Objective To evaluate the safety and efficacy of a self-developed micro-normothermic machine perfusion (NMP) system (micro-perfusion device) for preserving isolated porcine limbs. Methods Five healthy Landrace pigs were selected, and their left and right forelimbs were randomly divided into the NMP group and static cold storage (SCS) group. The NMP group was perfused with the self-developed micro-perfusion device and polymerized hemoglobin perfusate for 32 hours at normothermia, while the SCS group was preserved at 4 ℃. Hemodynamic parameters such as perfusion pressure and flow were monitored. The pH value, partial pressure of oxygen (PO₂), lactic acid (Lac), creatine kinase (CK) and lactate dehydrogenase (LDH) in the perfusate were measured. Hematoxylin-eosin staining was used to assess the muscle tissue structure, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was employed to evaluate muscle cell apoptosis, and immunohistochemistry staining was applied to detect the expressions of tumor necrosis factor (TNF)-α and interleukin (IL)-6. A mixed-effects model was used to analyze the effects of time and treatment methods on tissue structure, cell apoptosis and inflammatory factors. Results The device could stably maintain a perfusion pressure of (69±15) mmHg and a flow rate of (117±42) mL/min. The pH value and electrolytes of the perfusate were generally stable, with PO₂ maintained at a high level. Lac was maintained at 5.38(3.81, 6.45) mmol/L, while CK and LDH increased over time. After 32 hours of perfusion in the NMP group, both the myocyte spacing and apoptosis rate were better than those in the SCS group. Mixed-effects model analysis showed that there were statistically significant differences in the effects of NMP treatment and SCS treatment on myocyte spacing and apoptosis rate per unit time (both P < 0.05). There were no statistically significant differences in TNF-α and IL-6 between the two groups, and mixed-effects model analysis showed no statistically significant differences in the effects of NMP treatment and SCS treatment on TNF-α and IL-6 per unit time (both P > 0.05). Conclusions The micro-perfusion device used in this study may achieve 32-hour normothermic preservation in a porcine limb amputation model, maintain basic metabolism and ionic homeostasis, reduce muscle structural damage and cell apoptosis without inducing additional inflammatory responses. This technology is expected to significantly extend the time window for replantation of amputated limbs in disaster rescue and long-distance transportation, providing an important technical basis for clinical translation and subsequent replantation research.

Objective To systematically evaluate the similarities between six-gene-modified pig red blood cells and human red blood cells in multiple dimensions, including cell morphology, hematological parameters, microbial safety and serological compatibility, and to verify the feasibility of using six-gene-modified pig red blood cells as an alternative for blood transfusion in cases of traumatic hemorrhage. Methods Blood samples from O-type human donor, six-gene-modified O-type pig and wild-type O-type pig were collected. The pig gene-modified phenotype was identified by flow cytometry. The morphology was observed using field emission scanning electron microscopy and the diameter was measured. Hematological tests, including blood routine, osmotic fragility test, adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) content and partial pressure of oxygen at 50% hemoglobin saturation (P50), were conducted to assess the deformability of red blood cells. Real-time fluorescent quantitative polymerase chain reaction was used to screen for zoonotic viruses, and cross-matching was performed using column gel method, condensation amine method and saline tube method. Results The six-gene-modified pig red blood cells successfully knocked out the α-Gal, Neu5Gc and Sda antigens and expressed hCD55. Wild-type pig red blood cells expressed these three antigens but did not express human complement regulatory proteins. All three types of red blood cells presented a biconcave discoid morphology. The diameter of O-type human red blood cells was larger than that of the six-gene-modified porcine red blood cells and wild-type pig red blood cells (all P < 0.05). The average red blood cell volume and average red blood cell hemoglobin of O-type humans were higher than those of the six-gene-modified pig red blood cells, and the distribution width-variable coefficient of O-type human red blood cells was lower than that of the six-gene-modified pig red blood cells (all P < 0.05). There were no statistically significant differences in the permeability fragility, ATP content, 2,3-DPG content, P50 and red blood cell deformability indicators of the three types of red blood cells (all P > 0.05). The screening for zoonotic viruses in human and animal cells of the six-gene-modified pigs and wild-type pigs was negative. The cross-matching results showed that in the column gel method and condensation amine method, the primary and secondary sides of the six-gene-modified pig red blood cells did not agglutinate with human red blood cells, while the wild-type pig red blood cells agglutinated with human red blood cells in all cross-matching methods. Conclusions The six-gene-modified pig red blood cells are highly similar to human red blood cells in terms of morphology, key hematological parameters, energy metabolism, oxygen-carrying capacity, mechanical properties, microbial safety and serological compatibility, providing an experimental basis for the clinical transformation of xenotransfusion.