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Passaged cell lines represent currently an integral component in various studies of malignant neoplasms. These cell lines are utilized for drug screening both in monolayer cultures or as part of three-dimensional (3D) tumor models. They can also be used to model the tumor microenvironment in vitro and in vivo through xenotransplantation into immunocompromised animals. However, immortalized cell lines have some limitations of their own. The homogeneity of cell line populations and the extensive passaging in monolayer systems make these models distant from the original disease. Recently, there has been a growing interest among scientists in the use of primary cell lines, as these are passaged directly from human tumor tissues. In this case, cells retain the morphological and functional characteristics of the tissue from which they were derived, an advantage often not observed in passaged cultures. This review highlights the advantages and limitations of passaged and primary cell cultures, their similarities and differences, as well as existing test systems that are based on primary and passaged cell cultures for drug screening purposes.
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The impact of tyrosine kinase inhibitors (TKIs) on multidrug resistance (MDR) in non-small cell lung carcinoma (NSCLC) is a critical aspect of cancer therapy. While TKIs effectively target specific signaling pathways of cancer cells, they can also act as substrates for ABC transporters, potentially triggering MDR. The aim of our study was to evaluate the response of 17 patient-derived NSCLC cultures to 10 commonly prescribed TKIs and to correlate these responses with patient mutational profiles. Using an ex vivo immunofluorescence assay, we analyzed the expression of the MDR markers ABCB1, ABCC1, and ABCG2, and correlated these data with the genetic profiles of patients for a functional diagnostic approach. NSCLC cultures responded differently to TKIs, with erlotinib showing good efficacy regardless of mutation burden or EGFR status. However, the modulation of MDR mechanisms by erlotinib, such as increased ABCG2 expression, highlights the challenges associated with erlotinib treatment. Other TKIs showed limited efficacy, highlighting the variability of response in NSCLC. Genetic alterations in signaling pathways associated with drug resistance and sensitivity, including TP53 mutations, likely contributed to the variable responses to TKIs. The relationships between ABC transporter expression, gene alterations, and response to TKIs did not show consistent patterns. Our results suggest that in addition to mutational status, performing functional sensitivity screening is critical for identifying appropriate treatment strategies with TKIs. These results underscore the importance of considering drug sensitivity, off-target effects, MDR risks, and patient-specific genetic profiles when optimizing NSCLC treatment and highlight the potential for personalized approaches, especially in early stages.
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Anaplastic thyroid cancer (ATC) is one of the deadliest human cancers and represents <2% of thyroid carcinomas. A therapeutic target for ATC is represented by anaplastic lymphoma kinase (ALK) rearrangements, involved in tumor growth. Crizotinib is an oral small-molecule tyrosine kinase inhibitor of the ALK, MET, and ROS1 kinases, approved in ALK-positive non-small cell lung cancer. Until now, the effect of crizotinib in "primary human ATC cells" (pATCs) with transforming striatin (STRN)-ALK fusion has not been reported in the literature. In this study, we aimed to obtain pATCs with STRN-ALK in vitro and evaluate the in vitro antineoplastic action of crizotinib. Thyroid surgical samples were obtained from 12 ATC patients and 6 controls (who had undergone parathyroidectomy). A total of 10/12 pATC cultures were obtained, 2 of which with transforming STRN-ALK fusion (17%). Crizotinib inhibited proliferation, migration, and invasion and increased apoptosis in 3/10 pATC cultures (2 of which with/1 without STRN-ALK), particularly in those with STRN-ALK. Moreover, crizotinib significantly inhibited the proliferation of AF cells (a continuous cell line obtained from primary ATC cells). In conclusion, the antineoplastic activity of crizotinib has been shown in human pATCs (with STRN-ALK) in preclinical studies in vitro, opening the way to future clinical evaluation in these patients.
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Quinasa de Linfoma Anaplásico , Apoptosis , Proliferación Celular , Crizotinib , Inhibidores de Proteínas Quinasas , Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides , Humanos , Crizotinib/farmacología , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Carcinoma Anaplásico de Tiroides/patología , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico/genética , Quinasa de Linfoma Anaplásico/metabolismo , Proliferación Celular/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Masculino , Femenino , Antineoplásicos/farmacología , Persona de Mediana Edad , Movimiento Celular/efectos de los fármacos , Anciano , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Células Tumorales Cultivadas , Línea Celular Tumoral , Proteínas de Unión a Calmodulina , Proteínas de la Membrana , Proteínas del Tejido NerviosoRESUMEN
In adult olfactory epithelium (OE), ATP plays a role in constant cell turnover and post-injury neuroregeneration. We previously demonstrated that constitutive and ATP-evoked ATP release are present in neonatal mouse OE and underlie continuous cell turn-over and post-injury neuroregeneration, and that activation of purinergic P2X7 receptors is involved in the evoked release. We hypothesized that both releases are present in adult mouse OE. To study the putative contribution of olfactory sensory neurons to ATP release, we used olfactory sensory neuronal-like OP6 cells derived from the embryonic olfactory placode cells. Calcium imaging showed that OP6 cells and primary adult OE cell cultures express functional purinergic receptors. We monitored ATP release from OP6 cells and whole adult OE turbinates using HEK cells as biosensors and luciferin-luciferase assays. Constitutive ATP release occurs in OP6 cells and whole adult mouse OE turbinates, and P2X7 receptors mediated evoked ATP release occurs only in turbinates. The mechanisms of ATP release described in the present study might underlie the constant cell turn-over and post-injury neuroregeneration present in adult OE and thus, further studies of these mechanisms are warranted as it will improve our knowledge of OE tissue homeostasis and post-injury regeneration.
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Human metapneumovirus (HMPV), a member of the Pneumoviridae family, causes upper and lower respiratory tract infections in humans. In vitro studies with HMPV have mostly been performed in monolayers of undifferentiated epithelial cells. In vivo studies in cynomolgus macaques and cotton rats have shown that ciliated epithelial cells are the main target of HMPV infection, but these observations cannot be studied in monolayer systems. Here, we established an organoid-derived bronchial culture model that allows physiologically relevant studies on HMPV. Inoculation with multiple prototype HMPV viruses and recent clinical virus isolates led to differences in replication among HMPV isolates. Prolific HMPV replication in this model caused damage to the ciliary layer, including cilia loss at advanced stages post-infection. These cytopathic effects correlated with those observed in previous in vivo studies with cynomolgus macaques. The assessment of the innate immune responses in three donors upon HMPV and RSV inoculation highlighted the importance of incorporating multiple donors to account for donor-dependent variation. In conclusion, these data indicate that the organoid-derived bronchial cell culture model resembles in vivo findings and is therefore a suitable and robust model for future HMPV studies. IMPORTANCE: Human metapneumovirus (HMPV) is one of the leading causative agents of respiratory disease in humans, with no treatment or vaccine available yet. The use of primary epithelial cultures that recapitulate the tissue morphology and biochemistry of the human airways could aid in defining more relevant targets to prevent HMPV infection. For this purpose, this study established the first primary organoid-derived bronchial culture model suitable for a broad range of HMPV isolates. These bronchial cultures were assessed for HMPV replication, cellular tropism, cytopathology, and innate immune responses, where the observations were linked to previous in vivo studies with HMPV. This study exposed an important gap in the HMPV field since extensively cell-passaged prototype HMPV B viruses did not replicate in the bronchial cultures, underpinning the need to use recently isolated viruses with a controlled passage history. These results were reproducible in three different donors, supporting this model to be suitable to study HMPV infection.
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Metapneumovirus , Infecciones por Paramyxoviridae , Humanos , Animales , Metapneumovirus/fisiología , Citología , Replicación Viral , Infecciones por Paramyxoviridae/patología , Epitelio , Macaca , TropismoRESUMEN
Lung cancer remains the leading cause of cancer death globally, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. Multidrug resistance (MDR), often caused by ATP-binding cassette (ABC) transporters, represents a significant obstacle in the treatment of NSCLC. While genetic profiling has an important role in personalized therapy, functional assays that measure cellular responses to drugs are gaining in importance. We developed an automated microplate-based immunofluorescence assay for the evaluation of MDR markers ABCB1, ABCC1, and ABCG2 in cells obtained from NSCLC patients through high-content imaging and image analysis, as part of a functional diagnostic approach. This assay effectively discriminated cancer from non-cancer cells within mixed cultures, which is vital for accurate assessment of changes in MDR marker expression in different cell populations in response to anticancer drugs. Validation was performed using established drug-sensitive (NCI-H460) and drug-resistant (NCI-H460/R) NSCLC cell lines, demonstrating the assay's capacity to distinguish and evaluate different MDR profiles. The obtained results revealed wide-ranging effects of various chemotherapeutic agents on MDR marker expression in different patient-derived NSCLC cultures, emphasizing the need for MDR diagnostics in NSCLC. In addition to being a valuable tool for assessing drug effects on MDR markers in different cell populations, the assay can complement genetic profiling to optimize treatment. Further assay adaptations may extend its application to other cancer types, improving treatment efficacy while minimizing the development of resistance.
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Cell cultures are an important part of the research and treatment of autoimmune connective tissue diseases. By culturing the various cell types involved in ACTDs, researchers are able to broaden the knowledge about these diseases that, in the near future, may lead to finding cures. Fibroblast cultures and chondrocyte cultures allow scientists to study the behavior, physiology and intracellular interactions of these cells. This helps in understanding the underlying mechanisms of ACTDs, including inflammation, immune dysregulation and tissue damage. Through the analysis of gene expression patterns, surface proteins and cytokine profiles in peripheral blood mononuclear cell cultures and endothelial cell cultures researchers can identify potential biomarkers that can help in diagnosing, monitoring disease activity and predicting patient's response to treatment. Moreover, cell culturing of mesenchymal stem cells and skin modelling in ACTD research and treatment help to evaluate the effects of potential drugs or therapeutics on specific cell types relevant to the disease. Culturing cells in 3D allows us to assess safety, efficacy and the mechanisms of action, thereby aiding in the screening of potential drug candidates and the development of novel therapies. Nowadays, personalized medicine is increasingly mentioned as a future way of dealing with complex diseases such as ACTD. By culturing cells from individual patients and studying patient-specific cells, researchers can gain insights into the unique characteristics of the patient's disease, identify personalized treatment targets, and develop tailored therapeutic strategies for better outcomes. Cell culturing can help in the evaluation of the effects of these therapies on patient-specific cell populations, as well as in predicting overall treatment response. By analyzing changes in response or behavior of patient-derived cells to a treatment, researchers can assess the response effectiveness to specific therapies, thus enabling more informed treatment decisions. This literature review was created as a form of guidance for researchers and clinicians, and it was written with the use of the NCBI database.
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Enfermedades Autoinmunes , Enfermedades del Tejido Conjuntivo , Humanos , Leucocitos Mononucleares , Enfermedades Autoinmunes/terapia , Enfermedades del Tejido Conjuntivo/tratamiento farmacológico , Inflamación , Técnicas de Cultivo de CélulaRESUMEN
Culturing respiratory epithelial cells at an air-liquid interface (ALI) represents an established method for studies on infection or toxicology by the generation of an in vivo-like respiratory tract epithelial cellular layer. Although primary respiratory cells from a variety of animals have been cultured, an in-depth characterization of canine tracheal ALI cultures is lacking despite the fact that canines are a highly relevant animal species susceptible to various respiratory agents, including zoonotic pathogens such as severe acute respiratory coronavirus 2 (SARS-CoV-2). In this study, canine primary tracheal epithelial cells were cultured under ALI conditions for four weeks, and their development was characterized during the entire culture period. Light and electron microscopy were performed to evaluate cell morphology in correlation with the immunohistological expression profile. The formation of tight junctions was confirmed using transepithelial electrical resistance (TEER) measurements and immunofluorescence staining for the junctional protein ZO-1. After 21 days of culture at the ALI, a columnar epithelium containing basal, ciliated and goblet cells was seen, resembling native canine tracheal samples. However, cilia formation, goblet cell distribution and epithelial thickness differed significantly from the native tissue. Despite this limitation, tracheal ALI cultures could be used to investigate the pathomorphological interactions of canine respiratory diseases and zoonotic agents.
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Técnicas de Cultivo de Célula , Células Epiteliales , Animales , Perros , Células Cultivadas , Células Epiteliales/metabolismo , Microscopía ElectrónicaRESUMEN
Atomic force microscopy (AFM) recently burst into biomedicine, providing morphological and functional characteristics of cancer cells and their microenvironment responsible for tumor invasion and progression, although the novelty of this assay needs to coordinate the malignant profiles of patients' specimens to diagnostically valuable criteria. Applying high-resolution semi-contact AFM mapping on an extended number of cells, we analyzed the nanomechanical properties of glioma early-passage cell cultures with a different IDH1 R132H mutation status. Each cell culture was additionally clustered on CD44+/- cells to find possible nanomechanical signatures that differentiate cell phenotypes varying in proliferative activity and the characteristic surface marker. IDH1 R132H mutant cells compared to IDH1 wild-type ones (IDH1wt) characterized by two-fold increased stiffness and 1.5-fold elasticity modulus. CD44+/IDH1wt cells were two-fold more rigid and much stiffer than CD44-/IDH1wt ones. In contrast to IDH1 wild-type cells, CD44+/IDH1 R132H and CD44-/IDH1 R132H did not exhibit nanomechanical signatures providing statistically valuable differentiation of these subpopulations. The median stiffness depends on glioma cell types and decreases according to the following manner: IDH1 R132H mt (4.7 mN/m), CD44+/IDH1wt (3.7 mN/m), CD44-/IDH1wt (2.5 mN/m). This indicates that the quantitative nanomechanical mapping would be a promising assay for the quick cell population analysis suitable for detailed diagnostics and personalized treatment of glioma forms.
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Glioma , Receptores de Hialuranos , Isocitrato Deshidrogenasa , Humanos , Glioma/diagnóstico , Glioma/metabolismo , Glioma/patología , Receptores de Hialuranos/metabolismo , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Microscopía de Fuerza Atómica , Microambiente Tumoral , MutaciónRESUMEN
Anaplastic thyroid cancer (ATC) is a rare and rapidly fatal human cancer. Its usual treatment includes the combination of surgery, external hyperfractionated radiation therapy, and chemotherapy. These treatments permit achieving about 6-10 months of median survival. For this reason, it is challenging to predict the ATC patient clinical therapy responsiveness. Pazopanib is a multitarget tyrosine kinase inhibitor of VEGF receptors, PDGF, and c-Kit. Until now, the effect of pazopanib in primary human ATC cells (pATC) has not been reported in the literature. The aim of our study was to evaluate in vitro the antineoplastic effect of pazopanib in pATC. Surgical thyroidal tissues were collected from five patients with ATC, from thyroid biopsy at the moment of first surgical operation. An inhibition of proliferation, migration, and invasion, and an increase in apoptosis were demonstrated upon treating pATC cells with pazopanib (p < 0.05). Moreover, pazopanib was able to significantly decrease the VEGF expression in pATC cells (p < 0.05). To conclude, in this study, we demonstrate the antineoplastic activity of the antiangiogenic inhibitor, pazopanib, in human pATC in vitro.
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Antineoplásicos , Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides , Humanos , Carcinoma Anaplásico de Tiroides/patología , Neoplasias de la Tiroides/patología , Factor A de Crecimiento Endotelial Vascular/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéuticoRESUMEN
The Nobel Prize awarded gene editing system, CRISPR-Cas9, is probably one of the greatest achievements of the last decades. CRISPR-Cas9 can introduce irreversible genomic changes in its target DNA by simple specifying a 20-nucleotide sequence within its RNA guide. Due to its simplicity, efficacy, and relative low cost in comparison with other genome editing systems, it has become the most common gene editing system used in research laboratories. Here we describe a step-by-step protocol to produce genetically edited primary oral keratinocytes using the CRISPR-Cas9 system.
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Sistemas CRISPR-Cas , Queratinocitos , Sistemas CRISPR-Cas/genética , Edición Génica , Genómica , ARNRESUMEN
Modern society faces many biomedical challenges that require urgent solutions. Two of the most important include the elucidation of mechanisms of socially significant diseases and the development of prospective drug treatments for these diseases. Experimental cell models are a convenient tool for addressing many of these problems. The power of cell models is further enhanced when combined with gene technologies, which allows the examination of even more subtle changes within the structure of the genome and permits testing of proteins in a native environment. The list and possibilities of these recently emerging technologies are truly colossal, which requires a rethink of a number of approaches for obtaining experimental cell models. In this review, we analyze the possibilities and limitations of promising gene technologies for obtaining cell models, and also give recommendations on the development and creation of relevant models. In our opinion, this review will be useful for novice cell biologists, as it provides some reference points in the rapidly growing universe of gene and cell technologies.
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Sistemas CRISPR-Cas , Edición Génica , Genoma , Clonación MolecularRESUMEN
Angiotensin IV (Ang IV), a metabolite of Angiotensin II, is a bioactive hexapeptide that inhibits the insulin-regulated aminopeptidase (IRAP). This transmembrane zinc metallopeptidase with many biological functions has in recent years emerged as a new pharmacological target. IRAP is expressed in a variety of tissues and can be found in high density in the hippocampus and neocortex, brain regions associated with cognition. Ang IV is known to improve memory tasks in experimental animals. One of the most potent IRAP inhibitors known today is the macrocyclic compound HA08 that is significantly more stable than the endogenous Ang IV. HA08 combines structural elements from Ang IV and the physiological substrates oxytocin and vasopressin, and binds to the catalytic site of IRAP. In the present study we evaluate whether HA08 can restore cell viability in rat primary cells submitted to hydrogen peroxide damage. After damaging the cells with hydrogen peroxide and subsequently treating them with HA08, the conceivable restoring effects of the IRAP inhibitor were assessed. The cellular viability was determined by measuring mitochondrial activity and lactate dehydrogenase (LDH) release. The mitochondrial activity was significantly higher in primary hippocampal cells, whereas the amount of LDH was unaffected. We conclude that the cell viability can be restored in this cell type by blocking IRAP with the potent macrocyclic inhibitor HA08, although the mechanism by which HA08 exerts its effects remains unclear.
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Rising ocean temperatures due to climate change combined with the intensification of anthropogenic activity can drive shifts in the geographic distribution of species, with the risks of introducing new diseases. In a changing environment, new host-pathogen interactions or changes to existing dynamics represent a major challenge for native species at high latitudes. Notothenioid fish constitute a unique study system since members of this group are found inside and outside Antarctica, are highly adapted to cold and particularly sensitive to temperature increments. However, data about their immune response remains scarce. Here, we aimed to evaluate the innate immune response under thermal stress in two species of Notothenioid fish, Harpagifer antarcticus and Harpagifer bispinis. Adult individuals from both species were collected on King George Island (Antarctica), and Punta Arenas (Chile), respectively. Specimens were assigned to a control group or injected with one of two agents (LPS and Poly I:C) to simulate either a bacterial or viral infection, and subjected to three different temperatures 2, 5 and 8 °C for 1 week. In parallel, we established leukocytes primary cell cultures from head kidney, which were also subjected to the immunostimulants at the same three temperatures, and incubated for 0.5, 1, 3, 6, 12, 24, and 48 h. We evaluated the relative gene expression of genes involved in the innate immune response (TLR1, TLR3, NF-kB, MYD88, IFNGR e IL-8) through real time qPCR. We found differences between species mainly in vivo, where H. antarcticus exhibited upregulation at high temperatures and H. bispinis seemed to have reached their physiological minimum at 2 °C. Although temperature had a strong effect during the in vivo assay for both species, it was negligible for primary cell cultures, which responded primarily to condition and time. Moreover, while leukocytes responded with fluctuations across time points, in vivo both species manifested strong and clear patterns of gene expression. These results highlight the importance of evaluating the effect of multiple stressors and set a precedent for future research.
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Lipopolisacáridos , Perciformes , Adyuvantes Inmunológicos/metabolismo , Animales , Regiones Antárticas , Peces/metabolismo , Inmunidad Innata , Interleucina-8 , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/metabolismo , Perciformes/genética , Poli I-C/farmacología , Temperatura , Receptor Toll-Like 1/metabolismo , Receptor Toll-Like 3/metabolismoRESUMEN
Several studies have proved that glial cells, as well as neurons, play a role in pain pathophysiology. Most of these studies have focused on the contribution of central glial cells (e.g., microglia and astrocytes) to neuropathic pain. Likewise, some works have suggested that peripheral glial cells, particularly satellite glial cells (SGCs), and the crosstalk between these cells and the sensory neurons located in the peripheral ganglia, play a role in the phenomenon that leads to pain. Nonetheless, the study of SGCs may be challenging, as the validity of studying those cells in vitro is still controversial. In this study, a research protocol was developed to examine the potential use of primary mixed neuronal-glia cell cultures obtained from the trigeminal ganglion cells (TGCs) of neonate mice (P10-P12). Primary cultures were established and analyzed at 4 h, 24 h, and 48 h. To this purpose, phase contrast microscopy, immunocytochemistry with antibodies against anti-ßIII-tubulin and Sk3, scanning electron microscopy, and time-lapse photography were used. The results indicated the presence of morphological changes in the cultured SGCs obtained from the TGCs. The SGCs exhibited a close relationship with neurons. They presented a round shape in the first 4 h, and a more fusiform shape at 24 h and 48 h of culture. On the other hand, neurons changed from a round shape to a more ramified shape from 4 h to 48 h. Intriguingly, the expression of SK3, a marker of the SGCs, was high in all samples at 4 h, with some cells double-staining for SK3 and ßIII-tubulin. The expression of SK3 decreased at 24 h and increased again at 48 h in vitro. These results confirm the high plasticity that the SGCs may acquire in vitro. In this scenario, the authors hypothesize that, at 4 h, a group of the analyzed cells remained undifferentiated and, therefore, were double-stained for SK3 and ßIII-tubulin. After 24 h, these cells started to differentiate into SCGs, which was clearer at 48 h in the culture. Mixed neuronal-glial TGC cultures might be implemented as a platform to study the plasticity and crosstalk between primary sensory neurons and SGCs, as well as its implications in the development of chronic orofacial pain.
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Several animal species are susceptible to SARS-CoV-2 infection, as documented by case reports and serological and in vivo infection studies. However, the susceptibility of many animal species remains unknown. Furthermore, the expression patterns of SARS-CoV-2 entry factors, such as the receptor angiotensin-converting enzyme 2 (ACE2), as well as transmembrane protease serine subtype 2 (TMPRSS2) and cathepsin L (CTSL), cellular proteases involved in SARS-CoV-2 spike protein activation, are largely unexplored in most species. Here, we generated primary cell cultures from the respiratory tract of domestic and wildlife animals to assess their susceptibility to SARS-CoV-2 infection. Additionally, the presence of ACE2, TMPRSS2 and CTSL within respiratory tract compartments was investigated in a range of animals, some with unknown susceptibility to SARS-CoV-2. Productive viral replication was observed in the nasal mucosa explants and precision-cut lung slices from dogs and hamsters, whereas culture models from ferrets and multiple ungulate species were non-permissive to infection. Overall, whereas TMPRSS2 and CTSL were equally expressed in the respiratory tract, the expression levels of ACE2 were more variable, suggesting that a restricted availability of ACE2 may contribute to reduced susceptibility. Summarized, the experimental infection of primary respiratory tract cell cultures, as well as an analysis of entry-factor distribution, enable screening for SARS-CoV-2 animal reservoirs.
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COVID-19 , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2 , Animales , Animales Salvajes , Perros , Hurones , Humanos , Cultivo Primario de Células , Glicoproteína de la Espiga del CoronavirusRESUMEN
The field of intestinal biology is thirstily searching for different culture methods that complement the limitations of organoids, particularly the lack of a differentiated intestinal compartment. While being recognized as an important milestone for basic and translational biological studies, many primary cultures of intestinal epithelium (IE) rely on empirical trials using hydrogels of various stiffness, whose mechanical impact on epithelial organization remains vague until now. Here, we report the development of hydrogel scaffolds with a range of elasticities and their influence on IE expansion, organization, and differentiation. On stiff substrates (>5 kPa), mouse IE cells adopt a flat cell shape and detach in the short-term. In contrast, on soft substrates (80-500 Pa), they sustain for a long-term, pack into high density, develop columnar shape with improved apical-basal polarity and differentiation marker expression, a phenotype reminiscent of features in vivo mouse IE. We then developed a soft gel molding process to produce 3D Matrigel scaffolds of close-to-nature stiffness, which support and maintain a culture of mouse IE into crypt-villus architecture. Thus, the present work is up-to-date informative for the design of biomaterials for ex vivo intestinal models, offering self-renewal in vitro culture that emulates the mouse IE.
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Biomimética , Intestinos , Animales , Diferenciación Celular , Hidrogeles/metabolismo , Mucosa Intestinal/metabolismo , Ratones , OrganoidesRESUMEN
Thyroid cancer (TC) is the most prevalent endocrine malignancy. More than 90 % of TC is represented by differentiated TC (DTC) arising from the follicular thyroid cells. DTC includes papillary TC (PTC), follicular TC (FTC), and Hürthle cell TC. Anaplastic TC (ATC) accounts for 1% of TC, and it represents 15-40 % of TC death. Current treatment strategies are not completely effective against aggressive DTC or ATC, and mortality is one of the most important challenges. Recently, progresses have been obtained in the understanding of the molecular/genetic basis of TC progression, and new drugs have been introduced [i.e. tyrosine kinase inhibitors (TKIs)], able to block the oncogenic or signaling kinases, associated with cellular growth. Thyroid cell lines, obtained from tumoral cells and chosen for high proliferation in vitro, have been used as preclinical models. Actually, these cells lose the characteristic features of the primary tumor, because they adapt to in vitro growth conditions. For these reasons, the use of these cell lines has important limitations, and more recently human primary cell cultures have been established as monolayer cultures, and investigated for their biological behavior. Moreover, in the past, primary TC cells could be collected only through surgical biopsies, while recently human primary cell cultures can be established also from samples of fine-needle aspiration citology from aggressive dedifferentiated DTC or ATC. Testing in vitro different TKIs in each patient can help to develop new personalized treatments, without using ineffective drugs. In conclusion, personalized medicine and precise oncology, which consider both patients and their disease features, represent the future of the treatment approach, and further progress is needed in this direction.
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Adenocarcinoma Folicular/patología , Adenocarcinoma Folicular/terapia , Adenoma Oxifílico/terapia , Terapia Molecular Dirigida/métodos , Inhibidores de Proteínas Quinasas/uso terapéutico , Carcinoma Anaplásico de Tiroides/terapia , Neoplasias de la Tiroides/terapia , Adenocarcinoma Folicular/mortalidad , Adenoma Oxifílico/patología , Línea Celular Tumoral , Proliferación Celular , Humanos , Medicina de Precisión/métodos , Cultivo Primario de Células , Carcinoma Anaplásico de Tiroides/patología , Glándula Tiroides/patología , Neoplasias de la Tiroides/patología , Resultado del TratamientoRESUMEN
Marine litter is composed mainly of plastics and is recognized as a serious threat to marine ecosystems. Ecotoxicological approaches have started elucidating the potential severity of microplastics (MPs) in controlled laboratory studies with pristine materials but no information exists on marine environmental microlitter as a whole. Here, we characterized the litter in the coastal Northern Tyrrhenian sea and in the stomach of two fish species of socio-economic importance, and exposed primary cell cultures of mucosal and lymphoid organs to marine microlitter for evaluating possible cytotoxic effects. An average of 0.30 ± 0.02 microlitter items m-3 was found in water samples. µFT-IR analysis revealed that plastic particles, namely HDPE, polyamide and polypropylene were present in 100% and 83.3% of Merluccius merluccius and Mullus barbatus analyzed, which overall ingested 14.67 ± 4.10 and 5.50 ± 1.97 items/individual, respectively. Moreover, microlitter was confirmed as a vector of microorganisms. Lastly, the apical end-point of viability was found to be significantly reduced in splenic cells exposed in vitro to two microlitter conditions. Considering the role of the spleen in the mounting of adaptive immune responses, our results warrant more in-depth investigations for clarifying the actual susceptibility of these two species to anthropogenic microlitter.
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Plásticos , Contaminantes Químicos del Agua , Animales , Ecosistema , Monitoreo del Ambiente , Peces , Mar Mediterráneo , Contaminantes Químicos del Agua/análisisRESUMEN
Myotonic Dystrophy type 1 (DM1) is a muscular dystrophy with a multi-systemic nature. It was one of the first diseases in which repeat associated non-ATG (RAN) translation was described in 2011, but has not been further explored since. In order to enhance our knowledge of RAN translation in DM1, we decided to study the presence of DM1 antisense (DM1-AS) transcripts (the origin of the polyglutamine (polyGln) RAN protein) using RT-PCR and FISH, and that of RAN translation via immunoblotting and immunofluorescence in distinct DM1 primary cell cultures, e.g., myoblasts, skin fibroblasts and lymphoblastoids, from ten patients. DM1-AS transcripts were found in all DM1 cells, with a lower expression in patients compared to controls. Antisense RNA foci were found in the nuclei and cytoplasm of a subset of DM1 cells. The polyGln RAN protein was undetectable in all three cell types with both approaches. Immunoblots revealed a 42 kD polyGln containing protein, which was most likely the TATA-box-binding protein. Immunofluorescence revealed a cytoplasmic aggregate, which co-localized with the Golgi apparatus. Taken together, DM1-AS transcript levels were lower in patients compared to controls and a small portion of the transcripts included the expanded repeat. However, RAN translation was not present in patient-derived DM1 cells, or was in undetectable quantities for the available methods.