Computational fluid dynamic analysis of bioprinted self-supporting perfused tissue models

Natural tissues are incorporated with vasculature, which is further integrated with a cardiovascular system responsible for driving perfusion of nutrient‐rich oxygenated blood through the vasculature to support cell metabolism within most cell‐dense tissues. Since scaffold‐free biofabricated tissues being developed into clinical implants, research models, and pharmaceutical testing platforms should similarly exhibit perfused tissue‐like structures, we generated a generalizable biofabrication method resulting in self‐supporting perfused (SSuPer) tissue constructs incorporated with perfusible microchannels and integrated with the modular FABRICA perfusion bioreactor. As proof of concept, we perfused an MLO‐A5 osteoblast‐based SSuPer tissue in the FABRICA. Although our resulting SSuPer tissue replicated vascularization and perfusion observed in situ, supported its own weight, and stained positively for mineral using Von Kossa staining, our in vitro results indicated that computational fluid dynamics (CFD) should be used to drive future construct design and flow application before further tissue biofabrication and perfusion. We built a CFD model of the SSuPer tissue integrated in the FABRICA and analyzed flow characteristics (net force, pressure distribution, shear stress, and oxygen distribution) through five SSuPer tissue microchannel patterns in two flow directions and at increasing flow rates. Important flow parameters include flow direction, fully developed flow, and tissue microchannel diameters matched and aligned with bioreactor flow channels. We observed that the SSuPer tissue platform is capable of providing direct perfusion to tissue constructs and proper culture conditions (oxygenation, with controllable shear and flow rates), indicating that our approach can be used to biofabricate tissue representing primary tissues and that we can model the system in silico

A perfusion culture system for assessing bone marrow stromal cell differentiation on PLGA scaffolds for bone repair

Biomaterials development for bone repair is currently hindered by the lack of physiologically relevant in vitro testing systems. Here we describe the novel use of a bi-directional perfusion bioreactor to support the long term culture of human bone marrow stromal cells (BMSCs) differentiated on polylactic co-glycolic acid (PLGA). Primary human BMSCs were seeded onto porous PLGA scaffolds and cultured in static vs. perfusion culture conditions for 21 days in osteogenic vs. control media. PLGA scaffolds were osteoconductive, supporting a mature osteogenic phenotype as shown by the upregulation of Runx2 and the early osteocyte marker E11. Perfusion culture enhanced the expression of osteogenic genes Osteocalcin and Osteopontin. Extracellular matrix deposition and mineralisation were spatially regulated within PLGA scaffolds in a donor dependant manner. This, together with the observed upregulation of Collagen type X suggested an environment permissive for the study of differentiation pathways associated with both intramembranous and endochondral ossification routes of bone healing. This culture system offers a platform to assess BMSC behavior on candidate biomaterials under physiologically relevant conditions. Use of this system may improve our understanding of the environmental cues orchestrating BMSC differentiation and enable fine tuning of biomaterial design as we develop tissue-engineered strategies for bone regeneration

Flow dynamics control the effect of sphingosine-1-phosphate on endothelial permeability in a microfluidic vessel bifurcation model

Blood vessels are lined by endothelial cells that form a semipermeable barrier to restrict fluid flow across the vessel wall. The endothelial barrier is known to respond to various molecular mechanisms, but the effects of mechanical signals that arise due to blood flow remain poorly understood. Here, we report a microfluidic model that mimics the flow conditions and endothelial/extracellular matrix (ECM) architecture of a vessel bifurcation to enable systematic investigation of how flow dynamics that arise within bifurcating vessels guides the endothelial response to biochemical signals. Applying the strengths of our system, we further investigate the endothelial response to sphingosine-1-phosphate, a bioactive lipid that has demonstrated flow-dependent regulation of vascular permeability. We demonstrate that bifurcated fluid flow (BFF) that arises at the base of vessel bifurcations and laminar shear stress (LSS) that arises along downstream vessel walls induce a decrease in endothelial permeability. Furthermore, we identify that flow-dynamics and chaperone proteins regulate the endothelial response to S1P. Through pharmacological inhibition of S1P receptors 1 and 2, we report ligand-independent mechanical activation of S1P receptors 1 and 2, providing support for the role of G protein-coupled receptors as mechanosensors. These findings introduce BFF as an important regulator of vascular permeability, and establish flow dynamics as a determinant of the endothelial response to S1P.Pelotonia Fellowship ProgramBarry M. Goldwater Excellence in Education FoundationThe Ohio State University College of EngineeringA one-year embargo was granted for this item.Academic Major: Biomedical Engineerin

Progressive Hypoxia-on-a-chip: An In Vitro Oxygen Gradient Model for Capturing the Effects of Hypoxia on Primary Hepatocytes in Health and Disease

Oxygen is vital to the function of all tissues including the liver and lack of oxygen, that is, hypoxia can result in both acute and chronic injuries to the liver in vivo and ex vivo. Furthermore, a permanent oxygen gradient is naturally present along the liver sinusoid, which plays a role in the metabolic zonation and the pathophysiology of liver diseases. Accordingly, here, we introduce an in vitro microfluidic platform capable of actively creating a series of oxygen concentrations on a single continuous microtissue, ranging from normoxia to severe hypoxia. This range approximately captures both the physiologically relevant oxygen gradient generated from the portal vein to the central vein in the liver, and the severe hypoxia occurring in ischemia and liver diseases. Primary rat hepatocytes cultured in this microfluidic platform were exposed to an oxygen gradient of 0.3–6.9%. The establishment of an ascending hypoxia gradient in hepatocytes was confirmed in response to the decreasing oxygen supply. The hepatocyte viability in this platform decreased to approximately 80% along the hypoxia gradient. Simultaneously, a progressive increase in accumulation of reactive oxygen species and expression of hypoxia‐inducible factor 1α was observed with increasing hypoxia. These results demonstrate the induction of distinct metabolic and genetic responses in hepatocytes upon exposure to an oxygen (/hypoxia) gradient. This progressive hypoxia‐on‐a‐chip platform can be used to study the role of oxygen and hypoxia‐associated molecules in modeling healthy and injured liver tissues. Its use can be further expanded to the study of other hypoxic tissues such as tumors as well as the investigation of drug toxicity and efficacy under oxygen‐limited conditions

Microfluidics : the fur-free way towards personalised medicine in cancer therapy

Microfluidic technology has great potential for complementing and, in some instances, replacing the use of animal models in the testing of medicines and in developing personalised treatments for cancer patients. The maintenance of tissue in an in vivo-like state provides a platform upon which normal and diseased tissue biology can be investigated in a novel way. This review describes the use of microfluidic technology for the maintenance of tissue samples ex vivo and the current state of play for the use of this technology in the replacement of animal models, with a focus on cancer

Liver Sinusoid on a Chip: Long-Term Layered Co-Culture of Primary Rat Hepatocytes and Endothelial Cells in Microfluidic Platforms

We describe the generation of microfluidic platforms for the co-culture of primary hepatocytes and endothelial cells; these platforms mimic the architecture of a liver sinusoid. This paper describes a progressional study of creating such a liver sinusoid on a chip system. Primary rat hepatocytes (PRHs) were co-cultured with primary or established endothelial cells in layers in single and dual microchannel configurations with or without continuous perfusion. Cell viability and maintenance of hepatocyte functions were monitored and compared for diverse experimental conditions. When primary rat hepatocytes were co-cultured with immortalized bovine aortic endothelial cells (BAECs) in a dual microchannel with continuous perfusion, hepatocytes maintained their normal morphology and continued to produce urea for at least 30 days. In order to demonstrate the utility of our microfluidic liver sinusoid platform, we also performed an analysis of viral replication for the hepatotropic hepatitis B virus (HBV). HBV replication, as measured by the presence of cell-secreted HBV DNA, was successfully detected. We believe that our liver model closely mimics the in vivo liver sinusoid and supports long-term primary liver cell culture. This liver model could be extended to diverse liver biology studies and liver-related disease research such as drug induced liver toxicology, cancer research, and analysis of pathological effects and replication strategies of various hepatotropic infectious agents

Lab-on-Chip for Testing Myelotoxic Effect of Drugs and Chemicals

This paper was presented at the 4th Micro and Nano Flows Conference (MNF2014), which was held at University College, London, UK. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute, ASME Press, LCN London Centre for Nanotechnology, UCL University College London, UCL Engineering, the International NanoScience Community, www.nanopaprika.eu.In the last twenty years, one of the main goals in the drug discovery field has been the development of reliable in vitro models. In particular, in 2006 the European Centre for the Validation of Alternative Methods (ECVAM) has approved the Colony forming Unit-Granulocytes-Macrophages (CFU-GM) test, which is the first and currently unique test applied to evaluate the myelotoxicity of xenobiotics in vitro. The present work aimed at miniaturizing this in vitro assay by developing and validating a Lab-on-Chip (LoC) platform consisting of a high number of bioreactor chambers with screening capabilities in a high-throughput regime

MITK-ModelFit: A generic open-source framework for model fits and their exploration in medical imaging -- design, implementation and application on the example of DCE-MRI

Many medical imaging techniques utilize fitting approaches for quantitative parameter estimation and analysis. Common examples are pharmacokinetic modeling in DCE MRI/CT, ADC calculations and IVIM modeling in diffusion-weighted MRI and Z-spectra analysis in chemical exchange saturation transfer MRI. Most available software tools are limited to a special purpose and do not allow for own developments and extensions. Furthermore, they are mostly designed as stand-alone solutions using external frameworks and thus cannot be easily incorporated natively in the analysis workflow. We present a framework for medical image fitting tasks that is included in MITK, following a rigorous open-source, well-integrated and operating system independent policy. Software engineering-wise, the local models, the fitting infrastructure and the results representation are abstracted and thus can be easily adapted to any model fitting task on image data, independent of image modality or model. Several ready-to-use libraries for model fitting and use-cases, including fit evaluation and visualization, were implemented. Their embedding into MITK allows for easy data loading, pre- and post-processing and thus a natural inclusion of model fitting into an overarching workflow. As an example, we present a comprehensive set of plug-ins for the analysis of DCE MRI data, which we validated on existing and novel digital phantoms, yielding competitive deviations between fit and ground truth. Providing a very flexible environment, our software mainly addresses developers of medical imaging software that includes model fitting algorithms and tools. Additionally, the framework is of high interest to users in the domain of perfusion MRI, as it offers feature-rich, freely available, validated tools to perform pharmacokinetic analysis on DCE MRI data, with both interactive and automatized batch processing workflows.Comment: 31 pages, 11 figures URL: http://mitk.org/wiki/MITK-ModelFi

Through Skull Fluorescence Imaging of the Brain in a New Near-Infrared Window

To date, brain imaging has largely relied on X-ray computed tomography and magnetic resonance angiography with limited spatial resolution and long scanning times. Fluorescence-based brain imaging in the visible and traditional near-infrared regions (400-900 nm) is an alternative but currently requires craniotomy, cranial windows and skull thinning techniques, and the penetration depth is limited to 1-2 mm due to light scattering. Here, we report through-scalp and through-skull fluorescence imaging of mouse cerebral vasculature without craniotomy utilizing the intrinsic photoluminescence of single-walled carbon nanotubes in the 1.3-1.4 micrometre near-infrared window. Reduced photon scattering in this spectral region allows fluorescence imaging reaching a depth of >2 mm in mouse brain with sub-10 micrometre resolution. An imaging rate of ~5.3 frames/s allows for dynamic recording of blood perfusion in the cerebral vessels with sufficient temporal resolution, providing real-time assessment of blood flow anomaly in a mouse middle cerebral artery occlusion stroke model.Comment: 38 pages, 4 main figures and 11 Supplementary figures. published in Nature Photonics, 201