The third dimension: new developments in cell culture models for colorectal research

Cellular models are important tools in various research areas related to colorectal biology and associated diseases. Herein, we review the most widely used cell lines and the different techniques to grow them, either as cell monolayer, polarized two-dimensional epithelia on membrane filters, or as three-dimensional spheres in scaffoldfree or matrix-supported culture conditions. Moreover, recent developments, such as gut-on-chip devices or the ex vivo growth of biopsy-derived organoids, are also discussed. We provide an overview on the potential applications but also on the limitations for each of these techniques, while evaluating their contribution to provide more reliable cellular models for research, diagnostic testing, or pharmacological validation related to colon physiology and pathophysiology.

3D Printing and bioprinting of osteomimetic materials for 3D in vitro models and tissue repair applications

Bone disorders have severe impact on body functions and quality life, and no satisfying therapies exist yet. The current models for bone disease study are scarcely predictive and the options existing for therapy fail for complex systems. To mimic and/or restore bone, 3D printing/bioprinting allows the creation of 3D structures with different materials compositions, properties, and designs. In this study, 3D printing/bioprinting has been explored for (i) 3D in vitro tumor models and (ii) regenerative medicine. Tumor models have been developed by investigating different bioinks (i.e., alginate, modified gelatin) enriched by hydroxyapatite nanoparticles to increase printing fidelity and increase biomimicry level, thus mimicking the organic and inorganic phase of bone. High Saos-2 cell viability was obtained, and the promotion of spheroids clusters as occurring in vivo was observed. To develop new syntethic bone grafts, two approaches have been explored. In the first, novel magnesium-phosphate scaffolds have been investigated by extrusion-based 3D printing for spinal fusion. 3D printing process and parameters have been optimized to obtain custom-shaped structures, with competent mechanical properties. The 3D printed structures have been combined to alginate porous structures created by a novel ice-templating technique, to be loaded by antibiotic drug to address infection prevention. Promising results in terms of planktonic growth inhibition was obtained. In the second strategy, marine waste precursors have been considered for the conversion in biogenic HA by using a mild-wet conversion method with different parameters. The HA/carbonate ratio conversion efficacy was analysed for each precursor (by FTIR and SEM), and the best conditions were combined to alginate to develop a composite structure. The composite paste was successfully employed in custom-modified 3D printer for the obtainment of 3D printed stable scaffolds. In conclusion, the osteomimetic materials developed in this study for bone models and synthetic grafts are promising in bone field.

Coloured Petri nets and graphical simulation for the validation of a robotic cell in aircraft industry

This paper proposes a mixed validation approach based on coloured Petri nets and 3D graphic simulation for the design of supervisory systems in manufacturing cells with multiple robots. The coloured Petri net is used to model the cell behaviour at a high level of abstraction. It models the activities of each cell component and its coordination by a supervisory system. The graphical simulation is used to analyse and validate the cell behaviour in a 3D environment, allowing the detection of collisions and the calculation of process times. The motivation for this work comes from the aeronautic industry. The automation of a fuselage assembly process requires the integration of robots with other cell components such as metrological or vision systems. In this cell, the robot trajectories are defined by the supervisory system and results from the coordination of the cell components. The paper presents the application of the approach for an aircraft assembly cell under integration in Brazil. This case study shows the feasibility of the approach and supports the discussion of its main advantages and limits. (C) 2011 Elsevier Ltd. All rights reserved.

Effects of immunosuppressants on hepatocyte cell mitosis during liver regeneration in growing animal models of partial hepatectomy

Objective. We sought to evaluate the effects of immunosuppressant drugs (corticosteroid, cyclosporine [CsA], and tacrolimus [Tac]) on liver regeneration in growing animals submitted to 70% hepatectomy. Materials and Methods. Newborn and weaning rats were submitted to 70% hepatectomy receiving separately methylprednisolone, CsA, or Tac. All animals were sacrificed 24 hours after the procedure. The remnant liver lobes were subjected to histomorphometric analyses with determination of hepatocyte mitotic index. Results., Administration of immunosuppressants did not change the mitotic index of the regenerating liver in newborn animals. In weaning rats, methylprednisolone reduced the mitotic index (P = .01) and Tac caused a greater increase in this rate (P = .001). CsA had no effect on mitotic index. The number of hepatocyte mitoses in newborn animal livers was greater than that in weaning animal livers (P = .001). Conclusion. In situations in which intense, fast processes of liver regeneration are crucial, the advantages of the use of Tac must be considered, such as in pediatric transplant patients.

INTENSITY-MODULATED AND 3D-CONFORMAL RADIOTHERAPY FOR WHOLE-VENTRICULAR IRRADIATION AS COMPARED WITH CONVENTIONAL WHOLE-BRAIN IRRADIATION IN THE MANAGEMENT OF LOCALIZED CENTRAL NERVOUS SYSTEM GERM CELL TUMORS

Purpose: To compare the sparing potential of cerebral hemispheres with intensity-modulated radiotherapy (IMRT) and three-dimensional conformal radiotherapy (3D-CRT) for whole-ventricular irradiation (WVI) and conventional whole-brain irradiation (WBI) in the management of localized central nervous system germ cell tumors (CNSGCTs). Methods and Materials: Ten cases of patients with localized CNSGCTs and submitted to WVI by use of IMRT with or without a ""boost"" to the primary lesion were selected. For comparison purposes, similar treatment plans were produced by use of 3D-CRT (WVI with or without boost) and WBI (opposed lateral fields with or without boost), and cerebral hemisphere sparing was evaluated at dose levels ranging from 2 Gy to 40 Gy. Results: The median prescription dose for WVI was 30.6 Gy (range, 25.2-37.5 Gy), and that for the boost was 16.5 Gy (range, 0-23.4 Gy). Mean irradiated cerebral hemisphere volumes were lower for WVI with IMRT than for 3D-CRT and were lower for WVI with 3D-CRT than for WBI. Intensity-modulated radiotherapy was associated with the lowest irradiated volumes, with reductions of 7.5%, 12.2%, and 9.0% at dose levels., compared with 3D-CRT. Intensity-modulated radiotherapy provided of 20, 30, and 40 Gy, respectively statistically significant reductions of median irradiated volumes at all dose levels (p = 0.002 or less). However, estimated radiation doses to peripheral areas of the body were 1.9 times higher with IMRT than with 3D-CRT. Conclusions: Although IMRT is associated with increased radiation doses to peripheral areas of the body, its use can spare a significant amount of normal central nervous system tissue compared with 3D-CRT or WBI in the setting of CNSGCT treatment. (C) 2010 Elsevier Inc.

Linear models of simple cells: Correspondence to real cell responses and space spanning properties

Despite their limitations, linear filter models continue to be used to simulate the receptive field properties of cortical simple cells. For theoreticians interested in large scale models of visual cortex, a family of self-similar filters represents a convenient way in which to characterise simple cells in one basic model. This paper reviews research on the suitability of such models, and goes on to advance biologically motivated reasons for adopting a particular group of models in preference to all others. In particular, the paper describes why the Gabor model, so often used in network simulations, should be dropped in favour of a Cauchy model, both on the grounds of frequency response and mutual filter orthogonality.

Estimation of 3D shapes using active surface models

This paper addresses the estimation of surfaces from a set of 3D points using the unified framework described in [1]. This framework proposes the use of competitive learning for curve estimation, i.e., a set of points is defined on a deformable curve and they all compete to represent the available data. This paper extends the use of the unified framework to surface estimation. It o shown that competitive learning performes better than snakes, improving the model performance in the presence of concavities and allowing to desciminate close surfaces. The proposed model is evaluated in this paper using syntheticdata and medical images (MRI and ultrasound images).

Cell encapsulated hydrogel microspheres as "building blocks" for producing 3D constructs

Cell encapsulation within hydrogel microspheres shows great promise in the field of tissue engineering and regenerative medicine (TERM). However, the assembling of microspheres as building blocks to produce complex tissues is a hard task because of their inability to place along length scales in space. We propose a proof-of-concept strategy to produce 3D constructs using cell encapsulated as building blocks by perfusion based LbL technique. This technique exploits the â bindingâ potential of multilayers apart from coating

Skin-derived cell-sheets as powerful tools to engineer skin analogues

Cell/cell-extracellular matrix (ECM) dynamic interactions appear to have a major role in regulating communication through soluble signaling, directing cell binding and activating substrates that participate in the highly organized wound healing process. Moreover, these interactions are also crucial for in vitro mimicking cutaneous physiology. Herein we explore cell sheet (CS) engineering to create cellular constructs formed by keratinocytes (hKC), fibroblasts (hDFB) and dermal microvascular endothelial cells (hDMEC), to target skin wound healing but also the in vitro recreation of relevant models. Taking advantage of temperature-responsive culture surfaces, which allow harvesting cultured cells as intact sheets along with the deposited native ECM, varied combinations of homotypic and heterotypic three-dimensional (3-D) CS-based constructs were developed. Constructs combining one CS of keratinocytes as an epidermis-like layer plus a vascularized dermis composed by hDFB and hDMECs were assembled as skin analogues for advancing in vitro testing. Simultaneously both hKC and hDMEC were shown to significantly contribute to the re-epithelialization of full-thickness mice skin wounds by promoting an early epithelial coverage, while hDMEC significantly lead to increased vessels density, incorporating the neovasculature. Thus, although determined by the cellular nature of the constructs, these outcomes demonstrated that CS engineering appear as an unique technology that open the possibility to create numerous combinations of 3D constructs to target defective wound healing as well as the construction of in vitro models to further mimic cutaneous functions crucial for drug screening and cosmetic testing assays.

Exploring the role of O-Mannosylation in the modulation of E-Cadherin function in Gastric Cancer cell line models

Dissertação de mestrado em Bioquímica Aplicada – Biomedicina

Mathematical models of influenza A virus infection : from intracellular replication to virus growth in cell populations

Magdeburg, Univ., Fak. für Verfahrens- und Systemtechnik, Diss., 2015

Identification of particular groups of microRNAs that positively or negatively impact on beta cell function in obese models of type 2 diabetes.

AIMS/HYPOTHESIS: MicroRNAs are key regulators of gene expression involved in health and disease. The goal of our study was to investigate the global changes in beta cell microRNA expression occurring in two models of obesity-associated type 2 diabetes and to assess their potential contribution to the development of the disease. METHODS: MicroRNA profiling of pancreatic islets isolated from prediabetic and diabetic db/db mice and from mice fed a high-fat diet was performed by microarray. The functional impact of the changes in microRNA expression was assessed by reproducing them in vitro in primary rat and human beta cells. RESULTS: MicroRNAs differentially expressed in both models of obesity-associated type 2 diabetes fall into two distinct categories. A group including miR-132, miR-184 and miR-338-3p displays expression changes occurring long before the onset of diabetes. Functional studies indicate that these expression changes have positive effects on beta cell activities and mass. In contrast, modifications in the levels of miR-34a, miR-146a, miR-199a-3p, miR-203, miR-210 and miR-383 primarily occur in diabetic mice and result in increased beta cell apoptosis. These results indicate that obesity and insulin resistance trigger adaptations in the levels of particular microRNAs to allow sustained beta cell function, and that additional microRNA deregulation negatively impacting on insulin-secreting cells may cause beta cell demise and diabetes manifestation. CONCLUSIONS/INTERPRETATION: We propose that maintenance of blood glucose homeostasis or progression toward glucose intolerance and type 2 diabetes may be determined by the balance between expression changes of particular microRNAs.

Ammonium accumulation and cell death in a rat 3D brain cell model of glutaric aciduria type I.

Glutaric aciduria type I (glutaryl-CoA dehydrogenase deficiency) is an inborn error of metabolism that usually manifests in infancy by an acute encephalopathic crisis and often results in permanent motor handicap. Biochemical hallmarks of this disease are elevated levels of glutarate and 3-hydroxyglutarate in blood and urine. The neuropathology of this disease is still poorly understood, as low lysine diet and carnitine supplementation do not always prevent brain damage, even in early-treated patients. We used a 3D in vitro model of rat organotypic brain cell cultures in aggregates to mimic glutaric aciduria type I by repeated administration of 1 mM glutarate or 3-hydroxyglutarate at two time points representing different developmental stages. Both metabolites were deleterious for the developing brain cells, with 3-hydroxyglutarate being the most toxic metabolite in our model. Astrocytes were the cells most strongly affected by metabolite exposure. In culture medium, we observed an up to 11-fold increase of ammonium in the culture medium with a concomitant decrease of glutamine. We further observed an increase in lactate and a concomitant decrease in glucose. Exposure to 3-hydroxyglutarate led to a significantly increased cell death rate. Thus, we propose a three step model for brain damage in glutaric aciduria type I: (i) 3-OHGA causes the death of astrocytes, (ii) deficiency of the astrocytic enzyme glutamine synthetase leads to intracerebral ammonium accumulation, and (iii) high ammonium triggers secondary death of other brain cells. These unexpected findings need to be further investigated and verified in vivo. They suggest that intracerebral ammonium accumulation might be an important target for the development of more effective treatment strategies to prevent brain damage in patients with glutaric aciduria type I.