• KSBB Journal
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• v.24 no.3
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• pp.312-320
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• 2009

Stable cell preservation is an essential factor in the regenerative medicine for cell therapies and transplantation of biologic materials. In this study, we studied to provide more stable hypothermic preservation by protection of cell damage during the preservation at $4^{\circ}C$. The result of searching for key components that have excellent efficacy in hypothermic preservation of cells, we have identified the fact that the hypothermic preservation adding protein hydrolysates such as yeast hydrolysate is far superior to others. All protein hydrolysates that are derived from animal, plant and microbe sources have superior efficacy, especially the peptides which have molecular weights under 10 kDa have the best efficacy among the components of protein hydrolysate. The protein hydrolysates prevented the decrease of ATP level in the cells caused by hypothermic environment and they inhibited the generation of ROS. Adding antioxidants and control agents of osmotic pressure were showed to have more superior efficacy in hypothermic preservation. Finally, KUL261 solution (DMEM/F12 1 : 1 medium, yeastolate 1%, $\alpha$-tocopherol $100{\mu}M$, dextran 2.5%), the preservation solution developed in this study, showed the best efficacy in both cell viability and cell growth more than other conventional preservation solutions. In conclusion, the improved hypothermic preservation solution that contains the protein hydrolysates as a key component provide the best preservation efficacy. It provides better efficacy than other preservation solutions and will contribute to both the development of regenerative medicine and global commercialization in this therapeutic field.

• KSBB Journal
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• v.21 no.4
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• pp.306-311
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• 2006

Cell preservation is indispensable in animal cell culture process and should be established according to the cell characteristics. In this study, we experimented hypothermic preservation of CHO cells that is widely used in pharmaceutical industry to produce therapeutic proteins and established a stable method of preservation. The highest viability of CHO cells was obtained when the cells were preserved using rolling tube, which means the cells should be suspended to avoid the cell lumping during the preservation. Also, we obtained superior preservation result under the anaerobic condition. To evaluate the serum-free media as a preservation solution, we investigated cell growth after hypothermic preservation using serum-free media. High cell viability and normal cell growth was observed during 10 days using serum-free media. Moreover, we found that more effective preservation when ${\alpha}$-tocopherol and retinoic acid is added to media as an additive. In the case of 1 liter large scale hypothermic preservation using established protocol, cell viability and growth rate was obtained as good as small scale one. This study is considered to be helpful for hypothermic preservation of CHO cells and large scale hypothermic preservation may be available through the further studies.

• Journal of Chest Surgery
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• v.7 no.2
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• pp.145-152
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• 1974

The effect of reticuloendothelial hyperfunction on hypothermic preservation of lung was studied in dogs. In order to evaluate the viability after hemodynamic_ load in preserved isolated lung, observations were made on the rate of increase in weight, degree of edema,compliance and surface activity of lung. The results obtained as follows: l. In the group of activating of the reticuloendothelial system by injection of sodium thiosulfate intravenously before pneumonectomy and infusion of naphthionine through the pulmonary artery before hypothermic preservation of isolated lung the limit of preservation was eight hours whereas four hours in non-treated control group. 2.Therefore the method of activating of the reticuloendothelial system before and after pulmonary resection seems effective in preserving for prolonging the period of preservation of lung by means of inhibition of pulmonary edema. 3. Pulmonary surface activity is expected to be valuable as a method in evaluation of the viability of preserved lung along with compliance and rate of increase in weight of lung.

• Journal of Chest Surgery
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• v.28 no.6
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• pp.549-556
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• 1995

We investigated the effects of aprotinin, a protease inhibitor, on isolated rat heart subjected to cardioplegia and global ischemia for 4 hours and then reperfused for 40 minutes. Before ischemia, hearts were perfused with either aprotinin 1x105KIU/L[Aprotinin group,n=8 or no aprotinin[control group,n=8 added to Krebs-Henseleite solution for 30 minutes. Hemodynamic and biochemical parameters such as heart rate, LVP, dP/dt, coronary flow and creatine kinase were measured before cardioplegia and after reperfusion 10,20,30,40 minutes. After completion of experiment, wet and dry heart weight were measured for tissue water and water content evaluation. On reperfusion, recovery of LVP of aprotinin group at each time point was significantly better than that of control group[p<0.05 , and of dP/dt at reperfusion 40 minutes[p=0.034 . No statistically significant differences in heart rate, coronary flow and CK were observed between the two groups, but aprotinin group seemed to have better recovery. No significant differences in tissue water and water content were observed between the two group.These results suggest that pretreatment of aprotinin is effective in myocardial preservation in prolonged hypothermic ischemia and reperfusion.

• Journal of Chest Surgery
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• v.18 no.4
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• pp.759-770
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• 1985

This study was experimentally undertaken to evaluate the effect of hypothermic oxygenated cardioplegic solution on myocardial protection during prolonged aortic cross clamping under cardiopulmonary bypass. Dogs were divided into two groups control group [received hypothermic unoxygenated cardioplegic solution] and experimental group [received hypothermic oxygenated cardioplegic solution]. Coronary sinus effluent was obtained at once and 30, 60, 90 minutes after cross-clamping for the determination of pH, PCO2,PO2 and lactate level during the infusion of cardioplegic solution and myocardial biopsies were obtained after cessation of 90 minutes of aortic cross-clamping. The results obtained were as follows: 1. There was no significant differences in the pH and PCO2 between the oxygenated and unoxygenated cardioplegic solution but the PO2 of the oxygenated solution was 4 times greater than unoxygenated solution, and also the oxygenated solution had a significantly greater oxygen content [2.020.05 ml 02/min] and had much more oxygen delivery than unoxygenated solution. 2. The myocardial oxygen consumption and the myocardial oxygen extraction in oxygenated group were 1.63 ml 02/100 ml and 67.32% respectively, which was greater than those in unoxygenated group. 3. Regarding to pH and PCO2 of coronary sinus effluent, there was no significant differences between two groups in early period of infusion of cardioplegic solution, but the pH shifted to acidosis from 60 minutes, PCO2 increased from 90 minutes of aortic cross-clamping, and PO2 markedly decreased from 90 minutes of aortic cross-clamping in unoxygenated group. 4. The lactate concentration of coronary sinus effluent revealed relatively normal in both groups, but showed slight increase up to 27.54.56 mg/100 ml at 90 minutes of aortic cross-clamping in unoxygenated group. 5. On electron microscopic study, the ultrastructural integrity of myocardial cells in oxygenated group was well preserved within 90 minutes. Slight swelling and deformity of mitochondria, interfibrillar widening, and disarrangement of myofibrils were observed at 90 minutes after aortic cross-clamping in unoxygenated group. From these results, the use of hypothermic oxygenated cardioplegic solution seemed to be effective and better method for the preservation of ischemic myocardium during the prolonged aortic cross-clamping.

• Journal of Chest Surgery
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• v.29 no.10
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• pp.1066-1075
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• 1996

To compare the efficacy of cardiac preservation, we examined purine metabolites during 24 hours of cold storage($0^{\circ}C$) of the Korean ongrel dog hearts after using three different types of cardioplegic solutions. The hypothermic arrest with total cardiopulmonAry bypass method was employed in 51. Thomas solution(575) and blood cardioplegic solution(BCPS) preservation cases. Specimens were analyzed for levels of adenine nucleotides and their precursors by high performance liquid chromatography. The ATP content in the UW(University of Wisconsin) solution group tends to be higher than that of the combined hypothermic arrest group(575 and BCPS groups) after 2,4,8, and 12 hours of preservation respectively, but there were no significant differences between 575 and BCPS groups. The ADP contents in the UWS and BCPS groups were higher than that of the 575 group at 4,8, 12, and 24 hours, but the difference was not statistically significant between UWS and BCPS groups. The AMP contents did not change significantly in the three groups. The adenosine, Inosine, and hypoxanthine concentrations increased progressively, but the lev l of xanthine was very low in the three groups.

• Journal of Chest Surgery
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• v.25 no.5
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• pp.458-468
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• 1992

With the aid of extracorporeal circulation, nine dogs underwent orthotopic cardiopulmonary transplantation after preservation of the donor heart in a hypothermic amino acid[glutamate, aspartate] enriched high potassium extracellular solution, and preservation of the donor lung with hypothermic low potassium dextran solution from June 1990 to May 1991. The mean body weights of dogs were 20kg and the recipients` preoperative hematologic and hemodynamic pictures were within normal range except slightly decreased level of albumin and total protein, which was supposed to be due to malnutrition. The following modifications of the original Stanford technique were emphasized: [1] the posterior mediastinum is dissected as little as possible with meticulous hemostasis; [2] the surgical procedure is kept away from the phrenic and vagus nerves; [3] the tracheal anastomosis may be wrapped with recipient`s pulmonary artery flap or surrouding soft tissues. A combination of Cyclosporine, Azathioprine, corticosteroid was used as perioperative immunosuppressive therapy. Postoperatively all recipients could be weaned from extracorporeal circulation, showing favorable vital signs, but within 24 hours, irreversible congetive heart failure, ascites, arrhythmias developed with a mean survival time 13.6$\pm$6.6[n=9, range=6~26] hours. Hemoglobin and platelet counts were significantly[p<0.05] decreased postoperatively, which is thought to be attributed to blood damage by cardiopulmonary bypass and hemodilution. Postmortem finding included multiple subendocardial patch hemorrhage in both atrial and ventricular cavities, pulmonary and liver congestion, and all tracheal anastomoses were intact. Further consideration about quality control of the animal, infection, rejection, the effect of cardiopulmonary bypass on the experimental animal is required to improve the results.

• Journal of Chest Surgery
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• v.25 no.3
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• pp.211-219
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• 1992

Hypothermic cardioplegia is a well established method to optimize myocardial preservation during ischemic arrest, and it has been demonstrated that oxygenation of crystalloid cardioplegic solutions markedly enhances myocardial protection, The addition of a small amount of red blood cells to a crystalloid cardioplegic solutions improves capillary perfusion. Considering these results, we changed our cardioplegic solution from cold oxygenated crystalloid[Group 2] to cold oxygenated diluted blood[Group 1]. In this investigation, we examined the effects of two hypothermic potassium cardioplegic solutions on myocardial preservation in 50 patients[30 of Group 1 and 20 of Group 2] of child age group. Factors considered preoperatively included age, sex, body weight, preoperative diagnosis, and they showed no statistical differences, Intraoperative factors considered included duration of cardiopulmonary bypass, duration of aortic occlusion, operative mortality, which also revealed no statistically significant differences, We measured the serum levels of GOT[glutamate oxaloacetate transaminase] and CPK [creatine phosphokinase] during the first two days postoperatively, which, in both groups, showed significantly higher values until postoperative 1 day, and decreasing tendancy thereafter, however we failed to find any significant difference between two groups regarding the serum levels of those enzymes each day. Time for extubation and use of inotropics also revealed no significant differences. Defibrillation was needed less in Group 1 than in Group 2[p<0.05], and one case of supraventricular tachyarrhythmia occured in Group l. We conclude that cold oxygenated diluted blood cardioplegia provides no less preservation than does an oxygenated crystalloid cardioplegic solution in child age group.

• Journal of Chest Surgery
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• v.21 no.3
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• pp.425-446
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• 1988

After 24 hours of preservation under 15 mmHg perfusion pressure the recovery rates of isolated canine hearts were determined. Preservation was performed in a cold room maintained at 4*C with 4 different types of perfusates bubbled with a mixture of 95% 0y and 5% CO~ using a modified perfusion unit designed in our institute. The perfusates used were as follows; Group 1: Krebs-Henseleit solution, Group 2: Krebs solution added by albumin and PGE1. Group 3: Modified Wicomb*s solution, Group 4: Modified Collin*s solution. The extent of myocardial recovery was evaluated using a modified isolated carmine perfusion model by measuring heart rate, systolic arterial pressure, left atrial pressure[LAP] and cardiac output. In addition to the above hemodynamic parameters, biochemical and enzymatic assays from perfusates and electron microscopic changes of the myocardium were also studied. The results were as follows; 1] The heart recovery rates were 41.6%, 53.4% and 108.9% in groups 1, 2 and 3, respectively, and group 3 elicited the best result[p< 0.001]. The heart beat was never recovered in group 4. 2] Recovered systolic arterial pressures[mmHg] were 63.3% in group 1, 94.9% in group 2 and 94.3% in group 3. 3] LAPs[mmHg] were 20 in group 1, 13.5 in group 2 and 11.2 in group 3, which suggested that the best myocardial preservation was elicited in group 3[p< 0.05]. 4] Cardiac output, the sum of aortic stroke volume and coronary leakage, were 69.1% in group 2, and 90.7% in group 3, but these were not statistically significant[p=0.24]. No aortic stroke output was measured in group 1 and 4. 5] The degree of myocardial edema increase was 17.5` in group 1, 24.6% in group 2, 20.9% in group 3 and 55.3% in group 4. But there were no statistical differences in each group[p= 0.08]. 6] CPK-MB[U/L] levels were increased 750% and 332%[p< 0.05], glucose levels[mg/dl] 60.5% and 78.2% and SGOT[U/L] levels 523% and 333%, in groups 2 and 3, respectively. Biochemical and enzymatic assays could not be performed in group 1 and group 4, because of poor recovery of heart beat. 7] Electron microscopic findings in the myocardium of most groups revealed slight to moderate muscle cell and mitochondrial edema. But all these findings were within the limits of reversible change. From these above results, it is suggested that modified Wicomb*s solution seems to be the most useful physiologic salt solution for preservation of the heart. We propose that after further study and improvement, our portable continuous hypothermic perfusion system will contribute to the development of a better preservation method for donor hearts for human heart transplantation.

• Journal of Chest Surgery
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• v.17 no.4
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• pp.587-592
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• 1984

Potassium cardioplegia for myocardial preservation is being used extensively in open heart surgery. This study was designed to investigate the change in serum and urine potassium before, during, after the administration of cold blood potassium cardioplegic solution containing 28-30 mEq/L [28.6\ulcorner.56 mEq/L] of potassium chloride in 6 dogs. Total potassium dose used in the study was 14.9\ulcorner.89 mEq/L [1.2 mEq/Kg]. Mean potassium level in serum was within normal limits. Urinary excretion of potassium decreased during bypass but increased after bypass and eventually exceeded the amount of potassium infused as cold blood potassium cardioplegic solution. While cold blood potassium cardioplegia is a safe and efficient method of myocardial protection, postoperative potassium level must be observed carefully in order to detect obligatory potassium excretion following open heart surgery.