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Cryopreservation of immature-testicular-tissue (ITT) prior to gonadotoxic treatment, while experimental, is the only recommended option for fertility preservation in prepubertal boys. The handling and manipulation of ITT before cryopreservation could influence the functionality of cells during fertility restoration, which this study explored by evaluating cellular niche and quality of mouse ITT subjected to various temperatures and time durations in vitro. ITT from 6-day-old mice were handled at ultraprofound-hypothermic, profound-hypothermic, and mild-warm-ischemic temperatures for varying time periods prior to 14-day organotypic culture. Viability, functionality, synaptonemal complex and chromatin remodeling markers were assessed. Results have shown that cell viability, testosterone level, and in vitro proliferation ability did not change when ITT were held at ultraprofound-hypothermic-temperature up to 24 h, whereas cell viability was significantly reduced (P < 0.01), when held at profound-hypothermic-temperature for 24 h before culture. Further, cell viability and testosterone levels in cultured cells from profound-hypothermic group were comparable to corresponding ultraprofound-hypothermic group but with moderate reduction in postmeiotic cells (P < 0.01). In conclusion, holding ITT at ultraprofound-hypothermic-temperature is most suitable for organotypic culture, whereas short-term exposure at profound-hypothermic-temperature may compromise postmeiotic germ cell yield post in vitro culture. This data, albeit in mouse model, will have immense value in human prepubertal fertility restoration research. [ABSTRACT FROM AUTHOR]

Sprouting of surviving axons is one of the major reorganization mechanisms of the injured brain contributing to a partial restoration of function. Of note, sprouting is maturation as well as age-dependent and strong in juvenile brains, moderate in adult and weak in aged brains. We have established a model system of complex organotypic tissue cultures to study sprouting in the dentate gyrus following entorhinal denervation. Entorhinal denervation performed after 2 weeks postnatally resulted in a robust, rapid, and very extensive sprouting response of commissural/associational fibers, which could be visualized using calretinin as an axonal marker. In the present study, we analyzed the effect of maturation on this form of sprouting and compared cultures denervated at 2 weeks postnatally with cultures denervated at 4 weeks postnatally. Calretinin immunofluorescence labeling as well as time-lapse imaging of virally-labeled (AAV2-hSyn1-GFP) commissural axons was employed to study the sprouting response in aged cultures. Compared to the young cultures commissural/associational sprouting was attenuated and showed a pattern similar to the one following entorhinal denervation in adult animals in vivo. We conclude that a maturation-dependent attenuation of sprouting occurs also in vitro, which now offers the chance to study, understand and influence maturation-dependent differences in brain repair in these culture preparations.

Sprouting of surviving axons is one of the major reorganization mechanisms of the injured brain contributing to a partial restoration of function. Of note, sprouting is maturation as well as age-dependent and strong in juvenile brains, moderate in adult and weak in aged brains. We have established a model system of complex organotypic tissue cultures to study sprouting in the dentate gyrus following entorhinal denervation. Entorhinal denervation performed after 2 weeks postnatally resulted in a robust, rapid, and very extensive sprouting response of commissural/associational fibers, which could be visualized using calretinin as an axonal marker. In the present study, we analyzed the effect of maturation on this form of sprouting and compared cultures denervated at 2 weeks postnatally with cultures denervated at 4 weeks postnatally. Calretinin immunofluorescence labeling as well as time-lapse imaging of virally-labeled (AAV2-hSyn1-GFP) commissural axons was employed to study the sprouting response in aged cultures. Compared to the young cultures commissural/associational sprouting was attenuated and showed a pattern similar to the one following entorhinal denervation in adult animals in vivo. We conclude that a maturation-dependent attenuation of sprouting occurs also in vitro , which now offers the chance to study, understand and influence maturation-dependent differences in brain repair in these culture preparations. [ABSTRACT FROM AUTHOR]

Organotypic culture of human fetal testis has achieved fertilization-competent spermatids followed by blastocysts development. This study focuses on whether the organotypic culture of testicular tissue from infant boys with cryptorchidism could support the development of spermatogonia and somatic cells. Frozen-thawed tissues were cultured in two different media, with or without retinoic acid (RA), for 60 days and evaluated by tissue morphology and immunostaining using germ and somatic cell markers. During the 60-day culture, spermatocytes stained by boule-like RNA-binding protein (BOLL) were induced in biopsies cultured with RA. Increased AR expression (p < 0.001) and decreased AMH expression (p < 0.001) in Sertoli cells indicated advancement of Sertoli cell maturity. An increased number of SOX9-positive Sertoli cells (p < 0.05) was observed, while the percentage of tubules with spermatogonia was reduced (p < 0.001). More tubules with alpha-smooth muscle actin (ACTA, peritubular myoid cells (PTMCs) marker) were observed in an RA-absent medium (p = 0.02). CYP17A1/STAR-positive Leydig cells demonstrated sustained steroidogenic function. Our culture conditions support the initiation of spermatocytes and enhanced maturation of Sertoli cells and PTMCs within infant testicular tissues. This study may be a basis for future studies focusing on maintaining and increasing the number of spermatogonia and identifying different factors and hormones, further advancing in vitro spermatogenesis.

The numerous neurological syndromes associated with COVID-19 implicate an effect of viral pathogenesis on neuronal function, yet reports of direct SARS-CoV-2 infection in the brain are conflicting. We used a well-established organotypic brain slice culture to determine the permissivity of hamster brain tissues to SARS-CoV-2 infection. We found levels of live virus waned after inoculation and observed no evidence of cell-to-cell spread, indicating that SARS-CoV-2 infection was non-productive. Nonetheless, we identified a small number of infected cells with glial phenotypes; however, no evidence of viral infection or replication was observed in neurons. Our data corroborate several clinical studies that have assessed patients with COVID-19 and their association with neurological involvement.

Three dimensional (3D) organ-like (organotypic) culture models are a rapidly advancing area of in vitro biological science. In contrast to monolayer cell culture methods which were developed to achieve proliferation of animal cells in the beginning of in vitro biology, the advancements in 3D culture methods are designed to promote cellular differentiation, and to achieve in vivo–like 3D structure and organotypic functions. This project was conceived through the Society for In Vitro Biology to draw on the expertise of individual scientists with special expertise in organotypic cultures of selected tissues or associated interrogation methods to prepare individual-focused reviews in this series. This introductory manuscript will review the early achievements of animal cell culture in monolayer culture and the limitations of that approach to reproduce functioning organ systems. Among these are the nature and 3D architecture of the substrate on which or in which the cells are grown, physical and mechanical clues from the substrate, cell-cell interactions, and defined biochemical factors that trigger the induction of the 3D organotypic differentiation. The organoid culture requires a source of cells with proliferative capacity (ranging from tissue-derived stem or immortalized cells to the iPSC cultures), a suitable substrate or matrix with the mechanical and stimulatory properties appropriate for the organotypic construct and the necessary stimulation of the culture to drive differentiation of the cell population to form the functioning organotypic construct. Details for each type of organotypic construct will be provided in the following papers. [ABSTRACT FROM AUTHOR]

Introduction: Recent advances have enabled organotypic culture of beating human myocardial slices that are stable for weeks. However, human myocardial samples are rare, exhibit high variability and frequently originate from diseased hearts. Thus, there is a need to adapt long-term slice culture for animal myocardium. When applied to animal cardiac slices, studies in healthy or genetically modified myocardium will be possible. We present the culture of slices from rabbit hearts, which resemble the human heart in microstructure, electrophysiology and excitation-contraction coupling. Methods: Left ventricular myocardium from New Zealand White rabbits was cut using a vibratome and cultured in biomimetic chambers for up to 7 days (d). Electro-mechanical uncoupling agents 2,3-butanedione monoxime (BDM) and cytochalasin D (CytoD) were added during initiation of culture and effects on myocyte survival were quantified. We investigated pacing rates (0.5 Hz, 1 Hz, and 2 Hz) and hormonal supplements (cortisol, T3, catecholamines) at physiological plasma concentrations. T3 was buffered using BSA. Contractile force was recorded continuously. Glucose consumption and lactate production were measured. Whole-slice Ca 2+ transients and action potentials were recorded. Effects of culture on microstructure were investigated with confocal microscopy and image analysis. Results: Protocols for human myocardial culture resulted in sustained contracture and myocyte death in rabbit slices within 24 h, which could be prevented by transient application of a combination of BDM and CytoD. Cortisol stabilized contraction amplitude and kinetics in culture. T3 and catecholaminergic stimulation did not further improve stability. T3 and higher pacing rates increased metabolic rate and lactate production. T3 stabilized the response to β-adrenergic stimulation over 7 d. Pacing rates above 1 Hz resulted in progredient decline in contraction force. Image analysis revealed no changes in volume fractions of cardiomyocytes or measures of fibrosis over 7 d. Ca 2+ transient amplitudes and responsiveness to isoprenaline were comparable after 1 d and 7 d, while Ca 2+ transient duration was prolonged after 7 d in culture. Conclusions: A workflow for rabbit myocardial culture has been established, preserving function for up to 7 d. This research underscores the importance of glucocorticoid signaling in maintaining tissue function and extending culture duration. Furthermore, BDM and CytoD appear to protect from tissue damage during the initiation phase of tissue culture., Competing Interests: TS is shareholder of InVitroSys GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Baron, Sommer, Fiegle, Pfeuffer, Peyronnet, Volk and Seidel.)

Dopamine D(2) receptor antagonists modulate gene transcription in the striatum. However, the molecular mechanism underlying this effect remains elusive. Here we used the expression of Nur77, a transcription factor of the orphan nuclear receptor family, as readout to explore the role of dopamine, glutamate, and adenosine receptors in the effect of a dopamine D(2) antagonist in the striatum. First, we investigated D(2) antagonist-induced Nur77 mRNA in D(2L) receptor knockout mice. Surprisingly, deletion of the D(2L) receptor isoform did not reduce eticlopride-induced upregulation of Nur77 mRNA levels in the striatum. Next, we tested if an ibotenic acid-induced cortical lesion could block the effect of eticlopride on Nur77 expression. Cortical lesions strongly reduced eticlopride-induced striatal upregulation of Nur77 mRNA. Then, we investigated if glutamatergic neurotransmission could modulate eticlopride-induced Nur77 expression. A combination of a metabotropic glutamate type 5 (mGlu5) and adenosine A(2A) receptor antagonists abolished eticlopride-induced upregulation of Nur77 mRNA levels in the striatum. Direct modulation of Nur77 expression by striatal glutamate and adenosine receptors was confirmed using corticostriatal organotypic cultures. Taken together, these results indicate that blockade of postsynaptic D(2) receptors is not sufficient to trigger striatal transcriptional activity and that interaction with corticostriatal presynaptic D(2) receptors and subsequent activation of postsynaptic glutamate and adenosine receptors in the striatum is required. Thus, these results uncover an unappreciated role of presynaptic D(2) heteroreceptors and support a prominent role of glutamate in the effect of D(2) antagonists.

Cellular senescence has classically been associated with aging. Intriguingly, recent studies have also unraveled key roles for senescence in embryonic development, regeneration, and reprogramming. Developmental senescence has been reported during embryonic development in different organisms and structures, such as the endolymphatic duct during inner ear development of mammals and birds. However, there is no study addressing the possible role of senescence on otic neurogenesis. TGFβ/SMAD is the best-known pathway linked to the induction of developmentally programmed cell senescence. Here, we studied if TGFβ2 induces cellular senescence during acoustic-vestibular-ganglion (AVG) formation. Using organotypic cultures of AVG, and characterizing different stages of otic neurogenesis in the presence of TGFβ2 and a selective TGF-β receptor type-I inhibitor, we show that TGFβ2 exerts a powerful action in inner ear neurogenesis but, contrary to what we recently observed during endolymphatic duct development, these actions are independent of cellular senescence. We show that TGFβ2 reduces proliferation, and induces differentiation and neuritogenesis of neuroblasts, without altering cell death. Our studies highlight the roles of TGFβ2 and cellular senescence in the precise regulation of cell fate within the developing inner ear and its different cell types, being their mechanisms of action highly cell-type dependent.

By preserving cell viability and three-dimensional localization, organotypic culture stands out among the newest frontiers of cell culture. It has been successfully employed for the study of diseases among which neoplasias, where tumoral cells take advantage of the surrounding stroma to promote their own proliferation and survival. Organotypic culture acquires major importance in the context of the immune system, whose cells cross-talk in a complex and dynamic fashion to elicit productive responses. However, organotypic culture has been as yet poorly developed for and applied to primary and secondary lymphoid organs. Here we describe in detail the development of a protocol suitable for the efficient cutting of mouse spleen, which overcomes technical difficulties related to the peculiar organ texture, and for optimized organotypic culture of spleen slices. Moreover, we used microscopy, immunofluorescence, flow cytometry, and qRT-PCR to demonstrate that the majority of cells residing in spleen slices remain alive and maintain their original location in the organ architecture for several days after cutting. The development of this protocol represents a significant technical improvement in the study of the lymphoid microenvironment in both physiological and pathological conditions involving the immune system.

Abstract Background Patient-derived tumor models are the new standard for pre-clinical drug testing and biomarker discovery. However, the emerging technology of primary pancreatic cancer organoids has not yet been broadly implemented in research, and complex organotypic models using organoids in co-culture with stromal and immune cellular components of the tumor have yet to be established. In this study, our objective was to develop and characterize pancreatic cancer organoids and multi-cell type organotypic co-culture models to demonstrate their applicability to the study of pancreatic cancer. Methods We employed organoid culture methods and flow cytometric, cytologic, immunofluorescent and immunohistochemical methods to develop and characterize patient-derived pancreatic cancer organoids and multi-cell type organotypic co-culture models of the tumor microenvironment. Results We describe the culture and characterization of human pancreatic cancer organoids from resection, ascites and rapid autopsy sources and the derivation of adherent tumor cell monocultures and tumor-associated fibroblasts from these sources. Primary human organoids displayed tumor-like cellular morphology, tissue architecture and polarity in contrast to cell line spheroids, which formed homogenous, non-lumen forming spheres. Importantly, we demonstrate the construction of complex organotypic models of tumor, stromal and immune components of the tumor microenvironment. Activation of myofibroblast-like cancer associated fibroblasts and tumor-dependent lymphocyte infiltration were observed in these models. Conclusions These studies provide the first report of novel and disease-relevant 3D in-vitro models representing pancreatic tumor, stromal and immune components using primary organoid co-cultures representative of the tumor-microenvironment. These models promise to facilitate the study of tumor-stroma and tumor-immune interaction and may be valuable for the assessment of immunotherapeutics such as checkpoint inhibitors in the context of T-cell infiltration.

Epigallocatechin gallate (EGCG) is a major flavan-3-ol of green tea polyphenols that exhibits various beneficial health effects, including antioxidant, anti-bacterial, and anti-inflammatory properties. This study aimed to evaluate whether EGCG prevents scopolamine-induced learning and memory impairment in in vivo and ex vivo models. Male Sprague-Dawley (SD) rats were pre-treated with EGCG (5 mg/kg/day; intraperitoneal injection (i.p.)) for 10 days. Then, EGCG and scopolamine (1 mg/kg/day; i.p.) were applied 60 and 30 min before the behavioral tests, respectively, for another 9 days. EGCG alleviated the cognitive deficits in the Y-maze, passive avoidance, and Morris water maze tests. EGCG showed improved cholinergic functions by decreasing acetylcholinesterase activity in hippocampi dissected from the brain of the rats after the behavioral tests. EGCG also reduced oxidative stress, partly due to increased superoxide dismutase activity and decreased malondialdehyde level in the hippocampi of the rat brains after the behavioral tests. Furthermore, EGCG attenuated the scopolamine-induced blockade of long-term potentiation in organotypic hippocampal tissue of seven-day-old SD rats. Taken together, these results suggested that EGCG is a potential therapeutic agent for alleviating cognitive dysfunction.

Printed circuit board (PCB) technology is well known, reliable, and low-cost, and its application to biomedicine, which implies the integration of microfluidics and electronics, has led to Lab-on-PCB. However, the biocompatibility of the involved materials has to be examined if they are in contact with biological elements. In this paper, the solder mask (PSR-2000 CD02G/CA-25 CD01, Taiyo Ink (Suzhou) Co., Ltd., Suzhou, China) of a commercial PCB has been studied for retinal cultures. For this purpose, retinal explants have been cultured over this substrate, both on open and closed systems, with successful results. Cell viability data shows that the solder mask has no cytotoxic effect on the culture allowing the application of PCB as the substrate of customized microelectrode arrays (MEAs). Finally, a comparative study of the biocompatibility of the 3D printer Uniz zSG amber resin has also been carried out.

Cellular senescence has classically been associated with aging. Intriguingly, recent studies have also unraveled key roles for senescence in embryonic development, regeneration, and reprogramming. Developmental senescence has been reported during embryonic development in different organisms and structures, such as the endolymphatic duct during inner ear development of mammals and birds. However, there is no study addressing the possible role of senescence on otic neurogenesis. TGFβ/SMAD is the best-known pathway linked to the induction of developmentally programmed cell senescence. Here, we studied if TGFβ2 induces cellular senescence during acoustic-vestibular-ganglion (AVG) formation. Using organotypic cultures of AVG, and characterizing different stages of otic neurogenesis in the presence of TGFβ2 and a selective TGF-β receptor type-I inhibitor, we show that TGFβ2 exerts a powerful action in inner ear neurogenesis but, contrary to what we recently observed during endolymphatic duct development, these actions are independent of cellular senescence. We show that TGFβ2 reduces proliferation, and induces differentiation and neuritogenesis of neuroblasts, without altering cell death. Our studies highlight the roles of TGFβ2 and cellular senescence in the precise regulation of cell fate within the developing inner ear and its different cell types, being their mechanisms of action highly cell-type dependent. [ABSTRACT FROM AUTHOR]

Lung gene therapy requires efficient transduction of slow-replicating epithelia and stable expression of delivered transgenes in the respiratory tract. Lentiviral (LV) vectors have the ideal coding, expression, and transducing capacity required for gene therapy. A modified envelope glycoprotein from the Jaagsiekte Sheep Retrovirus, termed Jenv, is well suited for LV-mediated lung gene therapy due to its inherent lung tropism. Here, two novel Jenv-pseudotyped LVs that effectively transduce lung tissue and yield titers similar to the gold standard, vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped LVs, were generated. As the concentration efficiency of LVs was found to depend on envelope pseudotype, a large-scale production method tailored for Jenv-pseudotyped LVs was developed and the most appropriate method of concentration was determined. In contrast to VSVg and Ebola virus glycoprotein-pseudotyped LVs, ultracentrifugation through a sucrose cushion drastically reduced the yield of Jenv LVs, whereas polyethylene glycol precipitation and tangential flow filtration (TFF) proved to be more suitable methods for concentrating Jenv LVs. Importantly, pressure during TFF was found to be crucial for increasing LV recovery. Finally, a unique mouse model was developed to test the suitability of these novel Jenv-pseudotyped LVs for use in lung gene therapy applications. [ABSTRACT FROM AUTHOR]

Hedgehog signaling is often activated in tumors, yet it remains unclear how GLI2, a transcription factor activated by this pathway, acts as an oncogene. We show that GLI2 is a pleiotropic oncogene. The overexpression induces genomic instability and blocks differentiation, likely mediated in part by enhanced expression of the stem cell gene SOX2. GLI2 also induces transforming growth factor (TGF)B1-dependent transdifferentiation of foreskin and tongue, but not gingival fibroblasts into myofibroblasts, creating an environment permissive for invasion by keratinocytes, which are in various stages of differentiation having downregulated GLI2. Thus, upregulated GLI2 expression is sufficient to induce a number of the acquired characteristics of tumor cells; however, the stroma, in a tissue-specific manner, determines whether certain GLI2 oncogenic traits are expressed.

Kainate receptors are potent modulators of circuit excitability and have been repeatedly implicated in pathophysiological synchronization of limbic networks. While the role of aberrant GluK2 subunit containing KARs in generation of epileptiform hypersynchronous activity is well described, the contribution of other KAR subtypes, including GluK1 subunit containing KARs remain less well understood. To investigate the contribution of GluK1 KARs in developmental and pathological synchronization of the hippocampal neural network, we used multielectrode array recordings on organotypic hippocampal slices that display first multi-unit activity and later spontaneous population discharges resembling ictal-like epileptiform activity (IEA). Chronic blockage of GluK1 activity using selective antagonist ACET or lentivirally delivered shRNA significantly delayed developmental synchronization of the hippocampal CA3 network and generation of IEA. GluK1 overexpression, on the other hand, had no significant effect on occurrence of IEA, but enhanced the size of the neuron population participating in the population discharges. Correlation analysis indicated that local knockdown of GluK1 locally in the CA3 neurons reduced their functional connectivity, while GluK1 overexpression increased the connectivity to both CA1 and DG. These data suggest that GluK1 KARs regulate functional connectivity between the excitatory neurons, possibly via morphological changes in glutamatergic circuit, affecting synchronization of neuronal populations. The significant effects of GluK1 manipulations on network activity call for further research on GluK1 KAR as potential targets for antiepileptic treatments, particularly during the early postnatal development when GluK1 KARs are strongly expressed in the limbic neural networks. • Hippocampal slices generate epileptiform activity spontaneously during time in culture. • Endogenous GluK1 activity facilitates generation of ictal-like epileptiform activity. • GluK1 overexpression enhances spatial propagation of network activity. • GluK1 regulates functional connectivity between hippocampal regions. [ABSTRACT FROM AUTHOR]

Molecular mechanisms responsible for the development of human skin epithelial cells are incompletely understood. As a consequence, the efficiency to establish a pure skin epithelial cell population from human induced pluripotent stem cells (hiPSCs) remains poor. Using an approach including RNAi and high-throughput imaging of early epithelial cells, we identified candidate kinases involved in their differentiation from hiPSCs. Among these, we found HIPK4 to be an important inhibitor of this process. Indeed, its silencing increased the amount of generated skin epithelial precursors at an early time point, increased the amount of generated keratinocytes at a later time point, and improved growth and differentiation of organotypic cultures, allowing for the formation of a denser basal layer and stratification with the expression of several keratins. Our data bring substantial input regarding regulation of human skin epithelial differentiation and for improving differentiation protocols from pluripotent stem cells.

ABSTRACT Jason Bodily works in the field of tumor virology. In this mSphere of Influence article, he reflects on how “Inactivation of Rb in stromal fibroblasts promotes epithelial cell invasion” by Adam Pickard et al. (EMBO J 31:3092–3103, 2012, https://doi.org/10.1038/emboj.2012.153) has impacted his work by making him think about the role of stromal cells in human papillomavirus infections.

Aging is a multifactorial phenomenon that results in several changes at cellular and molecular levels and is considered the main risk factor for some neurodegenerative diseases. Several evidence show the participation of the kallikrein-kinin system (KKS) in neurodegeneration and this system has been associated with inflammation and immunogenic responses in the central and peripheral systems by the activation of the B1 and B2 receptors. Previous work by our group showed that bradykinin (BK) and the B2 receptor played a possible role in neuroprotection. Therefore, the objective of this study was to evaluate the participation of B2 receptors in cell viability, neuroinflammatory response and neuroplasticity in organotypic hippocampal cultures (OHCs) of 6- and 12-month-old mice. It was observed that activation of the B2 receptor by bradykinin decreased the inflammatory response and increased plasticity in 12-month-old slices. Conversely, there was an increase in the inflammatory response and a decrease in neural plasticity in the 6-month-old slices. In both ages, an increase in cell viability was observed. This data suggests that the function of the kinin B2 receptor in the hippocampus is modulated by age, providing neuroprotective action in old age.

Alcohol-related liver disease (ALD) encompasses a range of pathological conditions that are complex to study at the clinical and preclinical levels. Despite the global burden of ALD, there is a lack of effective treatments, and mortality is high. One of the reasons for the unsuccessful development of novel therapies is that experimental studies are hindered by the challenge of recapitulating this multifactorial disorder in vitro, including the contributions of hepatotoxicity, impaired lipid metabolism, fibrosis and inflammatory cytokine storm, which are critical drivers in the pathogenesis of ALD in patients and primary targets for drug development. Here, we present the unique characteristics of the culture of human precision-cut liver slices (PCLS) to replicate key disease processes in ALD. PCLS were prepared from human liver specimens and treated with ethanol alone or in combination with fatty acids and lipopolysaccharide (FA + LPS) for up to 5 days to induce hepatotoxic, inflammatory and fibrotic events associated with ALD. Alcohol insult induced hepatocyte death which was more pronounced with the addition of FA + LPS. This mixture showed a significant increase in the cytokines conventionally associated with the prototypical inflammatory response observed in severe ALD, and interestingly, alcohol alone exhibited a different effect. Profibrogenic activation was also observed in the slices and investigated in the context of slice preparation. These results support the versatility of this organotypic model to study different pathways involved in alcohol-induced liver damage and ALD progression and highlight the applicability of the PCLS for drug discovery, confirming their relevance as a bridge between preclinical and clinical studies.

It is a well‐known fact, that there is a close interconnection between vascular and neural structures in both embryonic development and postnatal life. Different models have been employed to dissect the mechanisms of these interactions, ranging from in vitro systems (e.g., co‐culture of neural and endothelial cells) to in vivo imaging of central neural system recovery in laboratory animals after artificially induced trauma. Nevertheless, most of these models have serious limitations. Here, we describe an ex vivo model, representing an organotypic co‐culture of aortic fragments (AF) with longitudinal slices of mouse neonatal spinal cord (SC) or dorsal root ganglia (DRG). The samples were co‐cultured in a medium adapted for SC tissue and lacking any pro‐angiogenic or neurotrophic growth factors. It was found, that cultivation of AFs in the SC injury zone (transversal dissection of a SC slice) resulted in the initiation of active aortic sprouting. Remarkably, the endothelial cells exiting the AFs never invaded the SC tissue, concentrating in a nearby area (negative taxis). In contrast, the DRGs, while also promoting the sprouting, were a target of active endothelial CD31+ cell invasion (positive taxis). Thus, the tissues of both central and peripheral nervous systems have a prominent positive effect on aortic sprouting, while the vector of endothelial cell expansion is strictly nervous‐tissue‐type dependent. The ex vivo AF co‐culture with SC or DRG appeared to be a useful and promising model for a further endeavor into the mechanisms driving the complex interactions between neural and endothelial tissues. [ABSTRACT FROM AUTHOR]

Human papillomaviruses (HPVs) infect squamous epithelia and cause several important cancers. Immune evasion is critical for viral persistence. Fibroblasts in the stromal microenvironment provide growth signals and cytokines that are required for proper epithelial differentiation, maintenance, and immune responses and are critical in the development of many cancers. In this study, we examined the role of epithelial-stromal interactions in the HPV16 life cycle using organotypic (raft) cultures as a model. Rafts were created using uninfected human foreskin keratinocytes (HFKs) and HFKs containing either wild-type HPV16 or HPV16 with a stop mutation to prevent the expression of the viral oncogene E5. Microarray analysis revealed significant changes in gene expression patterns in the stroma in response to HPV16, some of which were E5 dependent. Interferon (IFN)-stimulated genes (ISGs) and extracellular matrix remodeling genes were suppressed, the most prominent pathways affected. STAT1, IFNAR1, IRF3, and IRF7 were knocked down in stromal fibroblasts using lentiviral short hairpin RNA (shRNA) transduction. HPV late gene expression and viral copy number in the epithelium were increased when the stromal IFN pathway was disrupted, indicating that the stroma helps control the late phase of the HPV life cycle in the epithelium. Increased late gene expression correlated with increased late keratinocyte differentiation but not decreased IFN signaling in the epithelium. These studies show HPV16 has a paracrine effect on stromal innate immunity, reveal a new role for E5 as a stromal innate immune suppressor, and suggest that stromal IFN signaling may influence keratinocyte differentiation. [ABSTRACT FROM AUTHOR]

Aging is a multifactorial phenomenon that results in several changes at cellular and molecular levels and is considered the main risk factor for some neurodegenerative diseases. Several evidence show the participation of the kallikrein-kinin system (KKS) in neurodegeneration and this system has been associated with inflammation and immunogenic responses in the central and peripheral systems by the activation of the B1 and B2 receptors. Previous work by our group showed that bradykinin (BK) and the B2 receptor played a possible role in neuroprotection. Therefore, the objective of this study was to evaluate the participation of B2 receptors in cell viability, neuroinflammatory response and neuroplasticity in organotypic hippocampal cultures (OHCs) of 6- and 12-month-old mice. It was observed that activation of the B2 receptor by bradykinin decreased the inflammatory response and increased plasticity in 12-month-old slices. Conversely, there was an increase in the inflammatory response and a decrease in neural plasticity in the 6-month-old slices. In both ages, an increase in cell viability was observed. This data suggests that the function of the kinin B2 receptor in the hippocampus is modulated by age, providing neuroprotective action in old age. [ABSTRACT FROM AUTHOR]

Cervical cancer is a major public health problem and research using cell culture models has improved understanding of this disease. The human cervix contains three anatomic regions; ectocervix with stratified squamous epithelium, endocervix with secretory epithelium, and transformation zone (TZ) with metaplastic cells. Most cervical cancers originate within the TZ. However, little is known about the biology of TZ cells or why they are highly susceptible to carcinogenesis. The goal of this study was to develop and optimize methods to compare growth and differentiation of cells cultured from ectocervix, TZ or endocervix. We examined the effects of different serum‐free media on cell attachment, cell growth and differentiation, and cell population doublings in monolayer culture. We also optimized conditions for organotypic culture of cervical epithelial cells using collagen rafts with human cervical stromal cells. Finally, we present a step‐by‐step protocol for culturing cells from each region of human cervix. The goal of this study was to develop and optimize methods to compare growth and differentiation of cells cultured from ectocervix, transformation zone (TZ) or endocervix. We examined the effects of different serum‐free media on cell attachment, cell growth and differentiation, and cell population doublings in monolayer culture. In addition, we present a step‐by‐step protocol for culturing cells from each region of human cervix. [ABSTRACT FROM AUTHOR]

Peripheral nerve injury elicits an enduring increase in the excitability of the spinal dorsal horn. This change, which contributes to the development of neuropathic pain, is a consequence of release and prolonged exposure of dorsal horn neurons to various neurotrophins and cytokines. We have shown in rats that nerve injury increases excitatory synaptic drive to excitatory neurons but decreases drive to inhibitory neurons. Both effects, which contribute to an increase in dorsal horn excitability, appear to be mediated by microglia-derived BDNF. We have used multiphoton Ca2+ imaging and whole cell recording of spontaneous excitatory postsynaptic currents in defined-medium organotypic cultures of GAD67-GFP+ mice spinal cord to determine the receptor dependence of these opposing actions of BDNF. In mice, as in rats, BDNF enhances excitatory transmission onto excitatory neurons. This is mediated via presynaptic TrkB and p75 neurotrophin receptors and exclusively by postsynaptic TrkB. By contrast with findings from rats, in mice BDNF does not decrease excitation of inhibitory neurons. The cytokine macrophage colony-stimulating factor 1 (CSF-1) has also been implicated in the onset of neuropathic pain. Nerve injury provokes its de novo synthesis in primary afferents, its release in spinal cord, and activation of microglia. We now show that CSF-1 increases excitatory drive to excitatory neurons via a BDNF-dependent mechanism and decreases excitatory drive to inhibitory neurons via BDNF-independent processes. Our findings complete missing steps in the cascade of events whereby peripheral nerve injury instigates increased dorsal horn excitability in the context of central sensitization and the onset of neuropathic pain. NEW & NOTEWORTHY Nerve injury provokes synthesis of macrophage colony-stimulating factor 1 (CSF-1) in primary afferents and its release in the dorsal horn. We show that CSF-1 increases excitatory drive to excitatory dorsal horn neurons via BDNF activation of postsynaptic TrkB and presynaptic TrkB and p75 neurotrophin receptors. CSF-1 decreases excitatory drive to inhibitory neurons via a BDNF-independent processes. This completes missing steps in understanding how peripheral injury instigates central sensitization and the onset of neuropathic pain. [ABSTRACT FROM AUTHOR]

Investigating the pathophysiological mechanisms underlying brain disorders is a priority if novel therapeutic strategies are to be developed. In vivo studies of animal models and in vitro studies of cell lines/primary cell cultures may provide useful tools to study certain aspects of brain disorders. However, discrepancies among these studies or unsuccessful translation from animal/cell studies to human/clinical studies often occur, because these models generally represent only some symptoms of a neuropsychiatric disorder rather than the complete disorder. Human brain slice cultures from postmortem tissue or resected tissue from operations have shown that, in vitro, neurons and glia can stay alive for long periods of time, while their morphological and physiological characteristics, and their ability to respond to experimental manipulations are maintained. Human brain slices can thus provide a close representation of neuronal networks in vivo, be a valuable tool for investigation of the basis of neuropsychiatric disorders, and provide a platform for the evaluation of novel pharmacological treatments of human brain diseases. A brain bank needs to provide the necessary infrastructure to bring together donors, hospitals, and researchers who want to investigate human brain slices in cultures of clinically and neuropathologically well-documented material. [ABSTRACT FROM AUTHOR]

The current study used an ex vivo [embryonic day (E)18] chick femur defect model to examine the bone regenerative capacity of implanted 3-dimensional (3D) skeletal-endothelial cell constructs. Human bone marrow stromal cell (HBMSC) and HUVEC spheroids were implanted within a bone defect site to determine the osteogenic potential of the skeletal-endothelial cell unit. Cells were pelleted as co- or monocell spheroids and placed within 1-mm-drill defects in the mid-diaphysis of E18 chick femurs and cultured organotypically for 10 d. Micro-computed tomography analysis revealed significantly (P = 0.0001) increased levels of bone volume (BV) and BV/tissue volume ratio in all cell-pellet groups compared with the sham defect group. The highest increase was seen in BV in femurs containing the HUVEC and HBMSC monocell constructs. Type II collagen expression was particularly pronounced within the cell spheres containing HBMSCs and HUVECs, and CD31-positive cell clusters were prominent within HUVEC-implanted defects. These studies demonstrate the importance of the 3D osteogenic-endothelial niche interaction in bone regeneration. Elucidating the component cell interactions in the osteogenic-vascular niche and the role of exogenous factors in driving these osteogenic processes will aid the development of better bone reparative strategies. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • The ex vivo modeling of injury is a robust approach that confronts with less experimental and ethical challenges. • In this study contusive model of SCI was set up in cultured slices of adult rat. • This model provide a suitable platform to define underlying biology of SCI. Abstract Nowadays there are various models of spinal cord injury (SCI) that recreate mechanisms of human SCI. The ex vivo modeling of injury is a robust approach, confronts with less experimental and ethical challenges. Currently almost all ex vivo models are obtained either from embryonic or postnatal animals, which can hardly mimic features of human SCI. This study was designed to develop SCI in slice culture of adult rats. Here, the lumbar enlargement of an adult rat was sliced and cultured. After seven days in vitro , a weight was dropped to simulate the injury. The result showed that although the rate of cell death in first days of in vitro was high, it reduced after 7 days and dropping a weight at the time caused significant rate of cell death in slices. It was shown that injury can disturb histological features and neuronal integrity in the slices. Treating the injured slices with valproic acid resulted in a significant decrease of TNF-α and increase of BDNF expression. Collected data revealed obtained slices from adult rat were able to adjust to the culture environment after 7 days and dropping a weight at the time point could simulate the injury. Besides mimicking the disturbing features of human SCI, this model can response to VPA pharmacological treatment. [ABSTRACT FROM AUTHOR]

In the field of wound healing, stem cell-based strategies are gaining importance for their regenerative potential. Adipose-derived stem cells (ADSCs) are a particular subset of mesenchymal stem cells present in the stromal-vascular fraction of the adipose tissue, today considered very attractive for their relative abundance and accessibility in the human body. However, ADSCs are still not routinely used in normal clinical practice. Several studies have also reported ADSC transplantation in association with biomaterials in an attempt to enhance the local retention and growth rate of the cells. The aim of our study was to evaluate the ability of ADSCs to build a dermal scaffold to be potentially used as a dermal substitute in the field of wound healing, with optimal biocompatibility and mechanical properties. ADSCs were defined as CD90-, CD73-, and CD105-positive cells. ADSCs turned out to be capable of secreting all the main components of the extracellular matrix (ECM) upon stimulation, thus efficiently producing a collagen and fibronectin-containing dermal matrix. We also checked whether the ADSC-produced dermal scaffold could be seeded with keratinocytes. The scaffolding material directly produced by ADSCs has several advantages when compared to the commercially available ones: it is easily obtained from the patients and it is 100% biocompatible and supports cell-ECM interaction. Moreover, it represents a possible powerful therapeutic tool for patients with chronic ulcers since it appears to be potentially grafted with keratinocytes layers, thus bypassing the classical two-step grafting procedure. [ABSTRACT FROM AUTHOR]

Retinal degenerative diseases such as macular degeneration, glaucoma, and diabetic retinopathy constitute the leading cause of blindness in the industrialized world. There is a continuous demand in investigative ophthalmic research for the development of new treatment modalities for retinal therapy. Unfortunately, efforts to identify novel neuroprotective and neuroregenerative agents have often been hindered by an experimental model gap that exists between high-throughput methods via dissociated cells and preclinical animal models. Even though dissociated cell culture is rapid and high-throughput, it is limited in its ability to reproduce the in vivo conditions. In contrast, preclinical animal models may offer greater fidelity, albeit they lack efficiency and experimental control. Retina explant cultures provide an ideal bridge to close this gap and have been used to study an array of biological processes such as retinal development and neurodegeneration. However, it is often difficult to interpret findings from these studies due to the wide variety of experimental species and culture methods used. This review provides a comprehensive overview of current ex vivo neuroretina culture methods and assessments, with a focus on their suitability, advantages, and disadvantages, along with novel insights and perspectives on the organotypic culture model as a high-throughput platform for screening promising molecules for retinal regeneration. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Small interfering RNA (siRNA) can induce RNA interference, which leads to the knockdown of messenger RNA (mRNA) and protein. As a result, siRNA is often used in vitro and in vivo to unravel the function of genes and as a therapeutic agent to disrupt excessive expression of disease-related genes. However, there is a large gap between in vitro and in vivo models in terms of simplicity, flexibility, throughput, and translatability. This gap could be bridged by using precision-cut tissue slices, which represent viable explants prepared from animal or human tissue that can be cultured ex vivo. Previously, we demonstrated that self-deliverable siRNA (Accell siRNA) induced significant mRNA knockdown in lung slices. The goal of this study, however, was to investigate whether Accell siRNA also induced protein knockdown in murine lung slices. Slices were incubated for up to 96 h with no siRNA (untransfected), non-targeting siRNA (control), or gene-targeting siRNA (Gapdh , Ppib , Serpinh1 , and Bcl2l1). Overall, untransfected and transfected slices remained viable during an incubation of 96 h. In addition, gene-targeting siRNAs induced not only significant and specific mRNA knockdown but also protein knockdown. Finally, protein knockdown of fibrogenesis-related targets (Ppib , Serpinh1 , and Bcl2l1) was shown to influence fibrogenesis on mRNA level, thereby demonstrating this model its utility in functional genomics and translational research. [ABSTRACT FROM AUTHOR]

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder with no effective treatment currently available. Although the mechanisms of motoneuron (MN) death are still unclear, glutamate excitotoxicity and neuroinflammatory reaction are two main features in the neurodegenerative process of ALS. Neuregulin 1 (NRG1) is a trophic factor highly expressed in MNs and neuromuscular junctions. Several recent evidences suggest that NRG1 and their ErbB receptors are involved in ALS. However, further knowledge is still needed to clarify the role of the NRG1-ErbB pathway on MN survival. In this study we used an in vitro model of spinal cord organotypic cultures (SCOCs) subject to chronic excitotoxicity caused by DL-threo-β-hydroxyaspartic acid (THA) to characterize the effect of NRG1 on MN survival. Our results show that addition of recombinant human NRG1 (rhNRG1) to the medium significantly increased MN survival through the activation of ErbB receptors which was ablated with lapatinib (LP), an ErbB inhibitor, and reduced microglial reactivity overcoming the excitotoxicity effects. rhNRG1 activated the pro-survival PI3K/AKT pathway and restored the autophagic flux in the spinal cord culture. Moreover, addition of rhNRG1 to the medium promoted motor and sensory neurite outgrowth. These findings indicate that increasing NRG1 at the spinal cord is an interesting approach for promoting MN protection and regeneration.

Purpose: Meibomian glands (MGs) are crucial for maintaining tear film stability and ocular surface health. Here, we aim to establish a novel organotypic culture model of MGs and explore the risk factors of MG dysfunction (MGD)., Methods: We developed a novel organotypic culture model for MGs at the air-liquid interface. The viability and cell proliferation of MGs were assessed using CCK-8, immunofluorescence, and qPCR. Lipid accumulation was evaluated by Nile red staining and microscopic examination. Protein expression levels were evaluated by immunofluorescence and Western blot assay. EdU assay was employed to track the proliferation of acinar cells. The validity of the model was confirmed through culturing MGs from mice of different ages and incorporating certain drugs (Dex) into the culture system., Results: Utilizing the novel culture model, the MG tissue exhibited sustained viability, cellular division, and continuous production of lipids for a duration of 7 days. Lipid droplets formed were directly visualized using light field microscopy. Through the cultivation of aged mice's MGs, it was discovered that aging resulted in diminished proliferation and lipid synthesis, along with an aberrant increase in Krt10 expression. Further application of this model showed that Dex treatment diminished MG's proliferation and lipid synthesis. Finally, an in vivo study was conducted to provide additional confirmation of the phenomenon of Dex-induced abnormalities., Conclusions: In this study, a stable organotypic culture model of the MGs was established. The organotypic culture model offers a valuable tool to investigate the pathophysiological mechanisms and facilitate drug screening for MG-related diseases., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Purpose: Given that porcine and human retinas have similar structures and characteristics, ex vivo culture of porcine neuroretina provides an attractive model for studying mechanisms of human retinal injury and degenerative disease. Here, we describe the method that was used to establish and characterize an adult porcine retina culture system as a rapid screening tool for retinal survival in real time.Methods: Neuroretina explants 8 mm in diameter were harvested from adult swine and cultured on porous cell culture inserts with adjustable heights. Retina explant viability was evaluated at 1, 4, 7, 11, and 14 days of culture using a resazurin-based metabolic assay. The explants were analyzed morphologically through immunohistochemistry for glial activation and apoptosis. Morphometric analysis was also performed on hematoxylin and eosin-stained retina sections from each time point.Results: The viability of retina explants gradually decreased over time in culture. The laminar structure of the neuroretina was well preserved during the first 7 days. However, by day 14, most explants showed significant loss of cells in each laminar layer and obvious thinning. Overall, the progressive loss of retinal lamination and thickness, and increase in apoptotic nuclei with activated hypertrophic Müller cells were well correlated with the metabolic activity of the ex vivo neuroretina explants.Conclusions: This study was the first report to describe the use of a high-throughput and quantitative method for monitoring retina explant viability in real time. Ex vivo neuroretina cultures closely mimic the functional dynamics of the organ, and can be used efficiently to screen novel therapeutics for retinal neurodegenerative disease. [ABSTRACT FROM AUTHOR]

Background Slice cultures have been prepared from several organs. With respect to the brain, advantages of slice cultures over dissociated cell cultures include maintenance of the cytoarchitecture and neuronal connectivity. Slice cultures from adult human brain have been reported and constitute a promising method to study neurological diseases. Despite this potential, few studies have characterized in detail cell survival and function along time in short-term, free-floating cultures. New Method We used tissue from adult human brain cortex from patients undergoing temporal lobectomy to prepare 200 μm-thick slices. Along the period in culture, we evaluated neuronal survival, histological modifications, and neurotransmitter release. The toxicity of Alzheimer’s-associated Aβ oligomers (AβOs) to cultured slices was also analyzed. Results Neurons in human brain slices remain viable and neurochemically active for at least four days in vitro, which allowed detection of binding of AβOs. We further found that slices exposed to AβOs presented elevated levels of hyperphosphorylated Tau, a hallmark of Alzheimer’s disease. Comparison with Existing Method(s) Although slice cultures from adult human brain have been previously prepared, this is the first report to analyze cell viability and neuronal activity in short-term free-floating cultures as a function of days in vitro. Conclusions Once surgical tissue is available, the current protocol is easy to perform and produces functional slices from adult human brain. These slice cultures may represent a preferred model for translational studies of neurodegenerative disorders when long term culturing in not required, as in investigations on AβO neurotoxicity. [ABSTRACT FROM AUTHOR]

A Merkel cell‐neurite complex is a touch receptor composed of specialized epithelial cells named Merkel cells and peripheral sensory nerves in the skin. Merkel cells are found in touch‐sensitive skin components including whisker follicles. The nerve fibers that innervate Merkel cells of a whisker follicle extend from the maxillary branch of the trigeminal ganglion. Whiskers as a sensory organ attribute to the complicated architecture of the Merkel cell‐neurite complex, and therefore it is intriguing how the structure is formed. However, observing the dynamic process of the formation of a Merkel cell‐neurite complex in whiskers during embryonic development is still difficult. In this study, we tried to develop an organotypic co‐culture method of a whisker pad and a trigeminal ganglion explant to form the Merkel cell‐neurite complex in vitro. We initially developed two distinct culture methods of a single whisker row and a trigeminal ganglion explant, and then combined them. By dissecting and cultivating a single row from a whisker pad, the morphogenesis of whisker follicles could be observed under a microscope. After the co‐cultivation of the whisker row with a trigeminal ganglion explant, a Merkel cell‐neurite complex composed of Merkel cells, which were positive for both cytokeratin 8 and SOX2, Neurofilament‐H‐positive trigeminal nerve fibers and Schwann cells expressing Nestin, SOX2 and SOX10 was observed via immunohistochemical analyses. These results suggest that the process for the formation of a Merkel cell‐neurite complex can be observed under a microscope using our organotypic co‐culture method. [ABSTRACT FROM AUTHOR]

Background Insights into human brain diseases may emerge from tissue obtained after operations on patients. However techniques requiring transduction of transgenes carried by viral vectors cannot be applied to acute human tissue. New method We show that organotypic culture techniques can be used to maintain tissue from patients with three different neurological syndromes for several weeks in vitro. Optimized viral vector techniques and promoters for transgene expression are described. Results Region-specific differences in neuronal form, firing pattern and organization as well as pathological activities were maintained over 40–50 days in culture. Both adeno-associated virus and lentivirus based vectors were persistently expressed from ∼10 days after application, providing 30–40 days to exploit genetically expressed constructs. Different promoters, including hSyn, e/hSyn, CMV and CaMKII, provided cell-type specific transgene expression. The Ca probe GCaMP let us explore epileptogenic synchrony and a FRET-based probe was used to follow activity of the kinase mTORC1. Comparison with existing methods The use of a defined culture medium, with low concentrations of amino acids and no growth factors, permitted organotypic culture of tissue from humans aged 3–62 years. Epileptic activity was maintained and excitability changed relatively little until ∼6 weeks in culture. Conclusions Characteristic morphology and region-specific neuronal activities are maintained in organotypic culture of tissue from patients diagnosed with mesial temporal lobe epilepsy, cortical dysplasia and cortical glioblastoma. Viral vector techniques permit expression of probes for long-term measurements of multi-cellular activity and intra-cellular signaling. [ABSTRACT FROM AUTHOR]

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder with no effective treatment currently available. Although the mechanisms of motoneuron (MN) death are still unclear, glutamate excitotoxicity and neuroinflammatory reaction are two main features in the neurodegenerative process of ALS. Neuregulin 1 (NRG1) is a trophic factor highly expressed in MNs and neuromuscular junctions. Several recent evidences suggest that NRG1 and their ErbB receptors are involved in ALS. However, further knowledge is still needed to clarify the role of the NRG1-ErbB pathway on MN survival. In this study we used an in vitro model of spinal cord organotypic cultures (SCOCs) subject to chronic excitotoxicity caused by DL-threo-β-hydroxyaspartic acid (THA) to characterize the effect of NRG1 on MN survival. Our results show that addition of recombinant human NRG1 (rhNRG1) to the medium significantly increased MN survival through the activation of ErbB receptors which was ablated with lapatinib (LP), an ErbB inhibitor, and reduced microglial reactivity overcoming the excitotoxicity effects. rhNRG1 activated the pro-survival PI3K/AKT pathway and restored the autophagic flux in the spinal cord culture. Moreover, addition of rhNRG1 to the medium promoted motor and sensory neurite outgrowth. These findings indicate that increasing NRG1 at the spinal cord is an interesting approach for promoting MN protection and regeneration. [ABSTRACT FROM AUTHOR]

Arsenic is one of the most important human carcinogens and environmental pollutants. However, the evaluation of the underlying carcinogenic mechanisms is challenging due to the lack of suitable in vivo and in vitro models, as distinct interspecies differences in arsenic metabolism exist. Thus, it is of high interest to develop new experimental models of arsenic-induced skin tumorigenesis in humans. Consequently, aim of this study was to establish an advanced 3D model for the investigation of arsenic-induced skin derangements, namely skin equivalents, built from immortalized human keratinocytes (NHEK/SVTERT3-5). In contrast to spontaneously immortalized HACAT cells, NHEK/SVTERT3-5 cells more closely resembled the differentiation pattern of primary keratinocytes. With regard to arsenic, our results showed that while our new cell model was widely unaffected by short-time treatment (72 h) with low, non-toxic doses of ATO (0.05-0.25 µM), chronic exposure (6 months) resulted in distinct changes of several cell characteristics. Thus, we observed an increase in the G2 fraction of the cell cycle accompanied by increased nucleus size and uneven tubulin distribution. Moreover, cells showed strong signs of de-differentiation and upregulation of several epithelial-to-mesenchymal transition markers. In line with these effects, chronic contact to arsenic resulted in impaired skin-forming capacities as well as localization of ki67-positive (proliferating) cells at the upper layers of the epidermis; a condition termed Bowen's disease. Finally, chronically arsenic-exposed cells were characterized by an increased tumorigenicity in SCID mice. Taken together, our study presents a new model system for the investigation of mechanisms underlying the tumor-promoting effects of chronic arsenic exposure. [ABSTRACT FROM AUTHOR]

Interpenetrating polymer networks (IPNs) have gained great attention for a number of biomedical applications due to their improved properties compared to individual components alone. In this study, we investigated the capacity of newly-developed naturally-derived IPNs as potential biomaterials for tissue engineering. These IPNs combine the biologic properties of a fibrous fibrin network polymerized at the nanoscale and the mechanical stability of polyethylene oxide (PEO). First, we assessed their cytotoxicity in vitro on L929 fibroblasts. We further evaluated their biocompatibility ex vivo with a chick embryo organotypic culture model. Subcutaneous implantations of the matrices were subsequently conducted on nude mice to investigate their biocompatibility in vivo. Our preliminary data highlighted that our biomaterials were non-cytotoxic (viability above 90%). The organotypic culture showed that the IPN matrices induced higher cell adhesion (across all the explanted organ tissues) and migration (skin, intestine) than the control groups, suggesting the advantages of using a biomimetic, yet mechanically-reinforced IPN-based matrix. We observed no major inflammatory response up to 12 weeks post implantation. All together, these data suggest that these fibrin-based IPNs are promising biomaterials for tissue engineering. [ABSTRACT FROM AUTHOR]