Over the past few decades, there has been an increase in the development of new cellular therapies used for the treatment of various conditions. Thus, the rapid development of therapies requiring transfusion and transplantation of cells has resulted in a need to preserve these cellular therapy products. Cryopreservation is the only currently used method for the long-term storage of cells. The most commonly used cryoprotectants are 10% dimethyl sulfoxide (DMSO) for hematopoietic stem cells (HSCs) and 40% glycerol for red blood cells (RBCs). DMSO fails to protect the functionality of HSCs after cryopreservation and therefore, up to 20% of HSC transplantations fail to engraft. The glycerol in thawed RBC units must be removed to <1% to prevent intravascular hemolysis which is time-consuming. Thus, there is an urgent need to develop improved cryoprotectants for HSCs and RBCs. DMSO and glycerol are unable to control ice recrystallization which is a major source of cellular injury during cryopreservation. Therefore, compounds with the ability to inhibit ice recrystallization could represent a new class of cryoprotectant with a novel mechanism of action. This thesis focuses on the rational design of small-molecule ice recrystallization inhibitors. The key structural attributes required for ice recrystallization inhibition (IRI) activity are investigated. The amphiphilic balance required for IRI activity is explored. Furthermore, two new classes of small-molecule IRIs containing aromatic rings were rationally designed. As a result, several very highly IRI active molecules were discovered. The use of IRIs to improve the cryopreservation of HSCs and RBCs was explored. A number of IRIs improved the post-thaw functionality of HSCs. Supplementation of the current cryoprotectant solution with IRIs resulted in an increase in CFU recovery and frequency of multipotent progenitors. This would reduce the percentage of engraftment failure and allow for a larger proportion of cord blood banks’ inventory to provide an adequate dose for patients requiring transplants. Several IRIs were found to be effective cryoprotectants for RBCs with reduced amounts of glycerol. This could reduce the deglycerolization time for RBCs. These results demonstrate the potential of small-molecule IRIs to improve the current cryopreservation procedures for important cellular therapy products.

Current equine sperm cryopreservation methods fail to reliably prevent damages to important cellular structures such as the cell membrane and DNA. The objective of this study was to determine the effects of supplementing a stallion semen extender with 1 or 10 mM resveratrol on post-thaw sperm characteristics. Results showed that sperm death was increased with 10 mM compared to both the control and 1 mM (P < 0.05). DNA fragmentation was increased in the 1 mM treatment compared to the control (P < 0.05). ROS activity was reduced the most in the 10 mM with differences between all groups (P < 0.05). Membrane integrity was not different between groups (P > 0.05). Motility of the control was higher than the treatment groups (P < 0.05). Resveratrol was able to reduce ROS but was unable to preserve motility or viability at the concentrations tested.

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Intracellular ice formation (IIF), a major cause of cryoinjury in biological cells, is significantly more pronounced during freezing of tissue than during freezing of suspended cells. While extensive studies of IIF have been conducted for single cells in suspension, few have investigated IIF in tissue. Due to the increased complexity that arises from both cell-substrate and cell-cell interactions in tissue, knowledge of cryobiology of isolated cells cannot simply be extrapolated to tissue. Different theories have been hypothesized for the mechanisms of IIF in tissue, but none have been conclusively proven. Towards the goal of developing mathematical models to accurately predict the probability of IIF in tissues of one or more cell types, we have developed a novel high-speed video cryomicroscopy system capable of image acquisition at sampling rates up to 32,000 Hz. Specifically, the effects of cell adhesion to the substrate and cell-cell interactions were investigated with experimental (micropatterned endothelial cell constructs) and mathematical models (Monte Carlo simulations). We have reported the first direct observations of the IIF process recorded at unprecedented sub-millisecond and sub-micron resolution. For the majority of our experiments, IIF nucleation was determined to occur preferentially at the cell perimeter. This observation was not consistent with the commonly accepted hypotheses of ice nucleation in suspended cells and suggests that an alternative mechanism of IIF initiation is dominant in adherent cells. In addition, the kinetics of ice nucleation were shown to be influenced by time in culture, attached cell perimeter, fibronectin coating density, and degree of cell-cell contact. Moreover, an additional phenomenon, paracellular ice penetration was identified, and the frequency of formation was correlated with focal adhesion formation. The data and mathematical models presented in this thesis bring closer the goal of elucidating the primary mechanisms contributing to IIF in tissue; providing important contributions to both the fields of cryopreservation (minimizing IIF) and cryosurgery (maximizing IIF).

Cryopreservation of fish germ cells has important applications in aquaculture, conservation of endangered species and human genomic studies. Although investigations on cryopreservation of fish sperm and embryos have been carried out extensively, cryopreservation of fish oocytes has not been studied systematically. The objective of the present study was to develop successful cryopreservation protocol for zebrafish oocytes at temperature of liquid nitrogen (-196°C), or if unachieved, to investigate the limiting factors associated with fish oocytes cryopreservation. In this study, the effects of cryoprotectants exposure and enzymatic treatments on oocytes survival were studied, and new viability tests for zebrafish oocytes were developed. The effects of controlled slow cooling with different cryoprotective agents, in different freezing media and at different cooling rates on cryosurvival of zebrafish (D. rerio) oocytes were investigated. Cryomicroscopic observations on zebrafish oocytes were also carried out. Three reliable vital tests -trypan blue (TB) staining, ATP assay, and in vitro maturation followed by germinal vesicle breakdown observation (GVBD) were found suitable for assessment of oocytes viability. Vitellogenesis (stage III) was found to be the optimal developmental stage for cryopreservation. Methanol was found to be the best CPA for zebrafish oocytes. Combination of 4M methanol and 0.2M glucose in potassium chloride (KCI) buffer was found to be the optimal cryoprotective solution. Controlled slow cooling at 0.3°C/min rate, combined with seeding at -12.5°C and plunge to liquid nitrogen (LN) at-40°C were found to be the optimal conditions for cryopreservation of stage III oocytes. However, even with the optimal protocol, TB-assessed viability, Le. the ratio of oocytes with intact plasma membrane after cooling to -196°C was 19.6±8%. Furthermore, GVBD experiments showed that none of the cryopreserved oocytes can be matured in vitro, and their ATP levels were decreased dramatically, indicating that successful cryopreservation of fish oocytes at liquid nitrogen temperature still remains elusive. Cryomicroscopic observations demonstrated, that the damages of oocytes are associated with intracellular ice formation (lIF). IIF occurred simultaneously with extracellular ice formation (ElF) in nearly 100% of the cases, and formation of lethal hexagonal type of ice was observed. This study was the first systematic attempt to cryopreserve fish oocytes at liquid nitrogen temperature. The results provided will undoubtedly assist successful protocol design for cryopreservation of fish oocytes in the future.

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Orientador: José Antônio Dell'Aqua Júnior / Resumo: O espermatozoide exige um fornecimento constante de energia para a manutenção de suas funções celulares e quando desafiado por processos de criopreservação do sêmen, sofrem danos irreversíveis. Para o desenvolvimento de técnicas que visam o aumento da longevidade espermática, é necessário considerar que mesmo em metabolismo basal, o espermatozoide necessita de substratos para garantir sua motilidade e poder fecundante após a ejaculação. Para atender suas demandas energéticas, estudos recomendam o uso de nutrientes exógenos, como o piruvato de sódio e a coenzima Q10 (CoQ10), substratos fundamentais na bioenergética celular. Visto a importância da refrigeração de sêmen em garanhões e o potencial destas substâncias em melhorar os parâmetros seminais atuando como substrato energético e antioxidante, respectivamente, o presente trabalho tem por objetivo abordar aspectos relacionados ao metabolismo espermático, bem como o papel do piruvato e CoQ10 visando minimizar os efeitos deletérios da refrigeração sobre a qualidade do sêmen equino. Foram adicionadas diferentes concentrações de piruvato de sódio e da CoQ10 ao sêmen de garanhões considerados “good coolers” (GC) e “bad coolers” (BC). Primeiramente, foram estabelecidas as concentrações mais eficazes de piruvato e CoQ10 no diluente de refrigeração BotuSêmen® (Botupharma Botucatu/SP Brasil) para preservar os parâmetros espermáticos na refrigeração a 5° C por até 48 horas. Foi utilizado 1 ejaculado de 25 garanhões das raças Quarto de... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The spermatozoa require a constant supply of energy for the maintenance of their cellular functions and when challenged by processes of cryopreservation of the semen, they suffer irreversible damages. For the development of techniques that aim to increase sperm longevity, it is necessary to consider that even in basal metabolism, the spermatozoa require substrate to ensure their motility and fertilizing power after ejaculation. To attend their energy demands, studies recommend the use of exogenous nutrients, such as sodium pyruvate and coenzyme Q10 (CoQ10), key substrates in cellular bioenergetics. Considering the importance of semen cooling in stallions and the potential of these substances to improve seminal parameters acting as an energetic and antioxidant substrate, this review aims to address aspects related to cooling, as well as the role of sodium pyruvate and CoQ10 in minimizing the effects of cooling on the quality of equine semen. Different concentrations of sodium pyruvate and CoQ10 have been added to semen from good coolers (GC) and bad coolers (BC). First, the most effective concentrations of sodium pyruvate and CoQ10 in the BotuSêmen® extender (Botupharma Botucatu / SP Brazil) were established to preserve the sperm parameters at 5°C for up to 48 hours. Each ejaculate was split into 7 treatments, with sodium pyruvate and CoQ10 being added at concentrations of 1 mmol/l (P1), 2 mmol/l (P2), 3 mmol/l sodium pyruvate (P3), 25 µmol/l (Q25), 50 µmol/l (Q50) and 75 µmol... (Complete abstract click electronic access below) / Mestre

<p>Crypreservation of oocytes is recently being considered to be a valid choice in infertility treatments.Low survival and fertilization rates due to inefficient slow freeze protocols have been the outcome ofmany previous studies done in the field. However, introduction of the vitrification technique and itsapplication in reproductive medicine and to some extent new improved slow freeze protocols haveshown that oocytes can be cryopreserved with successful outcome.</p><p>In this project the survival rate of oocytes after vitrification with MediCult Vitrification andWarming Media has been studied. Also, a comparison of the carriers Cryoloop (an open system) andCryopette (a closed system) has been performed.</p><p>A total of 43 oocytes were vitrified and warmed according to MediCult's protocol, of which 21oocytes with Cryoloop and 22 with Cryopette. The cells were post-thaw incubated in a physiologicalenvironment for 24h. During that time the morphology and viability were observed and noted after 2h,over night and after 24h. No significant difference in survival rates of the oocytes' was observed usingthe two carriers, and the total survival rate of the oocytes was 83.7%. This indicates thatcryopreservation of oocytes is a valid choice in fertility treatments and that the new product Cryopettecan be used clinically with just as good results as the well established Cryoloop technique. This merits further studies on the applicability of vitrification on oocytes and the Cryopette technique.</p>

Cryopreservation of adherent cells may be advantageous for cell types that are difficult to preserve in suspension or when it is necessary to preserve characteristics of the adherent cultured cells. Vitrification is a promising procedure for the preservation of adherent cells that prevents ice crystal formation and the resulting dissociation and morphological damage. To successfully vitrify adherent cells, high concentrations of CPA are required which increases the likelihood of osmotic and toxic damage. In this dissertation, we describe a rational design strategy that predicts mathematically optimized CPA addition and removal procedures based on the minimization of a toxicity cost function. These rationally designed procedures rely on the accurate knowledge of cell biophysical parameters. We validate an in situ calcein fluorescence quenching method for the determination of membrane permeability parameters for adherent cells. We also describe the determination of osmotic tolerance limits for adherent cells. We use rational design strategies to determine CPA addition and removal procedures for adherent endothelial cells, neuronal cells, and induced pluripotent stem cells as well as oocytes. Also, we provide experimental support for the feasibility of these methods using adherent endothelial cells. The mathematical methods and experimental procedures outlined in this dissertation are important tools for the design of addition and removal procedures for concentrated CPA solutions. This dissertation is an important step toward successful design and implementation of vitrification strategies for adherent cells and tissues. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Feb. 14, 2013 - Feb. 14, 2015