561 Conjugation of human recombinant CD39 to primary human hepatocytes protects against thromboinflammation. /Users/sanas317/Desktop/151116-IPITA CD39 final.pptx
Tuesday November 17, 2015 from 15:30 to 17:00
Room 111-112

Sana Asif, Sweden

PhD student

Department of immunology, genetics and pathology

Uppsala University


Conjugation of human recombinant CD39 to primary human hepatocytes protects against thromboinflammation.

Sana Asif1, Nina Jonsson1,2, Yuji Teramura3, Elisabet Gustafson4, Kristina N Ekdahl1,2, Bo Nilsson1.

1Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden; 2Linnaeus Centre for Biomaterials Chemistry, Linnaeus University, Kalmar, Sweden; 3Department of Bioengineering, University of Tokyo, Tokyo, Japan; 4Department of Women’s and Children health, Uppsala University Hospital, Uppsala, Sweden

Introduction:  Cell transplantation is an attractive and safe alternative to whole organ transplantation. However, cells and materials induce a thromboinflammatory reaction, termed the instant blood mediated inflammatory reaction (IBMIR) upon blood exposure. IBMIR is an innate immune reaction characterized by complement, coagulation and platelet activation, cytokine production, PMN infiltration, and leukocyte-endothelial interaction leading to cell death, necrosis and transplant failure.
Herein, we have developed a novel coating technique by utilizing poly ethyl-glycol (PEG) lipid derivatives to provide thromboinflammatory regulation post transplantation. Primary human cells such as hepatocytes and mesenchymal stem cells (MSC’s) were coated with CD39 by using PEG phospholipid. CD39 is an NTPdase present on endothelial cells which functions to regulate platelet activation by ADP degradation and hence is a potential regulator of the IBMIR.
By this approach, we intend to create a cell surface that attenuates innate immune attack and thromboinflammation, and that could be used to reduce incompatibility reactions in cell transplantations. 
Methods: Recombinant human CD39 was produced in a mammalian expression system by using a plasmid kindly provided by Prof. Peter J. Cowan (Affiliation; University of Melbourne, Australia) and Prof. Simon Robson (Affiliation; Harvard medical school, USA). The binding of CD39 to PEG-lipid was characterized by Quartz crystal microbalance with dissipation monitoring (QCM-D). Isolation of primary human hepatocytes was performed by a modified two-step collagenase digestion procedure. Freshly isolated hepatocytes were coated with Maleimide-PEG-lipid, subsequent washing and coating with (250μg/ml) thiolated CD39 (n=3). Uncoated hepatocytes and only PEG lipid coated hepatocytes were used as controls. Effect of surface modification on hepatocyte viability and proliferation was analyzed by Allmar blue assay. Furthermore, validation of CD39 binding on hepatocyte surface was performed by immunohistochemistry. For hemocompatability, control and coated hepatocytes (10,000 cells/ml) were exposed to whole blood in a whole blood loop model for 60 min. Serological samples for measurement of thrombin-anti thrombin (TAT), C3a and sC5b2-9 were collected 60 min post blood exposure.  All data is presented as (mean±SEM) and was statistically analyzed by paired t test.
Results: A significant binding of CD39 to MAL-PEG lipid was measured by QCM-D (150ng/cm3). Likewise, immunofluorescence staining demonstrated uniform and non-toxic coating of CD39 on hepatocyte surface. Hepatocyte viability was unaffected by surface modification and for all experiments cell viability was 95% 6 hours post modification. Hemocompatability analysis demonstrated, a significant reduction in expression of TAT (266±60.4 vs. 2156±302, p<0.05), C3a (416±48 vs. 1640±82, p<0.05) and sC5b2-9 (92.3±30 vs. 1151±152, p<0,05) in the CD39-immobilized group in comparison to control (non-coated cells).
Conclusion: Surface modification of primary human hepatocytes with recombinant human CD39 attenuates thromboinflammation. This approach provides a potential therapeutic procedure to attenuate IBMIR in cell transplantations.

Lillemor Stenbeck Funke. Sussane Lindblom.

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