CRYOBIOLOGY - V
Chairman: A. Hubel
TRANSPLANTATION OF TRACHEA WITH CRYOPRESERVED ALLOGRAFTS IN DOGS
Heng Zhao, Yun-Zhong Zhou, and Yi Yang
General Thoracic Surgery Department, Shanghai Chest Hospital, Shanghai, P.R.China
Tse-Chao Hua He-Sheng Ren, and Qi-Feng Wang
Cryobiological Engineering Laboratory, University of Shanghai for Science and
Technology,Shanghai 200093, P.R.China
Grafting is required when primary reconstruction of trachea defect is not
feasible. To determine the viability and the nature of the healing process occurring in the
cryopreserved graft, we performed tracheal grafts at 4°C in the Euro-Collin's solution and
grafts cryopreserved for more than 3 weeks at -196°C, and using the grafts transplantation
We performed trachea transplantation in dogs using cryopreserved allografts.
The viability of each graft was evaluated serially by fiberoptic macroexamination and
pathological examination. In group A(n=10), the trachea was immersed in Euro-Collin's
solution at 4°C for 48-96 hours. In group B(n=20), a programmable cooler was used to
freeze the samples uniformly with predetermined controlled constant cooling rates of
1°C/min to -80°C, then put the sample into liquid nitrogen for storage.After three weeks,
using the tracheal grafts to transplantation.
Sufficient viability and good healing occurred in the dogs with cryopreserved
trachea allotransplants. One of the 20 dogs with cryopreserved allotransplants survived
more than 720 days and fifteen dogs survived for 100 days without immunosuppression.
CONCLUSIONS The cryopreservation of trachea allografts by programmable freezer for 3
weeks was shown to be feasible. Cryopreservation can prolonged the survival of
nonimmunosuppressed allotransplants in dogs.
MEASUREMENT OF CELL VOLUME LOSS IN THE LIQUID REGION
PRECEDING AN ADVANCING PHASE CHANGE INTERFACE
Heather R. Harmison1, Kenneth R. Diller1, John R. Walsh1,
Christopher M. Neils1 and Jerry J. Brand2
1Biomedical Engineering Program and
2Department of Botany, The University of Texas at Austin, Austin, TX 78712
It is well understood that the solidification of a solution results in a redistribution of solute
in the liquid zone. For the freezing of suspensions of cells it is anticipated that accumulation
of solute in the region leading a growing ice phase will cause an osmotic response in cells before
the ice phase reaches the cells. To measure this phenomenon in a specific algal species, the volume
changes in Chlorococcum texanum during freezing were studied using directional solidification
cryomicroscopy. The relative cell volume was tracked continuously as a function of temperature
and position as cells encountered the moving phase front. The loss of cell volume was initiated in
the liquid region containing concentrated solute ahead of the growing solid phase.
BIOPHYSICS OF FREEZING LIVER OF THE FREEZE-TOLERANT WOOD FROGa
Paul R. Barrattb, Ramachandra V. Devireddyb,
Kenneth B. Storeyc AND John C. Bischofb,d
b Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN
c Department of Biology, Carleton University, Ottawa K1S 5B6, Canada
The extreme winter climate in some areas of the world requires animals to develop
freeze tolerance in order to survive. This mechanism allows the wood frog, R. sylvatica, to
cool to temperatures of -8°C with up to 65% of the frog's water in the ice phase, without
incurring freezing injury over many weeks1-2. Current technology for the cryopreservation
of mammalian tissues does not allow for successful storage of organs under the same
conditions. If the mechanism whereby R. sylvatica survives freezing could be more fully
understood and exploited, it could have tremendous impact in the storage and banking of
frozen/viable mammalian tissues. Two new experimental methods, one using freeze
substitution low temperature microscopy3 and another using a differential scanning
calorimeter (DSC)4 have been developed which allow more accurate quantification of the
processes that occur during freezing in tissue systems. In this work, these methods will be
used to quantify water transport in the liver of the freeze tolerant wood frog R. sylvatica,
in the hopes of learning more about how these frogs freeze and thus perhaps suggest how
freeze tolerance is achieved.
The driving force for water transport across the cell membrane is the difference in
chemical potential of water between the unfrozen cell interior and the partially frozen
extracellular solution5. The water flux between the cell and vascular/extracellular spaces
has been modeled using a Krogh cylinder approach, where the permeability of the
membrane separating the cellular and vascular/extracellular spaces, Lp is given by6:
This permeability depends on the water transport parameters: Lpg the permeability of
the cell membrane to water at the reference temperature TR (273.15 K), and ELp which is
the activation energy for the process, T is the absolute temperature and R is the universal
gas constant. The scarcity of space precludes further description of the model, which is
described in detail elsewhere3,6.
MATERIALS AND METHODS
Freshly isolated liver samples (1mm3) of wood frogs (R. sylvatica) were frozen using a
directional solidification stage7 by one of three methods: (a) slam freezing (> 1000°C/min),
(b) two-step equilibrium (2°C/min) freezing, or (c) two-step dynamic freezing at 5°C/min3.
The frozen tissue samples were freeze substituted, embedded in resin, sectioned and
imaged under a light microscope fitted with a digitizing system3,8. Image analysis was
then performed with NIH Image software™ (NIH, Bethesda, MD), to obtain the required
cellular and vascular/extracellular volumes during freezing3.
The DSC protocol developed to extrapolate water transport from measured latent heat
releases during freezing was used on frog liver tissue, as previously described for a cell
suspension system4. DSC experiments were conducted at cooling rates of 2 and 5°C/min
on frog liver tissue (1.0 to 1.5 mg of tissue slices in 8 to 10 mg of PBS with 0.3 to 0.5 mg
of a natural ice nucleator Pseudomonas syringae) and the measured heat releases were
then translated to water transport data, as described elsewhere4.
RESULTS AND DISCUSSION
The slam tissue images (Fig. 1A) were analyzed using NIH Image software™ (NIH,
Bethesda, MD) to obtain the following Krogh model dimensions: average distance between
the sinusoids, DX=64µm; initial sinusoid radius, rvo=18.4µm; and the length of the Krogh
cylinder, L=0.71µm (assuming that an isolated frog hepatocyte has a diameter of 16µm).
Comparable dimensions in rat liver tissue are DX=22µm, rvo=3.8µm, and L=11.4µm3. In
addition, a higher magnification analysis of slam tissue showed that up to 24% of the frog
hepatocyte cells were bounded by other hepatocyte cells and the remaining 76% are next to
vasculature, as compared to mammalian (rat) liver tissue where 100% of the hepatocyte
cells are next to vascular spaces.
A Boyle-van't Hoff (BVH) plot was constructed by examining freeze dehydrated tissue
slices which were allowed to come to "equilibrium" with the extracellular ice at
temperatures of -4, -6, -8, -10 and -20°C. By extrapolating the BVH plot to infinite
osmolality (Osm = DT/1.858, where DT=273.15 - T, K), the osmotically inactive cell
volume, Vb = 0.4Vo, was obtained (data not shown).
The frog liver response to dynamic freezing at 5°C/min is shown in Fig, 1. As freezing
begins the cells dehydrate and water moves out of the cells and into the vasculature (Figs.
1B, C, and D). Cellular dehydration (and therefore water transport from the cells) appears
to cease at -10°C. In Figs. 1B, C, and D, white spaces that were equal to or less than the
size of the hepatocyte cell diameter (16 µm) were assumed to be intracellular ice. These
spaces were included as cellular spaces in the analysis of end volume represented in Fig. 2.
This observation was supported by the 5°C/min DSC data, which showed a secondary heat
release at -14 to -16°C that translates to ~20% of the total intracellular water volume. The
5°C/min dynamic water transport data from both the techniques is shown in Fig. 2.
Assuming a two compartment Krogh model, a nonlinear curve fitting technique9 was used
to predict the best fit biophysical parameters of water transport: Lpg = 1.76 µm/min-atm,
and ELp = 75.5 Kcal/mol. These are comparable to other mammalian (rat) liver tissue
parameters: Lpg = 1.86 µm/min-atm, and ELp = 69.3 Kcal/mol, estimated using a similar
technique and model3.
This study investigates the water transport characteristics during freezing in the liver
tissue of the freeze-tolerant wood frog R. sylvatica using low temperature microscopy and
differential scanning calorimetry. Stereological analysis of the tissue micrographs showed:
74% of the control tissue is cellular space (26% is vascular space) and Vb = 0.4Vo. The
biophysical parameters of water transport obtained in this study for frog liver tissue; Lpg=1.76 µm/min-atm, and ELp=75.5 Kcal/mol, are comparable to those obtained in rat
liver tissue3. Both the techniques confirmed that frog liver tissues do not dehydrate
completely at 5°C/min but do so when cooled at 2°C/min, as opposed to the rat liver tissue
which dehydrates completely at both these cooling rates3. The reason for water retention
at 5°C/min in the frog liver tissue appears to be, primarily due to the differences in the
morphological architecture of the frog liver vs. rat liver (i.e. differences in the Krogh
model dimensions, DX, rvo, L; and the fact that 24% of frog hepatocyte cells are not in
contact with the vasculature) and not due to altered membrane permeabilities (Lpg and
- Storey, K. B and Storey, J. M. 1988. Freeze Tolerance in Animals. Physiological
- Storey, K. B., and Storey, J. M. 1993. Cellular adaptations for freezing survival by
amphibians and reptiles. Advances in Low-Temperature Biology, Vol. 2 (Steponkus, P.
L., ed.) JAI Press, London, pp. 101-129.
- Pazhayannur, P. V., and Bischof, J. C. 1997. Measurement and simulation of water
transport during freezing in mammalian liver tissue. ASME Journal of Biomechanical
- Devireddy, R. V., Raha, D., And Bischof, J. C. 1998. Measurement of water transport
during freezing in cellular suspensions using a differential scanning calorimeter.
Cryobiology . 36(2):124-155.
- Mazur, P. 1984. Freezing of living cells: mechanisms and implications. J. Gen. Physiol.
- Rubinsky, B., and Pegg, D. E. 1988. A mathematical model for the freezing process in
biological tissue. Proceedings of the Royal Society of London - Series B: Biological
- Rubinsky, B., and Ikeda, M., 1985. A cryomicroscope using directional solidification
for controlled freezing of biological material. Cryobiology. 22:55-68.
- Echlin, P., 1992. Low Temperature Microscopy and Analysis, Plenum Press, New York -
London, Chaps. 3 & 7.
- Montgomery, D. C., and Runger, G. C. 1994. Applied statistics and probability for
engineers. John Wiley & Sons, Inc. New York. 471-529.
a This work was supported by NSF-CTS#941004 and a grant from Whitaker Foundation to JB.
d Address for correspondence: Department of Mechanical Engineering, University of Minnesota,
Minneapolis, MN 55455, USA. Phone. 612/625-5513; fax. 612/624-5230;
SENSITIVITY OF KIDNEY PERFUSION PROTOCOL DESIGN
TO PHYSICAL AND PHYSIOLOGICAL PARAMETERS
Charles A. Lachenbruch1, Kenneth R. Diller1 and David E. Pegg2
1 The University of Texas at Austin Biomedical Engineering Program, Austin, Texas 78712-1084
Ddepartment of Biology, University of York, York YOl SDD, UK
The introduction and removal of cryoprotective agents (CPA) to a kidney via vascualar perfusion may induce changes in cell volume
that are destructive to the tubular epithelial or capillary endothelial cells as well as causing significant increases in vascular
resistance that compromise the perfusion process. A network thermodynamic model of the coupled osmotic, hydrodynamic and elastic
properties of the kidney was applied to evaluate the sensitivity of these critical outputs to a set of physiological and perfusion
Simulation results suggest that in the design of perfusion protocols for CPAs such as glycerol it may be advantageous to: (a) select
a CPA with as high a cell membrane permeability as possible; (b) inerease the concentration of mannitol in the perfusate to about
200 mos/kg, beyond which there is no discernible benefit; (c) when glycerol is the CPA, limit the rate of reduction in the perfusate
during removal to 30 mM/min or less; (d) limit the perfusion pressure to 20 - 30 mm Hg, within the practical constraints of the
perfusion system; (e) increase the concentration of impenneant in the perfusate to perhaps 400 mos/kg, although it is recognized
that this departure from plasma-like composition might impose other problems that are not considered in this model.
Further, it was observed that the vascular membrane permeability plays a relatively minor role in controlling cellular osmotic
injury and vascular perfusion resistance and is thus not a critical parameter in the perfusion design process.