Fig 1.
Chemical structure of HPG or HES.
Table 1.
Molecular characteristics of HPGs used for the preparation of organ preservation solutions.
Fig 2.
The size-dependent protection of mouse hearts from tissue damage at 4°C by HPGs.
The hearts from naïve B6 mice were stored in HPG (3%, w/v) or UW solution (0.2 mL/organ) at 4°C for 24 h. (A) LDH release from mouse hearts during cold storage with different sizes of HPG (0.5–119 kDa). Data are presented as mean ± standard error of mean (SEM). Two-way ANOVA was used for statistical comparison between UW and HPG (vs. 0.5 kDa HPG, p = 0.008; vs. 1 kDa HPG, p < 0.0001; vs. 3.5 kDa HPG, p = 0.0001; vs. 8.7 kDa HPG, p = 0.0281; vs. 10 kDa HPG, p = 0.0976; vs. 25 kDa HPG, p = 0.5124; vs. 53 kDa, p = 0.0554; vs. 119 kDa, p = 0.8017. n = 4). (B) The tissue damage of the hearts during cold storage with different concentrations (1–6%) of 1 kDa HPG solution was determined by LDH release to the preservation solution. Data are presented as mean ± SEM. Two-way ANOVA was used for statistical analyses (p < 0.0001, HPG vs. UW, n = 4–7). (C) A representative image of EB-stained heart slices after 24-h cold preservation with 1 kDa HPG or UW solution. The fluorescence intensity in dead cells stained with EB was visualized with UV light.
Fig 3.
Cold preservation of donor hearts in HPG solution (1 kDa, 3%) enhances functional recovery after transplantation.
Donor hearts were harvested from naïve B6 mice, and were stored in HPG or UW solution (0.5 mL/organ) at 4°C for 24 h. After transplantation to syngeneic B6 recipient mice, the graft function that was determined by the clinical score of graft contraction/beating was examined at both 15 min and 24 h post-transplantation. Score 4: normal contraction (equal to < 30 minutes of cold preservation in UW solution). At 15 min, p < 0.0001 (t-test, HPG vs. UW). At 24 h, p = 0.0209 (t-test, HPG vs. UW).
Table 2.
Characteristics of the optimized HPG organ preservation solution in comparison to UW solution.
Fig 4.
Cold preservation with the HPG solution reduces cardiac inflammation and cell death in heart transplants.
Donor hearts were treated with prolonged cold preservation in HPG solution vs. UW solution and transplanted as described in Fig. 3. The grafts were harvested at 24 h after transplantation, and were formalin-fixed and paraffin-embedded. (A) The graft injury was examined in H&E stained sections. Data are presented as a typical image of light microscopy, showing perivascular inflammation and cardiaomyocyte necrosis. (B) Histological scores of the graft injury in HPG versus UW solution group. Data are presented as mean ± SEM in each group (p = 0.0347, t-test, HPG vs. UW, n = 9–10).
Fig 5.
The severity of graft injury was determined by the release of LDH and CK from heart transplants to the serum.
Sera were harvested from recipients at 24 h after transplantation, and serum levels of LDH (A) or CK (B) as a biochemical marker of cardiac graft injury were quantitatively measured using cytotoxicity detection kit. Data are presented as mean ± SEM of ten recipients in each group (LDH: p = 0.0381; CK: p = 0.0024. Two-tailed t-test, HPG vs. UW, n = 10).
Fig 6.
Cold preservation with the HPG solution reduces myeloperoxidase (MPO)-positive infiltration in heart transplants.
MPO-positive cells in the sections of cardiac isografts were detected by immunohistochemical stain with anti-MPO antibody. The data are presented as a typical microscopic view in each group: (A) UW group; (B) HPG group; and (C) Positive control, blood clot. Red arrows point MPO-positive cells in the sections. (D) The number of MPO-positive infiltrates counted using a microscope under 400 × magnification (high-powered field, or hpf). The view was not overlapped and was randomly selected. At least 25 views from two separate sections were counted and averaged for each graft. Data are presented as mean ± SEM of six grafts in each group (p = 0.0287, two-tailed t-test, HPG vs. UW, n = 6).
Fig 7.
Cold preservation with the HPG solution prolongs survival of cardiac allografts.
Donor hearts from naïve B6 mice were stored in HPG or UW solution (0.5 mL/organ) at 4°C for 24 h, and transplanted into allogeneic BALB/c mice that were treated with CsA daily. Graft survival was assessed by daily transabdominal palpation, and cessation of the graft beat was considered as graft failure. (A) Graft survival in HPG versus UW group (p = 0.0175, log-rank test, n = 9–10). (B) A typical microscopic view of H&E-stained sections of functioning grafts on day 20 post-transplantation.
Fig 8.
Cold preservation with HPG solution protects HUVECs or H9c2 cells from cell lysis at low temperature.
Monolayers of HUVECs or H9c2 cells in 24-well plates were incubated with HPG versus UW solution at 4°C for 24 h. Cell survival was determined by a trypan blue exclusion assay. (A) HUVECs, p = 0.0063 (Two-tailed t-test, HPG vs. UW, n = 4). (B) H9c2 cells, p < 0.0001 (Two-tailed t-test, HPG vs. UW, n = 7). Cell death in the same cultures was confirmed by the measurement of LDH release. LDH in the preservation solution as a marker of cell lysis was measured and was calculated as a percentage of total LDH in a corresponding positive control (UW solution containing 2% Triton-100). (C) HUVECs, p = 0.0002 (Two-tailed t-test, HPG vs. UW, n = 4). (D) H9c2 cells, p < 0.0001 (Two-tailed t-test, HPG vs. UW, n = 12). Data are presented as mean ± SEM of four separate experiments in each group.
Table 3.
Cold preservation with HPG solution maintains intracellular ATP pool in cultured HUVECs at 4°C.
Table 4.
Cold preservation with HPG solution maintains intracellular ATP pool in cultured H9c2 cells at 4°C.
Fig 9.
Cold preservation with HPG solution enhances cell membrane fluidity of HUVECs at low temperature.
The cell membrane fluidity of HUVECs in HPG versus UW solution at 4°C was monitored by pyrene eximer formation for a period of 4 h. The ratio of eximer-to-monomer (E/M) was calculated as an indictor for the membrane fluidity at the various time points. Data are presented as mean ± SEM of five separate experiments (p < 0.0001, two-way ANOVA, HPG vs. UW, n = 5).