Cryopreservation Procedures Research Articles

An Impedance Measurement Platform for Cryopreservation Applications

One of the beneficial methods being widely used to preserve genetic resources of imperiled animals or improved agricultural animals is cryopreservation of germplasm. During this process, the biological structure of the cells remains relatively intact by freezing and storing them at very low temperatures. Freezing condition plays an essential role in cryopreservation procedure since the exposure of the cells to low temperature can cause damage to the cells [1]. Therefore, accurate monitoring of the freezing process can improve the recovery rate of the cells after thawing. During the phase change phenomena, the dielectric and conduction properties of ice changes [2]. Herein we present, a portable platform for monitoring of the freezing and thawing processes based on the electrical impedance of the samples during cryopreservation. The system is composed of a portable temperature and impedance measurement system in conjunction with an impedance sensing probe fabricated with printed circuit board (PCB) technology. The impedance measurement circuit, temperature measurement circuit, control unit for data acquisition and wireless communication and touch screen LCD with a graphical user interface are the building blocks of the measurement system that enables users to perform impedance measurement at single or multiple frequencies with various excitation signals (Figure 1A). Utilizing impedance measurement instead of DC resistance measurement enable us to monitor both dielectric properties and resistance changes during phase change and avoids the polarization of the electrodes. In addition, an impedance sensing probe was developed by using PCB technology, which provides accurate electrodes fabrication at low cost. The probe has been designed to be used within 0.5 ml French straw which is a conventional container in cryopreservation application. Pair of parallel electrodes were fabricated on the probe at three detection zones (Figure 1B). The system was evaluated at different cryopreservation conditions including various sample solutions and cooling rates. Hank's Balanced Salt Solution (HBSS) is being widely used in cryopreservation to maintain cells in a viable state. The system was characterized by measuring the impedance in HBSS and DI water solutions solution. The 0.5 ml French straws were filled sample solution along with the probe and a T-type thermocouple. Next, the samples were placed on top of the liquid nitrogen with a 2 cm gap to freeze, and after the sample temperature reached lower than -40 oC, the samples were left at room temperature to thaw. During this process, the impedance and temperature were measured inside the samples. Figures 1C and 1D show the magnitude and phase of the measured impedance for HBSS and DI water sample respectively. When the HBSS sample starts to freeze, the impedance magnitude does not change before the latent heat removal stage. However, the impedance phase starts to increase after the sample cools down. It reaches a minimum value during nucleation and increases during latent heat removal. Therefore, the phase change phenomena can be detected with the imaginary part of the impedance. In the DI water case, the impedance starts to increase by cooling down the solution when the nucleation happens there it remains relatively constant, and it keeps increasing after that. This shows that the solution containing various ions has different impedance characteristics, which can be used as a parameter to monitor the cryopreservation phenomena. The system was also characterized by different solutions containing salts and cryoprotectants, frozen at various cooling rates, to further investigate the potential of impedimetric sensors in quality control for cell freezing protocols. Figure 1

GSTM3, but not IZUMO1, is a cryotolerance marker of boar sperm

BackgroundCryopreservation is currently the most efficient method for long-term preservation of mammalian gametes and is extensively used in swine artificial insemination (AI) centres. However, it is well-known that cryopreservation procedures induce changes in the water phase in both intra and extracellular compartments, which alter the content and localisation of several proteins and ends up curtailing the structural integrity of functional sperm (i.e., cryoinjuries). Alterations and deficiencies of sperm-oocyte binding proteins during gamete recognition are one of the causes of reproductive failure both in vitro and in vivo. In this sense, characterisation of cryopreservation effects upon oocyte-binding proteins of sperm, such as IZUMO1 and GSTM3, is essential when assessing the impact of this technique in swine reproduction.ResultsCryopreservation was found to induce changes in the localisation of IZUMO1 and GSTM3 in boar sperm. However, the relative content of both proteins was not altered after thawing. Furthermore, whereas IZUMO1 content was found not to be related to the cryotolerance of boar sperm, GSTM3 content was observed to be higher in poor (PFE) than in good (GFE) freezability ejaculates in both pre-frozen (1.00 INT·mm2 ± 0.14 INT·mm2 vs. 0.72 INT·mm2 ± 0.15 INT·mm2; P < 0.05) and post-thawed (0.96 INT·mm2 ± 0.20 INT·mm2 vs. 70 INT·mm2 ± 0.19 INT·mm2; P < 0.05) samples. Moreover, GSTM3 levels were found to be higher in those spermatozoa that exhibited low mitochondrial activity, high reactive oxygen species (ROS) production, and high membrane lipid disorder post-thaw (P < 0.05).ConclusionsThe difference in GSTM3 content between GFE and PFE, together with this protein having been found to be related to poor sperm quality post-thaw, suggests that it could be used as a cryotolerance marker of boar spermatozoa. Furthermore, both IZUMO1 and GSTM3 relocate during cryopreservation, which could contribute to the reduced fertilising capacity of frozen-thawed boar sperm.

In vitro survival of follicles in prepubertal ewe ovarian cortex cryopreserved by slow freezing or non-equilibrium vitrification.

Vitrification is a well-accepted fertility preservation procedure for cryopreservation of oocytes and embryos but little is known regarding ovarian tissue, for which slow freezing is the current convention. The aim of the present study was to assess the efficiency of non-equilibrium vitrification compared to conventional slow freezing for ovarian cortex cryopreservation. Using prepubertal sheep ovaries, the capacity of the tissue to sustain folliculogenesis following cryopreservation and in vitro culture was evaluated. Ovarian cortex fragments were cultured in wells for 9days, immediately or after cryopreservation by conventional slow freezing or non-equilibrium vitrification in straws. During culture, follicular populations within cortex were evaluated by histology and immunohistochemistry for PCNA and TUNEL. Steroidogenic activity of the tissue was monitored by assay for progesterone and estradiol in spent media. No significant differences in follicle morphology, PCNA, or TUNEL labeling were observed between cryopreservation methods at the initiation of culture. Similar decreases in the proportion of primordial follicle population, and increases in the proportion of growing follicles, were observed following culture of fresh or cryopreserved ovarian tissue regardless of cryopreservation method. At the end of culture, PCNA and TUNEL-positive follicles were not statistically altered by slow freezing or vitrification in comparison to fresh cultured fragments. Overall, for both cryopreservation methods, the cryopreserved tissue showed equal capacity to fresh tissue for supporting basal folliculogenesis in vitro. Taken together, these data confirm that both non-equilibrium vitrification and slow-freezing methods are both efficient for the cryopreservation of sheep ovarian cortex fragments.

A Japanese nationwide survey on the cryopreservation of embryos, oocytes and ovarian tissue for cancer patients.

The survival rates of cancer patients have greatly improved owing to the advances in oncology. The preservation of fertility in cancer patients is an important task. To determine the reality of cryopreservation of embryos, oocytes and ovarian tissue in cancer patients, large-scale survey analysis was performed in Japan. We sent 613 Japan Society of Obstetrics and Gynecology-certified assisted reproductive technology institutions a questionnaire about their experience of performing cryopreservation for cancer patients between January 2011 and December 2015. Subsequently, the institutions that conducted cryopreservation for cancer patients were sent a second questionnaire. We received replies from 481 (78.5%) institutions. Among them, 126 (26.2%) conducted cryopreservation for cancer patients. These 126 institutions were sent a second questionnaire. Of these, 108 (85.7%) institutions responded. At the 108 institutions, 1085 embryo or oocyte cryopreservation procedures and 122 ovarian tissue cryopreservation procedures were conducted for cancer patients. Cryopreservation was mainly performed for breast cancer patients (~70%), followed by patients with hematological malignancy. A total of 361 and 19 embryo transfer cycles were performed for patients whose embryos and oocytes were cryopreserved, respectively, and 42 and seven institutions reported pregnancy outcomes after embryo transfer in patients that underwent embryo and oocyte cryopreservation, respectively. However, pregnancy was not observed in the seven cases that underwent ovarian tissue transfer. Indications, age limits and ovarian stimulation protocols for cryopreservation widely varied between the institutions. A national registration system for oncofertility must be established to evaluate the safety and efficacy of the current system.

Effects of cryopreservation on cAMP-dependent protein kinase and AMP-activated protein kinase in Atlantic salmon (Salmo salar) spermatozoa: Relation with post-thaw motility

Sperm motility in fish with external fertilization is critical for reproductive efficiency in aquaculture, especially in salmonids. Gamete preservation techniques, such as cryopreservation, however, reduce sperm motility and fertilizing capacity. Very few studies have addressed cryodamage from energetic and cell signalling approaches. In this study, cAMP-dependent protein kinase (PKA) and AMP-activated kinase (AMPK) activities were quantified in fresh and cryopreserved spermatozoa of Atlantic salmon (Salmo salar); and the relation with motility was analysed. Results indicate there was a decrease in membrane integrity and motility in post-thawed spermatozoa compared to fresh samples, however, there was about 30% of cells with intact plasma membrane but incapable of motility. The PKA and AMPK activities were less after cryopreservation, indicating that loss of motility may be related to alteration of these key enzymes. Furthermore, PKA and AMPK activities were positively correlated with each other and with motility; and inhibition decreased motility, indicating there is a functional relationship between PKA and AMPK. The PKA inhibition also decreased AMPK activity, but results from protein-protein docking analyses indicated AMPK activation directly by PKA is unlikely, thus an indirect mechanism may exist. There have been no previous reports of these kinase actions in fish spermatozoa, making these findings worthy of assessment when there are future studies being planned, and may serve as base knowledge for optimization of cryopreservation procedures and development of biotechnologies to improve reproduction efficiency in the aquaculture industry.

Phenotypical and functional analysis of donor lymphocyte infusion products after long-term cryopreservation

Donor lymphocyte infusions, collected from peripheral blood by apheresis, are regularly used to re-establishing disease control in patients with impending or full relapse after allogeneic cell transplantation. The cryopreservation and thawing processes of the cellular products, required for clinical needs, result in a decreased cellular recovery. The aim of this study was to perform an integral analysis of phenotypic and functional characteristics in different cell populations, within cryopreserved products used for therapeutic purposes.A total of 77 cryopreserved products were analysed. Cell viability and subpopulations such as CD3, CD4, CD8, CD14 and CD56 cells were quantified by FACS. Cell proliferation, cytotoxic capacity and CD4 intracellular ATP content were evaluated. A significant loss of cell viability was observed. CD56 cells were significantly reduced when compared with mononuclear cells without cryopreservation. Cell proliferation was also significantly reduced in the cryopreserved products. Cytotoxic capacity was decreased as well although it did not reach statistical significance. However, CD4 intracellular ATP was increased in the cryopreserved products. The analysed functional cell properties showed a wide distribution range although the apheresis, cryopreservation and thawing procedures were similar in all the analysed samples. Our findings may be useful for an improved characterisation of cryopreserved products to be used as donor lymphocyte infusion for therapeutic purposes.

Effect of Fractionated Seminal Plasma on Sperm Characteristics Following Cryopreservation of Boar Semen

Abstract This study aimed to investigate the effect of fractionated seminal plasma (SP) on boar sperm characteristics following cryopreservation. Gel filtration chromatography yielded two fractions: SP1 with more than 40 kDa (&gt;40 kDa) and SP2 with less than 40 kDa (&lt;40 kDa). The fractionated SP (SP1 and SP2), whole seminal plasma (wSP) and Beltsville Thawing Solution (BTS) were used for the treatment of semen before freezing-thawing. Besides the analysis of sperm motility characteristics, plasma membrane integrity (PMI), acrosome integrity, and viability (Vybrant Apoptosis Assay) were analyzed in pre-freeze and post-thaw (PT) semen. Among the analyzed pre-freeze sperm parameters, rapid movement was markedly affected by boar and treatment. Furthermore, boar and treatment were significant sources of variations in PT semen quality. Treatment with wSP caused a marked reduction in PT semen quality compared with BTS, SP1 or SP2. Wide variations in PT acrosome integrity and viability were observed in spermatozoa treated with BTS and the fractionated SP, being significantly higher in the SP1- and SP2-treated samples. However, PT semen quality did not differ between semen samples treated with SP1 and SP2. Representative electrophoretic profiles of sperm proteins from each treatment showed quantitative and qualitative differences, indicating varying effects of the cryopreservation procedure on the sperm membrane integrity. The findings of this study indicated that the cryoprotective effects of the SP components varied among boars and that different components of the fractionated SP exerted varying effects on sperm functions following cryopreservation. It could be suggested that the variable protective protein components of either fractionated SP ameliorated alterations in the sperm membranes during cryopreservation, resulting in reduced susceptibility to cryo-damage.

Hair-follicle-associated pluripotent stem cells derived from cryopreserved intact human hair follicles sustain multilineage differentiation potential

The bulge area of the hair follicle contains hair-follicle-associated pluripotent (HAP) stem cells. Here, we present effective cryopreservation procedures of the human hair follicle that preserve the differentiation potential of HAP stem cells. Whole hair follicles isolated from human scalp were cryopreserved by a slow-rate cooling medium and stored in liquid nitrogen. A careful thawing method was used to collect the upper parts of the human hair follicles which were cultured for four weeks in a Dulbecco’s Modified Eagle’s Medium with fetal bovine serum (FBS). Proliferating hair follicle cells were then shifted to DMEM/Ham’s Nutrient Mixture F-12 medium without FBS and allowed to grow for one week. These proliferating cells were able to produce HAP stem cell colonies with multilineage differentiation capacity. They produced keratinocytes, smooth muscle cells, cardiac muscle cells, neurons and glial cells. Interestingly, these cryopreserved hair follicles produced pluripotent HAP stem cell colonies similar to fresh follicles. These findings suggest that the cryopreserved whole human hair follicle preserves the ability to produce HAP stem cells, which will enable any individual to preserve a bank of these stem cells for personalized regenerative medicine.

The cryoprotectant PVS2 plays a crucial role in germinating Passiflora ligularis embryos after cryopreservation by influencing the mobilization of lipids and the antioxidant metabolism

Cryopreservation is a process whereby biological structures are preserved in liquid nitrogen (−196 °C) without losing their viability. Many cryopreservation techniques use the Plant Vitrification Solution 2 (PVS2) for cryoprotection. This study will therefore evaluate the influence of different exposure times to the cryoprotectant PVS2 and discuss the importance of the mobilization of reserves and the antioxidant metabolism during the germination of cryopreserved Passiflora ligularis embryos. The composition of P. ligularis seeds was analytically determined. We tested the germination capacity and the Germination Speed Index (GSI) of embryos (that is, seeds without external tegument) which were exposed to different PVS2 exposure times (0, 30, 60 and 120 min) at 30 days after thawing. Proline content, hydrogen peroxide, activity of isocitrate lyase (ICL), malate synthase (MSy), lipid peroxidation and antioxidant enzyme activities (SOD, CAT, APX) were measured at 7, 14 and 21 days after cryopreservation. The germination from cryopreserved embryos was maximal (85%) after 60 min PVS2 exposure with a GSI of 0.6. At 60 min, the highest activity of the enzymes involved in the glyoxylate cycle, ICL and MSy were recorded. We hypothesize that a 60 min exposure to PVS2 accelerates the reserve mobilization which correlates positively with germination. Until 60 min, there was a positive correlation between the PVS2 exposure time and the proline content, as well as the activity of antioxidant enzymes (SOD, CAT, APX), and a negative correlation with the lipid peroxidation. This study enables us to optimize the long-term conservation of this species. In conclusion, fundamental research is necessary to optimize the cryopreservation procedure, and this study offers an effective and efficient workflow which can be extrapolated to other (oil-rich) species.

Cryopreservation of grapevine (Vitis spp.) shoot tips from growth chamber-sourced plants and histological observations

Many genebanks rely on cryopreservation as a method to preserve vulnerable field collections of vegetatively propagated crops. Effective cryopreservation procedures have been identified for Vitis; however, they usually use in vitro plantlets as the shoot tip source materials. It is costly to establish Vitis collections in vitro prior to cryopreservation. We sought to determine if growth chamber derived Vitis plants could serve as the source of shoot tips for cryopreservation. Nodal sections from growth chamber derived plants were surface-disinfected and placed in tissue culture on pre-treatment medium for 2 weeks. Uniform apical shoot tips (1 mm) were first obtained from the nodal sections and then precultured for 3 days on medium containing 0.3 M sucrose, salicylic acid, glutathione (reduced form), ascorbic acid and plant preservative mixture. Half-strength PVS2 was applied for 30 min at 22 °C, prior to full-strength PVS2 treatment at 0 °C. Cryopreserved shoot tips had the highest average regrowth of 50 and 55 % without and with cold-acclimation followed with a full-strength PVS2 exposure duration of 40 and 30 min at 0 °C, respectively. This cryopreservation protocol achieved high percentages of regrowth in V. vinifera 'Chardonnay' and 'Riesling' and V. hybrid 'Oppenheim'. Histological observations revealed that shoot tips from growth chamber plants had apical as well as multiple lateral meristems that survived LN immersion. The preservation of multiple meristems in each shoot tip may increase the capacity of shoot tip regeneration in cryopreserved Vitis that originates from ex vitro sources. The high percentage of regrowth after shoot tip cryopreservation using Vitis shoot tips derived from growth chamber source plants suggest that it may be possible to cryopreserve Vitis shoot tips without first introducing each accession into tissue culture.

Effect of the desiccation tolerance and cryopreservation methods on the viability of Citrus limon L. Burm cv. Eureka seeds

Cryopreservation of the germplasm for long-term periods is of great importance to maintain the genetic resource. Argentina is one of the world's highest lemon producing country. The performance of different cooling/warming rates in the cryopreservation method of Citrus limon L. Burm cv. Eureka seeds and their influence on the interval of optimal moisture content in the desiccation stage were analyzed. Water sorption isotherm was determined and modeled using D'Arcy & Watt equation; it provided important information concerning the amounts of water associated to strong, weak and multimolecular binding sites along the sorption isotherm. Seeds tolerated a wide range of desiccation conditions (0.1<aw<0.85) showing a high viability (>80%), however desiccation to 0.0526 g H2O g−1 d.b. (aw = 0.0901) produced a significant loss of viability. Differential Scanning Calorimetry was used to identify the thermal transitions of lipids and water in the seed; enthalpies were used to calculate the unfrozen water fraction (0.19 g H2O g−1 d.b. corresponding to aw = 0.64). Two cooling/warming rates were tested on desiccated seeds (0.11<aw<0.85): i) 200 °C min−1 (reached with seeds placed inside a closed cryogenic vial); ii) 1000 °C min−1 (reached with aluminum-foiled seeds placed in a perforated cryogenic vial). For both methods, viability was maximum (83.3%) at aw = 0.64. Lethal ice formation was responsible for the loss of viability at aw>0.64 corresponding to the unfrozen water fraction. The use of higher cooling/warming rates enables a wider range of desiccation conditions (0.33<aw<0.76) in cryopreservation procedures. This work contributes to the optimization of cryopreservation methods of economically important germplasm.

Survivability of rabbit amniotic fluid-derived mesenchymal stem cells post slow-freezing or vitrification

This work aimed to evaluate the effect of two distinct cryopreservation procedures - conventional slow-freezing and vitrification, on survivability and mesenchymal marker expression stability of rabbit amniotic fluid-derived mesenchymal stem cells (rAF-MSCs). Cells at passage 2 were slowly frozen, using 10% of dimethylsulfoxide, or vitrified, using 40% of ethylene glycol, 0.5 M sucrose and 18% Ficoll 70. After three months storage in liquid nitrogen, viability, chromosomal stability, ultrastructure, surface and intracellular marker expression and differentiation potential of cells were evaluated immediately post-thawing/warming and after additional culture for 48–72 h. Our results showed decreased (P ≤ 0.05) viability of cells post-thawing/warming. However, after additional culture, the viability was similar to those in fresh counterparts in both cryopreserved groups. Increase (P ≤ 0.05) in the population doubling time of vitrified cells was observed, while doubling time of slow-frozen cells remained similar to non-cryopreserved cells. No changes in karyotype (chromosomal numbers) were observed in frozen/vitrified AF-MSCs, and histological staining confirmed similar differentiation potential of fresh and frozen/vitrified cells. Analysis of mesenchymal marker expression by qPCR showed that both cryopreservation approaches significantly affected expression of CD73 and CD90 surface markers. These changes were not detected using flow cytometry. In summary, the conventional slow-freezing and vitrification are reliable and effective approaches for the cryopreservation of rabbit AF-MSCs. Nevertheless, our study confirmed affected expression of some mesenchymal markers following cryopreservation.

Phospholipase C zeta 1 mRNA as a marker of oocyte-activation and fertilization potential of water buffalo (Bubalus bubalis) semen

The present study aimed to detect and quantify the expression of buffalo PLCZ1 mRNA (buPLCZ1 mRNA) in buffalo semen and to determine its oocyte-activation and fertilizing ability through IVF. Buffalo semen samples were collected and subjected to standard subjective physical and microscopic evaluation of semen characteristics. Small amount of fresh semen sample was taken for molecular experiment. The rest of the samples were processed into frozen semen. Both fresh and frozen semen were subjected to Reverse Transcriptase quantitative PCR (RT-qPCR) technique to determine and quantify buPLCZ1 mRNA expression. Subsequently, 40 bulls with known buPLCZ1 mRNA content were evaluated for its oocyte-activation activity through IVF using frozen semen. Correlation analysis was done to determine the relationship between the expression of buPLCZ1 mRNA and percentage of IVF, and other semen variables. PLCZ1 mRNA expression in buffalo semen was observed variable among donor bulls, but was not significantly different (P > 0.05) between fresh and frozen semen. The result suggests that the cryopreservation procedure does not affect the expression of buPLCZ1 mRNA in semen. Statistical analysis showed that the expression of buPLCZ1 mRNA in fresh semen was strongly correlated with that of frozen semen (r = 0.992; P < 0.001). Similarly, expression of buPLCZ1 mRNA in frozen semen was positively correlated with percentage of IVF (r = 0.776; P < 0.001), but not with Initial (r = −0718; P > 0.05) nor with Post-Thaw Motility (r = −0.0313; P > 0.05). A strong relationship between buPLCZ1 mRNA concentration and IVF rate indicates that buPLCZ1 mRNA concentration can be used to assess oocyte-activation and fertilizing potential of the buffalo semen. The present study essentially demonstrated buPLCZ1 mRNA as biological marker for male fertility and that molecular technique serves as an objective approach of semen quality evaluation to enhance bull selection for genetic improvement in water buffaloes.

NEP-TC a rRNA Methyltransferase Involved on Somatic Embryogenesis of Tamarillo (Solanum betaceum Cav.).

Somatic embryogenesis (SE) is an important biotechnological tool for large-scale clonal propagation and for embryogenesis research. Moreover, genetic transformation and cryopreservation procedures in many species rely on efficient SE protocols. We have been studying different aspects related to SE induction and somatic embryo development in tamarillo (Solanum betaceum Cav.), a small tree from the Solanaceae family. Previous proteomic analyses identified a protein (NEP-TC, 26.5 kDa) consistently present in non-embryogenic calluses of tamarillo, but absent in the embryogenic ones. In this work, the role of NEP-TC during SE was assessed by gene expression analysis and immunolocalization. The results obtained demonstrated that NEP-TC is a putative member of the SpoU rRNA methylase family. This protein, present in the cytoplasm and nucleus, is expressed in non-embryogenic cells and not expressed in embryogenic cells. Slightly enhanced SE induction levels in tamarillo plants with NEP-TC down-regulated levels also supports the role of this protein on SE induction. Heterologous expression was used to confirm NEP-TC rRNA methyltransferase activity, with enhanced activity levels when rRNA was used as a substrate. These data relate a putative member of the SpoU methylase family with plant morphogenesis, in particular with SE induction.

Opportunities and challenges related to the implementation of sperm cryopreservation into breeding of salmonid fishes

The main aim of the present review is to present the opportunities and challenges associated with the application of cryopreserved sperm, which may improve the breeding of salmonid fishes. Cryopreservation of sperm has been used as a strategy for the conservation of biodiversity of fishes populations, the preservation of sperm from the most valuable breeding individuals and facilitate transportation of genomes, and providing a biological source of sperm regardless of the synchronisation of the maturity of broodstocks. Cryopreserved sperm can be used for the genetic improvement of salmonid fishes based on the programs of individual crossing of selected males with individual females. However, these opportunities have not yet been fully implemented at the conditions of hatchery practice. Despite the significant progress concerning the standardization of cryopreservation procedures, there are still more challenges than opportunities related to the implementation of sperm cryopreservation into breeding of salmonid fishes. The main challenge concerns the scaling up of the method towards fulfilling the requirements of fishes-breeders, in particular mass production of eyed eggs and fry. The present review shows knowledge gaps that should be considered in further studies, including development of methods to obtain sufficient amounts of sperm from numerous species of salmonids, scaling up the methods towards cryopreservation of high volumes of sperm and towards thawing high number of straws, and optimizing artificial fertilization in which oocytes are fertilized with high numbers of frozen/thawed sperm. Moreover, the implementation of technologies into hatchery practice will require special consideration to counteract the risk of sperm infection and its transmission to offsprings during cryopreservation and storage in liquid nitrogen.

Holm Oak Somatic Embryogenesis: Current Status and Future Perspectives.

Quercus ilex (holm oak) is one of the most representative trees in the Mediterranean basin, but now the sustainability of its ecosystems is at serious risk due to the lack of natural regeneration and to the presence of a severe disease called oak decline that has caused the death of thousands of trees. The application of biotechnological tools, such as somatic embryogenesis, allows programs of genetic improvement of the species to be speeded up and helps in the conservation of its ecosystems. Somatic embryogenesis is currently considered one of the main biotechnological techniques that has demonstrated significant benefits when has applied to forest tree species, providing significant advantages such as mass propagation, genetic transformation, application of synthetic seed, and cryopreservation of elite genotypes. In this report, the state of the art of somatic embryogenesis in holm oak is reviewed. Factors affecting the induction (plant donor age, type of explant, or plant growth regulators) and maintenance and proliferation of the embryogenic cultures are summarized. Advances on the conversion of somatic embryos into plants and on the acclimatization of these plantlets, as well as the results obtained on the application of the genetic transformation and the cryopreservation procedures to holm oak embryogenic cultures, are also presented.

Autotransplantation of Cryopreserved/Thawed Ovarian Tissue: Overview of Methods and Challenges

In recent years, an increasing number of women diagnosed with malignant or non-malignant diseases have been subjected to cytotoxic chemoradiotherapy. Women who face the possibility of premature or imminent ovarian failure caused by cytotoxic therapy may retain their fertility potential via ovarian tissue cryopreservation. Despite its advantages, this fertility preservation method for women at risk of losing reproductive function is considered experimental due to 1) a possible decrease in the ovarian follicular pool as a result of cryopreservation and thawing procedures (although this has been minimised by improved methods), or by ischemic damage occurring during ovarian transplantation; and 2) the risk of minimal residual disease (which is the most important factor). Although the indications for ovarian cryopreservation now extend beyond cancer, cancer survivors remain as the patient population who most commonly need this procedure. For these patients, the risk of minimal residual disease, which can be defined as reintroducing pre-existing cancer cells, is an important challenge for the application of this method. Even though the risk of reimplanting pre-existing cancer cells through ovarian transplantation is minimal or non-existent for most types of cancer, this risk must be ascertained according to cancer type and disease stage.

Experimental Strategies of Mesenchymal Stem Cell Propagation: Adverse Events and Potential Risk of Functional Changes.

Mesenchymal stem cells (MSCs) are attractive candidates for cell-based tissue repair approaches. Hundreds of clinical trials using MSCs have been completed and many others are still being investigated. For most therapeutic applications, MSC propagation in vitro is often required. However, ex vivo culture condition is not fully physiological and may affect biological properties of MSCs including their regenerative potential. Moreover, both cell cryopreservation and labelling procedure prior to infusion may have the negative impact on their expected effect in vivo. The incidence of MSC transformation during in vitro culture should be also taken into consideration before using cells in stem cell therapy. In our review, we focused on different aspects of MSC propagation that might influence their regenerative properties of MSC. We also discussed the influence of different factors that might abolish MSC proliferation and differentiation as well as potential impact of stem cell senescence and aging. Despite of many positive therapeutic effects of MSC therapy, one has to be conscious about potential cell changes that could appear during manufacturing of MSCs.

Challenges in the development of a widely applicable method for sugarcane (Saccharum spp.) shoot tip cryopreservation

The USDA-ARS National Plant Germplasm System (NPGS) maintains 946 accessions of sugarcane (Saccharum spp.) in the field at the Subtropical Horticulture Research Station in Miami, Florida. These accessions are particularly vulnerable to hurricanes, diseases, and other threats. We sought to identify a method whereby clonally propagated sugarcane accessions could be successfully introduced into tissue culture, multiplied, and then cryopreserved as shoot tips for long-term preservation at the National Laboratory for Genetic Resources Preservation in Fort Collins, Colorado. For many accessions, 70% isopropyl alcohol and 20% commercial bleach treatments, followed by three rinses of sterile water were sufficient to remove microbial contaminants during the introduction process. However, in some cases, cefotaxime was particularly effective for removing bacterial contamination. We found that antioxidant treatments of glutathione, glycine betaine, and ascorbic acid did not improve regrowth after liquid nitrogen exposure using either PVS2 or PVS3 as cryoprotectants in droplet vitrification cryopreservation methods. Exposure durations of PVS2 and PVS3 were optimized, with and without exposure to liquid nitrogen (LN), and shoot tip regrowth levels ranged from 0 to 37% after LN exposure. Published methods for encapsulation-dehydration and V-plate vitrification cryopreservation procedures were tested to determine if acceptable results could be obtained. Using these methods, shoot tip regrowth levels ranged from 0 to 50% after LN exposure. We conclude that the sugarcane cryopreservation methods that we tested are not yet ready for implementation in the NPGS.

Ovarian Tissue Transplantation: Experience From Germany and Worldwide Efficacy.

Extraction of ovarian tissue prior to oncologic therapy and subsequent transplantation is being performed increasingly often to preserve fertility in women. The procedure can be performed at any time of the cycle and, therefore, generally does not lead to any delay in oncological therapy. Success rates with transplantation of cryopreserved ovarian tissue have reached promising levels. More than 130 live births have been reported worldwide with the aid of cryopreserved ovarian tissue and the estimated birth rate is currently approximately 30%. In Germany, Austria, and Switzerland, the FertiPROTEKT consortium has successfully achieved 21 pregnancies and 17 deliveries generated after 95 ovarian tissue transplantations by 2015, one of the largest case series worldwide confirming that ovarian tissue cryopreservation and transplantation are successful. Approximately, more than 400 ovarian tissue cryopreservation procedures are performed each year in the FertiPROTEKT consortium, and the request and operations for ovarian tissue transplantation have increased in recent years. Therefore, recommendations for managing transplantation of ovarian tissue to German-speaking reproductive medicine centers were developed. In this overview, these recommendations and our experience in ovarian tissue transplantation are presented and discussed with international procedures.