Genipin crosslinking reduced the immunogenicity of xenogeneic decellularized porcine whole-liver matrices through regulation of immune cell proliferation and polarization

Abstract

Decellularized xenogeneic whole-liver matrices are plausible biomedical materials for the bioengineering of liver transplantation. A common method to reduce the inflammatory potential of xenogeneic matrices is crosslinking. Nevertheless, a comprehensive analysis of the immunogenic features of cross-linked decellularized tissue is still lacking. We aimed to reduce the immunogenicity of decellularized porcine whole-liver matrix through crosslinking with glutaraldehyde or genipin, a new natural agent, and investigated the mechanism of the immune-mediated responses. The histologic assessment of the host's immune reaction activated in response to these scaffolds, as well as the M1/M2 phenotypic polarization profile of macrophages, was studied in vivo. The genipin-fixed scaffold elicited a predominantly M2 phenotype response, while the glutaraldehyde-fixed scaffold resulted in disrupted host tissue remodeling and a mixed macrophage polarization profile. The specific subsets of immune cells involved in the responses to the scaffolds were identified in vitro. Crosslinking alleviated the host response by reducing the proliferation of lymphocytes and their subsets, accompanied by a decreased release of both Th1 and Th2 cytokines. Therefore, we conclude that the natural genipin crosslinking could lower the immunogenic potential of xenogeneic decellularized whole-liver scaffolds.

Figure 1. Analysis of decellularized liver materials.

(a,b) Representative H&E staining of the ultrastructure of native liver and decellularized liver matrix. Scale bars = 100 μm. DAPI staining (c,d) and Sirius Red staining (e,f) illustrate a lack of residual cellular components and the preservation of matrix components in the decellularized ECM. Scale bars = 200 μm. Immunohistochemical staining of native and decellularized liver tissues for α-Gal (g,h), SLA-2 (i,j), SLA-DRα (k,l) are shown. Scale bars = 100 μm. (m) Semi-quantitative PCR for the α-Gal, SLA-DRα, PERV, and SLA-2 antigens in the liver scaffolds. Na = native liver; De = decellularized liver matrix.

Figure 2. Morphological and quantitative analysis of liver materials.

(a–d) Macroscopic appearance of native liver, decellularized liver ECM, genipin-fixed ECM and glutaraldehyde-fixed ECM. (e–h) Histological images of liver samples stained with H&E after dehydration and sterilization processes. Scale bars = 100 μm. (i–l) Scanning electron microscopy of the liver materials. Scale bars = 20 μm. (m) The concentration of GAGs in the liver materials. (n) The concentration of collagen in the liver materials. The collagen and GAG contents were normalized to the initial dry weight of the sample. *p < 0.05 with respect to the native liver group. All data are given as the mean ± SEM.

Figure 3. Cells seeded on the uncrosslinked and crosslinked liver ECMs.

(a) Cell viability assay for rat primary hepatocytes seeded on liver ECMs at day 3. (b) The effect of liver ECMs on the cell viability of EA.hy926 endothelial cells at day 3. The normal single layer culture was used as control. (c–e) H&E staining of liver ECMs with rat primary hepatocytes. Scale bars = 50 μm. (f–h) H&E staining of human of liver matrices with EA.hy926 endothelial cells. Scale bars = 50 μm. (i) Albumin production of rat primary hepatocytes in each group at day 3, day 5 and day 7. (j) Urea production of rat primary hepatocytes at day 3, day 5 and day 7. De = decellularized; GP = genipin; GA = glutaraldehyde; 3D control = sandwich culture; 2D control = single layer culture. *p < 0.05 with respect to GA group, ^#p < 0.05 with respect to 3D control group, ^op < 0.05 with respect to 2D control group. All data are given as the mean ± SEM (n = 6/each group).

Figure 4. Histologic analysis of the liver xenografts.

(a–l) The host immune responses towards native liver, decellularized liver ECM, genipin-fixed ECM, and glutaraldehyde-fixed ECM at 7 days, 14 days, and 21 days post-surgery are shown. Scale bars = 100 μm. The dotted line indicates the border of the implants and surrounding tissue. Abbreviations: S = surrounding tissue; I = implanted porcine liver materials. (m,n) Quantification of neutrophil and lymphocyte infiltration. The numbers of cells were counted in at least five different areas for each slide. *p < 0.05 with respect to the native group compared to the decellularized group; ^#p < 0.05 with respect to the genipin group compared to the decellularized group; ^##p < 0.01 with respect to the genipin group compared to the decellularized group; ^op < 0.05 with respect to the glutaraldehyde group compared to the decellularized group; ^oop < 0.01 with respect to the glutaraldehyde group compared to the decellularized group. All data are given as the mean ± SEM (n = 3/each group).

Figure 5. M1/M2 macrophage phenotype distribution in the host immuneresponses to the porcine liver matrices at 7 days and 21 days post-surgery.

Representative immunofluorescent images presenting host macrophage polarization responses to liver matrix materials 7 days (a–h) and 21 days (i–p) post-implantation. M1 cells were co-stained with CCR7 and CD68, and M2 cells were co-stained with CD206 and CD68. Scale bars = 100 μm. CCR7/CD206 = red; CD68 = green; DAPI = blue.

Figure 6. Proliferation properties of PBMCs in co-culture with protein extracts in the CFSE-labeling assay.

(a–f) Representative FACS histograms of immune cells co-cultured in the presence of a low-dose OKT3 stimulus. Proliferation responses without co-culture with protein extracts were used as a negative control. Con A served as a positive control. Na = native liver; De = decellularized; GP = genipin; GA = glutaraldehyde. The depicted line defines the level of proliferated immune cells.

Figure 7. Impact of porcine liver matrices on T cell and B cell subpopulation proliferation and Th1/Th2 cytokine secretion in co-cultures in the presence of OKT3.

(a–f) The proliferation patterns of T cells, B cells and their subsets were analyzed using anti-human CD3, CD8, CD4, HLA-DR and CD19 antibodies. (g–k) Th1 and Th2 cytokine levels for TNF-α, IFN-γ, IL-5, IL-6 and IL-10 are shown for co-cultures of PBMCs alone or in combination with protein extracts of the porcine matrix. Negative controls without any proteins from the liver materials in OKT3-coated wells are included. Na = native liver; De = decellularized; GP = genipin; GA = glutaraldehyde. *p < 0.05 with respect to the native group; **p < 0.01 with respect to the native group; ^#p < 0.05 with respect to the decellularized group; ^##p < 0.01 with respect to the decellularized group; ^op < 0.05 with respect to the control group. All data are given as the mean ± SEM.

Figure 8. Th1/Th2 cytokine secretion profiles of material implants in vivo.

(a–g) Levels of TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-6 and IL-10 in abdominal wall implantsare shown to indicate the Th1/Th2 profile of each group. Na = native liver; De = decellularized; GP = genipin; GA = glutaraldehyde. *p < 0.05Na group with respect to the decellularized group; **p < 0.01 native group with respect to the decellularized group; ^#p < 0.05 genipin group with respect to the decellularized group; ^op < 0.05 glutaraldehyde group with respect to the decellularized group. All data are given as the mean ± SEM.