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

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.

Fibronectin Adsorption on Electrospun Synthetic Vascular Grafts Attracts Endothelial Progenitor Cells and Promotes Endothelialization in Dynamic In Vitro Culture

“…Monocytes were incompletely activated after co-culturing with the scaffold as indicated by only a weak tendency to upregulate the HLA-DR expression and to increase their TNFα release. From the literature, it is well known that the upregulation of CD80 and HLA-DR would be a hallmark of M1 macrophages [62,104] and that the fiber and pore size of electrospun scaffolds could impact the macrophage polarization state [105]. When analyzing the potential impact of the TPCU scaffolds on macrophage polarization, no clear trend to drive the process into a specific macrophage subtype could be determined.…”

“…Polymorph nuclear cells (PMNs) were isolated from freshly donated human blood and peripheral blood mononuclear cells (PBMCs) from buffy coats according to the ethical approval by the local ethics committee at the Charité Berlin (EA2/139/10 approved on 10th December 2010; EA1/226/14 approved on 24th July 2014) and as recently described [61]. Monocytes were magnetically sorted via CD14 beads (130-050-201, Miltenyi Biotec, Bergisch Gladbach, Germany) from PBMCs as previously described [62]. Monocytes were differentiated into M0 macrophages by adding 50 ng/mL of macrophage colony-stimulating factor (M-CSF) (130-096-491, Miltenyi Biotec) to the culture medium for 7 days.…”

“…For SEM imaging of the monocytes and macrophages, the cells were cultured for two days on uncoated (w/o), DCN-or FN-coated scaffolds, followed by preparation (as described in Reference [62]) and imaging with a JCM 6000 Benchtop (JEOL, Freising, Germany).…”

Bioengineered human acellular vessels recellularize and evolve into living blood vessels after human implantation

“…However, their potential is limited by availability, cost, processing techniques, and mixed clinical outcomes (4). Specifically, the methods used to cryopreserve, fix, sterilize, or decellularize allogeneic and xenogeneic tis-sues often affect their immunogenicity and biocompatibility, resulting in host inflammatory reactions, and ultimately, in material degradation and mechanical failure (5)(6)(7)(8). Although many of these tissues have mechanical strengths that match or exceed those of native autologous vascular tissue, the structure and composition of their matrix are not conducive for appropriate repopulation by host cells (9)(10)(11).…”

Immunological Aspects Involved in the Degeneration of Cryopreserved Arterial Allografts

“…This specificity becomes of paramount importance when trying to establish the immune response triggered by vascular transplantation. Failure to address the issue may partially explain the controversial results published by different authors regarding the antigenicity of cryopreserved arteries and veins, many times reporting opposite conclusions (17)(18)(19)(20).…”