Browsing by Subject "cryopreservation"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    Characterizing Freezing and Thawing Responses of Multiple Types of Cells Cryopreserved in Both DMSO and non-DMSO Cryoprotectants
    (2018-09) Yu, Guanglin
    Cryopreservation is the technology used to stabilize cells at subzero temperature for a variety of applications including diagnosis and treatment of disease, and the production of therapeutic proteins. Current theories of cell damage during freezing were developed in the 1960’s, and little has changed since then. However, our understanding of cell biology as well as tools to interrogate cell responses during freezing has improved in the last 50 years. Low temperature Raman spectroscopy has been used to verify, for the first time using chemical spectra, the presence of ice inside the cell during freezing. With this tool, it is possible to internally observe frozen cells, and identify specific chemical and morphological changes that result in cell life or death. In this work, we propose to use this powerful tool to test two hypotheses to enhance our understanding of the mechanism of cell damage during freezing and thawing, and the manner by which the damage can be mitigated to improve cryopreservation outcome. For the first part of this work, we hypothesize that not all intracellular ice formation (IIF) is lethal and the conditions of cell membrane, cytoskeleton and mitochondria play an important role in determining IIF and cryopreservation outcome. Freezing responses of single cells as well as multi-cellular system cryopreserved and thawed in dimethyl sulfoxide (DMSO) solution will be examined to test this hypothesis. DMSO-free cryopreservation has attracted much recent interest due to the toxicity of DMSO. For the second part of this work, we hypothesis that non-DMSO multicomponent osmolyte solutions can be used to preserve cell viability and one component, disaccharide, acts to protect the cell through multiple interactions. Freezing responses of cells cryopreserved in a combination of non-DMSO cryoprotectants such as sugars, sugar alcohols, and amino acids will be examined. Interactions among sucrose (a typical disaccharide), water and cell membrane at low temperature will be also be investigated in order to test the second hypothesis. Enhancing our understanding of freezing damage and strategies to mitigate damage will improve the methods of preserving cell therapy products and therefore enable the treatment to reach the patients who could benefit from them.
  • Thumbnail Image
    Item
    Data for 3D Printed Organisms Enabled by Aspiration-Assisted Adaptive Strategies
    (2024-06-17) Han, Guebum; Khosla, Kanav; Smith, Kieran T; Ng, Daniel Wai Hou; Lee, JiYong; Ouyang, Xia; Bischof, John C; McAlpine, Michael C; hanguebum@gmail.com; Han, Guebum; University of Minnesota McAlpine Research Group; University of Minnesota Bischof Research Group
    Devising an approach to deterministically position organisms could impact various fields such as bioimaging, cybernetics, cryopreservation, and organism-integrated devices. This requires continuously assessing the locations of randomly distributed organisms to collect and transfer them to target spaces without harm. Here we developed an aspiration-assisted adaptive printing system that tracks, harvests, and relocates living and moving organisms on target spaces via a pick-and-place mechanism that continuously adapts to updated visual and spatial information about the organisms and target spaces. These adaptive printing strategies successfully positioned a single static organism, multiple organisms in droplets, and a single moving organism on target spaces. Their capabilities were exemplified by printing vitrification-ready organisms in cryoprotectant droplets, sorting live organisms from dead ones, positioning organisms on curved surfaces, organizing organism-powered displays, and integrating organisms with materials and devices in customizable shapes. These printing strategies could ultimately lead to autonomous biomanufacturing methods to evaluate and assemble organisms for a variety of single and multi-organism-based applications.
  • Thumbnail Image
    Item
    DMSO-Free Cryopreservation of Therapeutic Cells by Agarose Hydrogel Encapsulation
    (2023-07) Wang, Mian
    In recent decades, the rapid advancement of cell therapy and regenerative medicine has generated an urgent need for efficient and safe cell/tissue cryopreservation techniques on both laboratory and industrial scales. The current use of dimethyl sulfoxide (DMSO) as a cryoprotective agent (CPA) presents toxicity concerns, prompting the development of alternative methods. The primary objective of this research is to develop a novel cryopreservation method for therapeutic cells using hydrogel encapsulation, which aims to minimize cryoinjuries while eliminating the need for toxic penetrating CPAs such as DMSO. The dissertation consists of three main parts. In the first part, molecular changes associated with warming injury in cryopreserved human white blood cells (WBCs) were analyzed. It was observed that slow warming led to irreversible dehydration of cell membrane lipids and denaturation of cellular proteins. Also, WBCs were found to be very susceptible to kinetic processes during warming, including eutectic crystallization/melting, devitrification, and ice recrystallization. The second part focuses on the development of an encapsulation cryopreservation method using the combination of agarose hydrogel and trehalose as an alternative to membrane-permeable CPAs. A comprehensive analysis of the kinetic and thermodynamic changes within the agarose-trehalose hydrogel during freeze/thaw was conducted. The combination of agarose and hydrogel was found to reduce ice phase volume with a less ordered structure, eliminate eutectic crystallization and melting, and inhibit ice recrystallization during warming. The third part of the dissertation involves the validation of the DMSO-free hydrogel encapsulation method for the cryopreservation of natural killer (NK) cells. High post-thaw cell viability was achieved, while decreased viability with compromised cytotoxicity was observed after cells were extracted from the hydrogel. Ongoing efforts are focused on optimizing the cell extraction process from the hydrogel.
  • Thumbnail Image
    Item
    Laser Nanowarming: A platform technology for ultra-rapid rewarming of cryopreserved zebrafish embryos
    (2019-06) KHOSLA, KANAV
    This work describes the development of a platform technology called Laser Nanowarming that has enabled the cryopreservation of Zebrafish embryos for the first time. By injecting propylene glycol (PG) and biocompatible gold nanorods (GNR) followed by rapid cooling (90,000 °C/min), embryos were cryogenically stabilized to liquid nitrogen temperatures. Since the effective concentration of PG inside the embryos is approximately 2M, the embryos require rapid rewarming, which was achieved by using a 1064nm powerful millisecond laser pulse that can generate rates up to 14 million °C/min. We leverage biocompatible and photonic GNR that can create rapid and uniform warming throughout the embryo and overcome the damage induced by ice crystallization. We have since adapted this technology to demonstrate successful outcomes in Human Dermal Fibroblasts (HDF) cells as well as Coral larvae (F. Scutaria) and continue to use it to enable the cryopreservation of Pancreatic Islets, Drosophila Embryos, Shrimp Nauplii and other fish embryos. Our future work is geared towards improving the long-term survival rate of biological specimens as well as developing efficient high throughput methods. If successful, this technology can transform the way germplasm are banked and create a huge impact in the fields of species conservation, biomedical research and aquaculture.