X-Message-Number: 31673 Date: Fri, 8 May 2009 12:03:17 -0700 (PDT) From: Subject: the least toxic cryoprotectant is... [Wheat proteins! Although the superiority of wheat proteins for cryopreservation was proven in 2006, only two follow-up articles have since been published. This well illustrates how resistent to new ideas the fellows who write grant cheques are, and also explains why the pace of scientific advance is so slow. I refer to this effect as the "Institutional Lemming Effect", and this occurs in all walks of life. If enough people say black is white, then this becomes orthodoxy, and a lot of followers will continue to jump off cliffs just to follow orthodoxy, long after any rational justification has vanished. Iraq has weapons of mass destruction, aging is completely inevitable, and the US banks are too big to fail are three other examples of this Institutional Lemming Effect. Although, from the standpoint of cryonics and organ cryopreservation, the high molecular weight of wheat proteins would restrict their direct application (except via liposome encapsulation), these proteins may offer an indirect benefit.. Elucidating why these proteins are so highly protective, while their constituent amino acids are not, might be what is needed to overcome the logjam of cryoprotectant toxicity which is currently blocking progress in this field. Similar comments apply both to polyphenol based cryopreservation, and magnetic field based cryopreservation.. Might a combination of alternative technologies eventually be able to eliminate the use of toxic cryoprotectants? Such a notion is regarded as heretical in some quarters...] Snip: "They are an efficient, nontoxic, economic natural product and universal cryoprotectant that is superior to DMSO, which has limitations because of cellular toxicity." Biotechnol Bioeng. 2009 Jun 15;103(3):582-91. Wheat proteins improve cryopreservation of rat hepatocytes. Grondin M, Hamel F, Averill-Bates DA, Sarhan F. Departement des Sciences Biologiques, Universite du Quebec a Montreal, Succursale Centre-Ville, Canada. Hepatocytes are an important physiological model for in vitro studies of drug metabolism and toxicity. However, fresh hepatocytes are not always available and hence cyopreservation is needed to preserve large quantities until they are needed for these applications. Hepatocytes are extremely sensitive to damage induced by the freeze-thaw process, even after addition of traditional cryoprotectants such as dimethyl sulfoxide (DMSO). Furthermore, they do not proliferate in culture. We previously demonstrated that a crude wheat extract protects rat hepatocytes during cryopreservation and could provide a promising alternative to DMSO. We have considerably improved this novel cryopreservation procedure by using wheat extracts that are partially purified by either ammonium sulphate or acetone precipitation, or by using recombinant wheat freezing tolerance-associated proteins such as WCS120, TaTIL, WCS19, and TaIRI-2. These improved procedures enhance long-term storage (2-12 months) and recovery of large quantities of healthy cells after cryopreservation, and maintain the differentiated functions of rat hepatocytes, compared to freshly isolated cells, as judged by viability (77-93%), adherence (77%) and metabolic functions of major cytochrome P450 isoforms CYP1A1/2, CYP2C6, CYP2D2, and CYP3A1/2. The advantage of using wheat proteins as cryopreservants is that they are non-toxic, natural products that do not require animal serum, and are economical and easy to prepare. 2009 Wiley Periodicals, Inc. PMID: 19219915 Drug Metab Dispos. 2008 Oct;36(10):2121-9. Epub 2008 Jul 10. Metabolic activity of cytochrome p450 isoforms in hepatocytes cryopreserved with wheat protein extract. Grondin M, Hamel F, Sarhan F, Averill-Bates DA. Universite du Quebec a Montreal, Departement des Sciences Biologiques, C.P. 8888, Succursale Centreville, Montreal, Quebec, Canada H3C 3P8. The drug discovery and development process requires adequate safety testing for drug toxicity before new drugs can be administered to patients. Hepatocytes are used in vitro to screen compounds for hepatotoxicity, induction of drug-metabolizing enzymes such as cytochrome P450 (P450) isoforms, drug-drug interactions, and establish human relevance for metabolism. Cryopreservation makes it possible to preserve a large quantity of functional hepatocytes. Techniques for cryopreservation of hepatocytes are mainly based on dimethyl sulfoxide (DMSO). However, analyses of metabolic capacities of cryopreserved hepatocytes are often limited by loss of functional integrity of hepatocytes after thawing. Therefore, it is necessary to improve techniques of cryopreservation. We have developed a new cryopreservation technology for mammalian cells based on a wheat protein extract (WPE). We determined whether the WPE can better preserve activities of major P450 isoforms both in suspension and monolayer cultures of hepatocytes. This was achieved by comparing basal and inducible or metabolic activities of isoforms CYP1A1, CYP1A2, CYP2C6, CYP2D2, and CYP3A in rat hepatocytes that were cryopreserved with WPE, relative to fresh cells and those cryopreserved with DMSO. We conclusively show that rat hepatocytes cryopreserved with WPE retain their metabolic competency and their ability to respond to classical P450 inducers when compared with freshly isolated hepatocytes. These findings clearly show that WPEs are an excellent cryopreservant for rat hepatocytes. They are an efficient, nontoxic, economic natural product and universal cryoprotectant that is superior to DMSO, which has limitations because of cellular toxicity. PMID: 18617602 Biotechnol Bioeng. 2006 Nov 5;95(4):661-70. Wheat extracts as an efficient cryoprotective agent for primary cultures of rat hepatocytes. Hamel F, Grondin M, Denizeau F, Averill-Bates DA, Sarhan F. Departement des Sciences Biologiques, Universite du Quebec a Montreal, C.P. 8888, Succursale Centre-ville, Montreal, Quebec H3C 3P8, Canada. Hepatocytes are an important physiological model for evaluation of metabolic and biological effects of xenobiotics. They do not proliferate in culture and are extremely sensitive to damage during freezing and thawing, even after the addition of classical cryoprotectants. Thus improved cryopreservation techniques are needed to reduce cell injury and functional impairment. Here, we describe a new and efficient cryopreservation method, which permits long-term storage and recovery of large quantities of healthy cells that maintain high hepatospecific functions. In culture, the morphology of hepatocytes cryopreserved with wheat protein extracts (WPE) was similar to that of fresh cells. Furthermore, hepatospecific functions such as albumin secretion and biotransformation of ammonium to urea were well maintained during 4 days in culture. Inductions of CYP1A1 and CYP2B in hepatocytes cryopreserved with WPEs were similar to those in fresh hepatocytes. These findings clearly show that WPEs are an excellent cryopreservant for primary hepatocytes. The extract was also found to cryopreserve other human and animal cell types such as lung carcinoma, colorectal adenocarcinoma, Chinese hamster ovary transfected with TGF-b1 cDNA, cervical cancer taken from Henrietta Lacks, intestinal epithelium, and T cell leukemia. WPEs have potential as a universal cryopreservant agent of mammalian cells. It is an economic, efficient and non-toxic agent. (c) 2006 Wiley Periodicals, Inc. PMID: 16927246 Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=31673