Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

Sfriso, Riccardo; Bongoni, Anjan; Banz Wälti, Yara; Klymiuk, Nikolai; Wolf, Eckhard; Rieben, Robert (2017). Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood. Journal of visualized experiments, 127(127) MYJoVE Corporation 10.3791/56227

Full text not available from this repository.

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the "natural" anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

Item Type:

Journal Article (Further Contribution)

Division/Institute:

04 Faculty of Medicine > Service Sector > Institute of Pathology > Cytopathology
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > Forschungsbereich Mu50 > Forschungsgruppe Herz und Gefässe
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > Forschungsbereich Mu50 > Forschungsgruppe Handchirurgie
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > Forschungsbereich Mu50
09 Interdisciplinary Units > Microscopy Imaging Center (MIC)

Graduate School:

Graduate School for Cellular and Biomedical Sciences (GCB)

UniBE Contributor:

Sfriso, Riccardo, Banz Wälti, Yara Sarah, Rieben, Robert

Subjects:

500 Science > 570 Life sciences; biology

ISSN:

1940-087X

Publisher:

MYJoVE Corporation

Language:

English

Submitter:

Riccardo Sfriso

Date Deposited:

15 Dec 2017 09:42

Last Modified:

02 Mar 2023 23:29

Publisher DOI:

10.3791/56227

PubMed ID:

28930996

URI:

https://boris.unibe.ch/id/eprint/105333

Actions (login required)

Edit item Edit item
Provide Feedback