X-Message-Number: 31105 Date: Wed, 8 Oct 2008 09:44:37 -0700 (PDT) From: Subject: The Cryonics Institute Makes Another Technical Disclosure II Biotechnol Lett. 2005 May;27(9):655-60. Protection of osteoblastic cells from freeze/thaw cycle-induced oxidative stress by green tea polyphenol. Han DW, Kim HH, Lee MH, Baek HS, Lee KY, Hyon SH, Park JC. Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul, 120-752, Korea. Green tea polyphenol (GTP) together with dimethylsulphoxide (DMSO) were added to a freezing solution of osteoblastic cells (rat calvarial osteoblasts and human osteosarcoma cells) exposed to repeated freeze/thaw cycles (FTC) to induce oxidative stress. When cells were subjected to 3 FTCs, freezing medium containing 10% (v/v) DMSO and 500 mug GTP ml(-1) significantly (p<0.05) suppressed cell detachment and growth inhibition by over 63% and protected cell morphology. Furthermore, the alkaline phosphatase activity of osteoblastic cells was appreciably maintained after 2 and 3 FTCs in this mixture. Polyphenols may thus be of use as a cell cryopreservant and be advantageous in such fields as cell transplantation and tissue engineering. PMID: 15977073 J Neurosci Methods. 2005 Jun 30;145(1-2):255-66. Optimal conditions for peripheral nerve storage in green tea polyphenol: an experimental study in animals. Matsumoto T, Kakinoki R, Ikeguchi R, Hyon SH, Nakamura T. Department of Orthopedic Surgery, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. Our previous study demonstrated successful peripheral nerve storage for 1 month using polyphenol solution. We here report two studies to solve residual problems in using polyphenols as a storage solution for peripheral nerves. Study 1 was designed to determine the optimal concentration of the polyphenol solution and the optimal immersion period for nerve storage. Rat sciatic nerve segments were immersed in polyphenol solution at three different concentrations (2.5, 1.0, and 0.5 mg/ml) for three different periods (1, 7, and 26 days). Electrophysiological and morphological studies demonstrated that nerve regeneration from nerve segments that had been immersed in 1mg/ml polyphenol solution for 1 week and in Dulbecco's modified Eagle's medium (DMEM) for the subsequent 3 weeks was superior to the regeneration in other treatment groups. In study 2, the permeability of nerve tissue to polyphenol solution was investigated using canine sciatic nerve segments stored in 1.0mg/ml polyphenol solution for 1 week and in DMEM for the subsequent 3 weeks. Electron microscopy revealed that the Schwann cell structure within 500-700 microm of the perineurium was preserved, but cells deeper than 500-700 microm were badly damaged or had disappeared. The infiltration limit for polyphenol solution into neural tissue is inferred to be 500-700 microm. PMID: 15922041 Kidney Int. 2003 Feb;63(2):554-63. Bioflavonoids attenuate renal proximal tubular cell injury during cold preservation in Euro-Collins and University of Wisconsin solutions. Ahlenstiel T, Burkhardt G, Kohler H, Kuhlmann MK. Department of Medicine, Division of Nephrology and Hypertension, University Hospital of Saarland, Homburg/Saar, Germany. BACKGROUND: Cold ischemia and reperfusion during kidney transplantation are associated with release of free oxygen radicals and damage of renal tubular cells. Bioflavonoids may diminish cold storage-induced injury due to antioxidant and iron chelating activities. This study was designed to delineate the renoprotective mechanisms of bioflavonoids and to define the structural features conferring cytoprotection from cold injury. METHODS: LLC-PK1 cells were preincubated for three hours with bioflavonoids and cold stored in University of Wisconsin (UW)- or Euro-Collins (EC)-solution for 20 hours. After rewarming, cell viability was assessed by the lactate dehydrogenase (LDH) release, MTT-test, and amino acid transport activity. Lipid peroxidation was assessed from the generation of thiobarbituric acid-reactive substances. RESULTS: Twenty-hours of cold storage of LLC-PK1 cells resulted in a substantial loss of cell integrity that was more pronounced in the EC (LDH release, 93.6 +/- 1.6%) than the UW solution (67.2 +/- 6.9%; P < 0.0001). Pretreatment with quercetin significantly enhanced cell survival (LDH release, 5.4 +/- 2.7% for UW and 8.4 +/- 4.2% for EC) in a concentration dependent manner. Structure-activity studies revealed similar renoprotection for kaempferol, luteolin and fisetin, unlike myricetin, morin, apigenin, naringenin, catechin, silibinin and rutin. Lipid peroxidation was reduced (UW alone, 2.7 +/- 1.2 vs. UW+quercetin 0.5 +/- 0.2 nmol/mg protein, P < 0.01), and l-threonine uptake completely sustained by pretreatment with quercetin, kaempferol, luteolin, and fisetin. However, renoprotection by fisetin was rapidly lost during rewarming. Protective properties of bioflavonoids were governed by the number and arrangement of hydroxyl substitutes, electron-delocalization, sterical planarity, and lipophilicity of the basic diphenylpyran skeleton. CONCLUSION: Cold storage-induced renal tubular cell injury is ameliorated by bioflavonoids. Renoprotective effects of bioflavonoids are defined by structure, suggesting that flavonoids are incorporated into membrane lipid bilayers and interfere with membrane lipid peroxidation. PMID: 12631120 J Biosci Bioeng. 2003;96(6):559-63. Novel function of rare catechin, epigallocatechin-3-(3''-O-methyl)gallate, against cold injury in primary rat hepatocytes. Kagaya N, Hara Y, Saijo R, Kamiyoshi A, Tagawa Y, Kawase M, Yagi K. Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan. Epigallocatechin-3-(3''-O-methyl)gallate (EGCg-3''-OMe) is a rare component in green tea leaf and its bioactivity is hardly known. In this paper, we report that EGCg-3''-OMe has the function for cold preservation of primary rat hepatocytes. Confluent primary cultured hepatocytes were suspended in a storage solution, culture medium or cell banker (CB). EGCg-3''-OMe was tested as a supplement in the storage solution together with a general cryoprotectant, dimethylsulfoxide (DMSO). After 24 h cold preservation of cells at 4 degrees C followed by 1 h rewarming, cell viability and urea-synthesizing activity, one of the most important liver functions, were measured. EGCg-3''-OMe dose-dependently maintained cell viability and this effect was equal to that of a commercial CB at the highest concentration. Cell viability was also maintained after a further 24 h incubation at 37 degrees C of the cold-preserved hepatocytes. Conversely, urea-synthesizing activity was dose-dependently reduced by EGCg-3''-OMe. Cell protection by EGCg-3''-OMe due to the decrease in metabolic activity in cold-preserved cells was suggested. The decreased hepatic function of cells caused by EGCg-3''-OMe was rescued after a further 24 h incubation of cells at 37 degrees C. PMID: 16233573 Biol Pharm Bull. 2002 Sep;25(9):1156-60. Enhancing effect of zinc on hepatoprotectivity of epigallocatechin gallate in isolated rat hepatocytes. Kagaya N, Kawase M, Maeda H, Tagawa Y, Nagashima H, Ohmori H, Yagi K. Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan. The influence of metal ions (Fe2+, Cu2+, Zn2+) on the hepatoprotective activity of epigallocatechin gallate (EGCG) against hepatotoxin-induced cell injury was investigated. Primary cultures of rat hepatocytes were treated with a well-known hepatotoxin, bromobenzene (BB), in the presence of EGCG only or EGCG plus each metal ion. After 24 h, 0.02 mM EGCG did not show protective activity on the cultured hepatocytes. In contrast, the hepatocytes were protected against BB in the presence of 0.02 mM EGCG and 0.02 mM zinc. The addition of only zinc could not protect hepatocytes against BB. These results suggest that the formation of the zinc-EGCG complex is very important in the enhancement of the hepatoprotective activity of EGCG. The complexation of EGCG with zinc was confirmed by UV-VIS absorption spectroscopy. PMID: 12230108 Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=31105