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Specifying systems
Cryo buffers: the move from PBS to HEPES
In the mid-1990s when Sydney IVF was eliminating patient serum from all its culture media, the serum component in the embryo freezing and thawing protocols was replaced by a solution of 45 mg/ml of HSA in normal saline (this being the albumin content of serum). However, in one lab the freezing solutions were based on a modified version of phosphate-buffered saline (PBS) that contained phenol red and it was noted that, when straws were being seeded, the medium column had turned from the normal pink colour to bright yellow. Clearly there was a problem with pH buffering during cooling in the absence of serum, and this was confirmed by pH measurements. Advice from Dr. John Critser led to the adoption of a TL-HEPES medium as the basal medium for embryo freezing and thawing in August 1996. Not only did the HEPES buffering allow for proper pH control during cooling, but the implantation rate per thawed embryo transferred went from 6.5% to 16.2% (Cullinan et al., 1998). Moreover, even when embryos that had been frozen in PBS-based solutions were thawed in TL-HEPES there was a significant improvement in implantation rate to 14.3%, indicating that the major damage was probably being done during thawing and washing to remove the cryoprotectant. Subsequently, a HEPES-buffered version of the cleavage medium (D. Mortimer et al., 2002) was developed and has been used since that time (Cook IVF, 1999). Research on the temperature stability of phosphate buffers has revealed that they are highly unstable at lower temperatures and therefore unsuitable for freezing and thawing media. Early success with embryo cryopreservation using PBS-based solutions was probably aided by the additional buffering capacity of the serum component, but with the replacement of serum by albumin it seems that embryo freezing and thawing solutions should not be based on PBS. The mechanism for the adverse effect of the low extracellular pH remains unproven, but it has been shown that thawed hamster embryos are unable to regulate their intracellular pH for several hours, until