Insights into Species Preservation: Cryobanking of Rabbit Somatic and Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) are obtained by genetically reprogramming adult somatic cells via the overexpression of specific pluripotent genes. The resulting cells possess the same differentiation properties as blastocyst-stage embryonic stem cells (ESCs) and can be used to produce new individuals by embryonic complementation, nuclear transfer cloning, or in vitro fertilization after differentiation into male or female gametes. Therefore, iPSCs are highly valuable for preserving biodiversity and, together with somatic cells, can enlarge the pool of reproductive samples for cryobanking. In this study, we subjected rabbit iPSCs (rbiPSCs) and rabbit ear tissues to several cryopreservation conditions with the aim of defining safe and non-toxic slow-freezing protocols. We compared a commercial synthetic medium (STEM ALPHA.CRYO3) with a biological medium based on fetal bovine serum (FBS) together with low (0-5%) and high (10%) concentrations of dimethyl sulfoxide (DMSO). Our data demonstrated the efficacy of a CRYO3-based medium containing 4% DMSO for the cryopreservation of skin tissues and rbiPSCs. Specifically, this medium provided similar or even better biological results than the commonly used freezing medium composed of FBS and 10% DMSO. The results of this study therefore represent an encouraging first step towards the use of iPSCs for species preservation.

A chemically defined medium for rabbit embryo cryopreservation.

This study evaluates a new synthetic substitute (CRYO3, Ref. 5617, Stem Alpha, France) for animal-based products in rabbit embryo cryopreservation solutions. This evaluation was performed using two approaches: a thermodynamic approach using differential scanning calorimetry and a biological approach using rabbit embryo slow-freezing. During the experiment, foetal calf serum (FCS) was used as a reference. Because FCS varies widely by supplier, three different FCS were selected for the thermodynamic approach. The rabbit embryo slow-freezing solutions were made from Dulbecco's phosphate buffer saline containing 1.5 M Dimethyl Sulfoxide and 18% (v.v(-1)) of CRYO3 or 18% (v.v(-1)) of FCS. These solutions were evaluated using four characteristics: the end of melting temperature, the enthalpy of crystallisation (thermodynamic approach) and the embryo survival rates after culture and embryo transfer (biological approach). In the thermodynamic approach, the solutions containing one of the three different FCS had similar mean thermodynamic characteristics but had different variabilities in the overall data with aberrant values. The solution containing CRYO3 had similar thermodynamic properties when compared to those containing FCS. Moreover, no aberrant value was measured in the solution containing CRYO3. This solution appears to be more stable than the solutions containing a FCS. In the biological approach, the in vitro embryo survival rates obtained with the solution containing CRYO3 (73.7% and 81.3%) and with the solution containing a FCS (77.6% and 71.9%) were similar (p = 0.7). Nevertheless, during the in vivo evaluation, the implantation rate (21.8%) and the live-foetuses rate (18.8%) of the CRYO3 group were significantly higher than the implantation rate (7.1%, p = 0.0002) and the live-foetuses rate (5.3%, p = 0.0002) of the FCS group. The pregnancy rate was also higher in the CRYO3 group compared to the FCS group (81.3% and 43.8%, respectively, p = 0.066). We conclude that CRYO3 can be used as a chemically defined substitute for animal-based products in rabbit embryo cryopreservation solutions.

Effect of antibiotics on thermodynamic properties of freezing media in rabbit species: A first calorimetric approach.

Semen freezing is an effective and safe solution for the cryopreservation of animal genetic resources and for the diffusion of the genetic progress. Actually, these techniques are not yet under control for the rabbit species partly because methods are not clearly defined. Thus, the aim of this work is to study the effect of antibiotics (Penicillin G, Streptomycin) routinely used in freezing semen on the thermodynamic properties of freezing media mainly used in rabbit species. Measurements realized by differential scanning calorimetry show that these antibiotics may change the temperature of crystallization and the quantity of ice formed in the freezing media considered. Our calorimetric approach underlined that the composition and the properties of the cryoprotective solutions should be studied more precisely.

Cryopreservation of the ovary by vitrification as an alternative to slow-cooling protocols.

OBJECTIVE: To study the thermal properties of a cryoprotectant solution, called VS4, and of VS4-impregnated whole sheep ovaries with pedicle. DESIGN: Physical and experimental animal study. SETTING: Academic research environment. ANIMAL(S): Five- to 6-month-old ewes. INTERVENTION(S): Thermal properties on cooling of a cryoprotectant solution called VS4 were measured by differential scanning calorimetry. VS4 contains 2.75 mol/L dimethyl sulfoxide, 2.76 mol/L formamide, and 1.97 mol/L propylene glycol. Whole sheep ovaries were collected at the slaughterhouse and prepared for a vitrification procedure. Cortex and vessels underwent histologic examination before and after vitrification, and the thermal properties of VS4-impregnated ovarian cortex were then studied. MAIN OUTCOME MEASURE(S): Critical cooling rates (V(ccr)), vitreous transition temperature (Tg), end-of-melting temperature (Tm), follicle viability assessment by trypan blue test, and histologic examination of ovary and vessel structure. RESULT(S): The critical cooling rate (V(ccr)) of VS4 solution was estimated to be 14.3 +/- 1.1 degrees C/min. Its vitreous transition temperature (Tg) was -125.2 +/- 0.2 degrees C, and its end-of-melting temperature (Tm) -34.3 +/- 0.1 degrees C. Following our vitrification procedure, immediate follicle viability was 61.4% +/- 2.2%. The percentage of normal primordial follicles remaining after vitrification was 48% +/- 3.8%. The V(ccr) of VS4-impregnated cortex could not be determined because of the quantity of ice forming as of the top programmed cooling rate (-300 degrees C/min). The mean ovarian cortex cooling rate actually attained experimentally during vitrification was -342.9 +/- 49.6 degrees C/min. CONCLUSION(S): Vitrification of entire organs, such as ovaries, is a great challenge in cryobiology and reproductive medicine. Physical studies seem indispensable for progress with this technique. Thus, our ovarian perfusion procedure needs improving to enhance ovarian cortex impregnation and bring down the V(ccr) rate in both tissue and VS4 solution.

Freezing or supercooling: how does an aquatic subterranean crustacean survive exposures at subzero temperatures?

Crystallization temperature (T(c)), resistance to inoculative freezing (IF), ice contents, bound water, protein and glycogen body contents were measured in the aquatic subterranean crustacean Niphargus rhenorhodanensis and in the morphologically close surface-dwelling aquatic crustacean Gammarus fossarum, both acclimated at 12 degrees C, 3 degrees C and -2 degrees C. Cold acclimation induced an increase in the T(c) values in both species but no survival was observed after thawing. However, after inoculation at high sub-zero temperatures, cold-acclimated N. rhenorhodanensis survived whereas all others, including the 3 degrees C and -2 degrees C acclimated G. fossarum died. In its aquatic environment, N. rhenorhodanensis is likely to encounter inoculative freezing before reaching the T(c) and IF tolerance appears as a highly adaptive trait in this species. Bound water and glycogen were found to increase in the 3 degrees C and -2 degrees C acclimated N. rhenorhodanensis, whereas no variation was observed in G. fossarum. Considering the hydrophilic properties of glycogen, such a rise may be correlated with the increased bound water measured in cold-acclimated N. rhenorhodanensis, and may be linked to the survival of this species when it was inoculated. The ecological significance of the survival of the aquatic subterranean crustacean to inoculative freezing is paradoxical, as temperature is currently highly buffered in its habitat. However, we assume that past geographical distribution and resulting life history traits of N. rhenorhodanensis are key parameters in the current cold-hardiness of the species.

Thermal study of simple amino-alcohol solutions.

Widely regarded as the most promising approach to long-term cryopreservation of organs for transplantation, vitrification is a process where liquid is transformed into a disordered solid state free from crystals, known as the amorphous state. The vitreous state is obtained by rapid cooling to cryogenic temperatures in the presence of antifreeze substances called cryoprotectants, such as polyalcohols, which are known to be very good vitrification agents. This work reports on the thermal properties of a new class of compounds, the amino-alcohols, studied for its similarity to the structure of the equivalent polyalcohols. We studied by differential scanning calorimetry the glass-forming tendency and stability of the amorphous state for de-ionized water solutions containing 2-amino-1-ethanol and 3-amino-1-propanol at the concentrations of 35%, 40%, 43%, and 45% (w/w). A comparison is made with previous results obtained by Mehl [Cryobiology 27 (1990) 687-688] on the same compounds under different experimental conditions. The results are also compared with those obtained by Boutron [Cryobiology 30 (1993) 87-97] for the corresponding dialcohols. A further comparison is made with a few results obtained for the 1-amino-2-propanol and the 2-amino-1-propanol tested under the same conditions.