Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1. Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3. This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts. The chamber and all parts of the circuit are easily sterilized with steam autoclaving In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (> 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo

Exact Hybrid Particle/Population Simulation of Rule-Based Models of Biochemical Systems

Detailed modeling and simulation of biochemical systems is complicated by the problem of combinatorial complexity, an explosion in the number of species and reactions due to myriad protein-protein interactions and post-translational modifications. Rule-based modeling overcomes this problem by representing molecules as structured objects and encoding their interactions as pattern-based rules. This greatly simplifies the process of model specification, avoiding the tedious and error prone task of manually enumerating all species and reactions that can potentially exist in a system. From a simulation perspective, rule-based models can be expanded algorithmically into fully-enumerated reaction networks and simulated using a variety of network-based simulation methods, such as ordinary differential equations or Gillespie's algorithm, provided that the network is not exceedingly large. Alternatively, rule-based models can be simulated directly using particle-based kinetic Monte Carlo methods. This "network-free" approach produces exact stochastic trajectories with a computational cost that is independent of network size. However, memory and run time costs increase with the number of particles, limiting the size of system that can be feasibly simulated. Here, we present a hybrid particle/population simulation method that combines the best attributes of both the network-based and network-free approaches. The method takes as input a rule-based model and a user-specified subset of species to treat as population variables rather than as particles. The model is then transformed by a process of "partial network expansion" into a dynamically equivalent form that can be simulated using a population-adapted network-free simulator. The transformation method has been implemented within the open-source rule-based modeling platform BioNetGen, and resulting hybrid models can be simulated using the particle-based simulator NFsim. Performance tests show that significant memory savings can be achieved using the new approach and a monetary cost analysis provides a practical measure of its utility. © 2014 Hogg et al

Fatal complications after stereotactic body radiation therapy for central lung tumors abutting the proximal bronchial tree

PURPOSE: Stereotactic body radiation therapy (SBRT) is associated with excess toxicity following treatment of central lung tumors. Risk-adapted fractionation appears to have mitigated this risk, but it remains unclear whether SBRT is safe for all tumors within the central lung zone, especially those abutting the proximal bronchial tree (PBT). We investigated the dependence of toxicity on tumor proximity to PBT and whether tumors abutting the PBT had greater toxicity than other central lung tumors after SBRT. MATERIALS AND METHODS: A total of 108 patients receiving SBRT for central lung tumors were reviewed. Patients were classified based on closest distance from tumor to PBT. Primary endpoint was SBRT-related death. Secondary endpoints were overall survival, local control, and grade 3+ pulmonary adverse events. We compared tumors abutting the PBT to nonabutting and those ≤1 cm and \u3e1 cm from PBT. RESULTS: Median follow-up was 22.7 months. Median distance from tumor to PBT was 1.78 cm. Eighty-eight tumors were primary lung and 20 were recurrent or metastatic; 23% of tumors were adenocarcinoma and 71% squamous cell. Median age was 77.5 years. Median dose was 4500 cGy in 5 fractions prescribed to the 100% isodose line. Eighteen patients had tumors abutting the PBT, 4 of whom experienced SBRT-related death. No other patients experienced death attributed to SBRT. Risk of SBRT-related death was significantly higher for tumors abutting the PBT compared with nonabutting tumors (P \u3c .001). Two patients with SBRT-related death received anti-vascular endothelial growth factor therapy and experienced pulmonary hemorrhage. Patients with tumors ≤1 cm from PBT had significantly more grade 3+ events than those with tumors \u3e1cm from PBT (P = .014). CONCLUSIONS: Even with risk-adapted fractionation, tumors abutting PBT are associated with a significant and differential risk of SBRT-related toxicity and death. SBRT should be used with particular caution in central-abutting tumors, especially in the context of anti-vascular endothelial growth factor therapy

The biocompatibility of titanium cardiovascular devices seeded with autologous blood-derived endothelial progenitor cells: EPC-seeded antithrombotic Ti Implants

Implantable and extracorporeal cardiovascular devices are commonly made from titanium (Ti) (e.g. Ti-coated Nitinol stents and mechanical circulatory assist devices). Endothelializing the blood-contacting Ti surfaces of these devices would provide them with an antithrombogenic coating that mimics the native lining of blood vessels and the heart. We evaluated the viability and adherence of peripheral blood-derived porcine endothelial progenitor cells (EPCs), seeded onto thin Ti layers on glass slides under static conditions and after exposure to fluid shear stresses. EPCs attached and grew to confluence on Ti in serum-free medium, without preadsorption of proteins. After attachment to Ti for 15 min, less than 5% of the cells detached at a shear stress of 100 dyne / cm2. Confluent monolayers of EPCs on smooth Ti surfaces (Rq of 10 nm), exposed to 15 or 100 dyne / cm2 for 48 h, aligned and elongated in the direction of flow and produced nitric oxide dependent on the level of shear stress. EPC-coated Ti surfaces had dramatically reduced platelet adhesion when compared to uncoated Ti surfaces. These results indicate that peripheral blood-derived EPCs adhere and function normally on Ti surfaces. Therefore EPCs may be used to seed cardiovascular devices prior to implantation to ameliorate platelet activation and thrombus formation

Increased yield of endothelial cells from peripheral blood for cell therapies and tissue engineering

10.2217/rme.15.2Regenerative Medicine104447-46