Type of protein supplement in cryopreservation solutions impacts on the degree of ultrastructural damage in frozen-thawed human oocytes

Protein sources used as supplements of IVF culture media are known to have several implications for the function and stability of embryo culture environment. In fact, they i) transport biologically active molecules ii) chelate heavy metals, iii) regulate media pH, iii) scavenge reactive oxygen species (ROS) and iv) attenuate osmotic stress to which cells are exposed in sub-optimal culture conditions. Instead, their specific relevance to the formulation of cryopreservation solutions used for gamete and embryo cryopreservation remains uncertain. In the present work, we tested the hypothesis that different protein supplements present in cryopreservation solutions, serum or plasma protein solution (PPS), or different concentrations of the same supplement (serum), are associated with different types and/or magnitude of cryopreservation-derived cell damage. To this end, using cryopreservation solutions containing serum or PPS, donated supernumerary human mature oocytes were frozen-thawed by slow freezing and compared with fresh controls. Ultrastructural markers of oocyte quality were adopted as objective measure to assess possible damage from cryopreservation. The study results indicate that the adoption of serum minimises cell damage induced by cryopreservation. Indeed, typical hallmarks of cryodamage in human oocytes, i.e. loss of cortical granules, zona pellucida hardening and above all vacuolization, were largely reduced in oocytes cryopreserved with solutions containing serum, especially if used a higher concentration. This suggest that oocyte cryopreservation still has significant margins of improvement that may derive also from composition of cryopreservation media.     

Impacts of trehalose and l-proline on the thermodynamic nonequilibrium phase change and thermal properties of normal saline

Understanding the phase change behavior and thermal properties of cryoprotective agents (CPAs) in biological solutions is essential for enhancing the success of cryopreservation and biobanking. In this study, the phase change behavior and thermal properties of normal saline added with trehalose or l-proline were investigated using differential scanning calorimeter (DSC) and cryomicroscope during freezing and warming. The addition of trehalose or l-proline can eliminate the eutectic formation in normal saline. Trehalose had significantly lower latent heat release than l-proline does at a high concentration of 1 M (P < 0.05), while unfrozen water content of trehalose is significantly lower than that of l-proline at all the concentrations (P < 0.05). It was also found that addition of 0.2 M, 0.3 M and 1 M trehalose can achieve partial vitrification in normal saline and that the glass transition temperature rises along with the increase in concentrations of trehalose. However, no vitrification was observed in normal saline with l-proline at any concentrations. Besides, rates of ice crystal growth in normal saline added with trehalose are slower than those in normal saline with l-proline at the same concentrations. These results suggest that both trehalose and l-proline can act as CPAs by avoiding eutectic formation and inhibiting ice formation in normal saline for cell cryopreservation. It could be useful for CPA selection and designing in the future.     

Cells isolated from cryopreserved dental follicle display similar characteristics to cryopreserved dental follicle cells

Dental follicle tissue is a promising resource of mesenchymal stem cells for cytotherapeutic approaches and tissue engineering applications. There are two procedures for banking of human dental follicle stem cells have been reported. Conventional method requires cell isolation, expansion and immediate cryopreservation. Whereas dental follicle stem cells can be isolated from cryopreserved dental follicle fragments. The aim of this study was to compare the characteristics of dental follicle cells isolated from cryopreserved fragments (DFCs-CF) with dental follicle cells recovered from cryopreserved cells (DFCs-CC). Dental follicle fragments obtained after mechanical disaggregation were divided into two parts, with one part maintained in culture, while another part underwent cryopreservation. Dental follicle fragments and dental follicle cells from fresh tissue were stored in liquid nitrogen for 3 months. After thawing, the isolation, morphology, proliferation, cell cycle, colony-forming-unit ability, stemness-related marker expression, apoptosis, and multi-lineage differentiation potential of DFCs-CF were tested compared with DFCs-CC. DFCs-CF expressed mesenchymal stem cells marker, proliferated well, showed similar levels of mRNA for stemness- and apoptosis-related genes and exhibited the capacity of multi-lineage differentiation similar to those of DFCs-CC. These results imply that cryopreservation of dental follicle fragments is an effective banking method for isolation of dental follicle cells.     

A novel approach for human sperm cryopreservation with AFPIII

Sperm cryopreservation causes different stresses including thermal shock, osmotic damage, and ice crystal formation, thereby reducing sperm quality. Few studies have evaluated the application of AFPs in cryopreservation. The effects of antifreeze protein III (AFP III) on human sperm cryopreservation is not fully understood therefore, we conducted this study to investigate the effects of AFPIII treatment on human sperm parameters following cryopreservation. First, for 20 semen samples the effects of various concentrations of AFPIII (0, 0.01, 0.1, 1, 5, 10 μg/ml) were evaluated. Sperm parameters, such as motility and viability were assessed in order to identify an optimal dose. Next, liquefied 20 semen samples were divided into three aliquots and diluted in glycerol-egg-yolk-citrate (GEYC) cryopreserved without AFPIII (control), with optimal dose of AFPIII, as well as fresh groups. After thawing, samples were evaluated for plasma membrane integrity (PMI), DNA fragmentation index (DFI), reactive oxygen species (ROS), and total antioxidant capacity (TAC) levels. Spermatozoa treatment with 0.01, 0.1 and 1 μg/ml AFPIII increased the sperm motility and viability compared to the control group, but the highest concentrations were ineffective. In conclusion, the results showed that the addition of AFPIII to GEYC at 1 μg/ml improved motility, PMI, viability and TAC, and decreased ROS and DNA fragmentation of cryopreserved human semen compared to the control group.     

Cardiac stem cells: translation to human studies

The discovery of multiple classes of cardiac progenitor cells in the adult mammalian heart has generated hope for their use as a therapeutic in heart failure. However, successful results from animal models have not always yielded similar findings in human studies. Recent Phase I/II trials of c-Kit (SCIPIO) and cardiosphere-based (CADUCEUS) cardiac progenitor cells have demonstrated safety and some therapeutic efficacy. Gaps remain in our understanding of the origins, function and relationships between the different progenitor cell families, many of which are heterogeneous populations with overlapping definitions. Another challenge lies in the limitations of small animal models in replicating the human heart. Cryopreserved human cardiac tissue provides a readily available source of cardiac progenitor cells and may help address these questions. We review important findings and relative unknowns of the main classes of cardiac progenitor cells, highlighting differences between animal and human studies     

Perfusion bioreactor-based cryopreservation of 3D human mesenchymal stromal cell tissue grafts

The generation of an off-the-shelf in vitro engineered living tissue graft will likely require cryopreservation. However, the efficient addition and removal of cryoprotective agents (CPA) to cells throughout the volume of a three-dimensional (3D) tissue graft remains a significant challenge. In this work, we assessed whether a perfusion bioreactor-based method could be used to improve the viability of cryopreserved mesenchymal stromal cell- (MSC) based tissue constructs as compared to using conventional diffusion-based methods. The bioreactor was first used to saturate 3D constructs with CPA under perfused flow. Following cryopreservation, the bioreactor was then also used for the efficient removal of the CPA from the 3D tissues. We demonstrate that addition and removal of CPA under perfused flow significantly increased the viability of MSC within cryopreserved 3D tissue constructs as compared to conventional diffusion-based methods.     

Acrosomal integrity, viability, and DNA damage of sperm from dasyurid marsupials after freezing or freeze drying

Dasyurids are a diverse group of Australian native carnivores and insectivores that contains several threatened species. Despite successful cryopreservation of sperm from several marsupials, only 3% postthaw motility is reported for dasyurid marsupials. This study examined sperm preservation in the fat-tailed dunnart (Sminthopsis crassicaudata), an experimental model, with supplementary observations on the eastern quoll (Dasyurus viverrinus) and northern quoll (Dasyurus hallucatus). In S. crassicaudata, a toxicity trial demonstrated that incubation with up to 40% glycerol did not reduce sperm viability, suggesting that glycerol is not toxic to dasyurids. On the basis of this finding, S. crassicaudata, D. viverrinus, and D. hallucatus sperm were extended to a final concentration of 20% or 40% glycerol in Tris-citrate fructose and frozen in liquid nitrogen vapor. Postthaw sperm from all three species were nonmotile, and vital staining (SYBR14 and propidium iodide) indicated that sperm were nonviable. However, there was no evidence suggesting disruption of normal gross morphology or loss of acrosomal integrity when assessed by Bryan's staining. After freeze drying, Bryan's staining indicated that approximately 80% of S. crassicaudata sperm had normal acrosomes and no head loss. Despite being nonviable, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling showed that S. crassicaudata sperm frozen in 40% glycerol or freeze-dried had no DNA damage compared with fresh controls. This study has described a method for preservation of the dasyurid sperm nuclei, but continued studies are required to achieve viable motile sperm and establish tools for the long-term storage of dasyurid sperm.     

Emerging applications of sperm, embryo and somatic cell cryopreservation in maintenance, relocation and rederivation of swine genetics

Advances in porcine assisted reproductive technology (ART) make it possible to use cryopreserved sperm, embryos and somatic cells in the maintenance, relocation and regeneration of swine genetics. In this review, development of key application-limiting technology is discussed in each cell type, focusing on the efficiencies, ease of storage and transportation, and minimization of pathogen transmission. Methods to regenerate swine genetics and/or models using frozen sperm, embryos and somatic cells in combination with other porcine ARTs, such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transplantation (SCNT), are also discussed. The applications of these ARTs utilizing cryopreserved cells will greatly increase the efficiency as well as biosecurity for maintenance, relocation and rederivation of swine genetics/models.     

The effect of Tribulus terrestris extract on motility and viability of human sperms after cryopreservation

BackgroundSemen cryopreservation produces significant amounts of reactive oxygen species (ROS), which may lead to impairment of sperm morphology, function, and ultimately, male fertility. Since Tribulus terrestris has antioxidant and free-radical-scavenging properties, this study aims to reveal the effect of the Tribulus terrestris extract on motility and vitality of human sperms after cryopreservation.Materials and methodsSemen specimens from 80 healthy volunteers were divided into eight groups: fresh control (group I), freeze control (group II), groups III, IV, and V, which had 20, 40, and 50 μg/mL doses of Tribulus terrestris extract added before cryopreservation, and groups VI, VII, and VIII, which were supplemented by these extract doses after the freeze-thaw process. To evaluate the effects of the Tribulus terrestris extract, the semen samples were incubated with the extract and evaluated with a light microscope for motility and viability.ResultsAfter cryopreservation, a significant improvement in spermatozoa viability was observed in group VII. In groups VII and VIII, motility, according to World Health Organization (WHO) criteria, increased considerably (p < 0.001). There was no significant difference among groups III, IV, and V.ConclusionThe present study demonstrated that the protective effects of Tribulus terrestris, which improves human sperm motility and viability, may be due to its antioxidant properties. On the basis of the results, the researchers concluded that Tribulus terrestris can be used as a safe therapeutic alternative to current modalities for the management of motility dysfunction in males.     

Construction and long term preservation of clonal transgenic silkworms using a parthenogenetic strain

For the functional analysis of insect genes as well as for the production of recombinant proteins for biomedical use, clonal transgenic silkworms are very useful. We examined if they could be produced in the parthenogenetic strain that had been maintained for more than 40 years as a female line in which embryogenesis is induced with nearly 100% efficiency by a heat shock treatment of unfertilized eggs. All individuals have identical female genotype. Silkworm transgenesis requires injection of the DNA constructs into the non-diapausing eggs at the preblastodermal stage of embryogenesis. Since our parthenogenetic silkworms produce diapausing eggs, diapause programing was eliminated by incubating ovaries of the parthenogenetic strain in standard male larvae. Chorionated eggs were dissected from the implants, activated by the heat shock treatment and injected with the transgene construct. Several transgenic individuals occurred in the daughter generation. Southern blotting analysis of two randomly chosen transgenic lines VTG1 and VTG14 revealed multiple transgene insertions. Insertions found in the parental females were transferred to the next generation without any changes in their sites and copy numbers, suggesting that transgenic silkworms can be maintained as clonal strains with homozygous transgenes. Cryopreservation was developed for the storage of precious genotypes. As shown for the VTG1 and VTG14 lines, larval ovaries can be stored in DMSO at the temperature of liquid nitrogen, transferred to Grace’s medium during defrosting, and then implanted into larvae of either sex of the standard silkworm strains C146 and w1-pnd. Chorionated eggs, which developed in the implants, were dissected and activated by the heat shock to obtain females (nearly 100% efficiency) or by a cold shock to induce development to both sexes in 4% of the eggs. It was then possible to establish bisexual lines homozygous for the transgene.