Colemansherrill9248

Three-dimensional (3D) cell cultures combining multipotent mesenchymal stromal cells (MSC), tendon extracellular matrix scaffolds, and mechanical stimulation by a bioreactor have been used to induce tenogenic differentiation in vitro. Yet, these conditions alone do not mimic the environment of acute inflammatory tendon disease adequately, thus the results of such studies are not representatives for tendon regeneration after acute injury. In this chapter, we describe two different approaches to introduce inflammatory stimuli, comprising co-culture with leukocytes and supplementation with the cytokines IL-1 β and TNF-α. The presented in vitro model of inflammatory tendon disease could be used to study musculoskeletal pathophysiology and regeneration in more depth.Human mesenchymal stromal cells (MSC) are adult stem cells, which feature hepatotropism by supporting liver regeneration through amelioration of hepatic inflammation and lipid accumulation in a mouse model of non-alcoholic steatohepatitis (NASH), a more advanced stage of fatty liver. It remains open, how MSC impact on hepatocytic lipid metabolism. To study MSC actions on fatty liver mechanistically, we established an in vitro model of co-culture comprising MSC and isolated mouse hepatocytes at a ratio of 11. Lipid storage in hepatocytes was induced by the treatment with medium deficiency of methionine and choline (MCD). The protocol can be adapted for the use of other lipid storage-inducing agents such as palmitic acid and linoleic acid. This co-culture model allows to study, e.g., whether MSC act indirectly via MSC-born paracrine mechanisms or through direct physical interactions between cells beside others. The protocol allows us to detect the formation of extensions (filopodia) from MSC to contact the fatty hepatocytes or other MSC within 24 h of co-culture. These structures may represent tunneling nanotubes (TNT), allowing for long-range intercellular communication.The gold standard for organ preservation before transplantation is static cold storage, which is unable to fully protect suboptimal livers from ischemia/reperfusion injury. An emerging alternative is normothermic machine perfusion (NMP), which permits organ reconditioning. The ex vivo NMP hypoxic Rat Liver Perfusion Model represents a feasible approach that allow pharmacological intervention on isolated rat livers by using a combination of NMP and infusion of a number of drugs and/or biological material (cells, microvesicles, etc.). The combination of these two techniques may not only be applied for tissue preservation purposes, but also to investigate the biological effects of molecules and treatment useful in tissue protection. The protocol describes an ex vivo murine model of NMP capable of maintaining liver function despite an ongoing hypoxic injury induced by hemodilution. Furthermore, with this NMP system it is possible to deliver cells treatment or pharmacological intervention to an ex vivo perfused liver and suggests that could represent an innovative approach to recondition organs.Ex vivo neuroretina cultures closely resemble in vivo conditions, retaining the complex neuroretina cells dynamics, connections, and functionality, under controlled conditions. Therefore, these models have allowed advancing in the knowledge of retinal physiology and pathobiology over the years. Furthermore, the ex vivo neuroretina models represent an adequate tool for evaluating stem cell therapies over neuroretinal degeneration processes.Here, we describe a physically separated co-culture of neuroretina explants with stem cells to evaluate the effect of stem cells paracrine properties on spontaneous neuroretinal degeneration.Umbilical cord blood of neonates is a precious source for many fields of research because of distinct unique features combined with easy accessibility at the time of birth. The number of applications are vast with an emphasis in the field of stem cell therapy and regenerative medicine since cord blood contains relatively large numbers of pluripotent cells. This chapter provides a protocol for developing an autologous co-culture of endothelial-like cells and peripheral blood mononuclear cells from umbilical cord blood of premature born babies and describes an experimental setting to investigate inflammatory processes that are a cornerstone of pathophysiology in the developing organs of preterm born babies.Mesenchymal stem cells (MSCs) have emerged as an attractive candidate for cell-based therapy. In the past decade, many animal and pilot clinical studies have demonstrated that MSCs are therapeutically beneficial for the treatment of obstructive lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). However, due to the scarcity of adult human MSCs, human-induced pluripotent stem cells mesenchymal stem cells (iPSCs) are now increasingly used as a source of MSCs. iPSCs are derived by reprogramming somatic cells from a wide variety of tissues such as skin biopsies and then differentiating them into iPSC-MSCs. One of the mechanisms through which MSCs exert their protective effects is mitochondrial transfer. Specifically, transfer of mitochondria from iPSC-MSCs to lung cells was shown to protect lung cells against oxidative stress-induced mitochondrial dysfunction and apoptosis and to reduce lung injury and inflammation in in vivo models of lung disease. In this chapter, we detail our methods to visualize and quantify iPSC-MSC-mediated mitochondrial transfer and to study its effects on oxidant-induced airway epithelial and smooth muscle cell models of acute airway cell injury.A co-culture model of mesenchymal stem cells (MSCs) and fibroblasts is an efficient and rapid method to evaluate the anti-fibrotic effects of MSCs-based cell therapy. Transforming growth factor (TGF)-β1 plays a key role in promotion of fibroblast activation and differentiation which can induce collagen deposition, increase ECM production in lung tissue, eventually resulted in pulmonary fibrosis. Here, we use this co-culture system and examine the ECM production in activated fibroblasts by western blot and quantitative real-time analysis to understand the therapeutic effects of MSCs.Acute Respiratory Distress Syndrome (ARDS) is a devastating clinical disorder with high mortality rates and no specific pharmacological treatment available yet. It is characterized by excessive inflammation in the alveolar compartment resulting in edema of the airspaces due to loss of integrity in the alveolar epithelial-endothelial barrier leading to the development of hypoxemia and often severe respiratory failure. Changes in the permeability of the alveolar epithelial-endothelial barrier contribute to excessive inflammation, the formation of lung edema and impairment of the alveolar fluid clearance. Tiplaxtinin ic50 In recent years, Mesenchymal Stromal Cells (MSCs) have attracted attention as a cell therapy for ARDS. MSCs are known to secrete a variety of biologically active factors (growth factors, cytokines, and extracellular vesicles). These paracrine factors have been shown to be major effectors of the anti-inflammatory and regenerative properties observed in multiple in vitro and in vivo studies. This chapter provides a simple protocol on how to investigate the paracrine effect of MSCs on the alveolar epithelial-endothelial barrier functions.In solid tumors, mesenchymal stem cells (MSCs) are recognized to establish complex intercommunication networks with cancer cells and to significantly influence their invasion and metastasis potential. Such bidirectional interplay occurs between both tissue resident/tumor-associated MSCs (TA-MSCs) and also tumor infiltrating MSCs (TM-MSCs) that migrate from distant sites such as the bone marrow. Interestingly, malignant cells interactions with MSCs in the tumor microenvironment extends beyond conventional exchanges of signaling factors and extracellular vesicles, including unconventional direct exchanges of intracellular components, or cancer cells cannibalism of MSCs. In the context of 3D in vitro tumor models, cell tracking assays making use of cell-labeling probes such as membrane penetrating dyes, can be leveraged to shed light on these events, and allow researchers to analyze overtime cell-to-cell spatial distribution, fusion, internal organization, and changes in co-cultured populations ratios. Herein, we describe a high-throughput compatible method through which MSCs positioning and permanence within in vitro 3D multicellular tumor spheroid models (3D-MCTS) can be tracked overtime. Although we have focused on the interactions of human bone marrow-derived MSCs (hBM-MSCs) within heterotypic lung cancer A549 3D-MCTS, these procedures can be implemented for other 3D tumor spheroid models and types of cells, taking into consideration that optimization steps are undertaken.Ionizing radiation is a critical component of glioblastoma (GBM) therapy. Recent data have implicated glioblastoma stem-like cells (GSCs) as determinants of GBM development, maintenance, and treatment response. Understanding the response of GSCs to radiation should thus provide insight into the development of improved GBM treatment strategies. Towards this end, in vitro techniques for the analysis of GSC radiosensitivity are an essential starting point. One such method, the clonogenic survival assay has been adapted to assessing the intrinsic radiosensitivity of GSCs and is described here. As an alternative method, the limiting dilution assay is presented for defining the radiosensitivity of GSC lines that do not form colonies or only grow as neurospheres. In addition to these cellular strategies, we describe γH2AX foci analysis, which provides a surrogate marker for radiosensitivity at the molecular level. Taken together, the in vitro methods presented here provide tools for defining intrinsic radiosensitivity of GSCs and for testing agents that may enhance GBM radioresponse.In an increasingly geriatric population, in which elderly people frequently face chronic diseases and degenerative conditions, cell therapies as part of novel regenerative medicine approaches are of great interest. Even though today's cell therapies mostly rely on adult stem cells like the mesenchymal stem cells or primary somatic cells, pluripotent stem cells represent an enormously versatile cell model to explore possible new avenues in the field of regenerative medicine due to their capacity to grow indefinitely and to differentiate into the desired cell types. The discovery of reprogramming somatic cells into induced pluripotent stem cells augmented the pool of applicable cell entities so that researchers nowadays can resort to embryonic stem cells, but also to a plethora of patient- and disease-specific induced pluripotent stem cells. The ease of targeted genome engineering is an additional benefit that allows using pluripotent stem cells for disease modeling, drug discovery, and the development of cell therapies. However, the task is still demanding as the generation of subpopulations and a sufficient cell maturation for some cell entities have yet to be achieved. Likewise, even though for some applications the cells of interest can be produced in the large-scale dimensions and purity that are required for clinical purposes, proper integration, and function in the host tissue remain challenging. Nonetheless, the immense progress that has been made over the last decades warrants the prominent role of pluripotent stem cells in regenerative medicine as in vitro models to broaden our knowledge of disease onset/progression and treatment as well as in vivo as a substitution of damaged/aged tissue.