Department of Clinical Research
Functional evaluation of immortalized porcine endothelial cells in a whole blood clotting assay
Riccardo Sfriso1, Pavan S Garimella1, Gisella Puga Yung2, Nikolay Klymiuk3, Eckhard Wolf3, David Ayares4, Joerg D Seebach2, Robert Rieben1.
1Department of Clinical Research, University of Bern, Bern, Switzerland; 2Division of Immunology and Allergology, University Hospital and Medical Faculty, Geneva, Switzerland; 3Institute of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University, Munich, Germany; 4Revivicor, Inc., Blacksburg, VA, United States
Background Immortalization of porcine aortic endothelial cells (PAEC) offers the advantage of an unlimited source of uniform cells for in vitro assays in the context of pig-to-human xenotransplantation. In this study we analyzed the anticoagulant properties of the PED2*3.51 cell line. These cells are derived from porcine bone marrow, immortalized by SV40, and alpha-Gal knockout (GalKO).
Methods PED2*3.51 were characterized by immunofluorescence and FACS in order to investigate the expression of typical endothelial cells markers: CD31, VWF and VE-cadherin. Cell ELISA was used to investigate complement deposition after incubation of wildtype PAEC, PAEC from GalKO/hCD46/hTM transgenic pigs, and PED2*3.51 cells, with normal human serum (NHS) at a concentration of 1:10 for 45 minutes. To investigate whether PED2*3.51 cells provide a natural anti-coagulant environment, a whole blood clotting assay was performed. The different types of PAEC were cultured on microcarrier beads to increase the endothelial surface-to-blood volume ratio and mimic the in vivo situation in small arterioles or venules. When PAEC were grown to confluence on the microcarrier beads, they were incubated with whole, non-anticoagulated human blood. Time until clotting was measured and the values obtained with the different types of PAEC compared.
Results Immunofluorescence staining and FACS analysis revealed that PED2*3.51 cells express endothelial cell markers, confirming that these cells still show an endothelial cell phenotype, even after the immortalization process. Cell ELISA after incubation with NHS showed a lower deposition of IgM, C4b/c and sC5b-9 on PED2*3.51 cells as compared with wildtype PAEC, which is in line with the GalKO phenotype. Clotting times in the microcarrier bead assay with non-anticoagulated human blood were 98 ± 9 min for PED2*3.51 and 72 ± 3 min for GalKO/hCD46/hTM transgenic PAEC (p=0.009 by t-test).
Conclusions Our data suggest that the immortalized PED2*3.51 cells carry a anticoagulant phenotype, delaying clotting in a whole blood assay significantly longer than GalKO/hCD46/hTM transgenic PAEC. It remains to be clarified whether this strongly anticoagulant phenotype is due to the nature of the original endothelial cells or due to the immortalization process itself. The availability of an immortalized PAEC cell line with anticoagulant properties will allow for in vitro experiments with non-anticoagulated human blood, for example to screen the effects of multiple transgenic modification strategies using the CRISPR-CAS technology.
11:00 - 12:30
|Inflammation & Coagulation||Functional evaluation of immortalized porcine endothelial cells in a whole blood clotting assay||Room 109|