Scaling up Cryopreservation from Cell Suspensions to Tissues: Challenges and Successes

This chapter covers the key physical, biological and practical challenges encountered when developing cryopreservation protocols for larger biological structures and examines areas where cryopreservation has been successful in scaling to larger structures. Results from techniques being used in attempts to overcome these challenges are reviewed together with the indicators for future development that arise from them. The scale-up of cryopreservation to tissues with diverse functions and cell types makes the control of freezing and thawing more challenging. Technology may—or may not—be available depending on the size of the material involved. To meet the challenge there must be innovation in technology, techniques and understanding of damage-limiting strategies. Diversity of cell structure, size, shape and expected function means a similarly diverse response to any imposed cryopreservation conditions and interaction with ice crystals. The increasing diffusion distances involved, and diversity of permeability properties, will affect solutes, solvents, heat and cryoprotectant (CPA) transfer and so add to the diversity of response. Constructing a single protocol for cryopreservation of a larger sample (organoids to whole organs) becomes a formidable challenge

Automated dry thawing of cryopreserved haematopoietic cells is not adversely influenced by cryostorage time, patient age or gender.

Cell therapies are becoming increasingly widely used, and their production and cryopreservation should take place under tightly controlled GMP conditions, with minimal batch-to-batch variation. One potential source of variation is in the thawing of cryopreserved samples, typically carried out in water baths. This study looks at an alternative, dry thawing, to minimise variability in the thawing of a cryopreserved cell therapy, and compares the cellular outcome on thaw. Factors such as storage time, patient age, and gender are considered in terms of cryopreservation and thawing outcomes. Cryopreserved leukapheresis samples from 41 donors, frozen by the same protocol and stored for up to 17 years, have been thawed using automated, water-free equipment and by conventional wet thawing using a water bath. Post-thaw viability, assessed by both trypan blue and flow cytometry, showed no significant differences between the techniques. Similarly, there was no negative effect of the duration of frozen storage, donor age at sample collection or donor gender on post-thaw viability using either thawing method. The implication of these results is that the cryopreservation protocol chosen initially remains robust and appropriate for use with a wide range of donors. The positive response of the samples to water-free thawing offers potential benefits for clinical situations by removing the subjective element inherent in water bath thawing and eliminating possible contamination issues

Etude des stress liés au procédé de cryopréservation via une approche globale et multi-échelle chez la bactérie lactique Lactobacillus delbrueckii subsp. Bulgaricus

Cryopreservation leads to variable degradation of the biological activity and functionality among lactic acid bacteria (LAB), particularly Lactobacillus delbrueckii subsp. bulgaricus, a dairy starter of industrial relevance. The aim of this work was to identify cellular markers of cryoresistance or cryosensitivity for better understanding the mechanisms of cell cryoinjury and increasing LAB industrial performances. Cryopreservation was here considered as a combination of cold and osmotic stresses. A particular focus was given to the analysis of the cell membrane, recognised as a primary site of cryoinjury, but also of the cell wall and proteins. Moreover, cells were analysed from the population level down to the single-cell level to quantify the heterogeneity of cell properties within populations. In the first part of this work, bacterial cultivation conditions were compared to identify two L. bulgaricus strains with markedly different cell cryoresistance. Moreover, a comparative genomic analysis of the strains was performed to provide some clues for the explanation of their different behaviours. In the second part of this work, the membrane properties were evaluated in response to the cold and osmotic stresses: fatty acid composition, organisation of fatty acyl and phospholipid headgroups, and fluidity.Subcellular membrane fluidity was also characterised by fluorescence microscopy using synchrotron radiation, enabling the quantification of inter- and intra-cellular heterogeneities. Finally, original methodological and technical developments were undertaken to achieve the analysis of individual bacterial cells in an aqueous environment by Fourier transform infrared (FTIR) spectroscopy, for the analysis of the biochemical signature of cells under native conditions. These complementary multidisciplinary approaches revealed different properties and organisation of the membrane of both L. bulgaricus strains. It was proposed that different types of interaction between cryoprotectants of the extracellular matrix and the membrane of both strains could be at the origin of cryoinjury for the sensitive strain. Moreover, a high population heterogeneity characterised the cryosensitive strain, ascribed to differences in terms of biochemical composition and organisation of the membrane and cell wall. Altogether, this work suggests some cellular markers to evaluate LAB cryoresistance and provides methods to characterize population biochemical heterogeneity. These could be applied to any other stressful step of their production process, and should be useful for future production of homogeneous populations of resistant LAB.La cryopréservation engendre des dégradations variables de l’activité biologique et des fonctionnalités des bactéries lactiques, notamment chez Lactobacillus delbrueckii subsp. bulgaricus, un starter de l’industrie laitière. Le but de ce travail a été d’identifier les marqueurs cellulaires de cryorésitance et de cryosensibilité afin de mieux comprendre les mécanismes de dégradation sous-jacents et d’améliorer les performances industrielles des bactéries lactiques. La cryopresérvation a ici été considérée comme une combination de deux stress majoritaires : froid et osmotique. Une attention particulière a été portée à l’analyse de la membrane cellulaire, un site majeur de dégradation lié à la congélation, mais également à la paroi cellulaire et aux protéines. De plus, les cellules ont été analysées à différentes échelles d’observation, de la population jusqu’à la cellule unique, afin de quantifier l’hétérogénéité des propriétés cellulaires existant au sein de populations. Dans une première partie de ce travail, des conditions de culture ont été comparées pour identifier deux souches de L. bulgaricus présentant des résistances contrastées vis-à-vis de la congélation. Une analyse génomique comparative des souches a également été menée dans le but de fournir des pistes de compréhension de ces comportements différents. Dans une seconde partie, des propriétés membranaires des cellules ont été évaluées en réponse aux stress froid et osmotique : composition en acides gras, organisation au niveau des chaînes d’acides gras et des têtes phospholipidiques, et fluidité.Leur fluidité membranaire a également été caractérisée à une échelle subcellulaire par microscopie de fluorescence au moyen du rayonnement synchrotron, permettant la quantification des hétérogénéités inter- et intra-cellulaires. Enfin, un développement technique et méthodologique a été entrepris afin de permettre l’analyse de bactéries individuelles en milieu aqueux par spectroscopie infrarouge à transformée de Fourier, et ainsi leur signature biochimique en conditions natives. Ces approches complémentaires et multidisciplinaires ont révélé l’existence de propriétés et d’organisation différentes de la membrane des deux souches de L. bulgaricus. Différents types d’interaction entre les molécules cryoprotectrices du milieu extracellulaire et la membrane des deux souches a été proposé, pouvant être à l’origine des dommages causés à la souche sensible. De plus, une hétérogénéité plus importante au sein de la population sensible a été identifiée, attribuée à des différences en termes de composition biochimique et d’organisation au niveau de la membrane et de la paroi. Finalement, ce travail suggère quelques marqueurs cellulaires d’évaluation de la cryorésistance des bactéries lactiques, et fournit des méthodes de caractérisation de l’hétérogénéité biochimique au sein des populations. Ceux-ci pourraient être appliqués à l’étude de toute autre étape critique du procédé de production des bactéries lactiques, et pourraient être utiles pour aller vers la production de ferments homogènes au niveau de leur résistance

Biophysical characterization of the Lactobacillus delbrueckii subsp. bulgaricus membrane during cold and osmotic stress and its relevance for cryopreservation

International audienceFreezing lactic acid bacteria often leads to cell death and loss of technological properties. Our objective was to provide an in-depth characterization of the biophysical properties of the Lactobacillus delbrueckii subsp. bulgaricus membrane in relation to its freeze resistance. Freezing was represented as a combination of cold and osmotic stress. This work investigated the relative incidence of increasing sucrose concentrations coupled or not with subzero temperatures without ice nucleation on the biological and biophysical responses of two strains with different membrane fatty acid compositions and freeze resistances. Following exposure of bacterial cells to the highest sucrose concentration, the sensitive strain exhibited a survival rate of less than 10 % and 5 h of acidifying activity loss. Similar biological activity losses were observed upon freeze-thawing and after osmotic treatment for each strain thus highlighting osmotic stress as the main source of cryoinjury. The direct measurement of membrane fluidity by fluorescence anisotropy was linked to membrane lipid organization characterized by FTIR spectroscopy. Both approaches made it possible to investigate the specific contributions of the membrane core and the bilayer external surface to cell degradation caused by cold and osmotic stress. Cold-induced membrane rigidification had no significant implication on bacterial freeze-thaw resistance. Interactions between extracellular sucrose and membrane phospholipid headgroups under osmotic stress were also observed. Such interactions were more evident in the sensitive strain and when increasing sucrose concentration, thus suggesting membrane permeabilization. The relevance of biophysical properties for elucidating mechanisms of cryoinjury and cryoprotection is discussed

Physical events occurring during the cryopreservation of immortalized human T cells

Cryopreservation is key for delivery of cellular therapies, however the key physical and biological events during cryopreservation are poorly understood. This study explored the entire cooling range, from membrane phase transitions above 0°C to the extracellular glass transition at -123°C, including an endothermic event occurring at -47°C that we attributed to the glass transition of the intracellular compartment. An immortalised, human suspension cell line (Jurkat) was studied, using the cryoprotectant dimethyl sulfoxide. Fourier transform infrared spectroscopy was used to determine membrane phase transitions and differential scanning calorimetry to analyse glass transition events. Jurkat cells were exposed to controlled cooling followed by rapid, uncontrolled cooling to examine biological implications of the events, with post-thaw viable cell number and functionality assessed up to 72 h post-thaw. The intracellular glass transition observed at -47°C corresponded to a sharp discontinuity in biological recovery following rapid cooling. No other physical events were seen which could be related to post-thaw viability or performance significantly. Controlled cooling to at least -47°C during the cryopreservation of Jurkat cells, in the presence of dimethyl sulfoxide, will ensure an optimal post-thaw viability. Below -47°C, rapid cooling can be used. This provides an enhanced physical and biological understanding of the key events during cryopreservation and should accelerate the development of optimised cryobiological cooling protocols

Cryopreservation-related stresses in Lactobacillus delbrueckii SUBSP. Bulgaricus: Global and multi-scale study

Cryopreservation-related stresses in Lactobacillus delbrueckii SUBSP. Bulgaricus: Global and multi-scale study. 55. Annual Meeting of the Society for Cryobiolog

Complementary analytical approaches improving knowledge on lactic acid bacteria cryoresistance

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 777657Complementary analytical approaches improving knowledge on lactic acid bacteria cryoresistance. 56. Annual Meeting of the Society for Cryobiolog

Determination of intracellular vitrification temperatures for unicellular micro organisms under conditions relevant for cryopreservation

During cryopreservation ice nucleation and crystal growth may occur within cells or the intracellular compartment may vitrify. Whilst previous literature describes intracellular vitrification in a qualitative manner, here we measure the intracellular vitrification temperature of bacteria and yeasts under conditions relevant to cryopreservation, including the addition of high levels of permeating and nonpermeating additives and the application of rapid rates of cooling. The effects of growth conditions that are known to modify cellular freezing resistance on the intracellular vitrification temperature are also examined. For bacteria a plot of the activity on thawing against intracellular glass transition of the maximally freeze-concentrated matrix (Tg') shows that cells with the lowest value of intracellular Tg' survive the freezing process better than cells with a higher intracellular Tg'. This paper demonstrates the role of the physical state of the intracellular environment in determining the response of microbial cells to preservation and could be a powerful tool to be manipulated to allow the optimization of methods for the preservation of microorganisms