SESSION 11

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

BACKGROUND

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 4C in the Euro-Collin's solution and grafts cryopreserved for more than 3 weeks at -196C, and using the grafts transplantation in dogs.

METHODS

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 4C 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 1C/min to -80C, then put the sample into liquid nitrogen for storage.After three weeks, using the tracheal grafts to transplantation.

RESULTS

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

ABSTRACT

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 55455, USA
c Department of Biology, Carleton University, Ottawa K1S 5B6, Canada

INTRODUCTION

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 -8C 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.

THEORETICAL BACKGROUND

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 (> 1000C/min), (b) two-step equilibrium (2C/min) freezing, or (c) two-step dynamic freezing at 5C/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 5C/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=64m; initial sinusoid radius, rvo=18.4m; and the length of the Krogh cylinder, L=0.71m (assuming that an isolated frog hepatocyte has a diameter of 16m). Comparable dimensions in rat liver tissue are DX=22m, rvo=3.8m, and L=11.4m3. 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 -20C. 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 5C/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 -10C. In Figs. 1B, C, and D, white spaces that were equal to or less than the size of the hepatocyte cell diameter (16 m) 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 5C/min DSC data, which showed a secondary heat release at -14 to -16C that translates to ~20% of the total intracellular water volume. The 5C/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 m/min-atm, and ELp = 75.5 Kcal/mol. These are comparable to other mammalian (rat) liver tissue parameters: Lpg = 1.86 m/min-atm, and ELp = 69.3 Kcal/mol, estimated using a similar technique and model3.

CONCLUSIONS

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 m/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 5C/min but do so when cooled at 2C/min, as opposed to the rat liver tissue which dehydrates completely at both these cooling rates3. The reason for water retention at 5C/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 ELp).

REFERENCES

  1. Storey, K. B and Storey, J. M. 1988. Freeze Tolerance in Animals. Physiological Reviews. 68(1):27-841
  2. 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.
  3. Pazhayannur, P. V., and Bischof, J. C. 1997. Measurement and simulation of water transport during freezing in mammalian liver tissue. ASME Journal of Biomechanical Engineering., 119:269-277.
  4. 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.
  5. Mazur, P. 1984. Freezing of living cells: mechanisms and implications. J. Gen. Physiol. 247:C125-C142.
  6. 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 Sciences. 234(1276):343-358.
  7. Rubinsky, B., and Ikeda, M., 1985. A cryomicroscope using directional solidification for controlled freezing of biological material. Cryobiology. 22:55-68.
  8. Echlin, P., 1992. Low Temperature Microscopy and Analysis, Plenum Press, New York - London, Chaps. 3 & 7.
  9. 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;
email: bischof@maroon.tc.umn.edu

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

ABSTRACT

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 variables.

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.

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