The choice of cryopreservation method affects immune compatibility of human cardiovascular matrices

Maria Schneider,Ulrich A Stock,Martina Seifert,Kelvin G M Brockbank,Christof Stamm

doi:10.1038/s41598-017-17288-z

Abstract

Conventional frozen cryopreservation (CFC) is currently the gold standard for cardiovascular allograft preservation. However, inflammation and structural deterioration limit transplant durability. Ice-free cryopreservation (IFC) already demonstrated matrix structure preservation combined with attenuated immune responses. In this study, we aim to explore the mechanisms of this diminished immunogenicity in vitro. First, we characterized factors released by human aortic tissue after CFC and IFC. Secondly, we analyzed co-cultures with human peripheral blood mononuclear cells, purified monocytes, T cells and monocyte-derived macrophages to examine functional immune effects triggered by the tissue or released cues. IFC tissue exhibited significantly lower metabolic activity and release of pro-inflammatory cytokines than CFC tissue, but surprisingly, more active transforming growth factor β. Due to reduced cytokine release by IFC tissue, less monocyte and T cell migration was detected in a chemotaxis system. Moreover, only cues from CFC tissue but not from IFC tissue amplified αCD3 triggered T cell proliferation. In a specifically designed macrophage-tissue assay, we could show that macrophages did not upregulate M1 polarization markers (CD80 or HLA-DR) on either tissue type. In conclusion, IFC selectively modulates tissue characteristics and thereby attenuates immune cell attraction and activation. Therefore, IFC treatment creates improved opportunities for cardiovascular graft preservation.

Highlights

Providing matrices for tissue replacements that fulfill all requirements for successful and appropriate treatment of cardiovascular diseases is still an important issue
To predict adaptive response mechanisms, we evaluated whether just the soluble factors released by conventional frozen cryopreservation (CFC) and ice-free cryopreservation (IFC) aortic tissue could induce or modulate T cell responses
Since T cell activation requires two signals, we provided the first signal by low dose stimulation with αCD3, and analyzed whether the conditioned medium (CM) was able to deliver a second co-stimulatory signal that would enhance the immune cell response

Summary

Introduction

Providing matrices for tissue replacements that fulfill all requirements for successful and appropriate treatment of cardiovascular diseases is still an important issue. After fixing, the tissue is not viable and lacks remodeling capacity[13,14] Another treatment option to reduce immune responses to xenogeneic tissue following implantation is the introduction of a decellularization www.nature.com/scientificreports/. Allografts, known as homografts, can be preserved and stored without the need for complex and long lasting pre-treatment procedures by conventional frozen cryopreservation (CFC). To prevent ice-crystal formation in cardiovascular tissues and to reduce the associated higher risk for calcification, an alternative ice-free cryopreservation (IFC) method was developed, which promotes ECM retention and loss of cell viability[27]. In an allogeneic sheep model it could be shown that this preservation method resulted in better performance, with less thickening of heart valve tissue and reduced immune cell infiltration after in vivo-implantation compared to conventional frozen grafts[31,32]

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