Cryobiology

of wild animals has been a formidable task: it is expensive, time consuming and even sometime harmful both for the animal and for the collector. From this standpoint, collection of the mortem (cadaveric, posthumous) sperm of recently died animal (Postmortem Semen Cryopreservation, PMSCP) for rare and endangered species can be a substantial addition, and sometime the method of choice in preservation of reproductive cells, both in the wild, at zoos and in national parks.

Applications for induced pluripotent stem cells are extensive and include basic biology, drug discovery, toxicological assessment, tissue regeneration, and cancer therapy, among many others. However, reprogramming of somatic cells is an intensive and time consuming process and karyotypic abnormalities are known to occur after extended periods in culture. Therefore, the ability to cryopreserve iPSCs with a high survival rate would be advantageous. Although stem cells are most frequently cryopreserved as small cell clumps in suspension, colony dissociation is known to cause cell loss and increased differentiation. Thus cryopreservation in the adherent state would alleviate any need to dissociate stem cells from the culture surface and prevent any damage that is a result of dissociation. Vitrification methods are particularly promising for cryopreservation of adherent cells because damage due to extracellular ice is prevented. However, vitrification methods require the use of high CPA concentrations which increase the risk of osmotic and toxic damage. Recently, we developed a rational design algorithm for designing toxicity-minimized CPA addition and removal procedures. (J.D. Benson, A.J. Kearsley, A.Z. Higgins, Cryobiology 64 To successfully apply these predicted procedures, accurate knowledge of cell biophysical parameters is required. The purpose of this study was to determine the necessary biophysical parameters for the rational design of CPA addition and removal procedures for vitrification of iPSCs. To determine membrane permeability parameters for human iPSCs, we adapted our calcein fluorescence quenching method (A.K. Fry, A.Z. Higgins, Cellular and Molecular Bioengineering 5 (2012) 287-298) for use with an automated plate reader. Permeability parameters were determined for dimethyl sulphoxide, ethylene glycol, glycerol, and propylene glycol at 4°C, 21°C, and 37°C. Most notably, glycerol permeation was significantly slower than the other CPA types. To determine the osmotic tolerance limits of iPSCs, cells were exposed to test solutions, which included varying concentrations of hypotonic buffer and hypertonic sucrose, for 15 mins. Cell yield was assessed 24 h after cells were returned to culture using PrestoBlueÒ. Osmotic tolerance limits were determined for single cell suspensions and adherent iPSCs at 4°C, 21°C, and 37°C. Using ANOVA analysis, the effect of the osmolality and temperature as well as the cross interaction between osmolality and temperature were significant for both hypotonic and hypertonic exposures. Also, the tolerable limits varied greatly depending on the test temperature. In particular, excessive cell volume changes were more damaging at 37°C than 4°C or 21°C. The permeability parameters and osmotic tolerance limits presented in this study enable rational design of CPA addition and removal procedures. The information in this study is an important step toward development of successful vitrification strategies for adherent human iPSCs.

Effects of curcumin as antioxidant in extender were evaluated on freezability of buffalo spermatozoa. Semen from each of the five bulls (n = 3 replicates, six ejaculates/bull, a total of 30 ejaculates) was diluted in Tris-citric acid extender containing curcumin (0.5, 1.0, 1.5 or 2.0 mM) or control. At pre-freezing and post-thawing, total antioxidant contents (μM/L) and lipid peroxidation levels (μM/ml) were higher (p < .05) and lower (p < .05) respectively, with 1.5 and 2.0 mM compared to 0.5 and 1.0 mM curcumin and control. At post-thawing, progressive motility (PM, %) and rapid velocity (RV, %) were higher (p < .05) with 1.5 mM compared to other doses of curcumin and control (except in case of RV, 1.5 was similar with 1.0 mM). Kinematics (average path velocity, μm/s; straight-line velocity, μm/s; curved-line velocity, μm/s; straightness, %; linearity, %), in vitro longevity (%, PM and RV) and DNA integrity (%) at post-thawing were higher (p < .05) with 1.5 mM compared to control. At post-thawing, supravital plasma membrane integrity (%) and viable spermatozoa with intact acrosome (%) were higher with 1.5 compared to 2.0 mM curcumin and control. We concluded that freezability of water buffalo spermatozoa is improved with the addition of 1.5 mM curcumin in extender.

Preservation of cell and tissue samples from endangered species is a part of biodiversity conservation strategy. Therefore, setting up proper cell and tissue cryopreservation methods is very important as these tissue samples and cells could be used to reintroduce the lost genes into the breeding pool by nuclear transfer. In this study, we investigated the effect of vitrification and slow freezing on cartilage cell and tissue viability for biobanking. Firstly, primary adult cartilage cells (ACCs) and fetal cartilage cells (FCC) were cryopreserved by vitrification and slow freezing. Cells were vitrified after a two-step equilibration in a solution composed of ethylene glycol (EG), Ficoll and sucrose. For slow freezing three different cooling rates (0.5, 1 and 2°C/min) were tested in straws. Secondly, the tissues taken from articular cartilage were cryopreserved by vitrification and slow freezing (1°C/min). The results revealed no significant difference between the viability ratios, proliferative activity and GAG synthesis of cartilage cells which were cryopreserved by using vitrification or slow freezing methods. Despite the significant decrease in the viability ratio of freeze-thawed cartilage tissues, cryopreservation did not prevent the establishment of primary cell cultures from cartilage tissues. The results revealed that the vitrification method could be recommended to cryopreserve cartilage tissue and cells from bovine to be used as alternative cell donor sources in nuclear transfer studies for biobanking as a part of biodiversity conservation strategy. Moreover, cartilage cell suspensions were successfully cryopreserved in straws by using a controlled-rate freezing machine in the present study.

Vitrification of sugar-based solutions plays an important role in cryopreservation, lyophilization, and the emerging field of anhydrous preservation. An understanding of the glass transition characteristics of such formulations is essential for determining an appropriate storage temperature to ensure an extended shelf life of vitrified products. To better understand the effect of salts on the glass transition temperature (T g ) of glass-forming sugars, we investigated several data-fitting models (Fox, Gordon-Taylor and Kwei) for sugar-salt formulations using data from the literature, as well as new data generated on blends of trehalose and choline dihydrogen phosphate (CDHP). CDHP has recently been shown to have promise as a stabilizing agent for proteins and DNA. The Kwei equation, which has a specific parameter characterizing intermolecular interactions, provides good fits to the T g data for sugar-salt blends, and complements other commonly used models that are frequently used to model T g data.

Les biobanques sont des infrastructures dédiées à la collecte, à la préparation et à la conservation au froid d’échantillons biologiques (sang, cellules, fragments de tumeurs, etc.) destinés à être utilisés pour la recherche biomédicale. Si les pratiques de conservation du vivant à des fins de connaissance sont très anciennes, le développement actuel de la médecine des données massives, personnalisée et de précision, rend indispensable la mise à disposition des chercheur.e.s d’échantillons biologiques standardisés et de haute qualité. Les biobanques ont ainsi acquis au cours des vingt dernières années une importance stratégique considérable.
Ce livre propose d’étudier les biobanques en croisant trois perspectives : celle de l’histoire des sciences, afin de comprendre comment les biobanques, tout en s’inscrivant dans l’histoire longue des collections du vivant, s’en démarquent pourtant ; celle de l’épistémologie, afin d’analyser la fabrique des connaissances biomédicales à partir des collections biologiques ; celle enfin de la philosophie des techniques, assumant que les échantillons gagnent à être étudiés comme de véritables objets techniques. Au carrefour de ces trois perspectives, il s’agit d’examiner les enjeux éthiques du biobanking ainsi que les relations inédites qui se nouent entre le vivant et la technique dans la biomédecine contemporaine. Pour cela, les études réunies ici font la part belle aux enquêtes de terrain ainsi qu’aux analyses des professionnels des biobanques.

The cryopreservation of algae could prevent genetic drift and minimize labor costs compared to the current method of maintenance and subculturing. Clear, simple protocols for cryopreservation of marine microalga, Nannochloropsis oculata were developed and cryoprotectant choice and concentration optimized. The viability of the microalga was assessed directly after thawing, and algal concentration was measured after 2-30 days of growth. Five cryoprotectants (dimethyl sulphoxide, Me 2 SO; ethylene glycol, EG; glycerol, Gly; methanol, MeOH; and propylene glycol, PG) at Wve concentrations (10, 20, 30, 40, and 50%; v/v) were evaluated to determine the toxicity of various cryoprotectants to N. oculata. The toxicity of cryoprotectant (Me 2 SO, EG, MeOH, and PG) was observed only at higher concentrations of CPAs: >20% for EG, >30% for Me 2 SO and methanol, and >40% for PG. Direct freezing of algae in liquid nitrogen resulted in a severe loss of viability and a modiWed cryopreservation protocol proved to be more appropriate for the preservation of N. oculata. Cryopreservation protocols developed and tested in the present study might be applied to cryopreserving other strains, or species, in this genus. 

The preservative effects of low temperature on biological materials have been long recognised, and cryopreservation is now widely used in biomedicine, including in organ transplantation, regenerative medicine and drug discovery. The lack of organs for transplantation constitutes a major medical challenge, stemming largely from the inability to preserve donated organs until a suitable recipient is found. Here, we review the latest cryopreservation methods and applications. We describe the main challenges-scaling up to large volumes and complex tissues, preventing ice formation and mitigating cryoprotectant toxicity-discuss advantages and disadvantages of current methods and outline prospects for the future of the field.

Vitrification of articular cartilage (AC) could enhance tissue availability but requires high concentrations of cyroprotective agents (CPAs). This study investigated relative injuries caused by commonly used CPAs. We hypothesized that the in situ chondrocyte dose-injury relationships of five commonly used CPAs are nonlinear and that relative injuries could be determined by comparing cell death after exposure at increasing concentrations. Human AC samples were used from four patients undergoing total knee arthroplasty surgery. Seventy lm slices were exposed in a stepwise protocol to increasing concentrations of 5 CPAs (max = 8 M); dimethyl sulfoxide (Me 2 SO), glycerol (Gly), propylene glycol (PG), ethylene glycol (EG), and formamide (FM). Chondrocyte viability was determined by membrane integrity stains. Statistical analysis included t-tests and nonlinear least squares estimation methods. The dose-injury to chondrocytes relationships for all CPAs were found to be nonlinear (sigmoidal best fit). For the particular loading protocol in this study, the data identified the following CPA concentrations at which chondrocyte recoveries statistically deviated significantly from the control recovery; 1 M for Gly, 4 M for FM and PG, 6 M for Me 2 SO, and 7 M for EG. Comparison of individual means demonstrated that Gly exposure resulted in the lowest recovery, followed by PG, and then Me 2 SO, FM and EG in no specific order. The information from this study provides an order of damage to human chondrocytes in situ of commonly used CPAs for vitrification of AC and identifies threshold CPA concentrations for a stepwise loading protocol at which chondrocyte recovery is significantly decreased. In general, Gly and PG were the most damaging while DMSO and EG were among the least damaging.

The latent heat during phase change of water-NaCl binary mixture was measured using a differential scanning calorimeter, and the magnitude for two distinct phase change events, water/ice and eutectic phase change, were analyzed considering the phase change characteristics of a binary mixture. During the analysis, the latent heat associated with each event was calculated by normalizing the amount of each endothermic peak with only the amount of sample participating in each event estimated from the lever rule for the phase diagram. The resulting latent heat of each phase change measured is 303.7 ± 2.5 J/g for water/ice phase change, and 233.0 ± 1.6 J/g for eutectic phase change, respectively regardless of the initial concentration of mixture. Although the latent heats of water/ice phase change in water-NaCl mixtures are closely correlated, further study is warranted to investigate the reason for smaller latent heat of water/ice phase change than that in pure water (335 J/g). The analysis using the lever rule was extended to estimate the latent heat of dihydrate as 115 J/g with the measured eutectic and water/ice latent heat values. This new analysis based on the lever rule will be useful to estimate the latent heat of water-NaCl mixtures at various concentrations, and may become a framework for more general analysis of latent heat of various biological solutions.

An ecomorphological analysis of the skeletal remains of large canids, Canis ( Xenocyon ) falconeri and Canis etruscus (Mammalia, Carnivora, Canidae), preserved in an assemblage of large mammals from the lower Pleistocene site at Venta Micena (Guadix-Baza Basin, Orce, Granada, southeastern Spain) is reported. Mean body mass of adult individuals was estimated to be around 10 kg for C. etruscus and approximately 28 kg for C. falconeri using multiple regression. A comparative study of tooth measurements in modern canids, using principal components and discriminant function analysis, infers quite different ecomorphological adaptations and feeding behavior for both fossil species. The craniodental morphology of C. falconeri is similar to that shown by extant hypercarnivorous canids whose diet include more than 70% of vertebrate meat, whereas C. etruscus shows a cranial morphology similar to those of modern omnivorous species, thus indicating a dietary niche in which vertebrate meat represented less than 70% of its diet, with other feeding resources making up the balance. These results suggest that there was a marked ecological segregation between both sympatric species of large canids. The find of a complete skull of C. falconeri showing bilateral asymmetry and marked dental anomalies could suggest high levels of genetic homozygosis in the population which inhabited this region during early Pleistocene times, possibly as a consequence of isolation and the low number of individuals. This may have subsequently led to the extinction of C. falconeri in the Western fringe of Europe. The survival of this pathological individual to adulthood indicates that this species may have developed cooperative behavior similar to that of modern African wild dogs. 0 C ANIS ( X ENOCYON ) FALCONERI , C ANIS ETRUSCUS , ecomorphology, principal component analysis, discriminant analysis, cranial fluctuating asymmetry, Venta Micena, lower Pleistocene. Paul Palmqvist [ Paul.Palmqvist@uma.es ] ,

Fertilizability of cryopreserved and cadaveric fish spermatozoa was attempted in the freshwater catfish Pangasius sutchi. Cryopreservation of spermatozoa was done with three cryoprotectants for short time storage (30 days). Whereas the spermatozoa obtained from the cadaveric fish were stored at À20°C (30 days) without any cryoprotectants. Cryoprotectant toxicity assay showed maximum motility of 88.53 AE 2.01% and viability of spermatozoa (96.19 AE 4.92%) with 15% of Dimethyl acetamide (DMA) at 15 min equilibration time. Whereas Dimethyl sulfoxide (DMSO) (15%) registered moderate level of motility and viability 79.23 AE 2.02% and 80.89 AE 2.1%, respectively. However, the methanol (MeOH) (20%) resulted in low percentage of motility (58.6 AE 0.9%) and viability (68.6 AE 0.9%). Scanning electron micrographs further showed no significant deformity on the surface topography of spermatozoa of cadaveric fish as well as cryopreserved with DMA (15%). The results indicated that 15% of DMA with hanks balanced salt solution (HBSS) extender at a dilution ratio of 1:10 at À80°C proved to be suitable for cryopreservation of spermatozoa in P. sutchi. This may be due to the osmolality of HBSS similar to seminal plasma of P. sutchi. Further studies on motility, viability and fertility potential of spermatozoa revealed 73.62 AE 1.61%, 88.34 AE 1.05% and 54 AE 2.2%, respectively, with DMA (15%). On the other hand, cadaveric fish sperm registered 57.12 AE 2.32%, 63.45 AE 0.94% and 25.33 AE 1.53% of motility, viability and fertilizability respectively. Thus, this study augments the feasibility of using cryopre-served as well as cadaveric fish spermatozoa for the seedling production in the fresh water catfish P. sutchi.