Crystalline ice as a cryoprotectant: theoretical calculation of cooling speed in capillary tubes

Abstract

It is generally assumed that vitrification of both cells and the surrounding medium provides the best preservation of ultrastructure of biological material for study by electron microscopy. At the same time it is known that the cell cytoplasm may provide substantial cryoprotection for internal cell structure even when the medium crystallizes. Thus vitrification of the medium is not essential for good structural preservation. In contrast, a high cooling rate is an essential factor for good cryopreservation because it limits phase separation and movement of cellular components during freezing, thus preserving the native-like state. Here we present calculations of freezing rates that incorporate the effect of medium crystallization, using finite difference methods. We demonstrate that crystallization of the medium in capillary tubes may increase the cooling rate of suspended cells by a factor of 25-300 depending on the distance from the center. We conclude that crystallization of the medium, for example due to low cryoprotectant content, may actually improve cryopreservation of some samples in a near native state.

Freezing in sealed capillaries for preparation of frozen hydrated sections

“…The small size of the cubic ice crystals can give an idea of the distance over which molecules can diffuse during freezing, which can be compared with predictions based on measured diffusion constants. The diffusion constant for dextran is on the order of tens of micrometres squared per second (Periasamy & Verkman, 1998), whereas the freezing front moves at tens of micrometres per millisecond and whole tubes freezes in tens of millisecond (Yakovlev & Downing, 2011). Thus, the distance a dextran molecule may diffuse is much less than 1 μm and diffusion can not significantly change the dextran concentration over distances on the scale of micrometres; in particular, dextran can not diffuse ahead of the cooling front to accumulate towards the centre of the tube to enhance amorphization.…”

“…Moreover, it is highly probable and to some degree supported in our images that, although the medium crystallizes, the interior of the cell vitrifies. It is also known that crystallization increases the cooling rate which may, thus, minimize cell dehydration due to crystallization (Yakovlev & Downing, 2011). The fact that the cell membrane and S‐layer show so much more structure in 10% dextran than in higher dextran samples is surprising, and may be a decisive factor for using reduced amount of the cryoprotectant even to the point that the sample crystallizes.…”

The Journal of Microscopy would like to thank all those referees who reviewed papers for the Journal during 2011. Your assistance and contribution to the Journal is greatly appreciated.

“…The diffusion constant for dextran is on the order of tens of microns squared per second (Periasamy & Verkman, 1998), while the freezing front moves at tens of microns per millisecond and whole tubes freezes in tens of millisecond (Yakovlev & Downing, 2011). Thus the distance a dextran molecule may diffuse is much less than one micron and diffusion can not significantly change the dextran concentration over distances on the scale of microns; in particular, dextran can not diffuse ahead of the cooling front to accumulate toward the center of the tube in order to enhance amorphization.…”

“…Moreover it is highly probable and to some degree supported in our images that, while the medium crystallizes, the interior of the cell vitrifies. It is also known that crystallization increases the cooling rate and may thus minimize cell dehydration due to crystallization (Yakovlev & Downing, 2011). The fact that the cell membrane and S-layer show so much more structure in 10% dextran than in higher dextran samples is surprising and may be a decisive factor for using reduced amount of the cryoprotectant even to the point that the sample crystallizes.…”

“…Some of these reasons still may create problems with 15% dextran. It is possible that using smaller capillaries will further reduce cryoprotectant requirements (Yakovlev & Downing, 2011). …”