Point-of-Care Rapid-Seeding Ventricular Assist Device with Blood-Derived Endothelial Cells to Create a Living Antithrombotic Coating.

The most promising alternatives to heart transplantation are left ventricular assist devices and artificial hearts; however, their use has been limited by thrombotic complications. To reduce these, sintered titanium (Ti) surfaces were developed, but thrombosis still occurs in approximately 7.5% of patients. We have invented a rapid-seeding technology to minimize the risk of thrombosis by rapid endothelialization of sintered Ti with human cord blood-derived endothelial cells (hCB-ECs). Human cord blood-derived endothelial cells were seeded within minutes onto sintered Ti and exposed to thrombosis-prone low fluid flow shear stresses. The hCB-ECs adhered and formed a confluent endothelial monolayer on sintered Ti. The exposure of sintered Ti to 4.4 dynes/cm for 20 hr immediately after rapid seeding resulted in approximately 70% cell adherence. The cell adherence was not significantly increased by additional ex vivo static culture of rapid-seeded sintered Ti before flow exposure. In addition, adherent hCB-ECs remained functional on sintered Ti, as indicated by flow-induced increase in nitric oxide secretion and reduction in platelet adhesion. After 15 day ex vivo static culture, the adherent hCB-ECs remained metabolically active, expressed endothelial cell functional marker thrombomodulin, and reduced platelet adhesion. In conclusion, our results demonstrate the feasibility of rapid-seeding sintered Ti with blood-derived hCB-ECs to generate a living antithrombotic surface.

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

AIM: Peripheral blood-derived endothelial cells (pBD-ECs) are an attractive tool for cell therapies and tissue engineering, but have been limited by their low isolation yield. We increase pBD-EC yield via administration of the chemokine receptor type 4 antagonist AMD3100, as well as via a diluted whole blood incubation (DWBI). MATERIALS & METHODS: Porcine pBD-ECs were isolated using AMD3100 and DWBI and tested for EC markers, acetylated LDL uptake, growth kinetics, metabolic activity, flow-mediated nitric oxide production and seeded onto titanium tubes implanted into vessels of pigs. RESULTS: DWBI increased the yield of porcine pBD-ECs 6.6-fold, and AMD3100 increased the yield 4.5-fold. AMD3100-mobilized ECs were phenotypically indistinguishable from nonmobilized ECs. In porcine implants, the cells expressed endothelial nitric oxide synthase, reduced thrombin-antithrombin complex systemically and prevented thrombosis. CONCLUSION: Administration of AMD3100 and the DWBI method both increase pBD-EC yield.

Isolation of functional human endothelial cells from small volumes of umbilical cord blood.

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 ECs 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.

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 stresses. 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. 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. 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).