Comparison of the effects of normothermic machine perfusion and cold storage preservation on porcine intestinal allograft regenerative potential and viability

Abstract

Intestinal transplantation (IT) is the final treatment option for intestinal failure. Static cold storage (CS) is the standard preservation method used for intestinal allografts. However, CS and subsequent transplantation induce ischemia-reperfusion injury (IRI). Severe IRI impairs epithelial barrier function, including loss of intestinal stem cells (ISC), critical to epithelial regeneration. Normothermic machine perfusion (NMP) preservation of kidney and liver allografts minimizes CS-associated IRI; however, it has not been used clinically for IT. We hypothesized that intestine NMP would induce less epithelial injury and better protect the intestine's regenerative ability when compared with CS. Full-length porcine jejunum and ileum were procured, stored at 4 °C, or perfused at 34 °C for 6 hours (T6), and transplanted. Histology was assessed following procurement (T0), T6, and 1 hour after reperfusion. Real-time quantitative reverse transcription polymerase chain reaction, immunofluorescence, and crypt culture measured ISC viability and proliferative potential. A greater number of NMP-preserved intestine recipients survived posttransplant, which correlated with significantly decreased tissue injury following 1-hour reperfusion in NMP compared with CS samples. Additionally, ISC gene expression, spheroid area, and cellular proliferation were significantly increased in NMP-T6 compared with CS-T6 intestine. NMP appears to reduce IRI and improve graft regeneration with improved ISC viability and proliferation.

Keywords: cold storage; intestinal stem cells; intestinal transplantation; ischemia-reperfusion injury; normothermic machine perfusion; preservation injury.

Figure 1.

Experimental outline. Jejunal and ileal biopsies were collected after graft procurement, after 6 hours of CS or NMP, 1 hour after in vivo reperfusion posttransplant, and after a 48-hour postoperative period. CS, cold storage; NMP, normothermic machine perfusion.

Figure 2.

Histologic images and pathologic injury scoring of the jejunum after 6 hours of cold storage (CS) or normothermic machine perfusion (NMP) and after 1 hour of reperfusion posttransplant. (A) Jejunal hematoxylin and eosin-stained sections of T0 (control), after 6 hours of CS or NMP (CS-T6, NMP-T6), and after 1 hour of reperfusion posttransplant were evaluated for differences in intestinal injury following each storage method. (B) NMP-T6 jejunum had elevated Chiu/Park injury grades compared with control (T0), consistent with mild to moderate villous epithelial loss. After 1 hour of reperfusion, transplanted CS jejunum had a significantly greater injury than that of control (T0) or after 6 hours of CS (CS-T6). Gen 2 and Gen 3 are the second and third generations of NMP protocols. T0 = 20 pigs, CS-T6 = 11 pigs, and NMP-T6 = 9 pigs (Gen 2 = 4 pigs, Gen 3 = 5 pigs). After 1 hour of reperfusion: CS = 7 pigs, NMP = 7 pigs (Gen 2 = 1 pig, Gen 3 = 6 pigs). Original magnification: 10×, scale bar: 100 μm. *P ≤.05, **P ≤ 0.01, ***P ≤.001, and ****P ≤.0001.

Figure 3.

Histologic images and pathologic injury scoring of the ileum after 6 hours of cold storage (CS) or normothermic machine perfusion (NMP) and after 1 hour of reperfusion posttransplant. (A) Ileal hematoxylin and eosin-stained sections of T0 (control), after 6 hours of CS or NMP (CS-T6, NMP-T6), and after 1 hour of reperfusion posttransplant were evaluated for differences in intestinal injury following each storage method. (B) The NMP-T6 ileum had elevated Chiu/Park injury grades compared with the control (T0) and ileum that underwent 6 hours of CS (CS-T6), consistent with mild villous epithelial loss. After 1 hour of reperfusion, transplanted CS ileum had significantly greater injury than that of the control (T0). Gen 3 is the third generation of NMP protocols. T0 = 15 pigs, CS-T6 = 7 pigs, and NMP-T6 = 4 pigs (all Gen 3). After 1 hour of reperfusion: CS = 7 pigs, NMP = 5 pigs (all Gen 3). Original magnification: 10×, scale bar: 100 μm. *P ≤.05, **P ≤.01, ***P ≤.001, and ****P ≤.0001.

Figure 4.

Gene expression analysis of intestinal stem cell and proliferating cell biomarkers after 6 hours of cold storage (CS) or normothermic machine perfusion (NMP). (A) At CS-T6, LGR5, SOX9, and PCNA were significantly increased in jejunal crypts compared with T0. In jejunum, at NMP-T6, OLFM4, HOPX, PCNA, KI67, and YAP1 all increased compared with T0. In jejunum, at CS-T6, SOX9 was increased compared with NMP-T6, whereas jejunal HOPX and KI67 were increased in NMP-T6 compared with CS-T6. (B) In the NMP-T6 ileum, KI67 and YAP1 were increased compared with T0. Ileal KI67, YAP1, and HOPX were increased in NMP-T6 compared with CS-T6. Jejunum and ileum: T0 = 12 pigs, CS-T6 = 7 pigs, NMP-T6 = 9 pigs. *P ≤.05, **P ≤.01, ***P ≤.001, and ****P .0001. CS-T6, 6 hours of cold storage; NMP-T6, 6 hours of normothermic machine perfusion.

Figure 5.

Immunofluorescence analysis of the jejunum after 6 hours of storage and after 1 hour of in vivo reperfusion posttransplant. (A) The expression of KI67 (green) and SOX9 (red) was evaluated between T0, CS-T6, and NMP-T6. (B) At NMP-T6, there was no significant difference in the number of KI67⁺ cells compared with T0, whereas the number of KI67⁺ cells was significantly lower at CS-T6 compared with T0. The number of SOX9⁺ cells was increased in NMP-T6 compared with T0. Both the number of NMP-T6 KI67⁺ and SOX9⁺ cells were significantly increased compared with CS-T6. (C) Expression of CC3 (red) was evaluated between T0, CS-T6, NMP-T6, and after 1 hour of in vivo reperfusion posttransplant. (D) The number of CC3⁺ cells was significantly increased at CS-T6 compared with T0 and NMP-T6, while the number of NMP-T6 CC3⁺ cells was only mildly elevated compared with T0. After 1 hour of reperfusion, there was a significantly increased number of CC3⁺ cells in the CS-T6 jejunum compared with T0 and NMP-T6. NMP-T6 was decreased in CC3⁺ cells compared with T0. All sampling timepoints KI67, SOX9: n = 7 pigs. T0 and T6 CC3: n = 7 pigs. After 1 hour of reperfusion CC3: n = 14 pigs. KI67, SOX9 original magnification: 40×, scale bar: 20 μm. CC3 original magnification: 10×, scale bar: 100 μm. *P ≤ .05, **P ≤ .01, ***P ≤.001, and ****P ≤ .0001. CC3, cleaved caspase 3; CS, cold storage; CS-T6, 6 hours of cold storage; NMP, normothermic machine perfusion; NMP-T6, 6 hours of normothermic machine perfusion; SOX9, sex-determining region Y-box 9; T0, control tissue.

Figure 6.

Jejunal intestinal stem cell culture spheroids and area measurements. (A) Images of 24- and 48-hour cell culture for spheroids from control (T0) jejunum, and jejunum stored for 6 hours (CS-T6 and NMP-T6). Spheroids cultured from transplanted jejunum that underwent 1 hour of in vivo reperfusion are not pictured. (B) At 24- and 48-hours of cell culture, jejunal spheroid area measurements were significantly larger from NMP-T6-derived crypts compared with crypts from T0 and CS-T6 jejunum. After 1 hour of reperfusion posttransplant, NMP-stored jejunal spheroid area measurements were significantly larger than T0, NMP-T6, and 1 hour reperfused CS jejunum. Additionally, after 1 hour of reperfusion posttransplant, CS jejunal spheroid area measurements were significantly larger than T0 and CS-T6. T0 = 15 pigs, CS-T6 = 11 pigs, and NMP-T6 = 10 pigs. After 1 hour of reperfusion: CS = 6 pigs, NMP = 4 pigs at 24 hours, and 5 pigs at 48 hours of cell culture. Original magnification: 20×, scale bar: 50 μm. *P ≤.05, **P ≤.01, ***P ≤.001, and ****P ≤.0001. CS, cold storage; CS-T6, 6 hours of cold storage; NMP, normothermic machine perfusion; NMP-T6, 6 hours of normothermic machine perfusion; T0, control tissue.

Figure 7.

Ileal intestinal stem cell culture spheroids and area measurements. (A) Images of 24- and 48-hour cell culture for spheroids from control (T0) ileum, and ileum stored for 6 hours (CS-T6 and NMP-T6). Spheroids cultured from transplanted ileum that underwent 1 hour of in vivo reperfusion are not pictured. (B) At 24- and 48-hours of cell culture, ileal spheroid area measurements were significantly larger from NMP-T6 derived crypts compared with crypts from T0 and CS-T6 ileum. After 1 hour of reperfusion posttransplant, NMP-stored ileal spheroid area measurements were significantly larger than T0 and reperfused CS jejunum. T0 = 14 pigs, CS-T6 = 11 pigs, and NMP-T6 = 9 pigs. After 1 hour of reperfusion: CS = 6 pigs and NMP = 5 pigs. Original magnification: 20×, scale bar: 50 μm. *P ≤.05, **P ≤.01, ***P ≤.001, and ****P ≤.0001. CS, cold storage; CS-T6, 6 hours of cold storage; NMP, normothermic machine perfusion; NMP-T6, 6 hours of normothermic machine perfusion; T0, control tissue.

Figure 8.

5-ethynyl-2’-deoxyuridine (EdU) proliferation assay images and the number of EdU⁺ cells after 6 hours of jejunal and ileal CS or NMP. (A) EdU (purple) whole mount immunofluorescence on jejunal and ileal T0, CS-T6, and NMP-T6 spheroids. (B) NMP-T6 jejunal and ileal spheroids contained a great number of EdU⁺ cells compared with T0 or CS-T6. T0 = 10 pigs, CS-T6 = 7 pigs, and NMP-T6 = 8 pigs. Original magnification: 20×, scale bar: 50 μm. *P ≤.05, **P ≤.01, ***P ≤.001, and ****P ≤.0001. CS, cold storage; CS-T6, 6 hours of cold storage; NMP, normothermic machine perfusion; NMP-T6, 6 hours of normothermic machine perfusion; T0, control tissue.