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.

Figure 1

Histological and metabolic characteristics of human aortic tissue after Conventional Frozen Cryopreservation (CFC) or Ice-Free Cryopreservation (IFC). (a) The tissue preparation and analysis is shown schematically. Punches of fresh aortic tissue were made and frozen according to either the CFC or IFC protocols (see methods). Tissue punches were stored at least 1 month in the vapor phase above liquid nitrogen (about −160 °C) or at −80 °C before use, respectively. Tissue samples were thawed and washed according to the CFC or IFC protocols for further analysis. Punches were incubated in cell culture medium and tissue viability (metabolism, apoptosis, and necrosis) and cytokine release were analyzed, and tissue specimens were histologically examined. (b) Human CD31+ staining (yellow) indicates the endothelial cell layer (white arrows) on the intimal side of both CFC and IFC aortic tissue. Nuclei were stained with DAPI (pseudo-colored white); while the extracellular matrix shows green autofluorescence. Scale bars represent 100 µm. (c) Metabolic activity of the tissue was assessed with an MTS assay. (d) Necrosis level was determined by the measurement of lactate dehydrogenase (LDH) activity in conditioned medium (CM). (e) Apoptosis of tissue cells was analyzed via measurement of caspase-3 and -7 activities in the CM. All measurements are normalized to the medium control (dotted line). Data are shown as the mean + SEM (n = 5–7) and analyzed with Mann Whitney test *p < 0.05. (f) TUNEL staining revealed some apoptotic cells (brown nuclei) highlighted by black arrows in the CFC tissue. The DNAse-treated positive control (Pos. Ctrl.), and a negative control (Neg. Ctrl.) are shown for comparison. Scale bars represent 75 µm.

Figure 2

IFC tissue releases lower cytokine and chemokine levels than CFC tissue, however more biologically active TGF-β is present. CFC and IFC aortic tissue punches were incubated in DMEM culture medium for 2 days. The cytokines and chemokines IL-6, MCP-1 IL-8, IL-10 (a) total TGF-β (active and latent form) and active TGF-β (b) were analyzed by ELISA or multiplex bead assay. Data are shown as the mean + SEM (n = 4–8) and analyzed with Mann Whitney test **p < 0.01, ***p < 0.01; n.s.: not significant; n.d.: not detectable. (c) Over a culture period of 6 days, total TGF-β (upper graph) and active TGF-β (lower graph) was measured by ELISA. The absolute amount of TGF-β in pg produced per tissue punch was calculated and a representative kinetic graph is shown.

Figure 3

Immune cell migration is triggered by soluble factors delivered by CFC tissue, but not IFC tissue. Monocytes (CD14+) and T cells (CD3+) were separated from human PBMC. Thirty thousand cells in diet-medium were seeded on the porous membrane of a chemotaxis system. CFC or IFC tissue conditioned diet-medium (CM) was placed in the lower well. After 3 h, the number of migrated monocytes (a) and T cells (b) was analyzed. Migrated cells were defined as cells in the lower well plus cells attached to the bottom side of the membrane. Pure diet-medium served as negative control (Neg. Ctrl.), to define the random cell migration without a chemotactic gradient. Data are shown as the mean + SEM (n = 5, 3 replicates each) and analyzed with one-way ANOVA (Kruskal-Wallis test) *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Soluble factors from CFC tissue but not from IFC tissue amplify αCD3 triggered T cell proliferation. Human CFSE-labeled PBMC were stimulated with a low dose of αCD3 antibody and subjected to CFC or IFC tissue conditioned medium (CM). After 4 days, PBMC were harvested, stained with fluorochrome-labeled human specific antibodies for T cell subset markers and analyzed by flow cytometry. Representative flow cytometry plots show the induction of CD4+ T cell (a) and CD8+ T cell (c) proliferation in PBMC cultures stimulated with either αCD3 alone (Ctrl.; left), or combined with CFC tissue CM (CFC-CM; middle) or IFC tissue CM (IFC-CM; right). Bar graphs show the levels of proliferated CD4+ T cells (b) and CD8+ T cells (d) in summary. Data are shown as the mean + SEM (n = 5–8) and analyzed with one-way ANOVA (Kruskal-Wallis test) *p < 0.05.

Figure 5

Macrophages cultured on CFC or IFC tissue show comparable adherence and appearance. (a) In a newly developed macrophage-tissue assay, macrophages were cultured directly on the aortic tissue surface. Monocytes were separated from human PBMC with MACS CD14 MicroBeads. Monocytes were differentiated to macrophages in vitro for 7 days with M-CSF and seeded directly on the surface (intimal side) of the human CFC or IFC treated aorta. A silicone ring held the tissue punch on the bottom of the culture well to ensure direct contact of macrophages and tissue. After a 2-day co-culture, the tissue punch was either analyzed with scanning electron microscopy (SEM), or the macrophages were harvested and their surface marker expression pattern was analyzed by flow cytometry. (b) Representative SEM pictures of the macrophage-tissue co-culture are depicted. The intima surface of CFC (upper row) and IFC (lower row) aortic tissue are shown with macrophages adhered to the tissue surface (black arrows). In higher magnification (right column) the attached macrophages are visible (black arrows). Scale bars represent 200 µm (left column) and 20 µm (right column).

Figure 6

Both types of cryopreserved tissue upregulate common surface molecules on macrophages, but do not influence their polarization state. Macrophages were cultured on the intima surface of CFC or IFC treated aortic tissue. Cells cultured on tissue culture plastic (TCP) served as control. After 2 days, macrophages were harvested, stained with fluorochrome-labeled human specific antibodies and analyzed by flow cytometry. Representative histograms and quantitative analyses of the mean fluorescence intensity (MFI) are shown for the surface markers CD16 (a) and CD14 (b), the M1 polarization marker CD80 (c) and HLA-DR (d) and the M2 polarization markers CD206 (e) and CD163 (f). The means of the data are shown (n = 11) and analyzed with one-way ANOVA (Kruskal-Wallis test) *p < 0.05, ***p < 0.01.

Figure 7

Hypothesized mechanism indicating how the cryopreservation methods affect the immune compatibility of human cardiovascular matrices. Potential effects of CFC and IFC treatments on the human immune response to cardiovascular tissue (vessel or heart valves) are illustrated. After CFC, some tissue cells are apoptotic or necrotic, but the tissue still releases high amounts of cytokines such as IL-6, MCP-1, IL-8, latent TGF-β, and smaller amounts of IL-10. These cytokines lead to a strong activation of blood immune cells. Monocytes and T cells are attracted to the tissue graft, where they can infiltrate or receive signals to proliferate. In contrast, after IFC treatment, tissue cells do not undergo apoptosis or necrosis, but have a diminished metabolic activity. The cytokines IL-6, MCP-1, IL-8, IL-10, and TGF-β are released only in small amounts and TGF-β is secreted in its biologically active form. The decreased levels of cytokine release and the presence of active TGF-β lead to an attenuated activation of immune cells. Particularly, migration and infiltration of T cells and monocytes is reduced and T cell proliferation is blocked, which results in a diminished human immune response to IFC tissue compared to CFC.