The advent of artificial tissues, cord blood therapies, cell transplantation procedures, and the desire to bank germ cells have driven the need to establish a means to reliably store biological materials. Cryopreservation is an effective means to store biological materials without functional degradation over a prolonged period of time. The challenge with successful cryopreservation is developing cooling protocols that avoid cellular dehydration and intracellular ice formation. While models have been developed to facilitate the development of cooling protocols, there is still a significant gap between model predictions and behavior in practice. It is our hypothesis that one significant reason for this gap is that current state-of-the-art models do not account for the difference in chemical composition between intracellular solution and extracellular media. We are proposing to develop an experimental tool and technique that will allow direct measurement of the intracellular phase diagram. We will then use that new phase diagram to develop new cryopreservation protocols. The measurement of the intracellular phase diagram will be accomplished by designing and building a micro-scale differential scanning calorimeter (?DSC) that is capable of detecting the phase transition of a single cell in media. The operation of the ?DSC is based on the Peltier effect, which allows precise temperature control during both heating and cooling modes. We have experimentally established that the proposed design concept is capable of detecting the latent heat released from a single porcine oocyte with a cell diameter of about 100 ?m. In order for true DSC measurements to be taken it is necessary for us to improve the repeatability and reliability of the ?DSC assembly process. We are proposing to design and build the structures using a combination of traditional and micro-fabrication techniques. Improved electronic control and fast measurement equipment will improve the measurement quality as well. After the ?DSC assembly process has been improved and the device calibrated using pure water as a standard, we will demonstrate the capabilities of the new measurement tool by measuring the intracellular phase diagram of mouse oocytes, mouse and rat preimplantation embryos, and zebrafish embryos. Using the phase diagrams thus obtained, we will develop a new cryopreservation protocols for each biological system. In order to develop the new protocols we will make adjustments to the current cryopreservation modeling approach, accounting for the phase diagram difference between the intracellular solution and the extracellular media. The overall success of the research project will be defined by the level of cryopreservation enhancement.
(provided by applicant): The metabolic rate of living cells diminishes dramatically at low temperatures, making cryopreservation an attractive long term storage option for biological cells and tissues. Cell transplantation is becoming more prevalent for treatment of acquired diseases and for correction of genetic defects. An aging population is demanding such procedures for improving their quality of life. Therefore the need for effective biological material storage has become increasingly important. Specific examples of storage needs include: - Banking of large quantity of living cells/tissues for typing to use in clinical settings and in case of terrorist or natural disasters. - Preservation of umbilical cord blood from newborns for the potential of generating perfectly matched genetic therapies. - Allowing sufficient time for the screening of transmissible diseases in donated biological materials. - Facilitating the transport of cells and tissues from one medical center to another - Increasing the shelf-life of engineered tissues, such as compliant heart valves, to reduce manufacturing costs. - Preserving the sperm and egg cells of endangered or transgenic species. Although cryopreservation is an attractive means to biopreservation, the freezing process is rather hazardous. Bringing the temperature of biological material from a nominal in vivo temperature of 37 oC to a storage temperature of -196oC causes the intracellular water to pass through its liquid/solid phase transition point. Depending on the rate at which cooling takes place and the chemistry of the cell media, the water inside a cell may form ice crystals, dehydrate, or vitrify (form a glass). Of those three mechanisms, the first two generally result in the death of the cell/tissue, while the glassy vitrification state provides a chemically and mechanically stable structure that increases the probability of cell/tissue survival. Thus, it is our goal to help understand if a biological sample will tend to vitrify, and to engineering cooling procedures to help encourage vitrification. Our work is focusing on developing a measurement tool, called a micro-differential scanning calorimeter, which will allow the measurement of internal cell ice formation under different cooling conditions and for different concentrations of cryoprotectant agents, such as glycerol. Currently available machines are macroscopic in nature and do not allow such fine measurement resolution. Our hypothesis is that by understanding the phase transitions at the scale of a cell, we will be able to modify mathematical models of cellular freezing to improve the cryopreservation process. We will test the new machine on mouse oocytes, mouse and rat embryos, and zebrafish embryos.
