled by the steroid hormone ecdysone, which acts through a well-conserved, nuclear hormone receptor complex. Ecdysone signaling determines the timing of border cell migration, but the molecular mechanisms governing this remain obscure. We found that border cell clusters expressing a dominant-negative form of ecdysone receptor extended ineffective protrusions. Additionally, these clusters had aberrant spatial distributions of E-cadherin (E-cad), apical domain markers and activated myosin that did not overlap. Remediating their expression or activity individually in clusters mutant for ecdysone signaling did not restore proper migration. We propose that ecdysone signaling synchronizes the functional distribution of E-cadherin, atypical protein kinase C (aPKC), Discs large (Dlg1) and activated myosin post-transcriptionally to coordinate adhesion, polarity and contractility and temporally control collective cell migration.

Telencephalic eversion in embryos and early larvae of four teleost species.

The telencephalon of ray-finned fishes undergoes eversion, which is very different to the evagination that occurs in most other vertebrates. Ventricle morphogenesis is key to build an everted telencephalon. Thus, here we use the apical marker zona occludens 1 to understand ventricle morphology, extension of the tela choroidea and the eversion process during early telencephalon development of four teleost species: giant danio (Devario aequipinnatus), blind cavefish (Astyanax mexicanus), medaka (Oryzias latipes), and paradise fish (Macroposus opercularis). In addition, by using immunohistochemistry against tubulin and calcium-binding proteins, we analyze the general morphology of the telencephalon, showing changes in the location and extension of the olfactory bulb and other telencephalic regions from 2 to 5 days of development. We also analyze the impact of abnormal eye and telencephalon morphogenesis on eversion, showing that cyclops mutants do undergo eversion despite very dramatic abnormal eye morphology. We discuss how the formation of the telencephalic ventricle in teleost fish, with its characteristic shape, is a crucial event during eversion.

Epithelial polarity-driven membrane separation but not cavitation regulates lumen formation of rat eccrine sweat glands

BackgroundEach eccrine sweat gland (ESG) is a single-tubular structure with a central lumen, and the formation of hollow lumen in the initial solid cell mass is a key developmental process. To date, there are no reports on the mechanism of native ESG lumen formation. MethodsTo investigate the lumen morphogenesis and the lumen formation mechanisms of Sprague-Dawley (SD) rat ESGs, SD rat hind-footpads at E20.5, P1–P5, P7, P9, P12, P21, P28 and P56 were obtained. The lumen morphogenesis of ESGs was examined by HE staining and immunofluorescence staining for polarity markers. The possible mechanisms of lumen formation were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay and autophagy marker LC3B immunofluorescence staining, and further explored by ouabain intervention experiment. ResultsIn SD rat ESGs, the microlumen was formed at P1, and the small intact lumen with apical-basal polarity appeared at P3. The expression of apical marker F-actin, basal marker Laminin, basolateral marker E-cadherin was consistent with the timing of lumen formation of SD rat ESGs. During rat ESG development, apoptosis and autophagy were not detected. However, inhibition of Na+-K+-ATPase (NKA) with ouabain resulted in decreased lumen size, although neither the timing of lumen formation nor the expression of polarity proteins was altered. ConclusionsEpithelial polarity-driven membrane separation but not cavitation regulates lumen formation of SD rat ESGs. NKA-regulated fluid accumulation drives lumen expansion.

P-103 Three-dimensional oviductal organoid model to study spermatozoa-oviduct interaction in humans

Abstract Study question Is it possible to establish an in vitro model that recapitulating human oviduct to investigate sperm-oviduct interaction Summary answer Three-dimensional (3D) oviductal organoid model was established from either primary human oviductal tissues or immortalized human OE-E6/67 cell line, based on our protocol. What is known already The sperm reservoir in oviduct is formed to maintain the fertilizing capacity of spermatozoa until ovulation by the binding of sperm to the epithelial lining of the oviduct, which is also significant to select sperm with normal chromatin structure and superior fertilization ability. The regulation and mechanism of sperm reservoir formation in human is still unknown, mainly due to the ethical issue and technical difficulty in obtaining enough human oviduct. We previously demonstrated that sperm fucosyltransferase-5 (sFUT5), a carbohydrate-binding protein on spermatozoa, is responsible for spermatozoa-oviduct interaction in humans. Study design, size, duration Human oviductal epithelial cell line immortalized by HPV 16 E6/E7 open reading frame by retroviral expression system was employed to establish the co-culture model. Fresh human samples (human spermatozoa,N = 30; 1-2 cm primary oviductal tissue,N = 15) were collected in hospital with signed consent. Fluorescent pre-labelled sperm were co-cultured with organoids for 2 hours. At least three replicates were conducted for each assay. Participants/materials, setting, methods Morphological characterization and markers expressions of the organoid were determined by immunohistochemistry and confocal microscopy analysis. The sperm binding capacity of the oviductal organoid was studied by co-culture system. Sperm chromatin structure of the organoid-bound sperm was assessed by flow cytometry-based TUNEL assay. Affinity chromatography followed by nano-liquid chromatography–mass spectrometry was used to identify the sFUT5-binding proteins on ovidutal epithelial cells (OECs) and their expressions on OECs were validated by immunostaining/flow cytometry. Main results and the role of chance The organoid exhibited a hollow structure surrounded by a single layer of OECs. Immunohistochemistry analysis revealed the high expressions of epithelial marker E-cadherin (basolateral membrane), secretory marker pair-box 8 (PAX8) and proliferative marker Ki67. Expression of ciliated cell marker, acetylated tubulin (AcTUB) can also be detected. The modification of culture environment successfully reversed the OEC organoids polarity as demonstrated by immunostaining using antibodies against ZO-1 (apical marker) and E-cadherin (basolateral marker), making the organoid accessible during the co-culture. These apical-out OEC organoids have significantly higher sperm binding capacities than their basal-out counterparts. Furthermore, OEC organoid-bound spermatozoa possessed more intact DNA than the unbound spermatozoa. The results indicate the relevance of using OEC organoid-spermatozoa co-culture model as a tool to assess spermatozoa-oviduct interaction in humans. By using immuno-affinity chromatography and mass spectrometry analysis, cell adhesion molecule 4 (CADM4) was then identified as a potential sFUT5-interacting protein on OECs. This result was further supported by flow cytometry and immunofluorescent staining. 3-D oviductal organoid model demonstrates profound potential for in vitro investigation of human oviduct physiology. Limitations, reasons for caution Our 3D model may not fully represent the entire cellular complexity and tubular architecture of the oviductal epithelium. Further investigation on the sperm morphology and DNA integrity and analysis on organoid transcriptome will enhance our understanding on oviduct in human reproduction. Wider implications of the findings The results from co-culture may give a clue to future study on whether oviductal organoid can be applied clinically for functional sperm selection in in vitro fertilization. Our apical-out organoid model may also be applied to study tubal ectopic pregnency. Trial registration number not applicable

A morphogenetic wave in the chick embryo lateral mesoderm generates mesenchymal-epithelial transition through a 3D-rosette intermediate.

Formation of epithelia through mesenchymal-epithelial transition (MET) is essential for embryonic development and for many physiological and pathological processes. This study investigates MET invivo in the chick embryo lateral mesoderm, where a multilayered mesenchyme transforms into two parallel epithelial sheets that constitute the coelomic lining of the embryonic body cavity. Prior to MET initiation, mesenchymal cells exhibit non-polarized distribution of multiple polarity markers, albeit not aPKC. We identified an epithelializing wave that sweeps across the lateral mesoderm, the wavefront of which is characterized by the accumulation of basal fibronectin and a network of 3D rosettes composed of polarized, wedge-shaped cells surrounding a central focus of apical markers, now including aPKC. Initiation of the MET process is dependent on extracellular matrix-integrin signaling acting through focal adhesion kinase and talin, whereas progression through the rosette phase requires aPKC function. We present a stepwise model for MET, comprising polarization, 3D-rosette, and epithelialization stages.

Diagnostic and Prognostic Role of Prostate-Specific Antigen Immunologic Staining in Prostate Cancer

Background: Prostatic adenocarcinoma is the second leading cause of death in males due to cancer. The prostate-specific antigen (PSA) serum level is the first marker for screening such patients. Objectives: The present study evaluated the comparison between the degree of staining of the PSA marker and some factors in the prognosis of prostate cancer. Methods: The current cross-sectional study was performed on 97 tissue blocks from patients with prostate adenocarcinoma collected in 2019 - 2020 from the Pathology Department of Golestan Ahvaz Hospital archives. Clinical information such as age, tumor size, grade of tumor, lymph node involvement, and vascular and perineural invasion was extracted from patients' pathology reports and recorded on a checklist. Then, PSA apical and intensity staining was studied using immunohistochemistry. Results: The mean age of patients was 72.76 ± 8.19 years. The mean serum PSA level was 39.2 ± 8.90. In the present study, the highest PSA staining intensity in the samples was related to grade 1 with a frequency of 39 (40.2%), and then it was related to grade 4 with a frequency of 38 (39.2%), while the lowest frequency of staining intensity was related to grade 2 was with a frequency of 9 (9.3%). Regarding PSA apical staining, 58 samples (59.8%) were positive, and 39 (40.2%) were negative. A statistically significant comparison was shown between Gleason scoring and PSA apical staining (P < 0.001). The results indicate that increasing Gleason scoring decreases the staining intensity, and this correlation is significant (P < 0.05). The mean age in samples with positive staining of apical marker PSA is lower than in samples with negative staining of apical marker PSA but is not statistically significant (P = 0.38). In contrast, the serum level of the PSA marker is statistically significantly higher in samples with positive staining of apical marker PSA than in samples with negative staining of PSA (P < 0.001). According to the Spearman correlation coefficient test, the intensity of staining of the PSA marker decreases with age, and this correlation is significant (P = 0.032). However, according to the Spearman correlation coefficient test, staining intensity increases with increasing serum PSA level, and this correlation is statistically significant (P < 0.001). Conclusions: It is also suggested that in future studies with a larger sample size, the comparison between apical marker expression and PSA marker staining intensity with clinicopathological factors, survival and mortality rates, response to treatment, etc., will be evaluated.

Matrix Stiffness Influences Tubular Formation in Renal Tissue Engineering

Tubular structures contribute to essential organ functions. Therefore, controlling tubulogenesis is essential for bottom-up tissue engineering approaches. Tissue engineering strategies to form tubular structures utilize extracellular matrix (ECM) components and micropatterned molds. To improve the tubular formation rate, we studied the substrate stiffness’s influence on the tubulogenesis of murine inner medullary collecting duct (mIMCD) cells. mIMCD cells were seeded in micropatterned molds with different compositions of polydimethylsiloxane (PDMS) (1:5, 1:10, 1:15, 1:20, 1:30) and agarose (1%, 2%, 3% 5%). We established the Young’s modulus of the PDMS and agarose substrates and determined the ideal substrate stiffness for tube formation to be between 277 kPa and 2610 kPa. Within our parameters, optimal tube formation was observed at 439.9 kPa, a value similar to the Young’s Modulus found in the basement membrane of the murine renal tubular compartment. We also found that different substrate concentrations of agarose or PDMS are associated with different expression levels of the apical polarization marker Zonula occludens 1 (ZO-1) in the generated tubular structures. In addition to the substrate stiffness, we observed that the tube formation differed based on the substrate material, with agarose showing a generally greater tube formation rate. While previous research demonstrated that ECM stiffness influences cellular behavior towards tube formation, our results suggest that the stiffness of the substrate influences tubular formation independently of the ECM.

BDE-209 disrupted the blood-testis barrier integrity by inhibiting estrogen receptorα signaling pathway in Sprague-Dawley rats.

Deca brominated diphenyl ether (BDE-209) is a widely used flame retardant with endocrine-disrupting activity which reportedly caused sperm quality decline and damaged blood-testis barrier (BTB). However, whether BDE-209 exposure led to BTB integrity dysfunction through affecting microtubule cytoskeletal organization and junctions was not well-elucidated. This study aimed to investigate the role of estrogen receptor α (ERα) in BDE-209-mediated perturbation of BTB integrity. Male rats and primary culture Sertoli cells were co-treated with BDE-209 and propylpyrazoletriol (PPT). The data demonstrated that BDE-209 impaired BTB integrity by reducing crucial tight junction-related proteins with ZO-1 and Occludin. Furthermore, the data suggested that BDE-209 diminished the apical ectoplasmic specialization markers with Eps8 and Formin1. In addition, BDE-209 damaged BTB ultrastructure including tight junctions and ectoplasmic specialization structures with broken tight junctions and the absence of actin microfilaments. Further experiments revealed that ERα was triggered in BDE-209-treated Sertoli cells. Unexpectedly, we found that PPT rescued BDE-209-mediated disruption of BTB integrity including tight junction and apical ectoplasmic specialization by activating ERα in Sertoli cells. Taken together, these findings indicated that intratesticular BDE-209 exposure perturbed BTB integrity and destroyed BTB structure by blocking ERα pathway. Our findings provide a new therapeutic target for male reproductive dysfunction.

Sequential changes in cellular properties accompanying amniote somite formation.

Somites are transient structures derived from the pre-somitic mesoderm (PSM), involving mesenchyme-to-epithelial transition (MET) where the cells change their shape and polarize. Using Scanning electron microscopy (SEM), immunocytochemistry and confocal microscopy, we study the progression of these events along the tail-to-head axis of the embryo, which mirrors the progression of somitogenesis (younger cells located more caudally). SEM revealed that PSM epithelialization is a gradual process, which begins much earlier than previously thought, starting with the dorsalmost cells, then the medial ones, and then, simultaneously, the ventral and lateral cells, before a somite fully separates from the PSM. The core (internal) cells of the PSM and somites never epithelialize, which suggests that the core cells could be 'trapped' within the somitocoele after cells at the surfaces of the PSM undergo MET. Three-dimensional imaging of the distribution of the cell polarity markers PKCζ, PAR3, ZO1, the Golgi marker GM130 and the apical marker N-cadherin reveal that the pattern of polarization is distinctive for each marker and for each surface of the PSM, but the order of these events is not the same as the progression of cell elongation. These observations challenge some assumptions underlying existing models of somite formation.

An Adaptable Physiological Model of Endocytic Megalin Trafficking in Opossum Kidney Cells and Mouse Kidney Proximal Tubule.

The cells that comprise the proximal tubule (PT) are specialized for high-capacity apical endocytosis necessary to maintain a protein-free urine. Filtered proteins are reclaimed via receptor-mediated endocytosis facilitated by the multiligand receptors megalin and cubilin. Despite the importance of this pathway, we lack a detailed understanding of megalin trafficking kinetics and how they are regulated. Here, we utilized biochemical and quantitative imaging methods in a highly differentiated model of opossum kidney (OK) cells and in mouse kidney in vivo to develop mathematical models of megalin traffic. A preliminary model based on biochemically quantified kinetic parameters was refined by colocalization of megalin with individual apical endocytic compartment markers. Our model predicts that megalin is rapidly internalized, resulting in primarily intracellular distribution of the receptor at steady state. Moreover, our data show that early endosomes mature rapidly in PT cells and suggest that Rab11 is the primary mediator of apical recycling of megalin from maturing endocytic compartments. Apical recycling represents the rate-limiting component of endocytic traffic, suggesting that this step has the largest impact in determining the endocytic capacity of PT cells. Adaptation of our model to the S1 segment of mouse PT using colocalization data obtained in kidney sections confirms basic aspects of our model and suggests that our OK cell model largely recapitulates in vivo membrane trafficking kinetics. We provide a downloadable application that can be used to adapt our working parameters to further study how endocytic capacity of PT cells may be altered under normal and disease conditions.

Co-axial printing of convoluted proximal tubule for kidney disease modeling

Despite the increasing incidence of kidney-related diseases, we are still far from understanding the underlying mechanisms of these diseases and their progression. This lack of understanding is partly because of a poor replication of the diseases in vitro, limited to planar culture. Advancing towards three-dimensional models, hereby we propose coaxial printing to obtain microfibers containing a helical hollow microchannel. These recapitulate the architecture of the proximal tubule (PT), an important nephron segment often affected in kidney disorders. A stable gelatin/alginate-based ink was formulated to allow printability while maintaining structural properties. Fine-tuning of the composition, printing temperature and extrusion rate allowed for optimal ink viscosity that led to coiling of the microfiber’s inner channel. The printed microfibers exhibited prolonged structural stability (42 days) and cytocompatibility in culture. Healthy conditionally immortalized PT epithelial cells and a knockout cell model for cystinosis (CTNS -/-) were seeded to mimic two genotypes of PT. Upon culturing for 14 days, engineered PT showed homogenous cytoskeleton organization as indicated by staining for filamentous actin, barrier-formation and polarization with apical marker α-tubulin and basolateral marker Na+/K+-ATPase. Cell viability was slightly decreased upon prolonged culturing for 14 days, which was more pronounced in CTNS -/- microfibers. Finally, CTNS -/- cells showed reduced apical transport activity in the microfibers compared to healthy PT epithelial cells when looking at breast cancer resistance protein and multidrug resistance-associated protein 4. Engineered PT incorporated in a custom-designed microfluidic chip allowed to assess leak-tightness of the epithelium, which appeared less tight in CTNS -/- PT compared to healthy PT, in agreement with its in vivo phenotype. While we are still on the verge of patient-oriented medicine, this system holds great promise for further research in establishing advanced in vitro disease models.

Rab39 and its effector UACA regulate basolateral exosome release from polarized epithelial cells.

Exosomes are small extracellular vesicles that originate from the intraluminal vesicles of multivesicular bodies (MVBs). We previously reported that polarized Madin-Darby canine kidney (MDCK) epithelial cells secrete two types of exosomes, apical and basolateral exosomes, from different MVBs. However, how these MVBs are selectively targeted to the apical or basolateral membrane remained unknown. Here, we analyze members of the Rab family small GTPases and show that different sets of Rabs mediate asymmetrical exosome release. Rab27, the best-known regulator of MVB transport for exosome release, is specifically but partially involved in apical exosome release, and Rab37, a close homolog of Rab27, is an additional apical exosome regulator. By contrast, Rab39 functions as a specific regulator of basolateral exosome release. Mechanistically, Rab39 interacts with its effector UACA, and UACA then recruits Lyspersin, a component of BLOC-1-related complex (BORC). Our findings suggest that the Rab39-UACA-BORC complex specifically mediates basolateral exosome release.

MUC1 and Polarity Markers INADL and SCRIB Identify Salivary Ductal Cells

Current treatments for xerostomia/dry mouth are palliative and largely ineffective. A permanent clinical resolution is being developed to correct hyposalivation using implanted hydrogel-encapsulated salivary human stem/progenitor cells (hS/PCs) to restore functional salivary components and increase salivary flow. Pluripotent epithelial cell populations derived from hS/PCs, representing a basal stem cell population in tissue, can differentiate along either secretory acinar or fluid-transporting ductal lineages. To develop tissue-engineered salivary gland replacement tissues, it is critical to reliably identify cells in tissue and as they enter these alternative lineages. The secreted protein α-amylase, the transcription factor MIST1, and aquaporin-5 are typical markers for acinar cells, and K19 is the classical ductal marker in salivary tissue. We found that early ductal progenitors derived from hS/PCs do not express K19, and thus earlier markers were needed to distinguish these cells from acinar progenitors. Salivary ductal cells express distinct polarity complex proteins that we hypothesized could serve as lineage biomarkers to distinguish ductal cells from acinar cells in differentiating hS/PC populations. Based on our studies of primary salivary tissue, both parotid and submandibular glands, and differentiating hS/PCs, we conclude that the apical marker MUC1 along with the polarity markers INADL/PATJ and SCRIB reliably can identify ductal cells in salivary glands and in ductal progenitor populations of hS/PCs being used for salivary tissue engineering. Other markers of epithelial maturation, including E-cadherin, ZO-1, and partition complex component PAR3, are present in both ductal and acinar cells, where they can serve as general markers of differentiation but not lineage markers.

High-throughput production of liver parenchymal microtissues and enrichment of organ-specific functions in gelatin methacrylamide microenvironment.

Liver parenchymal microtissues (LPMTs) are three-dimensional (3D) aggregates of hepatocytes that recapitulate in vivo-likecellular assembly. They are considered as a valuable model to study drug metabolism, disease biology, and serve as ideal building blocks for liver tissue engineering. However, their integration into the mainstream drug screening process has been hindered due to the lack of simple, rapid techniques to produce a large number of uniform microtissues and preserve their structural-functional integrity over the long term. Here, we present a high-throughput methodology to produce LPMTsin a novel, economic, and reusable Hanging-drop Culture Chamber (HdCC). A drop-on-demand bioprinting approach was optimized to generate droplets of HepG2 cell suspension on a polyethylene terephthalate substrate. The substrates carrying droplets were placed inside a novel HdCC and incubated to obtain 1600 LPMTs having a size of 200-300 μm. Tissue size, cell viability, cellular arrangement and polarity, and insulin-mediated glucose uptake by LPMTs were analyzed. The microtissues were viable and exhibited an active response to insulin stimulation. Cells within the microtissue reorganized to form hepatic plate-like structures and expressed apical (Multidrug Resistance Protein 2 [MRP2]) and epithelial (Zonula Occludens 1 [ZO1]) markers. Further to maintain the structural integrity and enhance the functional capabilities, LPMTs were sandwiched within gelatin methacrylamide (GelMA) hydrogel and the liver-specific functions were monitored for 2 weeks. The results showed that the 3D structure of LPMTs in GelMA sandwich was maintained while the albumin secretion, urea synthesis, and cytochrome P450 activity were enhanced compared with LPMTs in suspension. In conclusion, this study presents a novel culture chamber for mass production of microtissues and a method for enhancing organ-specific functions of LPMTs in vitro.

A rodent thyroid-liver chip to capture thyroid toxicity on organ function level.

Endocrine disruption by environmental chemicals continues to be a concern for human safety. The rat, a widely used model organism in toxicology, is very sensitive to chemical-induced thyroid perturbation, e.g., histopathological alterations in thyroid tissue. Species differences in the susceptibility to thyroid perturbation lead to uncertainty in human safety risk assessments. Hazard identification and characterization of chemically induced thyroid perturbation would therefore benefit from in vitro models addressing different mechanisms of action in a single functional assay, ideally across species. We here introduce a rat thyroid-liver chip that enables simultaneous identification of direct and indirect (liver-mediated) thyroid perturbation on organ-level functions in vitro. A second manuscript describes our work toward a human thyroid-liver chip (Kühnlenz et al., 2022). The presented microfluidic model consisting of primary rat thyroid follicles and liver 3D spheroids maintains a tissue-specific phenotype for up to 21 days. More precisely, the thyroid model exhibits a follicular architecture expressing basolateral and apical markers and secretes T4. Likewise, liver spheroids retain hepatocellular characteristics, e.g., a stable release of albumin and urea, the presence of bile canalicular networks, and the formation of T4-glucuronide. Experiments with reference chemicals demonstrated proficiency to detect direct and indirect mechanisms of thyroid perturbation through decreased thyroid hormone secretion and increased gT4 formation, respectively. Prospectively this rat thyroid-liver chip model, together with its human counterpart, may support a species-specific quantitative in vitro to in vivo extrapolation to improve a data-driven and evidence-based human safety risk assessment with significant contributions to the 3R principles.

Response of Olfactory Sensory Neurons to Mercury Ions in Zebrafish: An Immunohistochemical Study.

Olfactory sensory neurons (OSNs) of fish belong to three main types: ciliated olfactory sensory neurons (cOSNs), microvillous olfactory sensory neurons (mOSNs), and crypt cells. Mercury is a toxic metal harmful for olfaction. We exposed the olfactory epithelium of zebrafish to three sublethal Hg2+ concentrations. Molecular markers specific for the different types of OSNs were immunohistochemically detected. Image analysis of treated sections enabled counting of marked cells and measurement of staining optical density indicative of the response of OSNs to Hg2+ exposure. The three types of OSNs reacted to mercury in a different way. Image analysis revealed that mOSNs are more susceptible to Hg2+ exposure than cOSNs and crypt cell density decreases. Moreover, while the ratio between sensory/nonsensory epithelium areas is unchanged, epithelium thickness drops, and dividing cells increase in the basal layer of the olfactory epithelium. Cell death but also reduction of apical processes and marker expression could account for changes in OSN immunostaining. Also, the differential results between dorsal and ventral halves of the olfactory rosette could derive from different water flows inside the olfactory chamber or different subpopulations in OSNs.

Improvement of human embryonic stem cell-derived retinal pigment epithelium cell adhesion, maturation, and function through coating with truncated recombinant human vitronectin.

To explore an xeno-free and defined coating substrate suitable for the culture of H9 human embryonic stem cell-derived retinal pigment epithelial (hES-RPE) cells in vitro, and compare the behaviors and functions of hES-RPE cells on two culture substrates, laminin521 (LN-521) and truncated recombinant human vitronectin (VTN-N). hES-RPE cells were used in the experiment. The abilities of LN-521 and VTN-N at different concentrations to adhere to hES-RPE cells were compared with a high-content imaging system. Quantitative real-time polymerase chain reaction was used to evaluate RPE-specific gene expression levels midway (day 10) and at the end (day 20) of the time course. Cell polarity was observed by immunofluorescent staining for apical and basal markers of the RPE. The phagocytic ability of hES-RPE cells was identified by flow cytometry and immunofluorescence. The cell adhesion assay showed that the ability of LN-521 to adhere to hES-RPE cells was dose-dependent. With increasing coating concentration, an increasing number of cells attached to the surface of LN-521-coated wells. In contrast, VTN-N presented a strong adhesive ability even at a low concentration. The optimal concentration of LN-521 and VTN-N required to coat and adhesion to hES-RPE cells were 2 and 0.25 µg/cm2, respectively. Furthermore, both LN-521 and VTN-N could facilitate adoption of the desired cobblestone cellular morphology with tight junction and showed polarity by the hES-RPE cells. However, hES-RPE cells cultivated in VTN-N had a greater phagocytic ability, and it took less time for these hES-RPE cells to mature. VTN-N is a more suitable coating substrate for cultivating hES-RPE cells.

Investigating virus–host cell interactions: Comparative binding forces between hepatitis C virus-like particles and host cell receptors in 2D and 3D cell culture models

Cell cultures have been successfully used to study hepatitis C virus (HCV) for many years. However, most work has been done using traditional, 2-dimensional (2D) cell cultures (cells grown as a monolayer in growth flasks or dishes). Studies have shown that when cells are grown suspended in an extra-cellular-matrix-like material, they develop into spherical, ‘organoid’ arrangements of cells (3D growth) that display distinct differences in morphological and functional characteristics compared to 2D cell cultures. In liver organoids, one key difference is the development of clearly differentiated apical and basolateral surfaces separated and maintained by cellular tight junctions. This phenomenon, termed polarity, is vital to normal barrier function of hepatocytes in vivo. It has also been shown that viruses, and virus-like particles, interact very differently with cells derived from 2D as compared to 3D cell cultures, bringing into question the usefulness of 2D cell cultures to study virus–host cell interactions. Here, we investigate differences in cellular architecture as a function of cell culture system, using confocal scanning laser microscopy, and determine differences in binding interactions between HCV virus-like particles (VLPs) and their cognate receptors in the different cell culture systems using atomic force microscopy (AFM). We generated organoid cultures that were polarized, as determined by localization of key apical and basolateral markers. We found that, while uptake of HCV VLPs by both 2D and 3D Huh7 cells was observed by flow cytometry, binding interactions between HCV VLPs and cells were measurable by AFM only on polarized cells. The work presented here adds to the growing body of research suggesting that polarized cell systems are more suitable for the study of HCV infection and dynamics than non-polarized systems.