X-Message-Number: 32840
Date: Thu, 16 Sep 2010 18:49:19 -0700 (PDT)
From: 
Subject: slow freezing protocols


[Slow freezing protocols can offer in some cases higher whole organ viability 
than some vitrification protocols. Slow freezing protocols are subject to ice 
crystalization damage effects, while vitrification is subject to significant 
cryoprotectant toxicity effects. IMHO, which approach is the more promising for 
organ cryopreservation is an open question. Cryoprotectant toxicity is still 
something of a mystery, but membrane damage does appear to be the primary mode 
of damage from slow freezing. Additives to coat and thereby armor cell 
membranes, directional freezing, variable microwave fields, or variable magentic
fields could all act in concert to reduce or perhaps even eliminate  ice 
crystal damage.]

J Assist Reprod Genet. 2010 Sep 15. [Epub ahead of print]

Whole sheep ovary cryopreservation: evaluation of a slow freezing protocol with 
dimethylsulphoxide.

Milenkovic M, Wallin A, Ghahremani M, Brannstrom M. Department of Obstetrics and
Gynecology, Sahlgrenska Academy, University of Gothenburg, SE-41345, Goteborg, 
Sweden,
Abstract

    PURPOSE: To evaluate a slow freezing method for whole ovary cryopreservation
    by evaluating effects of added cryoprotectant.

METHODS: Sheep ovaries were isolated during surgery, flushed with either 
Ringer-Acetate or dimethylsulphoxide and cryopreserved by slow freezing. After 
rapid thawing, viability was assessed by ovarian in vitro perfusion, cell 
culture, histology and fluorescent live-dead assay.

RESULTS: Production of cyclic AMP and progesterone was slightly higher in the 
dimethylsulphoxide group. Cultured ovarian cells from 
dimethylsulphoxide-preserved ovaries secreted larger amounts of progesterone 
than cells from Ringer-Acetate preserved. Light microscopy of ovarian biopsies 
obtained after perfusion, revealed well-preserved tissue in the 
dimethysulphoxide group but not in the Ringer-Acetate group. The density of 
small follicles and ovarian cell viability were higher in dimethysulphoxide 
ovaries compared to Ringer-Acetate ovaries.

CONCLUSIONS: Equilibrium with its protective effect can be achieved by slow 
freezing protocol, with an additional protective effect by the presence of 
dimethylsulphoxide.
PMID: 20842419


[Wheat protein extracts are an example of a cryoprotectant substitute which 
apparently functions primarily by coating cell membranes to improve the freeze 
tolerance of cells.]

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.
Abstract

    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.
PMID: 19219915

Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=32840