Abstract: TH-PO643
Towards the Use of Immortalized Perfusate-Isolated Human Endothelial Cells for the Screening of Non-human Leukocyte Antigen (Non-HLA) Antibodies in Kidney Transplant Recipients
Session Information
- Transplantation: Basic
November 03, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Transplantation
- 2001 Transplantation: Basic
Authors
- Altulea, Dania, Universitair Medisch Centrum Groningen, Groningen, Groningen, Netherlands
- Lammerts, Rosa G.m., Universitair Medisch Centrum Groningen, Groningen, Groningen, Netherlands
- Dam, Wendy, Universitair Medisch Centrum Groningen, Groningen, Groningen, Netherlands
- van den Born, Jacob, Universitair Medisch Centrum Groningen, Groningen, Groningen, Netherlands
- Sanders, Jan-Stephan, Universitair Medisch Centrum Groningen, Groningen, Groningen, Netherlands
- Figueiredo, Constanca, Medizinische Hochschule Hannover, Hannover, Niedersachsen, Germany
- Berger, Stefan P., Universitair Medisch Centrum Groningen, Groningen, Groningen, Netherlands
Background
Endothelial cells (ECs) are important target cells for both cellular and antibody mediated rejection in transplanted kidneys. Non-HLA antibodies may play an important role in immunity to the allograft, yet screening for non-HLA antibodies is not routinely performed prior to transplantation due to uncertainty about the clinical relevance and the lack of validated detection assays. Recently, cell-based crossmatching assays have been described for the screening of non-HLA antibodies using either primary ECs such as human umbilical vein endothelial cells (HUVECs), Tie-2+ endothelial precursor cells, or cell lines such as the conditionally immortalized human glomerular endothelial cells (CiGeNCs) as the target cells. However, despite the promising results of these assays, HUVECs and Tie- 2+ ECs are not organ specific, and CiGeNCs, despite being kidney-specific, are derived from a single donor and thus lack heterogeneity in protein expression. Therefore, the development of an ideal EC crossmatching assay with organ and donor specificity is warranted. We aimed to establish a cell bank of kidney-derived ECs covering a broad array of HLA and non-HLA targets, upon which, an EC-based crossmatch assays will be developed using ECs isolated from the liquid of machine-perfused kidneys.
Methods
Human cells were collected and cultured from the perfusate after post-mortem kidney donation and perfusion, then, ECs were isolated based on the expression of CD31.Transduction of the ECs was performed utilizing a lentiviral vector encoding for the SV40 large T antigen and mCherry as reporter gene.
Results
To assess the success of the transduction, the expression of the reporter gene was assessed using florescence microscopy. The positive cells were cultured for a period of 5-8 weeks until a suitable expansion level, then checked for the expression of ECs markers including CD31, CD34, VEGFR-2, ET-1, and vWF with flow cytometry.
Conclusion
Our results showed that the transformed cells expressed the selected common ECs markers even after a long period of culturing, confirming their endothelial characteristics. Additional flow cytometry analysis for a broad array of EC markers, as well as single-clone expansion and HLA-silencing is planned in the near future.
Funding
- Government Support – Non-U.S.