X-Message-Number: 17122
Date: Fri, 27 Jul 2001 02:02:51 -0700 (PDT)
From: Doug Skrecky <>
Subject: cryoprotective solutions should be sodium free

<1> of <2>
Title
  Cryopreservation of unfertilized mouse oocytes: the effect of replacing
  sodium with choline in the freezing medium.
Source
  Cryobiology.  37(4):346-54, 1998 Dec.
Abstract
  Although embryo cryopreservation has become commonplace in many species,
  effective methods are not available for routine freezing of unfertilized
  eggs. Cryopreservation-induced damage may be caused by the high concentration
  of sodium ions in conventional freezing media. This study investigates the
  effect of a newly developed low-sodium choline-based medium
  (CJ2) on the ability of unfertilized, metaphase II mouse eggs to survive
  cryopreservation and develop to the blastocyst stage in vitro. Specifically,
  the effects of cooling to subzero temperatures, thawing rate, LN2 plunge
  temperature, and equilibration with a low-sodium medium prior to freezing are
  examined. In contrast to cooling to 23, 0, or -7.0 degreesC in a sodium-based
  freezing medium (ETFM), cooling in CJ2 had no significant negative effect on
  oocyte survival or development. Oocytes frozen in CJ2 survived plunging into
  LN2 from -10, -20, or -33 degreesC at significantly higher rates than oocytes
  frozen in ETFM. With the protocol used (1.5 M PrOH, 0.1 M sucrose, -0.3
  C/min, plunging at -33 degreesC) rapid thawing by direct submersion in 30
  degreesC water was more detrimental to oocyte survival than holding in air
  for 30 or 120 s prior to transfer to water. Equilibration of unfertilized
  oocytes with a low-sodium medium prior to cryopreservation in CJ2
  significantly increased survival and blastocyst development. These results
  demonstrate that the high concentration of sodium in conventional freezing
  media is detrimental to oocyte cryopreservation and show that
  choline is a promising replacement. Reducing the sodium
  content of the freezing medium to a very low level or eliminating sodium
  altogether may allow oocytes and other cells to be frozen more effectively.
  Copyright 1998 Academic Press.

<2>
Title
  Detrimental effects of sodium during mouse oocyte cryopreservation.
Source
  Biology of Reproduction.  59(2):395-400, 1998 Aug.
Abstract
  Cryopreservation is an established way of storing embryos, but effective
  methods are not available for freezing eggs. Most freezing damage is caused
  by high solute concentration (solution effects) and intracellular ice. Sodium
  salts are the major components of cryopreservation media, and the main
  contributor to the solution effects. The present experiments examine the
  effect of substituting choline for sodium as the major
  extracellular cation in the cryopreservation of mouse eggs. The effects of
  serum and various cryoprotectants were also examined. Survival,
  fertilization, and development were inversely related to the concentration of
  sodium in the freezing medium. Oocytes frozen in a
  choline-based medium had the highest (p < 0.001) survival
  and development rates. The absence of serum during thawing inhibited
  fertilization, whereas exposure to serum or opening the zona allowed
  fertilization to reach the control level. Dimethyl sulfoxide was as effective
  as 1,2 propanediol for obtaining high survival and fertilization rates. These
  results support the hypothesis that the high concentration of sodium in
  conventional freezing media is detrimental to cells and show that
  choline is a promising replacement for sodium. Reducing or
  eliminating sodium may allow oocytes and other cells to be frozen more
  efficiently.

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