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Kenneth R. Diller
Advances in medical science make it possible to transplant many of the major tissues and organs of the human body. Unfortunately, lack of donor tissue frequently limits a patient's chances for a life-changing transplant. Although many suitable tissues become available on a daily basis, two critical factors severely limit their use. First, the immunological and physiological properties of the donor tissue must match the recipient's to minimize rejection. Second, all tissues deteriorate rapidly when removed from a living body, creating a short window of opportunity for transplantation.
The long-term banking of frozen tissues and organs (a process called cryopreservation, cryo being derived from the Greek word for "cold") offers the potential for alleviating both of these limiting factors.
Cryopreservation enables the stockpiling of large numbers of donor tissues and organs which are immunologically typed and screened for communicable diseases. Further, the ultralow temperatures of cryopreservation (typically hundreds of degrees below zero) stops the biological deterioration processes and creates an unlimited shelf life of tissues. Tissue banks can optimally match donor tissue with recipient needs and ship the frozen tissue to virtually anywhere in the world for transplantation.
In spite of this potential, it is well-known that living cells frozen in their native state suffer nearly total destruction and are nonviable upon thawing. Scientists have searched for the key to reversibly freezing living systems for many centuries, but discovery of the secret of cryopreservation remains an elusive goal.
Cryopreservation Methods. The first successful trial of retrieving living cells from cryopreservation for human transplantation occurred approximately fifty years ago. Researchers in England discovered by accident that the addition of a cryoprotective agent, such as glycerol, prior to freezing enabled cells to withstand the rigors of freezing and thawing. Within a short time, human red blood cells were cryopreserved and transfused with virtually no apparent loss in viability. Clinical procedures were quickly worked out for the frozen banking of blood cells and other single cell suspensions such as sperm.
Expectations ran high that cryopreservation of virtually all cells, tissues, and organs would soon follow. Unfortunately, history has shown that simple initial successes do not easily lead to the solution of the whole problem. It was first necessary to gain a rigorous understanding of the underlying physical, chemical, and physiological phenomena that govern the behavior of a complex biological system. At this juncture bioengineers began to make significant contributions to the emerging science of cryobiology.
Biological systems consist predominately of water, with a small amount of electrolytes and macromolecules in solution. When a biological system is frozen, the electrolyte and macromolecular solutes become highly concentrated and impose a large osmotic stress on the cells. Without a cryoprotective agent added prior to freezing, the solutes increase in concentration by a factor of twenty-five or more. Most human cells cannot withstand exposure to solutions concentrated only three or four times above normal. Cooling rates also affect tissue cell preservation. Slow cooling rates increase the electrolyte concentration. Rapid cooling rates trap water inside the cells and cause intracellular ice formation. Both are lethal, so a moderate rate of cooling during cryopreservation minimizes the effects of these two injury factors. The extreme processes illustrated in Figures 1 and 2 lead directly to cell death.
Note that no dimensions are indicated on the cooling rate scale in Figure 3. The optimum range of cooling rates may be unique to each cell species. The primary property determining a cell's optimal freezing conditions is its membrane permeability to water, and particularly, how it changes at temperatures below 0°C. The permeability may differ by more than a thousand-fold among different cell types, so no standard recipe can be applied to design cryopreservation protocols for every cell.
Although we know much about cryopreservation methods, many important problems remain to be solved before cryopreservation can provide a large and readily available source of tissues and organs for human transplantation. In this article I will review several significant breakthroughs achieved by bioengineers, particularly at The University of Texas, in solving the problem of optimal cryopreservation design and the prospects for future success.
Cryopreservation Process. Two distinct engineering practices have had a major impact on advances in cryobiology: first, an emphasis on rigorous quantitative measurement and analysis of processes, and second, the synthesis of nature's fundamental principles into the design of novel devices and processes. Designing optimal cryopreservation methods requires identifying the cooling rate that produces maximum survival. This rate is a direct function of the cell membrane permeability at subzero temperatures. Measuring cell membrane permeability at temperatures below 0°C has been one of the primary and most challenging goals of bioengineers working in cryobiology.
First, bioengineers quantified the cell dehydration and intracellular ice formation processes. The most direct approach to this task is to observe the cells under a microscope to monitor and record intracellular ice formation and alterations in size and shape during freezing and thawing. Although researchers have used low temperature light microscopy since the mid-1800s to observe the events during cell freezing, their data had little practical application since the experiments were limited to strictly qualitative observations.
Since the early 1970s our UT laboratory has pioneered the development of quantitative instrumentation for control and measurement of the thermal and chemical parameters of the cryopreservation process. This has transformed cryomicroscopy into a precise technique now used by researchers around the world. Figures 1 and 2, for example, were taken on an early version of our cryomicroscope which accurately and repeatably regulates the cooling rate over the entire range of freezing temperatures. We also introduced the use of computer imaging techniques to measure the changes in size and shape of cells during slow cooling and to detect the occurrence of intracellular ice formation during rapid cooling.
With quantitative data describing details of individual cell response to the entire range of cooling rates, it is now possible to predict the behavior of cells to specific cryopreservation protocols ahead of time. This capability requires analytical models for both the osmotic dehydration and the intracellular ice formation processes in cells. These models have been devised, first for individual cells and later for tissues and whole organs, in our laboratory and by several other groups of bioengineers.
We match the analytical model to the experimental data to measure the properties of cells and tissues that govern their response to freezing. Using this technique, called inverse solution, we made the first ever measurements of the membrane permeability of many cell types at subfreezing temperatures. This information can be applied to predict the continual state of osmotic stress on cells during any defined freezing scenario. In combination with thermodynamic parameters from ice nucleation theory, we can predict the conditions for intracellular ice formation.
These new tools provide the ability to design an optimal cryopreservation protocol for a specific cell type based on its intrinsic physiological and thermodynamic properties. To date the greatest success in developing clinical techniques for cryopreservation has been for systems consisting of individual cells in suspension (blood cells, gametes, bone marrow) or multicellular tissues with a relatively simple morphology (embryos, cornea). However, the success rate drops dramatically for the cryopreservation of more complex biological systems.
Since the mid-1980s our laboratory has been focused primarily on understanding freezing and thawing processes in multicellular tissues and organs. We have worked most actively with the pancreas islet of Langerhans, which contains the ß cells secreting insulin in response to elevated blood glucose.
I began working with this model at the University of Stuttgart (Germany) and continued at the University of Cambridge. The pancreas islet is an important model because of its potential for clinical transplantation to ameliorate the diabetic state. The islet is also a useful bioengineering model since it contains more than 1,000 cells arranged in a tissue with an overall diameter somewhat smaller than a human hair. It therefore presents a system with a rather simple morphology. On the other hand, it has a large enough volume so that cells in the interior are not in direct diffusive communication with cryoprotective agents or ice in the extraislet space. Islets behave as an aggregate tissue in which both intra- and intercellular transport and storage properties are important. It represents a step upward in complexity from single cells to a whole organ.
UT Austin Work. Together with Dr. Robson Freitas, a UT Austin graduate, I developed more sophisticated system models to account for the multicellular function of the islet. These can be applied to new cryomicroscopy data for osmotic response and intracellular ice formation during freezing. This work was performed with our medical collaborator, Dr. Ray Rajotte of the University of Alberta in Edmonton. Dr. Rajotte is the world's leading researcher in the isolation of islets from the human pancreas and in transplantation of islets to diabetic patients.
For our collaborative research, Dr. Rajotte prepared freshly isolated islets in Edmonton, then shipped them to Austin via overnight air express. The next day Dr. Freitas would perform experiments with the islets on our special cryomicroscope to measure their properties. Since it is necessary to culture the islets overnight after they are isolated and before they can be used for experiments, our international collaboration was nearly as effective as if we had resided in the same institution (see Figures 4 and 5).
The combination of the experimental data and model for a multicellular tissue enabled us to obtain values for the cell membrane permeability and the interstitial diffusion properties of islets. Identification of these properties, along with the thermodynamic parameters for ice nucleation, helped us develop the first design tool that can be applied to optimize the cryopreservation protocol for a multicellular tissue in terms of the thermal and chemical manipulations that can be applied.
At the next level of complexity, we are working on design techniques to optimize the cryopreservation of whole organs. Our collaborator is Dr. David Pegg, formerly at Cambridge and now at the University of York. Another UT graduate, Dr. Charles Lachenbruch, has developed a model for the much more complex behavior of a kidney. His model sends a cryoprotective agent through the vascular system to introduce it into cells throughout the entire organ.
Flowing a cryoprotective agent through the circulatory system of an organ creates many problems. At low temperatures the viscosity increases, making it difficult for the cryoprotective agent solution to flow through the tiny vessels and to diffuse through the interstitial tissue to the cells. The cryoprotective agent presents an osmotically active agent to the organ. As it diffuses into the extravascular space, the organ takes on extra water causing it to swell. Since the organ has elastic properties, it resists swelling, which increases the internal interstitial pressure and makes it more difficult for the cryoprotective agent to enter the tissues. Swelling also occurs in the endothelial cells which line the vessel walls. The endothelial swelling, in combination with elevated interstitial pressure, causes the vessel cross-sections to diminish and increases resistance to perfusion of the cryoprotective agent. As you can see, a complex interplay occurs among the hydrodynamic, osmotic, and mechanical energy domains of the organ when adding the cryoprotective agent.
Dr. Lachenbruch developed the first model that accounts for all of these simultaneous effects. In conjunction with kidney information supplied by Dr. Pegg, he was able to strengthen the design of the protocol to minimize damage to the organ. UT biomedical engineering professor Linda Hayes and her students worked with us to model the associated heat transfer processes that govern the subsequent cryopreservation process. We hope to develop methods to control the temperature and concentration fields that form in an organ during cryopreservation.
Our recent work is in a new branch of cryobiology collaborating with UT botany professor Jerry Brand. This project deals with the cryopreservation of a plant genetic base consisting of more than 2,000 algae strains. The algae present a different challenge to cryopreservation. They are surrounded by a wall that provides an elastic resistance to volume changes in addition to the semipermeable membrane. Certain algae are studied in molecular detail for understanding genetics. With cryopreservation, we can freeze algae and stabilize the material in a frozen state to prevent genetic modification. This allows us to study the unchanged genetic material at any time in the laboratory.
My doctoral student, John Walsh, applied techniques of cryomicroscopy and network thermodynamic modeling to discover how the combined osmomechanical properties of the algae control its response to freezing, and he successfully measured these properties at subzero temperatures. His data demonstrated a clear biphasic behavior of the cells in which there are separate domains governed by the mechanical properties and by the osmotic properties. The division between these domains is defined by the plasmolysis of the cells in which the membrane separates from the wall. Based on this collaboration between bioengineers and life scientists, Dr. Brand's team of researchers has been able to cryopreserve more than 500 different strains of algae, which far exceeds the success rate ever achieved before.
Summary. We are now prepared to apply the principles of bioengineering analysis to the solution of long standing problems in cryobiology, as well as to many other fields for biology and medicine. It is indeed possible to engineer an optimal design of processes for biological systems. New developments in instrumentation and analysis are enabling researchers to measure tissue properties that were heretofore inaccessible and to apply this information in the rational determination of protocols for the cryopreservation of important cells, tissues, and organs. We expect successful cyropreservation of the kidney to occur in perhaps ten years, with cyropreservation of the heart and liver following. A bright future surrounds bioengineering and the contributions and impact the discipline will have on life and the medical sciences.
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|March 16, 1998
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