An ex vivo posttreatment model of sepsis: Effect on inflammation by adding complement- and CD14-inhibitors after E. coli to human whole blood

Abstract Background Since autologous veins are unavailable when needed in more than 20% of cases in vascular surgery, the production of personalized biological vascular grafts for implantation has become crucial. Surface modification of decellularized xenogeneic grafts with vascular cells to achieve physiological luminal coverage and eventually thromboresistance is an important prerequisite for implantation. However, ex vivo thrombogenicity testing remains a neglected area in the field of tissue engineering of vascular grafts due to a multifold of reasons. Methods After seeding decellularized bovine carotid arteries with human endothelial progenitor cells and umbilical cord-derived mesenchymal stem cells, luminal endothelial cell coverage (LECC) was correlated with glucose and lactate levels on the cell supernatant. Then a closed loop whole blood perfusion system was designed. Recellularized grafts with a LECC > 50% and decellularized vascular grafts were perfused with human whole blood for 2 h. Hemolysis and complete blood count evaluation was performed on an hourly basis, followed by histological and immunohistochemical analysis. Results While whole blood perfusion of decellularized grafts significantly reduced platelet counts, platelet depletion from blood resulting from binding to re-endothelialized grafts was insignificant (p = 0.7284). Moreover, macroscopic evaluation revealed thrombus formation only in the lumen of unseeded grafts and histological characterization revealed lack of CD41 positive platelets in recellularized grafts, thus confirming their thromboresistance. Conclusion In the present study we were able to demonstrate the effect of surface modification of vascular grafts in their thromboresistance in an ex vivo whole blood perfusion system. To our knowledge, this is the first study to expose engineered vascular grafts to human whole blood, recirculating at high flow rates, immediately after seeding.

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Quantification of Drug-Induced mRNA in Human Whole Blood ex vivo

Clinical medicine Blood disorders ◽

10.4137/cmbd.s507 ◽

2008 ◽

Vol 1 ◽

pp. CMBD.S507 ◽

Cited By ~ 4

Author(s):

Masato Mitsuhashi ◽

Katsuya Endo ◽

Kazuhiko Obara ◽

Hiroshi Izutsu ◽

Taishi Ishida ◽

...

Keyword(s):

Whole Blood ◽

Kinase Inhibitor ◽

Ex Vivo ◽

Fold Increase ◽

Drug Induced ◽

Human Whole Blood ◽

Cyclin Dependent Kinase Inhibitor ◽

Sensitivity Tests ◽

Data Points ◽

Mrna Markers

Apoptosis was induced in heparinized human whole blood by 3 different ways (radiation, bleomycin, or etoposide), and various mRNA were quantified using the method we reported (Clin. Chem. 2006; 52:634-642). We found that cyclin-dependent kinase inhibitor 1A (p21) and p53 upregulated modulator of apoptosis (PUMA) were the most sensitive and universal mRNA markers of apoptosis in leukocytes. In order to define positive and negative responses, a synthetic RNA was spiked into the lysis buffer and the fold increase was calculated. As a result, 837/880 (95.1%) of data points stayed between 0.75 and 1.5 fold increase, and 874/880 (99.3%) were within 0.5-2.0 fold increase. When blood samples from 40 healthy adults were stimulated with 22 different drugs, more than 75% of the samples responded to bleomycin (1 μM), idarubicin (2 μM), vincristine (1 μM), daunorubicin (2 μM), cytarabine (10 μM), to induce p21 and/or PUMA mRNA, and approximately 25% showed no induction. Significant correlation was found between p21 and PUMA mRNA responses, and between daunorubicin and cytarabine, idarubicin, and vincristine for both p21 and PUMA. The quantification of drug-induced mRNA in whole blood will be considered as ex vivo, and is a suitable platform for biomarker screening as well as a model system for drug sensitivity tests in future.

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