The development of effective methods to cryopreserve precious cell types has had tremendous impact on regenerative and transfusion medicine. Hematopoietic stem cell (HSC) transplants from cryopreserved umbilical cord blood (UCB) have been used for regenerative medicine therapies to treat conditions including hematological cancers and immodeficiencies. Red blood cell (RBC) cryopreservation in blood banks extends RBC storage time from 42 days (for hypothermic storage) to 10 years and can overcome shortages in blood supplies from the high demand of RBC transfusions. Currently, the most commonly utilized cryoprotectants are 10% dimethyl sulfoxide (DMSO) for UCB and 40% glycerol for RBCs. DMSO is significantly toxic both to cells and patients upon its infusion. Glycerol must be removed to <1% post-thaw using complicated, time consuming and expensive deglycerolization procedures prior to transfusion to prevent intravascular hemolysis. Thus, there is an urgent need for improvements in cryopreservation processes to reduce/eliminate the use of DMSO and glycerol. Ice recrystallization during cryopreservation is a significant contributor to cellular injury and reduced cell viability. Compounds capable of inhibiting this process are thus highly desirable as novel cryoprotectants to mitigate this damage. The first compounds discovered that were ice recrystallization inhibitors were the biological antifreezes (BAs), consisting of antifreeze proteins and glycoproteins (AFPs and AFGPs). As such, BAs have been explored as potential cryoprotectants, however this has been met with limited success. The thermal hysteresis (TH)activity and ice binding capabilities associated with these compounds can facilitate cellular damage, especially at the temperatures associated with cryopreservation. Consequently, compounds that possess “custom-tailored” antifreeze activity, meaning they exhibit the potent ice recrystallization inhibition (IRI) activity without the ability to bind to ice or exhibit TH activity,are highly desirable for potential use in cryopreservation. This thesis focuses on the rational design of potent ice recrystallization inhibitors and on elucidating important key structural motifs that are essential for potent IRI activity. While particular emphasis in on the development of small molecule IRIs, exploration into structural features that influence the IRI of natural and synthetic BAs and BA analogues is also described as these are some of the most potent inhibitors known to date. Furthermore, this thesis also investigates the use of small molecule IRIs for the cryopreservation of various different cell types to ascertain their potential as novel cryoprotectants to improve upon current cryopreservation protocols, in particular those used for the long-term storage of blood and blood products. Through structure-function studies the influence of (glyco)peptide length, glycosylation and solution structure for the IRI activity of synthetic AFGPs and their analogues is described. This thesis also explores the relationship between IRI, TH and cryopreservation ability of natural AFGPs, AFPs and mutants of AFPs. While these results further demonstrated that BAs are ineffective as cryoprotectants, it revealed the potential influence of ice crystal shape and growth progression on cell survival during cryopreservation. One of the most significant results of this thesis is the discovery of alkyl- and phenolicglycosides as the first small molecule ice recrystallization inhibitors. Prior to this discovery, all reported small molecules exhibited only a weak to moderate ability to inhibit ice recrystallization. To understand how these novel small molecules inhibit this process, structure-function studies were conducted on highly IRI active molecules. These results indicated that key structural features, including the configuration of carbons bearing hydroxyl groups and the configuration of the anomeric center bearing the aglycone, are crucial for potent activity. Furthermore, studies on the phenolic-glycosides determined that the presence of specific substituents and their position on the aryl ring could result in potent activity. Moreover, these studies underscored the sensitivity of IRI activity to structural modifications as simply altering a single atom or functional group on this substituent could be detrimental for activity. Finally, various IRI active small molecules were explored for their cryopreservation potential with different cell types including a human liver cell line (HepG2), HSCs obtained from human UCB, and RBCs obtained from human peripheral blood. A number of phenolic-glycosides were found to be effective cryo-additives for RBC freezing with significantly reduced glycerol concentrations (less than 15%). This is highly significant as it could drastically decrease the deglycerolization processing times that are required when RBCs are cryopreserved with 40% glycerol. Furthermore, it demonstrates the potential for IRI active small molecules as novel cryoprotectants that can improve upon current cryopreservation protocols that are limited in terms of the commonly used cryoprotectants, DMSO and glycerol.

O curto tempo de armazenamento dos hemocomponentes é um dos fatores que dificulta e limita a quantidade de sangue que pode ser efetivamente armazenada, o que é uma desvantagem na medicina veterinária, pois o acesso a doadores é restrito e a demanda é contínua e cada vez maior na prática de clínicas e hospitais veterinários. Durante o armazenamento do sangue em baixas temperaturas, seja sob a forma de sangue total ou concentrado de eritrócitos, há uma queda intensa de metabólitos importantes para a viabilidade e funcionalidade dos eritrócitos. O desenvolvimento de meios e soluções de preservação sanguínea possibilitou o armazenamento dos eritrócitos e, consequentemente, facilitou o trabalho dos bancos de sangue. Portanto, a busca por melhores formas e soluções para preservação capazes de evitar ou diminuir estes efeitos prejudiciais durante o seu armazenamento é contínua, para que ao final se obtenha uma melhor qualidade do sangue transfundido. O presente trabalho avaliou o concentrado de eritrócitos felino armazenado na solução de CPDA-1 e CPD/SAGM durante 35 dias. Os dados laboratoriais foram comparados entre grupos e ao longo do tempo. Neste experimento foram utilizadas 10 bolsas de concentrado de eritrócitos felino divididos em dois grupos de cinco para avaliação de cada um dos aditivos. Os parâmetros laboratoriais K+, Na+, Cl-, lactato, HCO3-, amônia, glicose e pH foram avaliados nos dias 1, 7, 14, 21, 28 e 35 após a coleta. Vários parâmetros (K+, lactato, HCO3, glicose e cloreto) demonstraram que a solução CPD/SAGM manteve o metabolismo energético do eritrócito mais estável. Com este trabalho, foi possível entender melhor as alterações metabólicas sofridas pelos eritrócitos felinos durante o armazenamento. Concluímos que, apesar da solução CPD/SAGM se mostrar mais eficaz in vitro, são necessários mais estudos com relação aos hemocomponentes em gatos e à sua viabilidade pós-transfusional. / The short shelf life of blood products is one factor that complicates and limits the amount of blood that can be effectively stored, and it is a disadvantage in veterinary practice, because the access to donors is restricted and the demand is continuous and increasing at veterinary clinics and hospitals. During blood storage at low temperatures, either as whole blood or as packed red cells, there is a significant decrease of metabolites that are important for the viability and functionality of erythrocytes. The development of blood preservation solutions has enabled the storage of red blood cells and improved the service at the blood banks. Therefore, the search for better ways and blood preservation solutions to avoid or reduce these harmful effects during the storage conditions is continuous, in order to obtain the best blood product to be transfused. This study evaluated 10 bags of feline erythrocyte concentrate divided into two groups, stored in CPDA-1 and CPD/SAGM solutions during 35 days. The laboratory data were compared between groups and over time. K+, Na+, Cl-, lactate, HCO3-, ammonia, glucose and pH were assessed on days 1, 7, 14, 21, 28, and 35 after collection. On various parameters (K+, Cl-, HCO3-, glucose and lactate) solution of CPD/SAGM kept the energy metabolism of red blood cells more stable. With these results we can better understand the biochemical changes of feline erythrocytes during storage. We conclude that, although the CPD/SAGM solution shown to be more effective, more studies are needed to improve knowledge of feline blood components and post-transfusion viability.

In this work a Raman flow cytometer is presented. It is a whole new microfluidic device that takes advantage of basic principles of Raman spectroscopy and fluorescent flow cytometry mixed together in a system of particularly shaped channels. These are indeed composed by specific shape and sizes – thanks to which cells can flow one-by-one – and a trap by means of which cells are trapped in order to perform Raman analysis on single ones in a constant and passive way. In this sense the microfluidic device promotes a fast method to look for single cells in a whole multicellular sample. It is a label-free analysis and this means that, on the contrary of what happens with fluorescent flow cytometry, the sample does not need to undergo any particular time-consuming pretreatment before being analyzed. Moreover it gives a complete information about the biochemical content of the sample thanks to the involvement of Raman spectroscopy as method of analysis. Many thought about a device like this, but eventually it is the first one being designed, fabricated and tested. The materials involved in the production of the Raman flow cytometer are chosen wisely. In particular the chip – the most important component of the device – is multilayered, being composed by a slide of calcium fluoride (which gives a negligible signal in Raman analyses), a photosensitive resist containing a pattern with channels and another slide of calcium fluoride in order for the channels to be sealed on both sides. The chip is, in turn, connected to gaskets and external frames. Several fabrication processes are followed to ultimately get the complete Raman flow cytometer and experiments on red blood cells demonstrate its validity in this field.

Ce travail a été réalisé dans l’I. R. P. H. E. (Institut de Recherche sur les Phénomènes Hors Équilibre), unité de recherche de l’Université d’Aix-Marseille en collaboration avec l’Université de la Sarre, la Faculté de Physique Expérimentale. Cette étude est consacrée à une meilleure compréhension de la microcirculation du sang in vitro, ainsi que des phénomènes collectifs qui prennent place dans la microcirculation. Il se concentre principalement sur la margination en fonction du contrast de rigidité dans une suspension de globules rouges. L’expérience modale a été développée pour étudier la margination, causée exclusivement par le contraste de la déformabilité entre les deux sous-populations de globules rouges: les saines et les rigidifiées / This work was carried out in collaboration between I.R.P.H.E. (Institut de Recherche sur les Phénomènes Hors Équilibre), research unit of Aix-Marseille University and University of Saarland, Faculty of Experimental Physics (Naturwissenschaftlich-Technische Fakultät der Universität des Saarlandes) and aims to investigate microcirculatory hydrodynamics of blood in vitro. The study is dedicated to better understanding of complex collective phenomena that take place in microcirculation of blood through microfluidic in vitro experiments. It mainly focuses rigidity based margination in suspension of RBCs. For this purpose, model experiment was developed to examine margination caused exclusively by contrast of deformability between two sub-populations of RBCs

The acoustic microscope is an efficient non-invasive tool that can explore the acoustic properties and the related mechanical microstructure of a wide diversity of materials, including biomedical and biological samples, which are, nowadays, among the most intriguing targets for investigations. In the presented work, an acoustic microscope with vector contrast is used to image and characterize the acousto-mechanical properties of chitosan, an abundant natural derivative of chitin known to be a biodegradable, nontoxic and versatile biopolymer that suits many biomedical applications such as its usage in tissue engineering. The work also presents key measurements for the study of the acousto-mechanical properties that are subject to variations during the life span of red blood cells (RBCs). The characteristic signature of fixed cells from groups of three different ages, fractionated according to mass density, is obtained from the acoustic microscope images. The analysis of these data enabled the quantitative comparisons between the acousto-mechanical properties (velocity and attenuation of ultrasound propagating in the cells, mass density, and bulk modulus of compression). Comparison of the contrasts in the acoustic micrographs for the cells of the different age groups is exploited to generate a model that determines the age of the individual cells in a sample of red blood cells collected from a healthy person. The dependence of the parameters of the cells including density, velocity and attenuation of longitudinal polarized ultrasonic waves travelling in the cells on the age of the cell is also presented. The output signal in dependence on the thickness of the sample, the so called V(d), represented as polar graph was exploited as the method of analysis of the data extracted from the acoustic micrographs imaged with ultrasound of a center frequency of 1.2 GHz. This procedure allows for the extraction of the quantitative information from a single image in magnitude and phase contrast and allows for height profiling with so called super resolution, relating to resolution below the diffraction limit, based on the developed modeling, beside of other advantages concerning the acoustic characterization of biomedical and biological samples. This method and the applications are presented and discussed together with the developed or adapted modeling.

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