Objective To explore the construction of heart preservation model of empty beating donor based on extracorporeal membrane oxygenation (ECMO). Methods From January 2022 to August 2023, 20 Guangxi Bama miniature pigs weighing 25-30 kg were selected, half male and half female. Under general anesthesia and heparinization, a midline thoracotomy was performed. The pericardium was cut after freeing the anterior and posterior vena cavae, and a perfusion needle was inserted near the brachiocephalic artery in the ascending aorta, connected to a blood collection bag to collect 500-600 mL of blood. The anterior and posterior vena cavae were ligated, the aorta was blocked and perfused with HTK solution to stop the heart beating. The superior and inferior vena cavae were cut off, the right pulmonary vein was decompressed, the aorta and left and right pulmonary arteries and veins were cut off, and the whole heart was removed. An ECMO device was used to continuously perfuse a cardioprotective solution mainly composed of oxygenated warm blood, maintaining the isolated pig heart beating for 8 hours, monitoring (once/hour) ECMO perfusion parameters, blood gas indicators, perfusate electrolytes, inflammatory factors, myocardial enzymes, myoglobin, and troponin levels. Myocardial tissue was taken for hematoxylin-eosin (HE) staining to observe myocardial cell damage and evaluate the quality of heart preservation. Results Among the 20 isolated beating pig hearts, 17 successfully resumed beating, 3 experienced ventricular fibrillation, resuscitated after intracardiac electrical defibrillation, and all 20 pig hearts successfully beat for 8 hours. There was no statistical difference in ECMO perfusion parameters, blood gas indicators, perfusate electrolytes, and inflammatory factors at each time point (P>0.05). There were statistical increases in myocardial enzymes, myoglobin, and troponin levels (P<0.05). HE staining results suggested that there was no severe myocardial damage. Conclusion ECMO technology can be used for pig heart preservation with good results, and this study provides experimental evidence for improving heart preservation research in clinical heart transplantation.

Objective To investigate the regulatory effect of Xuebijing injection on inflammatory reaction during the preservation of isolated empty beating pig hearts with extracorporeal membrane oxygenation.Methods Twelve healthy Guangxi Bama miniature pigs were randomly divided into the Xuebijing group(n=6)and normal saline group(n=6).After the models were established in the Xuebijing group,Xuebijing injection was given at a dose of 5 mL/h through micropump in membrane oxygenator.In the normal saline group,an equivalent amount of 0.9％sodium chloride injection was pumped.Continuous pumping was performed for 8 h in both groups.The time of cardiac resuscitation and perfusion pressure,heart rate,perfusion flow rate after 8 h preservation were recorded in two groups.Pathological and ultrastructural changes of myocardial tissues in the left ventricular wall of hearts with cardiac arrest were observed after 8 h preservation.Serum levels of myocardial injury markers and inflammatory cytokines were detected in two groups at the beginning(T0),2 h(T2),4 h(T4),6 h(T6)and 8 h(T8)after model establishment,respectively.The expression levels of NOD-like receptor protein 3(NLRP3),cysteinyl aspartate specific proteinase-1(Caspase-1),apoptosis-associated speck-like protein containing a CARD(ASC)messenger RNA(mRNA)in myocardial tissues were measured at T0,T2,T4,T6 and T8,respectively.Results There were no significant differences in the time of cardiac resuscitation and perfusion pressure,heart rate,perfusion flow rate after 8 h preservation between two groups(all P＞0.05).Compared with the normal saline group,the levels of lactate dehydrogenase(LDH)at T4,creatine kinase(CK),LDH and α-hydroxybutyrate dehydrogenase(α-HBDH)at T6 and T8,tumor necrosis factor(TNF)-α at T4,T6 and T8,and interleukin(IL)-6,IL-18 and IL-1 β at T0,T2,T4,T6 and T8 were lower,and the mRNA relative expression levels of NLRP3 and Caspase-1 at T2,T4 and T6,and Caspase-1 and ASC at T8 were lower in the Xuebijing group,respectively(all P＜0.05).Hematoxylin-eosin staining and transmission electron microscopy showed that the degree of myocardial injury in the Xuebijing group was slighter than that in the normal saline group.Conclusions Xuebijing injection may effectively mitigate inflammatory response and exert certain myocardial protection effect during the ECMO preservation of isolated empty beating pig hearts.

BACKGROUND:Cel cryopreservation is required for clinical use of stem cel s, and the current process of cryopreservation however may be harmful to cel viability, pluripotency and differentiation capacity.
OBJECTIVE:To explore the effect of fructose and dithiothreitol on pluripotency and osteogenesis of cryopreserved bone marrow mesenchymal stem cel s.
METHODS:Bone marrow mesenchymal stem cel s were isolated from the bone marrow of Sprague-Dawley rats and pretreated with fructose (200μmol/L), dithiothreitol (500μmol/L) or combined components before cryopreservation. Then the cel s were cryopreseved for 6 months and the morphology of cel s was observed by inverted microscopy. The cel viability was evaluated by MTT, and real-time PCR was used to detect the mRNA expression of Nanog, OCT4 and Sox2. Alkaline phophatase activity assay and alizarin red staining were utilized to detect the osteogenic capacity of bone marrow mesenchymal stem cel s.
RESULTS AND CONCLUSION:Images captured by inverted microscopy showed no significant difference in cel morphology between groups. The MTT results indicated that fructose and combined pretreatment could promote the cel viability of bone marrow mesenchymal stem cel s after cryopreservation, while the real-time PCR results demonstrated that dithiothreitol significantly facilitated the expression of Naogo and Sox2 in bone marrow mesenchymal stem cel s. Moreover, ALP activity assay and alizarin red staining confirmed the positive effects of fructose, dithiothreitol and combined pretreatment on osteogenic capacity of bone marrow mesenchymal stem cel s after cryopreservation, and the best effects were found after pretreatment with dithiothreitol and combined components. Overal , these findings indicate that fructose pretreatment is beneficial for cel viability of cryopreseved bone marrow mesenchymal stem cel s, and dithiothreitol contributes to maintaining the pluripotency and osteogenesis capacity of cryopreseved bone marrow mesenchymal stem cel s.