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 stresses 1 .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 EPCs 2,3 . 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 microscopy 5 . 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) . Video LinkThe video component of this article can be found at https://www.jove.com/video/3349/ Protocol 1. Endothelial progenitor cell isolation 1. Prior to any collection of peripheral human blood, submit your research protocol to your Institutional Review Board (IRB), and after its approval, obtain the volunteer donors' informed consent (peripheral blood collection and EPC isolation had been approved by the Duke University IRB and is in full compliance with U.S. regulatory requirements related to the protection of human research participants). 2. When working with animal-derived EPCs, have your research protocol approved by your Institutional Animal Care and Use Committee (IACUC). All our porcine experiments had been approved by the Duke University IACUC and were conducted in accordance with the highest standards of humane care. 3. For isolation of endothelial progenitor cells, collect 50 ml of peripheral blood via standard phlebotomy technique from a consented volunteer donor into blood collection bags filled with the anticoagulant citrate phosphate dextrose and dilute the solution 1:1 with Hank's buffered salt solution (without CaCl 2 , MgCl 2 , MgSO 4 ) and layer on equal volumes of Histopaque to create well-defined layers. 4. Centrifuge (30 min, 740 g, low break setting) and colle...

Endothelial cells (ECs) isolated from endothelial progenitor cells in blood have great potential as a therapeutic tool to promote vasculogenesis and angiogenesis and treat cardiovascular diseases. However, current methods to isolate ECs are limited by a low yield with few colonies appearing during isolation. In order to utilize blood-derived ECs for therapeutic applications, a simple method is needed that can produce a high yield of ECs from small volumes of blood without the addition of animal-derived products. For the first time, we show that human endothelial cells can be isolated without the prior separation of blood components through the technique of diluted whole blood incubation (DWBI) utilizing commercially available human serum. We isolated ECs from small volumes of blood (~ 10 ml) via DWBI and characterized them with flow cytometry, immunohistochemistry, and uptake of DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL). These ECs are functional as demonstrated by their ability to form tubular networks in Matrigel, adhere and align with flow under physiological fluid shear stress, and produce increased nitric oxide under fluid flow. An average of 7.0 ± 2.5 EC colonies that passed all functional tests described above were obtained per 10 ml of blood as compared to only 0.3 ± 0.1 colonies with the traditional method based on density centrifugation. The time until first colony appearance was 8.3 ± 1.2 days for ECs isolated with the DWBI method and 12 ± 1.4 days for ECs isolated with the traditional isolation method. A simplified method, such as DWBI, in combination with advances in isolation yield could enable the use of blood-derived ECs in clinical practice.

Approximately 10% of all deep vein thromboses occur in the upper extremity, and that number is increasing due to the use of peripherally inserted central catheters. Sequelae of upper extremity deep vein thrombosis (UEDVT) are similar to those for lower extremity deep vein thrombosis (LEDVT) and include postthrombotic syndrome and pulmonary embolism. In addition to systemic anticoagulation, there are multiple interventional treatment options for UEDVT with the potential to reduce the incidence of these sequelae. To date, there have been no randomized trials to define the optimal management strategy for patients presenting with UEDVT, so many conclusions are drawn from smaller, single-center studies or from LEDVT research. In this article, the authors describe the evidence for the currently available treatment options and an approach to a patient with acute UEDVT.

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