Browsing by Subject "Cryopreservation"

Now showing 1 - 13 of 13

Algorithm Optimization of non-DMSO Cryopreservation Protocols to Improve Mesenchymal Stem Cell Post-Thaw Function
(2016-09) Pollock, Kathryn
Mesenchymal stem cells (MSCs) are a common transfusion cell therapy that have been used in over 300 clinical trials to treat over 2000 patients with diseases ranging from Crohn’s disease to heart failure. These cells are frequently cryopreserved to better coordinate the timing of cell administration with patient care regimes and to accommodate transport of samples between different sites of collection, processing, and administration. However, cryopreservation with DMSO (the current gold standard) can result in poor cell function post-thaw and adverse reactions upon infusion. We hypothesize that non-DMSO cryopreservative molecules, including sugars, sugar alcohols, amino acids, and other small molecule additives, can be used in combination to protect cell viability and function post-thaw. This research demonstrates that some combinations of non-DMSO cryopreservatives preserve cell functionality better than others, and these effects are dependent not on osmotic or physical changes in solution, but on biological changes that affect the cell during the freezing process. We observe that there is likely a sweet spot concentration combination that produces maximum recovery for each combination of molecules, and demonstrate that an evolutionary algorithm can be used to identify optimized combinations of molecules that yield high cell recovery post-thaw. Additionally, we demonstrate that these novel solutions maintain MSC functionality when evaluated using surface markers, attachment, proliferation, actin alignment, RNA expression, and DNA hydroxymethylation. These advances in cryopreservation can improve cell therapy, and ultimately patient care.
Cryopreservation of Pancreatic Islets Experimental Data Repository 2022
(2022-01-18) Bischof, John, C; Finger, Erik, B; efinger@umn.edu; Finger, Erik, B; University of Minnesota Organ and Tissue Preservation Group
Examination of Post-Thaw Behavior between DMSO and Non-DMSO Cyopreserved Bone Marrow Mesenchymal Stem Cells
(2016-12) Stumbras, Aron
Mesenchymal stem cells (MSCs) provide great potential for off the shelf therapeutics because of their immunomodulatory paracrine effects. Clinical trials have utilized MSCs to treat autoimmune diseases but low efficacy, possibly due to cryopreservation methods, has limited trial progression. MSCs cryopreserved in DMSO survive well but have been shown to exhibit functional differences compared to fresh cells. This has created a need for DMSO-free cryoprotectants and for defined assays to test their functionality. To test functional aspects of MSCs post thaw we utilized recovery, viability, attachment and proliferation assays as well as pSTAT1 activity in an attempt to highlight the effects of DMSO cryopreservation on freshly thawed cells. Additionally, we provide evidence through the addition of a DMSO-free cryopreservation solutions that there may be alternatives to freezing cells than the industry standard DMSO. One solution specifically, SGI showed similar behavior to DMSO frozen samples in all metrics. The discovery and definition of DMSO-free cryoprotectants may help increase efficacy in clinical trials and help move current MSCs treatments closer to off the shelf therapies.
Introducing Cost-effective Approaches for Fabricating and Characterizing Multifunctional Magnetic Nanowires for Advancing Nanobiotechnology
(2021-03) Zamani Kouhpanji, Mohammad Reza
Nanobiotechnology often requires a significant amount of nanoparticles with controlled morphology and properties that cannot be achieved using the current state of the arts. In this dissertation, we introduce a scalable approach for the mass production of elongated nanoparticles by harnessing the current distribution during template-assisted electrodeposition technique. This approach not only substantially reduces the synthesis cost and time (by a factor of 4X) but also significantly enhances monodispersity and fabrication yield (by a factor of 80X to 100X). Interestingly, this scalable approach can unlimitedly be scaled up for several magnitudes of orders higher fabrication yields. Practically, high-yielding fabrication methods usually do not allow perfectly identical nanoparticles, leading to variation in properties and functionalities. In this context, we advised a novel, fast, and universal characterization technique, named the projection method, that can potentially be used for characterizing hysteretic behaviors (dependency of a property of a system on its history) of magnetic nanoparticles. The projection method not only speeds up extracting magnetic signatures by a factor of 20X to 100X but also removes the trade-off between accuracy and characterization speed, thus beneficial for both research development and industrial quality control levels. Lastly, extensive characterizations and surface chemistry modifications of numerous magnetic nanowires have been conducted to advance Nanobiotechnology by tackling challenges in quantitative biolabeling and nanobarcoding, cell manipulation, selective detection and stimulation of cancer cells in multimodal therapeutic platforms, cancer hyperthermia, and cryopreservation applications. Specifically, this dissertation addresses long-standing obstacles, including magnetic nanowires agglomeration, surface biofunctionalization, colloidal stability in biological media, and selective detection of biological entities, to boost the progression of Nanobiotechnology. The proven success of magnetic nanowires in the aforementioned applications opens new directions to transform the future of Nanobiotechnology, where my scalable synthesis and universal characterization techniques are the cornerstones.
Mechanisms and models of dehydration and slow freezing damage to cell membranes
(2010-10) Ragoonanan, Vishard
Cell preservation is accomplished primarily by two methods: cryopreservation and dehydration, with the former being the standard technique used. In order to optimize and develop cell preservation protocols for cells that are difficult to preserve or whose end application is incompatible with current cell preservation protocls and to advance preservation by dehydration, a better understanding of the freeze- and dehydration-induced changes to the cell membrane is required. Despite a large body of literature on the topic, the mechanisms of damage to cells during slow freezing and dehydration are still ambiguous. The objective of this study is to investigate the mechanisms of damage to the cell membrane during slow freezing and dehydration and expand our outlook beyond the cell membrane to its underlying support, the cytoskeleton. In this study, we used several model systems to investigate slow freezing and dehydration. We used a liposome model to gather basic information on changes that can occur to a simple membrane system during freezing. This study revealed that eutectic formation was capable of dehydrating the membrane at low temperatures which may be contribute to alteration of the post-thaw membrane structure. We used a bacteria model to investigate the role of the phase transition and immediate versus slow osmotic stress on post-rehydration viability. This study revealed that going through a lyotropic membrane phase transition was detrimental to post-rehydration viability. This study also demonstrated that a rapidly applied osmotic stress was more detrimental to the structure/ organization of the membrane than gradual osmotic stress. We then subjected a model mammalian cell to both hyperosmotic stress and freeze-thaw and investigated both the membrane and cytoskeletal responses. Osmotic stress experiments suggested that alterations in membrane structure (i.e., surface defects and lipid dissolution) were directly dependent on the change in the chemical potential of water. These experiments also suggest that cell shrinkage and the resulting formation of membrane protrusions negatively affect viability upon return to isotonic conditions. It was found that membrane morphology in the dehydrated state and post-hyperosmotic viability was dependent on the stiffness of the cytoskeleton. Freeze/ thaw experiments suggested that ice-cell interaction decreases post-thaw viability. However, similar to osmotic stress experiments, cell shrinkage and cytoskeletal stiffness negatively impact post-thaw viability. We suggest the resulting membrane morphology due to cell shrinkage is also responsible for damage during freeze/ thaw. The various mechanisms discovered and the models proposed can be used in developing new protocols for cell preservation and for cell destruction (e.g. cryosurgery).
On A General Theory Of Phase Change, Nucleation, And Growth, And The Formation Of Ice In Cryopreserved Systems
(2023-01) Kangas, Joseph
In this work we address longstanding gaps in understanding in phase change theories linkingthe nucleation rate, growth rate, growth geometry, and transformed fraction of phase. We take a first principles approach whereby a fundamental understanding of the relationships between these properties can be derived without obfuscation by previous efforts. This is carried out by examining a growing region of space with some prescribed geometry which is transforming from one phase to another, tracking its volume as it grows and intersects with other transforming regions of space. Using this approach, we derive both ordinary and partial differential equations linking the nucleation rate, growth rate, fractal dimension, transformed fraction, phase size distribution, and initial distributions of phase for a system undergoing phase change. We then show that solutions to these equations under special conditions yield methods for extracting nucleation and growth rates for heat release curves, as well as more detailed descriptions of growth geometries. These nucleation and growth rates are important for understanding systems hindered by phase change, including cryobiology, metallurgy, pharmacology, and food science, among others. Extensions to gas phase allow for a deeper understanding of aerosol science and cavitation dynamics as well. Ice crystallization is studied in cryoprotectant agents (CPAs) in low concentrations via direct quenching and laser calorimetry. Critical cooling rates were measured by examining the temperature-time profiles during the direct quenching of droplets of CPA into liquid nitrogen. Critical warming rates were measured by examining ice crystallization in vitrified droplets of CPAs and plasmonic gold nanoparticles during high energy laser irradiation. High-speed imaging allowed for accurate measurements of the temperature rates necessary for avoiding ice formation on rewarming from a vitrified state. A model linking the critical cooling and warming rates in mixtures of CPA was also developed and verified. Additionally, the phase change theory we derived allow for corroboration of the rates necessary for the vitrification of pure water. The laser warming process was also studied numerically via Monte Carlo simulations of light transport in scattering media. The effect of system geometry, absorption coefficient, scattering coefficient, scattering anisotropy, and domain partitioning were studied for a variety of systems including the laser warming of spherical and hemispherical droplets laden with zebrafish embryos and coral nanofragments. Warming uniformity was the main focus of optimization as it is the driving factor in post-warming survival in laser warmed cryopreserved specimens. Laser warming in multi-laser systems is also briefly discussed.
Quantifying the Diffusivity of Cryoprotective Agents in Swine Skeletal Muscle Tissue
(2024-05-15) Saida, Agam; Kraft, Casey; Upchurch , Weston; Iaizzo, Paul A.; Bischof, John
Thermal Analysis of Cryoprotectants for Cryopreservation
(2017-02) Phatak, Shaunak
Cryopreservation by vitrification is a promising technique for preservation of biomaterials such as organs for long term storage. Crystallization while cooling and warming is an important hurdle for a successful cryopreservation. This problem can be addressed by the use of cryoprotectant solutions (CPAs) which help in inhibiting crystallization. The cooling and warming rates needed to prevent crystallization in these CPAs are called Critical Cooling Rate (CCR) and Critical Warming Rate (CWR) respectively. Thermal modeling is an important tool which can help to study this process and predict subsequent cooling and warming rates needed to avoid crystallization. Temperature dependent thermal properties such as thermal conductivity, specific heat capacity and density are needed in order to develop an accurate model. This work involved the measurement of specific heat capacity (Cp) of high concentration CPAs (> 6M) that are used to study vitrification. The thermal properties were then used in a numerical model to predict cooling and warming rates encountered in a cylindrical geometry of CPAs. Chapter 1 provides a review of the thermal properties (thermal conductivity and specific heat capacity) of various biomaterials available in the literature in the sub-zero and supra-zero temperature ranges. Thermal properties of biomaterials are highly temperature dependent. In addition to dependence on temperature, these properties are affected by crystallization and vitrification at sub-zero temperatures (<0°C) and protein denaturation and water loss at supra-zero temperatures (>0°C). Finally, a modeling case study (Bischof and Han 2002) has been provided to highlight the significance of using temperature dependent thermal properties for accurately predicting thermal history. Chapter 2 focusses on experimental measurements of specific heat capacity (cp) of five high concentration CPAs (> 6M) — VS55 (with and without sucrose), DP6 (with and without sucrose) and M22. Further, the effect of cooling / warming rate (1, 5 and 10 °C/min) on crystallization and vitrification has been studied. It was observed that the addition of 0.6 M sucrose to two CPAs viz., VS55 and DP6 suppressed their crystallization for all the three cooling and warming rates. Chapter 3 involves thermal modeling of cooling and warming in a COMSOL Multiphysics package. Thermal properties from Chapters 1 & 2 were used in order to predict the cooling and warming rates for three conditions, viz. convective cooling, convective warming and nano warming. These simulations were carried out in a cylindrical geometry for an increasing size, i.e. the radius of the cylinder. The objective was to find the size limit beyond which cooling and warming rates would not exceed the CCR and CWR respectively.
Thermobiomechanics of arteries
(2008-11) Venkatasubramanian, Ramji T.
Conventional treatments for arterial diseases, such as balloon angioplasty, often result in restenosis or re-narrowing of the arteries. In the last few years, the clinical importance of thermal therapies for atherosclerosis involving both freezing (cryoplasty) and heating (in-stent heating) has increased significantly because of their potential to control or minimize restenosis. An alternative to these therapies includes replacing the diseased artery through preserved arterial grafts which brings with it the need to effectively preserve them. Cryopreservation, i.e. preservation of tissues by freezing to very low temperatures, has therefore become an important problem in medicine. As mechanical properties of arteries play a large role in blood flow, a complete understanding of the biomechanical changes following thermal treatments and the underlying mechanisms is essential for further optimization of these treatments through controlling biomechanical changes. The objective of this dissertation was to quantify the biomechanical changes and investigate the underlying mechanisms post freeze-thaw. In this dissertation, the following specific aims were pursued: 1. Quantification of freeze-thaw induced biomechanical changes in arteries 2. Investigation of underlying mechanisms of thermobiomechanics SA1 involved quantification of freeze-thaw induced mechanical property changes in arteries using both uniaxial tensile tests and indentation. While uniaxial tensile tests were chosen for relatively easy sample preparation and testing, indentation was performed in order to study a more localized biomechanical response while characterizing the diseased artery response. SA2 involved investigation of the mechanisms underlying the biomechanical changes. This primarily involved understanding the changes to the collagen matrix and SMCs following thermal treatments. Changes to collagen matrix stability were assessed by quantifying the changes to the amide-III band using the FTIR spectroscopy. Changes in SMC function were studied from the response of arteries to norepinephrine and acetylcholine. Finally, MD simulations were performed as a tool to further investigate dehydration induced increase in thermal stability of the collagen matrix due to freeze-thaw at the molecular level. The important conclusions of this dissertation research are: 1. Freeze-thaw causes significant stiffening of the arteries. While, significant increase in the physiological elastic modulus (and reduction in toe region) was observed in the uniaxial tensile response, the peak and equilibrium modulus measured from indentation increased significantly following freeze-thaw. 2. Freeze-thaw induces significant changes in the collagen matrix and smooth muscle cells (SMCs) that are arguably the most important components of an artery. While dehydration accompanied by increased thermal stability was observed following freeze-thaw in the collagen matrix, it caused complete destruction of SMCs measured through loss in function. 3. At the molecular level, dehydration due to freeze-thaw (or any osmotic treatments) results in formation of new sidechain-backbone hydrogen bonds that are typically absent under hydrated conditions. These newly formed intra-protein hydrogen bonds in the absence of water molecules increase the thermal stability of the tropocollagen molecule.
Understanding physiochemical changes in Jurkat cells at different layers during freezing using Raman Spectroscopy
(2016-04) Yu, Guanglin; Yap, Yan Rou; Hubel, Allison
Cryopreservation is a process where cells or tissues are preserved by cooling to sub-zero temperatures, where any enzymatic or chemical reaction is stopped due to lack of thermal energy. Previous experiments studied the changes in cells at one specific layer. However, formation of ice on the surface, then into cell during the cooling process will affect image analysis. In this study, the physiochemical changes in Jurkat cells at three different layers were studied based on the distribution of ice, cryoprotectant, and cytochrome C using Raman Spectroscopy.
Understanding the benefits and limitations of magnetic nanoparticle heating for improved applications in cancer hyperthermia and biomaterial cryopreservation
(2013-12) Etheridge, Michael Laurence
The current work focused on the ability of magnetic nanoparticles to produce heat in the presence of an applied alternating magnetic field. Magnetic nanoparticle hyperthermia applications utilize this behavior to treat cancer and this approach has received clinical approval in the European Union, but significant developments are necessary for this technology to have a chance for wider-spread acceptance.Here then we begin by investigating some of the important limitations of the current technology. By characterizing the ability of superparamagnetic and ferromagnetic nanoparticles to heat under a range of applied fields, we are able to determine the optimal field settings for clinical application and make recommendations on the highest impact strategies to increase heating. In addition, we apply these experimentally determined limits to heating in a series of heat transfer models, to demonstrate the therapeutic impact of nanoparticle concentration, target volume, and delivery strategy.Next, we attempt to address one of the key questions facing the field- what is the impact of biological aggregation on heating? Controlled aggregate populations are produced and characterized in ionic and protein solutions and their heating is compared with nanoparticles incubated in cellular suspensions. Through this investigation we are able to demonstrate that aggregation is responsible for up to a 50% decrease in heating. However, more importantly, we are able to demonstrate that the observed reductions in heating correlate with reductions in longitudinal relaxation (T1) measured by sweep imaging with Fourier transformation (SWIFT) magnetic resonance imaging (MRI), providing a potential platform to account for these aggregation effects and directly predict heating in a clinical setting.Finally, we present a new application for magnetic nanoparticle heating, in the thawing of cryopreserved biomaterials. A number of groups have demonstrated the ability to rapidly cool and preserve tissues in the vitreous state, but crystallization and cracking failures occur upon the subsequent thaw. Magnetic nanoparticles offer a potential solution to these issues, through their ability to provide rapid, uniform heating, and we illustrate this through heating in several cryoprotectant solutions and by modeling the effects of heating at the bulk and micro-scales.
Universal robot for automated microinjection with applications in transgenesis and cryopreservation
(2023-01) Joshi, Amey
Microinjection is the process of injecting a small amount of solution into biological organisms at a microscopic level using a glass micropipette. It is a widely utilized technique with a wide range of applications in both fundamental research and clinical settings. However, microinjection is an extremely laborious and manual procedure, which makes it a critical bottleneck in the field and thus ripe for automation. In this thesis, we introduce a simple computer vision-guided robot that uses off-the-shelf components to fully automate the microinjection procedure in different model organisms. The robot uses machine learning models that have been trained to detect individual embryos on agar plates and serially performs microinjection at a particular site in each detected embryo with no human interaction. We deployed three such robots operated by expert and novice users to perform automated microinjections in zebrafish (Danio rerio) and Drosophila melanogaster. We conducted survivability studies to better understand the impact of microinjection on zebrafish embryos and the fundamental mechanisms by which microinjection affects zebrafish embryos. We were able to use the robot to examine the speed of the micropipette, the volume of the microinjectant, the micropipette geometry, and the rate of the volume delivered. These results helped us in determining the optimum settings for automated microinjection into zebrafish embryos. We used transgenesis studies to compare microinjection efficiency to manual microinjection utilizing optimum settings for automated microinjection. Further, we demonstrated that robotic microinjection of cryoprotective agents in zebrafish embryos significantly improves vitrification rates and post-thaw survivability of cryopreserved embryos compared to manual microinjection, opening the door to large-scale cryo-banking of various aquatic species on an industrial scale. We anticipate that this robotic microinjection can be readily adapted to other organisms and applications.
Using Computational Tools to Understand Interactions between Osmolytes and Optimize the Preservation of Heterogeneous Populations of Primary Cells
(2019-11) Pi, Chia-Hsing
Immunotherapies such as chimeric antigen receptor (CAR) T-cell therapy are emerging therapies for the treatment of cancers and persistent viral infections. It is common for immunotherapy products to be collected in one site and processed in another site. Cryopreservation is the technology to stabilize cells at a low temperature for a variety of applications including diagnosis and treatment of disease. However, cryopreservation with the current gold standard, DMSO, can result in poor post-thaw recoveries and adverse reactions to patients upon transfusion. In this work, we propose to understand and optimize DSMO-free cryoprotectants with combinations of non-toxic and natural osmolytes including sugars, sugar alcohols and amino acids. The post-thaw recoveries of Jurkat cells display comparable performance to DMSO and non-linear interactions between osmolytes. Raman spectroscopy observes different protective properties of osmolytes, and statistical modeling characterizes the importance of osmolytes and their interactions. The differential evolution algorithm was applied to optimize the formulations of cryoprotectants previously, but the suboptimal control parameters reduce the performance. The influence of control parameters and four types of differential evolution algorithms are examined and optimized for DMSO-free cryoprotectants specifically. Additionally, we demonstrate that these DMSO-free cryoprotectants can cryopreserve human peripheral blood mononuclear cells as good as conventional DMSO. The advantages of DMSO-free cryoprotectants can improve the accessibility of cell therapy. It will also be critical to providing a methodology to develop multiple component DMSO-free cryoprotectants for other cell types.

University Digital Conservancy

Browse by Subject

Browsing by Subject "Cryopreservation"