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1
Tsupykov, O., I. Lushnikova, Y. Nikandrova, et al. "A novel model of periventricular leukomalacia on mouse organotypic brain slice culture." Cell and Organ Transplantology 4, no. 2 (2016): 188–93. http://dx.doi.org/10.22494/cot.v4i2.60.
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The creation of adequate in vitro and in vivo models of neural tissue injury is essential to assess the therapeutic effect of pharmacological agents and regenerative potential of various types of stem cells in diseases of the central nervous system. The aim of this work was to create a novel model of cerebral white matter lesions – periventricular leukomalacia (PVL) – on murine organotypic brain slice culture.Materials and methods. The PVL model was developed on cultured organotypic mice brain slices subjected to oxygen-glucose deprivation (OGD) followed by addition of endotoxin lipopolysaccharide (LPS) in the culture medium. To analyze the degree of tissue injury within PVL simulation, we used spectrophotometric method for estimation of cytosolic enzyme lactate dehydrogenase (LDH) in the culture medium and immunohistochemical analysis of the slices using antibodies to Rip, GFAP and Iba-1 protein markers of oligodendrocyte, astroglia and microglia, respectively.Results. It was shown that the combined effect of OGD and lipopolysaccharide resulted in a significant release of the cytosolic enzyme LDH in culture medium, decrease of Rip-immunoreactivity and a pronounced reactive astro- and microgliosis in murine organotypic brain slice culture.Conclusions. Our model of PVL developed on cultured organotypic mice brain slices is novel and promising tool to study pathogenic mechanisms of cerebral white matter lesions and ways of neuroprotection in this pathology, including pharmacological agents and transplantation of stem cells.
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Kemppainen, Susanna, Nadine Huber, Roosa-Maria Willman, et al. "Organotypic Hippocampal Slice Cultures from Adult Tauopathy Mice and Theragnostic Evaluation of Nanomaterial Phospho-TAU Antibody-Conjugates." Cells 12, no. 10 (2023): 1422. http://dx.doi.org/10.3390/cells12101422.
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Organotypic slice culture models surpass conventional in vitro methods in many aspects. They retain all tissue-resident cell types and tissue hierarchy. For studying multifactorial neurodegenerative diseases such as tauopathies, it is crucial to maintain cellular crosstalk in an accessible model system. Organotypic slice cultures from postnatal tissue are an established research tool, but adult tissue-originating systems are missing, yet necessary, as young tissue-originating systems cannot fully model adult or senescent brains. To establish an adult-originating slice culture system for tauopathy studies, we made hippocampal slice cultures from transgenic 5-month-old hTau.P301S mice. In addition to the comprehensive characterization, we set out to test a novel antibody for hyperphosphorylated TAU (pTAU, B6), with and without a nanomaterial conjugate. Adult hippocampal slices retained intact hippocampal layers, astrocytes, and functional microglia during culturing. The P301S-slice neurons expressed pTAU throughout the granular cell layer and secreted pTAU to the culture medium, whereas the wildtype slices did not. Additionally, cytotoxicity and inflammation-related determinants were increased in the P301S slices. Using fluorescence microscopy, we showed target engagement of the B6 antibody to pTAU-expressing neurons and a subtle but consistent decrease in intracellular pTAU with the B6 treatment. Collectively, this tauopathy slice culture model enables measuring the extracellular and intracellular effects of different mechanistic or therapeutic manipulations on TAU pathology in adult tissue without the hindrance of the blood–brain barrier.
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Pourcel, Violaine, Emile Létourneau, Dominique Jean, and Marilyne Labrie. "Abstract A101: A novel ovarian cancer organotypic tumor slice culture model." Cancer Research 84, no. 5_Supplement_2 (2024): A101. http://dx.doi.org/10.1158/1538-7445.ovarian23-a101.
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Abstract High-grade serous ovarian carcinoma (HGSOC) is the most common form of ovarian cancer (OC). This heterogeneous disease is associated with various molecular alterations influencing the disease course. Unfortunately, the impact of those molecular alterations on the response to therapy is still misunderstood. Few OC models allow control of genetic background in the cancer cells while preserving the tumor's architecture and microenvironment, including the immune cells. Furthermore, there is currently no high-throughput model that retains those characteristics and is suitable for drug screens. This project aims to develop a high-throughput syngeneic murine organotypic tumor slice culture model to study the impact of common HGSOC molecular alterations on anticancer drug responses. The pre-established Trp53−/− syngeneic OC mouse model has been genetically engineered to represent molecular alterations frequently found in HGSC. Ten variants were produced by overexpressing Tp53R172h and overexpressing oncogenes such as Ccne, Brd4, Myc, Ndrg1, and Pik3ca and/or deleting Brca1, Pten, and Nf1. After validation and characterization of the cells in vitro, we performed allografts in immunocompetent C57/bl6 mice and obtained tumors with HGSOC histology. The tumors were used to develop an organotypic tumor slice culture model. Briefly, each tumor is collected and thinly sliced with a vibratome. Each slice can be cultured for several days without significant cell death induction and can be used for a drug screen. Our preliminary data shows that the model preserves the HGSOC histology over six days and remains viable. A viability assay has also been developed to assess the sensitivity of the tumor slices to various anticancer drugs. In conclusion, this project will allow the development of a platform for screening anticancer therapies against HGSOC and will lead to a better understanding of the relationship between HGSOC's common molecular alterations and the responses to therapy. Citation Format: Violaine Pourcel, Emile Létourneau, Dominique Jean, Marilyne Labrie. A novel ovarian cancer organotypic tumor slice culture model [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A101.
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Pantazopoulou, Vasiliki, Tracy Berg, and Alexander Pietras. "TAMI-71. EFFECTS OF THE TREATED MICROENVIRONMENT ON GLIOMA CELL PROPERTIES IN AN ORGANOTYPIC BRAIN SLICE MODEL." Neuro-Oncology 23, Supplement_6 (2021): vi213. http://dx.doi.org/10.1093/neuonc/noab196.853.
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Abstract Glioblastoma is the most aggressive primary brain tumor. Despite treatment all patients invariably recur. Treatment resistance is attributed to the presence of glioma stem-like cells. Initially thought to be a distinct and static cell population, it is becoming increasingly clear that the glioma stem-like cell phenotype represents one of many cellular states and that glioma cells show plasticity between stem-like and non-stem like states. These plastic cell states are affected by the tumor microenvironment. In our lab we have shown that irradiated and hypoxic astrocytes increase the stem-like cell properties of glioma cells. In this study, we aim to evaluate how the treated microenvironment alters glioma cell properties and use ex vivo organotypic brain slices generated from tumor bearing and tumor naïve mice to assess all aspects of the microenvironment. We first characterized organotypic brain slices cultured in different oxygen tensions. We saw that tumor-bearing slices survive for at least 14 days in culture at 21%, 5% or 1% oxygen tension (O2). Tumor cells were more viable in all culture conditions and timepoints compared to non-tumor cells. Moreover, we found that astrocytes seem to be attracted to tumor areas in both 5% and 1% O2 cultures. We then used the organotypic glioma slice culture system to address how preconditioning the microenvironment using radiation or temozolomide affects the properties of glioma cells that are seeded in these pretreated, tumor naïve slices. We saw that fluorescently labelled glioma cells seeded in treated slices can be isolated after two days of culture in the slices and can be used for downstream analyses, such as temozolomide or radiation treatment and colony formation. This study will elucidate the effect of the treated microenvironment on glioma cell properties by using the medium throughput method of organotypic slice cultures.
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Negussie, Mikias, Saritha Krishna, Andy Daniel, Cesar Nava Gonzales, and Shawn Hervey-Jumper. "TMOD-16. ZUCLOPENTHIXOL REDUCES ACTIVITY-DEPENDENT GLIOBLASTOMA PROLIFERATION." Neuro-Oncology 26, Supplement_8 (2024): viii322. http://dx.doi.org/10.1093/neuonc/noae165.1280.
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Abstract A major challenge in modeling glioblastoma in the laboratory is the complex microenvironment in which tumors evolve in humans. The varied architecture and immune privilege of the brain limits the external validity of many experiments performed in modern cell biology laboratories. Organotypic human cortical slices present an opportunity to model glioblastoma and test the effect of drugs against glioma in ex-vivo human tissue. Cortical slice cultures maintain the cellular organization of the human brain, more closely approximating glioblastoma proliferation in patients. In this study, we test the effect zuclopenthixol, a repurposed antipsychotic inhibitor of glioblastoma-induced neuronal hyperexcitability, on the proliferation of glioblastoma cells in an organotypic human cortical slice culture. Primary patient-derived glioblastoma cultures were established, and human cortical tissues were emersed in oxygenated ACSF agarose gel before preparing 250 µm thick slices. Slices were either exogenously seeded with glioblastoma cells or stained for endogenous tumor infiltration. Slices with either endogenous or exogenous seeding were then treated with zuclopenthixol or regular media change and cultured for 10 days. Proliferation was measured with Ki67 staining and quantified via automated pixel summation using the Pillow library in Python. 0.1 µM zuclopenthixol treated slices had significantly lower proliferation index in both the endogenous tumor condition (0.04 vs 0.30, P < 0.05) and the exogenous seeding condition (0.0013 vs 0.028, P = 0.033). Reduced tissue integrity was observed beginning day 8 of culture, as tissue surrounding tumor seeding sites eroded. Here, we model glioblastoma proliferation in live human tissue and quantify the anti-proliferative effect of zuclopenthixol. We show that zuclopenthixol treatment significantly reduced the proliferation index. Our experimental model allowed the optimization of organotypic human cortical slice cultures for immunofluorescence studies and live-cell imaging.
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Biancotti, Juan C., Kendal A. Walker, Guihua Jiang, Julie Di Bernardo, Lonnie D. Shea, and Shaun M. Kunisaki. "Hydrogel and neural progenitor cell delivery supports organotypic fetal spinal cord development in an ex vivo model of prenatal spina bifida repair." Journal of Tissue Engineering 11 (January 2020): 204173142094383. http://dx.doi.org/10.1177/2041731420943833.
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Studying how the fetal spinal cord regenerates in an ex vivo model of spina bifida repair may provide insights into the development of new tissue engineering treatment strategies to better optimize neurologic function in affected patients. Here, we developed hydrogel surgical patches designed for prenatal repair of myelomeningocele defects and demonstrated viability of both human and rat neural progenitor donor cells within this three-dimensional scaffold microenvironment. We then established an organotypic slice culture model using transverse lumbar spinal cord slices harvested from retinoic acid–exposed fetal rats to study the effect of fibrin hydrogel patches ex vivo. Based on histology, immunohistochemistry, gene expression, and enzyme-linked immunoabsorbent assays, these experiments demonstrate the biocompatibility of fibrin hydrogel patches on the fetal spinal cord and suggest this organotypic slice culture system as a useful platform for evaluating mechanisms of damage and repair in children with neural tube defects.
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Rappoldt, Liam, Adrienne Weeks, Rodney Ouellete, et al. "TMOD-26. ESTABLISHING A PATIENT-DERIVED, IN-VITRO ORGANOTYPIC SLICE CULTURE MODEL OF GBM." Neuro-Oncology 22, Supplement_2 (2020): ii233. http://dx.doi.org/10.1093/neuonc/noaa215.976.
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Abstract Glioblastoma Multiforme (GBM) is the most common primary malignant brain tumour. This tumour is universally fatal with a median survival of 15 months. Driving this pathology is an extremely heterogeneic tumour and complex tumour microenvironment. GBM research is primarily conducted using immortalized or primary cell lines due to their practicality and reproducibility. However, these cell lines do not effectively recapitulate the tumour microenvironment. Mouse models address these shortcomings but are laborious and expensive. We propose to utilize a patient derived organotypic culture model of GBM as an intermediary. We have utilized this model to test genetic manipulation via lentiviral transduction and the feasibility of utilizing this model to understand patient derived extracellular vesicles (EVs). We have sectioned and cultured patient derived organotypic models for 14 days without loss of viability. To determine if these organotypic cultures are amenable to lentiviral manipulation, tissue sections were transduced with far-red fluorescent lentivirus and efficiency determined by confocal laser scanning microscopy (CLSM) and flow cytometry (FC). To determine feasibility as a model for EVs, media obtained from patient-derived organotypic cultures was analyzed by western blot, nanoparticle tracking analysis (NTA), and nanoFlow Cytometry (nFC). In the future these EVs will be compared to those found in patient serum. The model of GBM has been lentivirally transduced to express a far-red fluorescent vector in approximately 15% of the slice, quantified by CLSM and FC. EV-sized particles positive for canonical EV markers have been identified in the media by NTA, nFC and western blot. Using lentiviral-mediated genetic engineering and emerging EV science, this organotypic slice culture models yields exciting utility in GBM research. The established organotypic slice culture model will likely be a valuable tool in the study of GBM biology and EV dynamics.
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Schwerdtfeger, Luke A., Elizabeth P. Ryan, and Stuart A. Tobet. "An organotypic slice model for ex vivo study of neural, immune, and microbial interactions of mouse intestine." American Journal of Physiology-Gastrointestinal and Liver Physiology 310, no. 4 (2016): G240—G248. http://dx.doi.org/10.1152/ajpgi.00299.2015.
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Organotypic tissue slices provide seminatural, three-dimensional microenvironments for use in ex vivo study of specific organs and have advanced investigative capabilities compared with isolated cell cultures. Several characteristics of the gastrointestinal tract have made in vitro models for studying the intestine challenging, such as maintaining the intricate structure of microvilli, the intrinsic enteric nervous system, Peyer's patches, the microbiome, and the active contraction of gut muscles. In the present study, an organotypic intestinal slice model was developed that allows for functional investigation across regions of the intestine. Intestinal tissue slices were maintained ex vivo for several days in a physiologically relevant environment that preserved normal enterocyte structure, intact and proliferating crypt cells, submucosal organization, and muscle wall composure. Cell death was measured by a membrane-impermeable DNA binding indicator, ethidium homodimer, and less than 5% of cells were labeled in all regions of the villi and crypt epithelia at 24 h ex vivo. This tissue slice model demonstrated intact myenteric and submucosal neuronal plexuses and functional interstitial cells of Cajal to the extent that nonstimulated, segmental contractions occurred for up to 48 h ex vivo. To detect changes in physiological responses, slices were also assessed for segmental contractions in the presence and absence of antibiotic treatment, which resulted in slices with lesser or greater amounts of commensal bacteria, respectively. Segmental contractions were significantly greater in slices without antibiotics and increased native microbiota. This model renders mechanisms of neuroimmune-microbiome interactions in a complex gut environment available to direct observation and controlled perturbation.
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Elfarrash, Sara, Nanna Møller Jensen, Nelson Ferreira, et al. "Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates." Acta Neuropathologica Communications 7, no. 1 (2019): 213. https://doi.org/10.1186/s40478-019-0865-5.
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Here we describe the use of an organotypic hippocampal slice model for studying α-synuclein aggregation and inter-neuronal spreading initiated by microinjection of pre-formed α-synuclein fibrils (PFFs). PFF injection at dentate gyrus (DG) templates the formation of endogenous α-synuclein aggregates in axons and cell bodies of this region that spread to CA3 and CA1 regions. Aggregates are insoluble and phosphorylated at serine-129, recapitulating Lewy pathology features found in Parkinson's disease and other synucleinopathies. The model was found to favor anterograde spreading of the aggregates. Furthermore, it allowed development of slices expressing only serine-129 phosphorylation-deficient human α-synuclein (S129G) using an adeno-associated viral (AAV) vector in α-synuclein knockout slices. The processes of aggregation and spreading of α-synuclein were thereby shown to be independent of phosphorylation at serine-129. We provide methods and highlight crucial steps for PFF microinjection and characterization of aggregate formation and spreading. Slices derived from genetically engineered mice or manipulated using viral vectors allow testing of hypotheses on mechanisms involved in the formation of α-synuclein aggregates and their prion-like spreading.
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Haque, Azizul, Donald C. Shields, Arabinda Das, Abhay Varma, Russel J. Reiter, and Narendra L. Banik. "Melatonin receptor-mediated attenuation of excitotoxic cell death in cultured spinal cord slices." Melatonin Research 4, no. 2 (2021): 336–47. http://dx.doi.org/10.32794/mr11250098.
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Recent studies suggest ex vivo modeling of neuronal injury is a robust approach for the mechanistic study of neurodegeneration. Melatonin, an indolamine, is a versatile molecule with antioxidative, antiapoptotic, neuroprotective, and anti-inflammatory properties. While melatonin has been studied as a therapeutic agent for spinal cord injury (SCI) related neuronal cell loss, its actions in organotypic slice cultures approximating SCI effects are less well understood. The actions of melatonin were therefore examined following exposure of cultured rat spinal cord slices to glutamate excitotoxicity. Exposure to glutamate (500 μM) for 4 hours induced neuronal degeneration that was prevented by 0.5 μM melatonin (applied immediately or 4 hours following glutamate exposure). Decreased internucleosomal DNA fragmentation, Bax:Bcl-2 and calpain:calpastatin ratios, caspase 8, 9 and 3 activities in slice cultures were measured following melatonin treatment. Melatonin receptor (MTR1, MTR2) mRNA levels were increased in the melatonin treated spinal cord slices. To confirm melatonin receptor-mediated protection, slice cultures were treated with 10 or 25 μM luzindole (melatonin receptor antagonist) at 0 and 4 hours, respectively, after glutamate exposure. Luzindole significantly decreased the ability of melatonin to prevent cell death in the sliced culture model. These results suggest melatonin receptors may provide a pathway for therapeutic applications to prevent penumbral neuron loss following SCI.
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Croft, Cara L., and Wendy Noble. "Preparation of organotypic brain slice cultures for the study of Alzheimer’s disease." F1000Research 7 (May 15, 2018): 592. http://dx.doi.org/10.12688/f1000research.14500.1.
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Alzheimer's disease, the most common cause of dementia, is a progressive neurodegenerative disorder characterised by amyloid-beta deposits in extracellular plaques, intracellular neurofibrillary tangles of aggregated tau, synaptic dysfunction and neuronal death. There are no cures for AD and current medications only alleviate some disease symptoms. Transgenic rodent models to study Alzheimer’s mimic features of human disease such as age-dependent accumulation of abnormal beta-amyloid and tau, synaptic dysfunction, cognitive deficits and neurodegeneration. These models have proven vital for improving our understanding of the molecular mechanisms underlying AD and for identifying promising therapeutic approaches. However, modelling neurodegenerative disease in animals commonly involves aging animals until they develop harmful phenotypes, often coupled with invasive procedures. In vivo studies are also resource, labour, time and cost intensive. We have developed a novel organotypic brain slice culture model to study Alzheimer’ disease which brings the potential of substantially reducing the number of rodents used in dementia research from an estimated 20,000 per year. We obtain 36 brain slices from each mouse pup, considerably reducing the numbers of animals required to investigate multiple stages of disease. This tractable model also allows the opportunity to modulate multiple pathways in tissues from a single animal. We believe that this model will most benefit dementia researchers in the academic and drug discovery sectors. We validated the slice culture model against aged mice, showing that the molecular phenotype closely mimics that displayed in vivo, albeit in an accelerated timescale. We showed beneficial outcomes following treatment of slices with agents previously shown to have therapeutic effects in vivo, and we also identified new mechanisms of action of other compounds. Thus, organotypic brain slice cultures from transgenic mouse models expressing Alzheimer’s disease-related genes may provide a valid and sensitive replacement for in vivo studies that do not involve behavioural analysis.
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Croft, Cara L., and Wendy Noble. "Preparation of organotypic brain slice cultures for the study of Alzheimer’s disease." F1000Research 7 (June 27, 2018): 592. http://dx.doi.org/10.12688/f1000research.14500.2.
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Alzheimer's disease, the most common cause of dementia, is a progressive neurodegenerative disorder characterised by amyloid-beta deposits in extracellular plaques, intracellular neurofibrillary tangles of aggregated tau, synaptic dysfunction and neuronal death. Transgenic rodent models to study Alzheimer’s mimic features of human disease such as age-dependent accumulation of abnormal beta-amyloid and tau, synaptic dysfunction, cognitive deficits and neurodegeneration. These models have proven vital for improving our understanding of the molecular mechanisms underlying AD and for identifying promising therapeutic approaches. However, modelling neurodegenerative disease in animals commonly involves aging animals until they develop harmful phenotypes, often coupled with invasive procedures. We have developed a novel organotypic brain slice culture model to study Alzheimer’s disease using 3xTg-AD mice which brings the potential of substantially reducing the number of rodents used in dementia research from an estimated 20,000 per year. Using a McIllwain tissue chopper, we obtain 36 x 350 micron slices from each P8-P9 mouse pup for culture between 2 weeks and 6 months on semi-permeable 0.4 micron pore membranes, considerably reducing the numbers of animals required to investigate multiple stages of disease. This tractable model also allows the opportunity to modulate multiple pathways in tissues from a single animal. We believe that this model will most benefit dementia researchers in the academic and drug discovery sectors. We validated the slice culture model against aged mice, showing that the molecular phenotype closely mimics that displayed in vivo, albeit in an accelerated timescale. We showed beneficial outcomes following treatment of slices with agents previously shown to have therapeutic effects in vivo, and we also identified new mechanisms of action of other compounds. Thus, organotypic brain slice cultures from transgenic mouse models expressing Alzheimer’s disease-related genes may provide a valid and sensitive replacement for in vivo studies that do not involve behavioural analysis.
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Raju, E. N. Sanjaya, Jan Kuechler, Susanne Behling, et al. "Maintenance of Stemlike Glioma Cells and Microglia in an Organotypic Glioma Slice Model." Neurosurgery 77, no. 4 (2015): 629–43. http://dx.doi.org/10.1227/neu.0000000000000891.
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Abstract BACKGROUND: The therapeutic resistance of gliomas is, at least in part, due to stemlike glioma cells (SLGCs), which self-renew, generate the bulk of tumor cells, and sustain tumor growth. SLGCs from glioblastomas (GB) have been studied in cell cultures or mouse models, whereas little is known about SLGCs from lower grade gliomas. OBJECTIVE: To compare cell and organotypic slice cultures from GBs and lower grade gliomas and study the maintenance of SLGCs. METHODS: Cells and tissue slices from astrocytomas, oligodendrogliomas, oligoastrocytomas, and GBs were cultivated in (1) serum-free medium supplemented with the growth factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), (2) medium containing 10% serum plus EGF and bFGF (F+GF medium), or (3) medium containing 10% fetal calf serum (F medium). Maintenance of cells and cytoarchitecture was addressed, using several candidate SLGC markers (Nestin, Sox2, CD133, CD44, CD49f/integrin α6, and Notch) as well as CD31 (endothelial cells), ionized calcium-binding adapter molecule 1 (microglia), and vimentin. Cell vitality was determined. RESULTS: SLGCs were present in tissue slices from lower and higher grade gliomas. Preservation of the cytoarchitecture in slices was possible for >3 weeks. Maintenance of SLGCs required the presence of EGF/bFGF in cell and slice cultures, in which F+GF appeared superior to N medium. Constraints were observed regarding the preservation of the microglia but not of the endothelial cells. Maintenance of the microglia was improved by addition of the cytokine macrophage colony-stimulating factor. CONCLUSION: Medium supplemented with serum and growth factors EGF, bFGF, and macrophage colony-stimulating factor permits the preservation of SLGCs and non-SLGCs in the original glioma microenvironment.
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Weitz, Jonathan Robert, Herve Tiriac, Tatiana Hurtado de Mendoza, Alexis Wascher, and Andrew M. Lowy. "Using Organotypic Tissue Slices to Investigate the Microenvironment of Pancreatic Cancer: Pharmacotyping and Beyond." Cancers 13, no. 19 (2021): 4991. http://dx.doi.org/10.3390/cancers13194991.
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Organotypic tissue slices prepared from patient tumors are a semi-intact ex vivo preparation that recapitulates many aspects of the tumor microenvironment (TME). While connections to the vasculature and nervous system are severed, the integral functional elements of the tumor remain intact for many days during the slice culture. During this window of time, the slice platforms offer a suite of molecular, biomechanical and functional tools to investigate PDAC biology. In this review, we first briefly discuss the development of pancreatic tissue slices as a model system. Next, we touch upon using slices as an orthogonal approach to study the TME as compared to other established 3D models, such as organoids. Distinct from most other models, the pancreatic slices contain autologous immune and other stromal cells. Taking advantage of the existing immune cells within the slices, we will discuss the breakthrough studies which investigate the immune compartment in the pancreas slices. These studies will provide an important framework for future investigations seeking to exploit or reprogram the TME for cancer therapy.
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Sullivan, Breandan L., David Leu, Donald M. Taylor, Christian S. Fahlman, and Philip E. Bickler. "Isoflurane Prevents Delayed Cell Death in an Organotypic Slice Culture Model of Cerebral Ischemia." Anesthesiology 96, no. 1 (2002): 189–95. http://dx.doi.org/10.1097/00000542-200201000-00033.
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Background General anesthetics reduce neuronal death caused by focal cerebral ischemia in rodents and by in vitro ischemia in cultured neurons and brain slices. However, in intact animals, the protective effect may enhance neuronal survival for only several days after an ischemic injury, possibly because anesthetics prevent acute but not delayed cell death. To further understand the mechanisms and limitations of volatile anesthetic neuroprotection, the authors developed a rat hippocampal slice culture model of cerebral ischemia that permits assessment of death and survival of neurons for at least 2 weeks after simulated ischemia. Methods Survival of CA1, CA3, and dentate gyrus neurons in cultured hippocampal slices (organotypic slice culture) was examined 2-14 days after 45 min of combined oxygen-glucose deprivation at 37 degrees C (OGD). Delayed cell death was serially measured in each slice by quantifying the binding of propidium iodide to DNA with fluorescence microscopy. Results Neuronal death was greatest in the CA1 region, with maximal death occurring 3-5 days after OGD. In CA1, cell death was 80 +/- 18% (mean +/- SD) 3 days after OGD and was 80-100% after 1 week. Death of 70 +/- 16% of CA3 neurons and 48 +/- 28% of dentate gyrus neurons occurred by the third day after OGD. Both isoflurane (1%) and the N-methyl-D-aspartate antagonist MK-801 (10 microm) reduced cell death to levels similar to controls (no OGD) for 14 days after the injury. Isoflurane also reduced cell death in CA1 and CA3 caused by application of 100 but not 500 microm glutamate. Cellular viability (calcein fluorescence) and morphology were preserved in isoflurane-protected neurons. Conclusions In an in vitro model of simulated ischemia, 1% isoflurane is of similar potency to 10 microm MK-801 in preventing delayed cell death. Modulation of glutamate excitotoxicity may contribute to the protective mechanism.
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Spahr-Schopfer, Isabelle, Lazlo Vutskits, Nicholas Toni, Pierre-Alain Buchs, Lorena Parisi, and Dominique Muller. "Differential Neurotoxic Effects of Propofol on Dissociated Cortical Cells and Organotypic Hippocampal Cultures." Anesthesiology 92, no. 5 (2000): 1408–17. http://dx.doi.org/10.1097/00000542-200005000-00032.
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Background Propofol is a widely used anesthetic agent for adults and children. Although extensive clinical use has demonstrated its safety, neurologic dysfunctions have been described after the use of this agent. A recent study on a model of aggregating cell cultures reported that propofol might cause irreversible lesions of gamma-aminobutyric acid-mediated (GABAergic) neurons when administered at a critical phase of brain development. We investigated this issue by comparing the effects of long-term propofol treatment on two models of brain cultures: dissociated neonatal cortical cell cultures and organotypic slice cultures. Methods Survival of GABAergic neurons in dissociated cultures of newborn rat cortex (postnatal age, 1 day) treated for 3 days with different concentrations of propofol was assessed using histologic and cytochemical methods. For hippocampal organotypic slice cultures (postnatal age, 1 and 7 days), cell survival was assessed by measuring functional and morphologic parameters: extracellular and intracellular electrophysiology, propidium staining of dying cells, and light and electron microscopy. Results In dissociated neonatal cell cultures, propofol induced dose-dependent lesions of GABAergic neurons and of glial cells. In contrast, no evidence for neurotoxic effects of propofol were found after long-term treatment of organotypic slice cultures. Excitatory transmission was not affected by propofol, and inhibitory transmission was still functional. Histologic preparations showed no evidence for cell degeneration or death. Conclusion Although long-term applications of propofol to dissociated cortical cell cultures produced degeneration and death of GABAergic neurons and glial cells, no such lesions were found when using a model of postnatal organotypic slice cultures. This conclusion is based on both functional and morphologic tests.
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Weitz, Jonathan, Tatiana Hurtado de Mendoza, Herve Tiriac, et al. "Abstract 289: A novel ex-vivo organotypic culture platform for functional interrogation of human appendiceal neoplasms." Cancer Research 82, no. 12_Supplement (2022): 289. http://dx.doi.org/10.1158/1538-7445.am2022-289.
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Abstract Appendiceal neoplasms are rare and often clinically present with peritoneal metastasis. While surgical tumor resection is effective for some types of primary appendiceal cancers, patients with metastatic disease have poor prognostic outcomes. Models to study appendix cancer biology are limited, given that 1) no mouse models exist and 2) reliable in vitro models are unavailable. As such, we have developed an ex-vivo organotypic slice model to examine cellular interactions between tumor cells and their local microenvironment. Tumor specimens from human appendiceal cancer patients were cut using a vibratome to make 200 μm organotypic slices. Slices were cultured on transwell inserts and tested for changes in morphological, cellular and functional characteristics over a seven-day period. Organotypic slices maintained their cellular composition in regard to the proportion of epithelial, immune cells and fibroblasts. Live cell [Ca2+]i imaging of long term cultured slices confirmed that immune cells remain functionally active when stimulated with extracellular ATP. Lasty, using tumor biopsies from human donors, we have identified a diverse immunological profile of appendiceal tumors not previously identified. Our study illustrates a novel approach for studying the pathophysiology of appendiceal cancer, a notoriously difficult disease to model. Citation Format: Jonathan Weitz, Tatiana Hurtado de Mendoza, Herve Tiriac, Joel Baumgartner, Kaitlyn Kelly, Jula Veerapong, Andrew Lowy. A novel ex-vivo organotypic culture platform for functional interrogation of human appendiceal neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 289.
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GIANINAZZI, C., M. SCHILD, N. MÜLLER, et al. "Organotypic slice cultures from rat brain tissue: a new approach forNaegleria fowleriCNS infectionin vitro." Parasitology 132, no. 6 (2005): 797–804. http://dx.doi.org/10.1017/s0031182005008619.
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The free-living amoebaNaegleria fowleriis the aetiological agent of primary amoebic meningoencephalitis (PAM), a disease leading to death in the vast majority of cases. In patients suffering from PAM, and in corresponding animal models, the brain undergoes a massive inflammatory response, followed by haemorrhage and severe tissue necrosis. Both,in vivoandin vitromodels are currently being used to study PAM infection. However, animal models may pose ethical issues, are dependent upon availability of specific infrastructural facilities, and are time-consuming and costly. Conversely, cell cultures lack the complex organ-specific morphology foundin vivo, and thus, findings obtainedin vitrodo not necessarily reflect the situationin vivo. The present study reports infection of organotypic slice cultures from rat brain withN. fowleriand compares the findings in this culture system within vivoinfection in a rat model of PAM, that proved complementary to that of mice. We found that brain morphology, as presentin vivo, is well retained in organotypic slice cultures, and that infection time-course including tissue damage parallels the observationsin vivoin the rat. Therefore, organotypic slice cultures from rat brain offer a newin vitroapproach to studyN. fowleriinfection in the context of PAM.
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Pandamooz, Sareh, M. S. Saied, M. Nabiuni, L. Dargahi, and M. Pourghasem. "Evaluation of Epidermal Neural Crest Stem Cells in Organotypic Spinal Cord Slice Culture Platform." Folia Biologica 62, no. 6 (2016): 263–67. http://dx.doi.org/10.14712/fb2016062060263.
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Among various strategies employed for spinal cord injury, stem cell therapy is a potential treatment. So far, a variety of stem cells have been evaluated in animal models and humans with spinal cord injury, and epidermal neural crest stem cells represent one of the attractive types in this area. Although these multipotent stem cells have been assessed in several spinal cord injury models by independent laboratories, extensive work remains to be done to ascertain whether these cells can safely improve the outcome following human spinal cord injury. Among the models that closely mimic human spinal cord injury, the in vitro model of injury in organotypic spinal cord slice culture has been identified as one of the faithful platforms for injury-related investigations. In this study, green fluorescent protein-expressing stem cells were grafted into injured organotypic spinal cord slice culture and their survival was examined by confocal microscope seven days after transplantation. Data obtained from this preliminary study showed that these stem cells can survive on top of the surface of injured slices, as observed on day seven following their transplantation. This result revealed that this in vitro model of injury can be considered as a suitable context for further evaluation of epidermal neural crest stem cells before their application in large animals.
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Sypecka, Joanna, Sylwia Koniusz, Maria Kawalec, and Anna Sarnowska. "The Organotypic Longitudinal Spinal Cord Slice Culture for Stem Cell Study." Stem Cells International 2015 (2015): 1–10. http://dx.doi.org/10.1155/2015/471216.
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The objective of this paper is to describe in detail the method of organotypic longitudinal spinal cord slice culture and the scientific basis for its potential utility. The technique is based on the interface method, which was described previously and thereafter was modified in our laboratory. The most important advantage of the presented model is the preservation of the intrinsic spinal cord fiber tract and the ventrodorsal polarity of the spinal cord. All the processes occurring during axonal growth, regeneration, synapse formation, and myelination could be visualized while being culturedin vitrofor up to 4-5 weeks after the slices had been isolated. Both pups and adult animals can undergo the same, equally efficient procedures when going by the protocol in question. The urgent need for an appropriatein vitromodel for spinal cord regeneration results from a greater number of clinical trials concerning regenerative medicine in the spinal cord injury and from still insufficient knowledge of the molecular mechanisms involved in the neuroreparative processes. The detailed method of organotypic longitudinal spinal cord slice culture is accompanied by examples of its application to studying biological processes to which both the CNS inhabiting and grafted cells are subjected.
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Duff, Karen, Wendy Noble, Kayte Gaynor, and Yasuji Matsuoka. "Organotypic Slice Cultures from Transgenic Mice as Disease Model Systems." Journal of Molecular Neuroscience 19, no. 3 (2002): 317–20. http://dx.doi.org/10.1385/jmn:19:3:317.
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Ravikumar, Madhumitha, Seema Jain, Robert H. Miller, Jeffrey R. Capadona, and Stephen M. Selkirk. "An organotypic spinal cord slice culture model to quantify neurodegeneration." Journal of Neuroscience Methods 211, no. 2 (2012): 280–88. http://dx.doi.org/10.1016/j.jneumeth.2012.09.004.
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Mann, Breanna, Noah Bell, Denise Dunn, Scott Floyd, Shawn Hingtgen, and Andrew Satterlee. "TMOD-31. AN ORGANOTYPIC TISSUE PLATFORM TO BRIDGE IN VITRO AND IN VIVO ASSAYS FOR BRAIN CANCER TREATMENT." Neuro-Oncology 23, Supplement_6 (2021): vi222. http://dx.doi.org/10.1093/neuonc/noab196.892.
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Abstract Brain cancers remain one of the greatest medical challenges. The lack of experimentally tractable models that recapitulate brain structure/function represents a major impediment. Platforms that enable functional testing in high-fidelity models are urgently needed to accelerate the identification and translation of therapies to improve outcomes for patients suffering from brain cancer. In vitro assays are often too simple and artificial while in vivo studies can be time-intensive and complicated. Our live, organotypic brain slice platform can be used to seed and grow brain cancer cell lines, allowing us to bridge the existing gap in models. These tumors can rapidly establish within the brain slice microenvironment, and morphologic features of the tumor can be seen within a short period of time. The growth, migration, and treatment dynamics of tumors seen on the slices recapitulate what is observed in vivo yet is missed by in vitro models. Additionally, the brain slice platform allows for the dual seeding of different cell lines to simulate characteristics of heterogeneous tumors. Furthermore, live brain slices with embedded tumor can be generated from tumor-bearing mice. This method allows us to quantify tumor burden more effectively and allows for treatment and retreatment of the slices to understand treatment response and resistance that may occur in vivo. This brain slice platform lays the groundwork for a new clinically relevant preclinical model which provides physiologically relevant answers in a short amount of time leading to an acceleration of therapeutic translation.
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Back, Adam, Kelsey Y. Tupper, Tao Bai, et al. "Ammonia-induced brain swelling and neurotoxicity in an organotypic slice model." Neurological Research 33, no. 10 (2011): 1100–1108. http://dx.doi.org/10.1179/1743132811y.0000000046.
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Mayer, Daniel, Heike Fischer, Urs Schneider, Bernd Heimrich, and Martin Schwemmle. "Borna Disease Virus Replication in Organotypic Hippocampal Slice Cultures from Rats Results in Selective Damage of Dentate Granule Cells." Journal of Virology 79, no. 18 (2005): 11716–23. http://dx.doi.org/10.1128/jvi.79.18.11716-11723.2005.
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ABSTRACT In the hippocampus of Borna disease virus (BDV)-infected newborn rats, dentate granule cells undergo progressive cell death. BDV is noncytolytic, and the pathogenesis of this neurodevelopmental damage in the absence of immunopathology remains unclear. A suitable model system to study early events of the pathology is lacking. We show here that organotypic hippocampal slice cultures from newborn rat pups are a suitable ex vivo model to examine BDV neuropathogenesis. After challenging hippocampal slice cultures with BDV, we observed a progressive loss of calbindin-positive granule cells 21 to 28 days postinfection. This loss was accompanied by reduced numbers of mossy fiber boutons when compared to mock-infected cultures. Similarly, the density of dentate granule cell axons, the mossy fiber axons, appeared to be substantially reduced. In contrast, hilar mossy cells and pyramidal neurons survived, although BDV was detectable in these cells. Despite infection of dentate granule cells 2 weeks postinfection, the axonal projections of these cells and the synaptic connectivity patterns were comparable to those in mock-infected cultures, suggesting that BDV-induced damage of granule cells is a postmaturation event that starts after mossy fiber synapses are formed. In summary, we find that BDV infection of rat organotypic hippocampal slice cultures results in selective neuronal damage similar to that observed with infected newborn rats and is therefore a suitable model to study BDV-induced pathology in the hippocampus.
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Warner, Jennifer D., Christian G. Peters, Rudel Saunders, et al. "Visualizing form and function in organotypic slices of the adult mouse parotid gland." American Journal of Physiology-Gastrointestinal and Liver Physiology 295, no. 3 (2008): G629—G640. http://dx.doi.org/10.1152/ajpgi.90217.2008.
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An organotypic slice preparation of the adult mouse parotid salivary gland amenable to a variety of optical assessments of fluid and protein secretion dynamics is described. The semi-intact preparation rendered without the use of enzymatic treatment permitted live-cell imaging and multiphoton analysis of cellular and supracellular signals. Toward this end we demonstrated that the parotid slice is a significant addition to the repertoire of tools available to investigators to probe exocrine structure and function since there is currently no cell culture system that fully recapitulates parotid acinar cell biology. Importantly, we show that a subpopulation of the acinar cells of parotid slices can be maintained in short-term culture and retain their morphology and function for up to 2 days. This in vitro model system is a significant step forward compared with enzymatically dispersed acini that rapidly lose their morphological and functional characteristics over several hours, and it was shown to be long enough for the expression and trafficking of exogenous protein following adenoviral infection. This system is compatible with a variety of genetic and physiological approaches used to study secretory function.
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Renic, Marija, Suresh N. Kumar, Debebe Gebremedhin, et al. "Protective effect of 20-HETE inhibition in a model of oxygen-glucose deprivation in hippocampal slice cultures." American Journal of Physiology-Heart and Circulatory Physiology 302, no. 6 (2012): H1285—H1293. http://dx.doi.org/10.1152/ajpheart.00340.2011.
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Recent studies have indicated that inhibitors of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) may have direct neuroprotective actions since they reduce infarct volume after ischemia reperfusion in the brain without altering blood flow. To explore this possibility, the present study used organotypic hippocampal slice cultures subjected to oxygen-glucose deprivation (OGD) and reoxygenation to examine whether 20-HETE is released by organotypic hippocampal slices after OGD and whether it contributes to neuronal death through the generation of ROS and activation of caspase-3. The production of 20-HETE increased twofold after OGD and reoxygenation. Blockade of the synthesis of 20-HETE with N-hydroxy- N′-(4-butyl-2-methylphenol)formamidine (HET0016) or its actions with a 20-HETE antagonist, 20-hydroxyeicosa-6( Z),15( Z)-dienoic acid, reduced cell death, as measured by the release of lactate dehydrogenase and propidium iodide uptake. Administration of a 20-HETE mimetic, 20-hydroxyeicosa-5( Z),14( Z)-dienoic acid (5,14-20-HEDE), had the opposite effect and increased injury after OGD. The death of neurons after OGD was associated with an increase in the production of ROS and activation of caspase-3. These effects were attenuated by HET0016 and potentiated after the administration of 5,14-20-HEDE. These findings indicate that the production of 20-HETE by hippocampal slices is increased after OGD and that inhibitors of the synthesis or actions of 20-HETE protect neurons from ischemic cell death. The protective effect of 20-HETE inhibitors is associated with a decrease in superoxide production and activation of caspase-3.
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Chong, Harvey K., Ziang Ma, Kendrew Ka Chuon Wong, Andrew Morokoff, and Chris French. "An In Vitro Brain Tumour Model in Organotypic Slice Cultures Displaying Epileptiform Activity." Brain Sciences 13, no. 10 (2023): 1451. http://dx.doi.org/10.3390/brainsci13101451.
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Brain tumours have significant impacts on patients’ quality of life, and current treatments have limited effectiveness. To improve understanding of tumour development and explore new therapies, researchers rely on experimental models. However, reproducing tumour-associated epilepsy (TAE) in these models has been challenging. Existing models vary from cell lines to in vivo studies, but in vivo models are resource-intensive and often fail to mimic crucial features like seizures. In this study, we developed a technique in which normal rat organotypic brain tissue is implanted with an aggressive brain tumour. This method produces a focal invasive lesion that preserves neural responsiveness and exhibits epileptiform hyperexcitability. It allows for real-time imaging of tumour growth and invasion for up to four weeks and microvolume fluid sampling analysis of different regions, including the tumour, brain parenchyma, and peritumoral areas. The tumour cells expand and infiltrate the organotypic slice, resembling in vivo behaviour. Spontaneous seizure-like events occur in the tumour slice preparation and can be induced with stimulation or high extracellular potassium. Furthermore, we assess extracellular fluid composition in various regions of interest. This technique enables live cell confocal microscopy to record real-time tumour invasion properties, whilst maintaining neural excitability, generating field potentials, and epileptiform discharges, and provides a versatile preparation for the study of major clinical problems of tumour-associated epilepsy.
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Su, X., D. Fang, Y. Liu, et al. "Three-dimensional organotypic culture of human salivary glands: the slice culture model." Oral Diseases 22, no. 7 (2016): 639–48. http://dx.doi.org/10.1111/odi.12508.
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Michael, A., G. Falgari, N. Annels, et al. "Organotypic slice ovarian cancer model as a platform to test novel therapeutics." Annals of Oncology 27 (October 2016): vi535. http://dx.doi.org/10.1093/annonc/mdw392.29.
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Roux, Amandine, Xinhe Wang, Katelyn Becker та Jiyan Ma. "Modeling α-Synucleinopathy in Organotypic Brain Slice Culture with Preformed α-Synuclein Amyloid Fibrils". Journal of Parkinson's Disease 10, № 4 (2020): 1397–410. http://dx.doi.org/10.3233/jpd-202026.
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Background: Synucleinopathy is a group of neurodegenerative disorders characterized by neurodegeneration and accumulation of alpha-synuclein (α-syn) aggregates in various brain regions. The detailed mechanism of α-syn-caused neurotoxicity remains obscure, which is partly due to the lack of a suitable model that retains the in vivo three-dimensional cellular network and allows a convenient dissection of the neurotoxic pathways. Recent studies revealed that the pre-formed recombinant α-syn amyloid fibrils (PFFs) induce a robust accumulation of pathogenic α-syn species in cultured cells and animals. Objective: Our goal is to determine whether PFFs are able to induce the pathogenic α-syn accumulation and neurotoxicity in organotypic brain slice culture, an ex vivo system that retains the in vivo three-dimensional cell-cell connections. Methods/Results: Adding PFFs to cultured wild-type rat or mouse brain slices induced a time-dependent accumulation of pathogenic α-syn species, which was indicated by α-syn phosphorylated at serine 129 (pα-syn). The PFF-induced pα-syn was abolished in brain slices prepared from α-syn null mice, suggesting that the pα-syn is from the phosphorylation of endogenous α-syn. Human PFFs also induced pα-syn in brain slices prepared from mice expressing human α-syn on a mouse α-syn-null background. Furthermore, the synaptophysin immunoreactivity was inversely associated with pα-syn accumulation and an increase of neuronal loss was detected. Conclusion: PFF-treatment of brain slices is able to induce key pathological features of synucleinopathy: pα-syn accumulation and neurotoxicity. This model will be useful for investigating the neurotoxic mechanism and evaluating efficacy of therapeutic approaches.
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Liu, Yan, Ping Wu, Yin Wang, et al. "Application of Precision-Cut Lung Slices as an In Vitro Model for Research of Inflammatory Respiratory Diseases." Bioengineering 9, no. 12 (2022): 767. http://dx.doi.org/10.3390/bioengineering9120767.
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The leading cause of many respiratory diseases is an ongoing and progressive inflammatory response. Traditionally, inflammatory lung diseases were studied primarily through animal models, cell cultures, and organoids. These technologies have certain limitations, despite their great contributions to the study of respiratory diseases. Precision-cut lung slices (PCLS) are thin, uniform tissue slices made from human or animal lung tissue and are widely used extensively both nationally and internationally as an in vitro organotypic model. Human lung slices bridge the gap between in vivo and in vitro models, and they can replicate the living lung environment well while preserving the lungs’ basic structures, such as their primitive cells and trachea. However, there is no perfect model that can completely replace the structure of the human lung, and there is still a long way to go in the research of lung slice technology. This review details and analyzes the strengths and weaknesses of precision lung slices as an in vitro model for exploring respiratory diseases associated with inflammation, as well as recent advances in this field.
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van Lier, Ben, Andreas Hierlemann, and Frédéric Knoflach. "Parvalbumin expression and gamma oscillation occurrence increase over time in a neurodevelopmental model of NMDA receptor dysfunction." PeerJ 6 (September 19, 2018): e5543. http://dx.doi.org/10.7717/peerj.5543.
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Dysfunction of the N-methyl-d-aspartate receptor (NMDAR) is thought to play a role in the pathophysiology of neurodevelopmental diseases like schizophrenia. To study the effects of NMDAR dysfunction on synaptic transmission and network oscillations, we used hippocampal tissue of NMDAR subunit GluN2A knockout (KO) mice. Field excitatory postsynaptic potentials were recorded in acute hippocampal slices of adult animals. Synaptic transmission was impaired in GluN2A KO slices compared to wild-type (WT) slices. Further, to investigate whether NMDAR dysfunction would alter neurodevelopment in vitro, we used organotypic hippocampal slice cultures of WT and GluN2A KO mice. Immunostaining performed with cultures kept two, seven, 14, 25 days in vitro (DIV) revealed an increasing expression of parvalbumin (PV) over time. As a functional readout, oscillatory activity induced by the cholinergic agonist carbachol was recorded in cultures kept seven, 13, and 26 DIV using microelectrode arrays. Initial analysis focused on the occurrence of delta, theta, beta and gamma oscillations over genotype, DIV and hippocampal area (CA1, CA3, dentate gyrus (DG)). In a follow-up analysis, we studied the peak frequency and the peak power of each of the four oscillation bands per condition. The occurrence of gamma oscillations displayed an increase by DIV similar to the PV immunostaining. Unlike gamma occurrence, delta, theta, and beta occurrence did not change over time in culture. The peak frequency and peak power in the different bands of the oscillations were not different in slices of WT and GluN2A KO mice. However, the level of PV expression was lower in GluN2A KO compared to WT mice. Given the role of PV-containing fast-spiking basket cells in generation of oscillations and the decreased PV expression in subjects with schizophrenia, the study of gamma oscillations in organotypic hippocampal slices represents a potentially valuable tool for the characterization of novel therapeutic drugs.
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Reid, Alexandra. "MODL-18. ORGANOTYPIC BRAIN SLICE CULTURES: A COMPREHENSIVE MODEL FOR PRIMARY CNS TUMOR STUDIES." Neuro-Oncology 24, Supplement_7 (2022): vii294—vii295. http://dx.doi.org/10.1093/neuonc/noac209.1146.
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Abstract Inducing an immunosuppressive environment has been one of the most notable subversive tactics employed by aggressive primary brain tumors like Glioblastoma (GBM). It is vital that we garner a better understanding of the cell-cell interactions occurring within the tumor microenvironment (TME) in situ. Current in vitro and in vivo models sorely limit our ability to study the cell-cell interactions within the TME while accounting for its complexity and the spatial orientation of its components. Unfilled, this gap in physiologically relevant models greatly hinders the translation of impactful research findings to successful treatment options for patients diagnosed with GBM and other primary CNS tumors. We have devised a novel organotypic brain slice culture (BSC) model study using tumor-bearing C57BL/6J mice which brings the potential to not only determine the cell-cell interactions within the TME but also investigate the underlined mechanisms which govern them. Obtaining BSCs from tumor-bearing mice at various time points post-implantation also allows for analysis of the trafficking of various immune cells across the blood-brain barrier (BBB). We demonstrated outcomes similar to those seen in vivo following the introduction of parallel conditions, further supporting the physiological relevancy of this model. Thus, organotypic brain slice cultures from tumor-bearing mouse models may serve as a credible and incredibly versatile model for the study of primary CNS tumors which may bridge the gap between in vitro and in vivo studies.
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Baeza-Kallee, Nathalie, Raphaël Bergès, Victoria Hein, et al. "Deciphering the Action of Neuraminidase in Glioblastoma Models." International Journal of Molecular Sciences 24, no. 14 (2023): 11645. http://dx.doi.org/10.3390/ijms241411645.
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Glioblastoma (GBM) contains cancer stem cells (CSC) that are resistant to treatment. GBM CSC expresses glycolipids recognized by the A2B5 antibody. A2B5, induced by the enzyme ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyl transferase 3 (ST8Sia3), plays a crucial role in the proliferation, migration, clonogenicity and tumorigenesis of GBM CSC. Our aim was to characterize the resulting effects of neuraminidase that removes A2B5 in order to target GBM CSC. To this end, we set up a GBM organotypic slice model; quantified A2B5 expression by flow cytometry in U87-MG, U87-ST8Sia3 and GBM CSC lines, treated or not by neuraminidase; performed RNAseq and DNA methylation profiling; and analyzed the ganglioside expression by liquid chromatography–mass spectrometry in these cell lines, treated or not with neuraminidase. Results demonstrated that neuraminidase decreased A2B5 expression, tumor size and regrowth after surgical removal in the organotypic slice model but did not induce a distinct transcriptomic or epigenetic signature in GBM CSC lines. RNAseq analysis revealed that OLIG2, CHI3L1, TIMP3, TNFAIP2, and TNFAIP6 transcripts were significantly overexpressed in U87-ST8Sia3 compared to U87-MG. RT-qPCR confirmed these results and demonstrated that neuraminidase decreased gene expression in GBM CSC lines. Moreover, neuraminidase drastically reduced ganglioside expression in GBM CSC lines. Neuraminidase, by its pleiotropic action, is an attractive local treatment against GBM.
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Chang, J., C. Lim, W. Lee, and I. Park. "Response of pancreatic ductal adenocarcinoma to gemcitabine using human organotypic slice culture model." Pancreatology 20 (November 2020): S124—S125. http://dx.doi.org/10.1016/j.pan.2020.07.226.
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Pitoulis, Fotios G., Raquel Nunez-Toldra, Worrapong Sam Kit-Anan, et al. "Exploring Mechanical Load-Induced Cardiac Remodelling Using a Novel Organotypic Myocardial Slice Model." Biophysical Journal 118, no. 3 (2020): 425a. http://dx.doi.org/10.1016/j.bpj.2019.11.2392.
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Vinukonda, Govindaiah, Furong Hu, Chirag Upreti, et al. "Novel organotypic in vitro slice culture model for intraventricular hemorrhage of premature infants." Journal of Neuroscience Research 90, no. 11 (2012): 2173–82. http://dx.doi.org/10.1002/jnr.23102.
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Sharma, Kapil, and Kumlesh K. Dev. "The Effects of Antipsychotics in Experimental Models of Krabbe Disease." Biomedicines 11, no. 5 (2023): 1313. http://dx.doi.org/10.3390/biomedicines11051313.
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The role of altered myelin in the onset and development of schizophrenia and changes in myelin due to antipsychotics remains unclear. Antipsychotics are D2 receptor antagonists, yet D2 receptor agonists increase oligodendrocyte progenitor numbers and limit oligodendrocyte injury. Conflicting studies suggest these drugs promote the differentiation of neural progenitors to oligodendrocyte lineage, while others report antipsychotics inhibit the proliferation and differentiation of oligodendrocyte precursors. Here, we utilised in-vitro (human astrocytes), ex-vivo (organotypic slice cultures) and in-vivo (twitcher mouse model) experimental study designs of psychosine-induced demyelination, a toxin that accumulates in Krabbe disease (KD), to investigate direct effects of antipsychotics on glial cell dysfunction and demyelination. Typical and atypical antipsychotics, and selective D2 and 5HT2A receptor antagonists, attenuated psychosine-induced cell viability, toxicity, and morphological aberrations in human astrocyte cultures. Haloperidol and clozapine reduced psychosine-induced demyelination in mouse organotypic cerebellar slices. These drugs also attenuated the effects of psychosine on astrocytes and microglia and restored non-phosphorylated neurofilament levels, indicating neuroprotective effects. In the demyelinating twitcher mouse model of KD, haloperidol improved mobility and significantly increased the survival of these animals. Overall, this study suggests that antipsychotics directly regulate glial cell dysfunction and exert a protective effect on myelin loss. This work also points toward the potential use of these pharmacological agents in KD.
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Rybachuk, O., V. Кyryk, P. Poberezhny, G. Butenko, G. Skibo, and T. Pivneva. "Effect of the bone marrow multipotent mesenchimal stromal cells to the neural tissue after ischemic injury in vitro." Cell and Organ Transplantology 2, no. 1 (2014): 74–78. http://dx.doi.org/10.22494/cot.v2i1.38.
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Stem cells application in neural system injuries is an actual and prospective scientific field of modern regenerative medicine. In recent years much attention has been paid for study of regenerative effects of multipotent mesenchymal stromal cells (MMSCs) from different sources on injured tissues.The aim of our study was to determine the level of tissue damage in hippocampus after in vitro model of ischemia and to investigate the effect of bone marrow MMSСs in non-contact co-culture with ischemic neural tissue. The ischemic injury of neural tissue in vitro was modeling in organotypic hippocampal slice culture (OHCs) by oxygen-glucose deprivation (OGD).Immunohistochemical analysis after 24 hours of BM-MMSCs co-cultivation with OHCs after ischemia showed a significant reduction of caspase-3-positive dead neural cells, as compared to those in ischemic damage without BM-MMSCs co-cultivation, and reducing of glial cells activation. After co-cultivation of OHCs after OGD with BM-MMSCs there remained cytoarchitectonics of the neural tissue.Analyzing of our data, the neuroprotective effects of BM-MMSCs in non-contact co-cultivation with ischemic hippocampal organotypic slice culture is shown.
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Korde, Dhwani S., and Christian Humpel. "A Combination of Heavy Metals and Intracellular Pathway Modulators Induces Alzheimer Disease-like Pathologies in Organotypic Brain Slices." Biomolecules 14, no. 2 (2024): 165. http://dx.doi.org/10.3390/biom14020165.
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Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is characterized by amyloid-beta (Aβ) plaques and tau neurofibrillary tangles (NFT). Modelling aspects of AD is challenging due to its complex multifactorial etiology and pathology. The present study aims to establish a cost-effective and rapid method to model the two primary pathologies in organotypic brain slices. Coronal hippocampal brain slices (150 µm) were generated from postnatal (day 8–10) C57BL6 wild-type mice and cultured for 9 weeks. Collagen hydrogels containing either an empty load or a mixture of human Aβ42 and P301S aggregated tau were applied to the slices. The media was further supplemented with various intracellular pathway modulators or heavy metals to augment the appearance of Aβ plaques and tau NFTs, as assessed by immunohistochemistry. Immunoreactivity for Aβ and tau was significantly increased in the ventral areas in slices with a mixture of human Aβ42 and P301S aggregated tau compared to slices with empty hydrogels. Aβ plaque- and tau NFT-like pathologies could be induced independently in slices. Heavy metals (aluminum, lead, cadmium) potently augmented Aβ plaque-like pathology, which developed intracellularly prior to cell death. Intracellular pathway modulators (scopolamine, wortmannin, MHY1485) significantly boosted tau NFT-like pathologies. A combination of nanomolar concentrations of scopolamine, wortmannin, MHY1485, lead, and cadmium in the media strongly increased Aβ plaque- and tau NFT-like immunoreactivity in ventral areas compared to the slices with non-supplemented media. The results highlight that we could harness the potential of the collagen hydrogel-based spreading of human Aβ42 and P301S aggregated tau, along with pharmacological manipulation, to produce pathologies relevant to AD. The results offer a novel ex vivo organotypic slice model to investigate AD pathologies with potential applications for screening drugs or therapies in the future.
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Zehendner, Christoph M., Heiko J. Luhmann, and Christoph RW Kuhlmann. "Studying the Neurovascular Unit: An Improved Blood–Brain Barrier Model." Journal of Cerebral Blood Flow & Metabolism 29, no. 12 (2009): 1879–84. http://dx.doi.org/10.1038/jcbfm.2009.103.
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The blood–brain barrier (BBB) closely interacts with the neuronal parenchyma in vivo. To replicate this interdependence in vitro, we established a murine coculture model composed of brain endothelial cell (BEC) monolayers with cortical organotypic slice cultures. The morphology of cell types, expression of tight junctions, formation of reactive oxygen species, caspase-3 activity in BECs, and alterations of electrical resistance under physiologic and pathophysiological conditions were investigated. This new BBB model allows the application of techniques such as laser scanning confocal microscopy, immunohistochemistry, fluorescent live cell imaging, and electrical cell substrate impedance sensing in real time for studying the dynamics of BBB function under defined conditions.
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Reid, Alexandra, Dan Jin, Bayli DiVita-Dean, et al. "Abstract 4611: Leveraging organotypic brain slice models to enhance adoptive cellular therapies." Cancer Research 83, no. 7_Supplement (2023): 4611. http://dx.doi.org/10.1158/1538-7445.am2023-4611.
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Abstract Glioblastoma (GBM) is an incredibly aggressive and prevalent primary CNS tumor with dismal survival outcomes. GBM’s intra-tumor heterogeneity and lack of anti-tumor immune cell infiltration have proved to be formidable challenges to the development of effective therapies. These hurdles may be overcome by garnering a better understanding of the cell-cell interactions occurring within the tumor microenvironment (TME). Accounting for the complexity and the spatial orientation of the components within the TME may offer insight into the multitiered layers of immunosuppression capitalized on by GBM tumors, thus elucidating the factors responsible for poor response to therapy and uncovering potential therapeutic targets that may offer more favorable therapeutic results for immunotherapies. Current in vitro and in vivo models, however, sorely limit our ability to probe these distinct attributes while preserving the intricate nature of the TME. To bridge this gap, we have devised a novel organotypic brain slice culture (BSC) model using tumor-bearing C57BL/6J mice, allowing us to characterize the tumor milieu in situ. We have identified infiltration of myeloid-derived suppressor cells (MDSC) and microglia polarized to the M2 anti-inflammatory phenotype within the TME. Leveraging this platform, we sought to define further the factors contributing to the dynamic impacts of GBM immunosuppression, such as the tolerogenic response in dendritic cells (DC) upon interaction with the TME. We have demonstrated outcomes similar to those seen in vivo following the introduction of DCs to tumor-bearing BSCs, further supporting the physiological relevancy of this model, and have since worked to unravel elements responsible for this response. Citation Format: Alexandra Reid, Dan Jin, Bayli DiVita-Dean, Laura Falceto-Font, John Figg, Connor Francis, Catherine Flores. Leveraging organotypic brain slice models to enhance adoptive cellular therapies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4611.
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Bausch, Suzanne B., and James O. McNamara. "Synaptic Connections From Multiple Subfields Contribute to Granule Cell Hyperexcitability in Hippocampal Slice Cultures." Journal of Neurophysiology 84, no. 6 (2000): 2918–32. http://dx.doi.org/10.1152/jn.2000.84.6.2918.
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Limbic status epilepticus and preparation of hippocampal slice cultures both produce cell loss and denervation. This commonality led us to hypothesize that morphological and physiological alterations in hippocampal slice cultures may be similar to those observed in human limbic epilepsy and animal models. To test this hypothesis, we performed electrophysiological and morphological analyses in long-term ( postnatal day 11; 40–60 days in vitro) organotypic hippocampal slice cultures. Electrophysiological analyses of dentate granule cell excitability revealed that granule cells in slice cultures were hyperexcitable compared with acute slices from normal rats. In physiological buffer, spontaneous electrographic granule cell seizures were seen in 22% of cultures; in the presence of a GABAA receptor antagonist, seizures were documented in 75% of cultures. Hilar stimulation evoked postsynaptic potentials (PSPs) and multiple population spikes in the granule cell layer, which were eliminated by glutamate receptor antagonists, demonstrating the requirement for excitatory synaptic transmission. By contrast, under identical recording conditions, acute hippocampal slices isolated from normal rats exhibited a lack of seizures, and hilar stimulation evoked an isolated population spike without PSPs. To examine the possibility that newly formed excitatory synaptic connections to the dentate gyrus contribute to granule cell hyperexcitability in slice cultures, anatomical labeling and electrophysiological recordings following knife cuts were performed. Anatomical labeling of individual dentate granule, CA3 and CA1 pyramidal cells with neurobiotin illustrated the presence of axonal projections that may provide reciprocal excitatory synaptic connections among these regions and contribute to granule cell hyperexcitability. Knife cuts severing connections between CA1 and the dentate gyrus/CA3c region reduced but did not abolish hilar-evoked excitatory PSPs, suggesting the presence of newly formed, functional synaptic connections to the granule cells from CA1 and CA3 as well as from neurons intrinsic to the dentate gyrus. Many of the electrophysiological and morphological abnormalities reported here for long-term hippocampal slice cultures bear striking similarities to both human and in vivo models, making this in vitro model a simple, powerful system to begin to elucidate the molecular and cellular mechanisms underlying synaptic rearrangements and epileptogenesis.
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Elfarrash, Sara, Nanna Møller Jensen, Nelson Ferreira, et al. "Polo-like kinase 2 inhibition reduces serine-129 phosphorylation of physiological nuclear alpha-synuclein but not of the aggregated alpha-synuclein." PLOS ONE 16, no. 10 (2021): e0252635. http://dx.doi.org/10.1371/journal.pone.0252635.
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Accumulation of aggregated alpha-synuclein (α-syn) is believed to play a pivotal role in the pathophysiology of Parkinson’s disease (PD) and other synucleinopathies. As a key constituent of Lewy pathology, more than 90% of α-syn in Lewy bodies is phosphorylated at serine-129 (pS129) and hence, it is used extensively as a marker for α-syn pathology. However, the exact role of pS129 remains controversial and the kinase(s) responsible for the phosphorylation have yet to be determined. In this study, we investigated the effect of Polo-like kinase 2 (PLK2) inhibition on formation of pS129 using an ex vivo organotypic brain slice model of synucleinopathy. Our data demonstrated that PLK2 inhibition has no effect on α-syn aggregation, pS129 or inter-neuronal spreading of the aggregated α-syn seen in the organotypic slices. Instead, PLK2 inhibition reduced the soluble pS129 level in the nuclei. The same finding was replicated in an in vivo mouse model of templated α-syn aggregation and in human dopaminergic neurons, suggesting that PLK2 is more likely to be involved in S129-phosphorylation of the soluble physiological fraction of α-syn. We also demonstrated that reduction of nuclear pS129 following PLK2 inhibition for a short time before sample collection improves the signal-to-noise ratio when quantifying pS129 aggregate pathology.
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Ding, Lina, Kristin Sullivan, Chensheng Zhou, et al. "292 Ex vivo profiling of PD-1 blockade using an organotypic tissue slice model in solid tumors." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (2021): A316. http://dx.doi.org/10.1136/jitc-2021-sitc2021.292.
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BackgroundTumor explant models provide a powerful ex vivo tool to evaluate complex biological mechanisms in a controlled environment. Ex vivo models retain much of the original tumor biology, heterogeneity, and tumor microenvironment, and therefore provide a useful preclinical platform and functional approach to assess drug responses rapidly and directly.MethodsTo explore mechanisms of resistance to cancer immunotherapy, we established an organotypic tissue slice Air-Liquid Interface (ALI) ex vivo system utilizing surgical tumor specimens from patients to assess the impact of the clinically utilized anti-PD-1 antibody nivolumab (OPDIVO). In the present study, we built a real-world patient cohort comprised of six tumor types: non-small cell lung cancer, melanoma, pancreatic ductal adenocarcinoma, breast cancer, prostate cancer, and colorectal cancer. We assessed tissue morphology, histology, PD-L1 IHC (CPS and TPS), CD8 T cell topology, proliferation in the tumor and stromal compartments, and secretome profiling.ResultsOur tumor slice model highly recapitulated features of the original tumor, including tumor architecture, immune phenotypes, and the prognostic markers. To identify responses to aPD-1 treatment, we compared baseline values for the cultured tumor slices with values at different timepoints post treatment. Secretome profiling of tissue explant supernatants using a panel of 94 analytes, revealed alterations to cytokines produced in the tumor microenvironment in response to aPD-1 treatment. We found that soluble expression patterns were associated with T-cell patterns (inflamed, excluded and desert) and PD-L1 score (CPS and TPS) in tumor tissues. These cytokines mediate critical functions across the immune cell cycle. Ongoing efforts to characterize T cell activation, exhaustion, tumor intrinsic responses and microenvironment composition using Imaging Mass Cytometry will be presented.ConclusionsIn this study, we demonstrated the feasibility of using fresh, surgically resected human tumors to test aPD-1 responses in an ex vivo system. Further, this model system has the potential to drive discovery and translational efforts by evaluating mechanisms of resistance to cancer immunotherapy and evaluate new single agent or combination therapies in the ex vivo setting.
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Mewes, Agneta, Heike Franke, and David Singer. "Organotypic Brain Slice Cultures of Adult Transgenic P301S Mice—A Model for Tauopathy Studies." PLoS ONE 7, no. 9 (2012): e45017. http://dx.doi.org/10.1371/journal.pone.0045017.
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Guldimann, Claudia, Beatrice Lejeune, Sandra Hofer, et al. "Ruminant organotypic brain-slice cultures as a model for the investigation of CNS listeriosis." International Journal of Experimental Pathology 93, no. 4 (2012): 259–68. http://dx.doi.org/10.1111/j.1365-2613.2012.00821.x.
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Srinivasaiah, Sriveena, Giuseppe Musumeci, Tamilselvan Mohan та ін. "A 300 μm Organotypic Bone Slice Culture Model for Temporal Investigation of Endochondral Osteogenesis". Tissue Engineering Part C: Methods 25, № 4 (2019): 197–212. http://dx.doi.org/10.1089/ten.tec.2018.0368.
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Chong, Seon-Ah, Silvia Balosso, Catherine Vandenplas, et al. "Intrinsic Inflammation Is a Potential Anti-Epileptogenic Target in the Organotypic Hippocampal Slice Model." Neurotherapeutics 15, no. 2 (2018): 470–88. http://dx.doi.org/10.1007/s13311-018-0607-6.
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