Cryopreservation remains the only practical option for extending human fertility in modern clinical practice. We are applying nano- and micro-scale technologies to develop a novel method for automated mammalian germ cell cryopreservation. The primary problem with current cryopreservation technologies is that they either leave large mammalian cells (e.g., oocytes) susceptible to intracellular damage secondary to the formation of intracellular ice crystals, or require the use of very high levels of toxic cryoprotectant agents (CPA's). The problem of how to balance the risk of intracellular ice crystal formation with cytotoxicity from high intracellular CPA levels has proven to be one of the defining problems in the field of biopreservation. ? ? A second problem with existing germ cell freezing technologies is that all current procedures are highly labor- and time-intensive, requiring the full attention of a well-trained clinical embryologist. The reliance on highly skilled human labor is not only inefficient, but also adds an additional level of variability to the process. Third, all current cryopreservation protocols expose cells to large, step-wise changes in the CPA concentrations as the sample is manually transferred from one solution to another. This approach has two major drawbacks: (1) each abrupt change in CPA concentration causes osmotic stress to the cells; (2) manually transferring cells between different media causes shearing and other mechanical stress. ? ? The ideal cryopreservation protocol would combine the reduced chemical toxicity of conventional slow freezing with the resistance to intracellular ice crystal formation of vitrification. At the same time, the ideal protocol would load and unload CPA's in a continuous manner in order to avoid osmotic shock. Lastly, it would minimize handling of cells in order to reduce mechanical stress, diminish the labor burden on the embryology lab, and lessen (or even eliminate) human error and test-to-test variability. We believe we can develop a disposable microfluidic chip that would accomplish all of these goals. ? ? (a) sufficiently high rate of cooling to allow the use of CPA concentrations as low as those utilized in slow freezing protocols; and, (b) greatly reduced osmotic and mechanical stress on cells due to a combination of the progressive loading/unloading of CPA's and a minimum level of handling. Cryopreservation remains the only practical option for extending human fertility in modern clinical practice. The problem of how to balance the risk of intracellular ice crystal formation with cytotoxicity from high intracellular CPA levels has proven to be one of the defining problems in the field of biopreservation. We are applying nano- and micro-scale technologies to develop a novel method for automated mammalian germ cell cryopreservation that has potential to decrease intracellular damage and use lower levels of toxic cryoprotectant agents (CPA's). Further our approach has the potential to decrease laborious and time-intensive techniques and additional level of variability to the process due to the human factor. ? ? ?
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