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Concept: Cryoprotectant

167

Development of techniques to isolate, culture, and transplant human spermatogonial stem cells (SSCs) has the future potential to treat male infertility. To maximize the efficiency of these techniques, methods for SSC cryopreservation need to be developed to bank SSCs for extended periods of time. Although, it has been demonstrated that SSCs can reinitiate spermatogenesis after freezing, optimal cryopreservation protocols that maximize SSC proliferative capacity post-thaw have not been identified. The objective of this study was to develop an efficient cryopreservation technique for preservation of SSCs. To identify efficient cryopreservation methods for long-term preservation of SSCs, isolated testis cells enriched for SSCs were placed in medium containing dimethyl sulfoxide (DMSO) or DMSO and trehalose (50 mM, 100 mM, or 200 mM), and frozen in liquid nitrogen for 1 week, 1 month, or 3 months. Freezing in 50 mM trehalose resulted in significantly higher cell viability compared to DMSO at all thawing times and a higher proliferation rate compared to DMSO for the 1 week freezing period. Freezing in 200 mM trehalose did not result in increased cell viability; however, proliferation activity was significantly higher and percentage of apoptotic cells was significantly lower compared to DMSO after freezing for 1 and 3 months. To confirm the functionality of SSCs frozen in 200 mM trehalose, SSC transplantation was performed. Donor SSCs formed spermatogenic colonies and sperm capable of generating normal progeny. Collectively, these results indicate that freezing in DMSO with 200 mM trehalose serves as an efficient method for the cryopreservation of SSCs.

Concepts: Organ transplant, Food preservation, Dimethyl sulfoxide, Cryobiology, Cryopreservation, Cryoprotectant, Sulfoxide, Dimethyl sulfide

148

Vitrification of endothelial cells (MHECT-5) has not previously been compared with controlled slow freezing methods under standardized conditions. To identify the best cryopreservation technique, we evaluated vitrification and standardized controlled-rate -1°C/minute cell freezing in a -80°C freezer and tested four cryoprotective agents (CPA), namely dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), and glycerol (GLY), and two media, namely Dulbecco’s modified Eagle medium Ham’s F-12 (DMEM)and K+-modified TiProtec (K+TiP), which is a high-potassium-containing medium. Numbers of viable cells in proliferation were evaluated by the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega Corporation, Mannheim, Germany). To detect the exact frozen cell number per cryo vial, DNA content was measured by using Hoechst 33258 dye prior to analysis. Thus, results could be evaluated unconstrained by absolute cell number. Thawed cells were cultured in 25 cm2 cell culture flasks to confluence and examined daily by phase contrast imaging. With regard to cell recovery immediately after thawing, DMSO was the most suitable CPA combined with K+TiP in vitrification (99 ±0.5%) and with DMEM in slow freezing (92 ±1.6%). The most viable cells in proliferation after three days of culture were obtained in cells vitrificated by using GLY with K+TiP (308 ±34%) and PG with DMEM in slow freezing (280 ±27%).

Concepts: Ethylene glycol, Ethylene, Diol, Propylene glycol, Dimethyl sulfoxide, Glycerol, Cryobiology, Cryoprotectant

68

Vitrification, a kinetic process of liquid solidification into glass, poses many potential benefits for tissue cryopreservation including indefinite storage, banking, and facilitation of tissue matching for transplantation. To date, however, successful rewarming of tissues vitrified in VS55, a cryoprotectant solution, can only be achieved by convective warming of small volumes on the order of 1 ml. Successful rewarming requires both uniform and fast rates to reduce thermal mechanical stress and cracks, and to prevent rewarming phase crystallization. We present a scalable nanowarming technology for 1- to 80-ml samples using radiofrequency-excited mesoporous silica-coated iron oxide nanoparticles in VS55. Advanced imaging including sweep imaging with Fourier transform and microcomputed tomography was used to verify loading and unloading of VS55 and nanoparticles and successful vitrification of porcine arteries. Nanowarming was then used to demonstrate uniform and rapid rewarming at >130°C/min in both physical (1 to 80 ml) and biological systems including human dermal fibroblast cells, porcine arteries and porcine aortic heart valve leaflet tissues (1 to 50 ml). Nanowarming yielded viability that matched control and/or exceeded gold standard convective warming in 1- to 50-ml systems, and improved viability compared to slow-warmed (crystallized) samples. Last, biomechanical testing displayed no significant biomechanical property changes in blood vessel length or elastic modulus after nanowarming compared to untreated fresh control porcine arteries. In aggregate, these results demonstrate new physical and biological evidence that nanowarming can improve the outcome of vitrified cryogenic storage of tissues in larger sample volumes.

Concepts: Blood, Extracellular matrix, Blood vessel, Fourier transform, Glass transition, Cryobiology, Cryopreservation, Cryoprotectant

28

The conservation of endangered fish is of critical importance. Cryobanking could provide an effective backup measure for use in conjunction with the conservation of natural populations; however, methodology for cryopreservation of fish eggs and embryos has not yet been developed. The present study established a methodology capable of deriving functional eggs and sperm from frozen type A spermatogonia (ASGs). Whole testes taken from rainbow trout were slowly frozen in a cryomedium, and the viability of ASGs within these testes did not decrease over a 728-d freezing period. Frozen-thawed ASGs that were intraperitoneally transplanted into sterile triploid hatchlings migrated toward, and were incorporated into, recipient genital ridges. Transplantability of ASGs did not decrease after as much as 939 d of cryopreservation. Nearly half of triploid recipients produced functional eggs or sperm derived from the frozen ASGs and displayed high fecundity. Fertilization of resultant gametes resulted in the successful production of normal, frozen ASG-derived offspring. Feasibility and simplicity of this methodology will call for an immediate application for real conservation of endangered wild salmonids.

Concepts: Salmon, Salmonidae, Rainbow trout, Freezing, Cryobiology, Cryopreservation, Cryoprotectant, Ex-situ conservation

28

This study dealt with the development of cryopreservation protocol for Nandus nandus, which entailed a number of experiments. Sperm was collected by sacrificing males. The collected sperm was suspended in extenders. Activation of sperm motility was evaluated in different osmolalities of NaCl. Motility of sperm decreased as the osmolality of the extender increased and was completely inhibited at almost 319mOsmol/kg. To evaluate the toxicity of cryoprotectant, sperm was incubated with DMSO, methanol and ethanol at 5%, 10% and 15% concentrations, respectively, for 5-35min. Five and ten percent of cryoprotectants produced better motility during 5 and 10min incubation. Sperm incubated with 15% cryoprotectant seemed to be toxic and this concentration was excluded in the subsequent trials. Three extenders, namely, Alsever’s solution, egg-yolk citrate and urea egg-yolk and three cryoprotectants, DMSO, methanol and ethanol were employed to preserve the sperm. Alsever’s solution with 10% DMSO showed best performance producing 90.0±1.8% and 75.0±2.5% equilibration and post-thaw motility followed by that of 82.5±4.2% and 62.5±5.5% with Alsever’s solution plus methanol, respectively. Between two diluents, sperm preserved with Alsever’s solution plus DMSO produced highest fertilization (76.7±3.3%) and hatching (43.8±7.9%) while fresh sperm yielded 83.3±6.7% and 64.0±10.4% fertilization and hatching, respectively. The protocol developed through the study can be applied for long-term conservation of genetic materials of the endangered fish N. nandus and the cryopreserved sperm can be used in artificial breeding for generating new individuals.

Concepts: Concentration, Solvent, Cryogenics, Cryobiology, Cryopreservation, Cryoprotectant, Cryonics, Ex-situ conservation

27

The intracellular ice formation (IIF) behavior of Haliotis diversicolor (small abalone) eggs is investigated in this study, in relation to controlling the cooling rate and the concentration of dimethyl sulfoxide (DMSO). The IIF phenomena are monitored under a self-developed thermoelectric cooling (TEC) cryomicroscope system which can achieve accurate temperature control without the use of liquid nitrogen. The accuracy of the isothermal and ramp control is within ±0.5°C. The IIF results indicate that the IIF of small abalone eggs is well suppressed at cooling rates of 1.5, 3, 7 and 12°C/min with 2.0, 2.5, 3.0 and 4.0 M DMSO in sea water. As 2.0 M DMSO in sea water is the minimum concentration that has sufficient IIF suppression, it is selected as the suspension solution for the cryopreservation of small abalone eggs in order to consider the solution’s toxicity effect. Moreover, IIF characteristics of the cumulative probability of IIF temperature distribution are shown to be well fitted by the Weibull probabilistic distribution. According to our IIF results and the Weibull distribution parameters, we conclude that cooling at 1.5°C/min from 20 to -50°C with 2.0 M DMSO in sea water is more feasible than other combinations of cooling rates and DMSO concentrations in our experiments. Applying this protocol and observing the subsequent osmotic activity, 48.8% of small abalone eggs are osmotically active after thawing. In addition, the higher the cooling rate, the less chance of osmotically active eggs. A separate fertility test experiment, with a cryopreservation protocol of 1.5°C/min cooling rate and 2.0 M DMSO in sea water, achieves a hatching rate of 23.7%. This study is the first to characterize the IIF behavior of small abalone eggs in regard to the cooling rate and the DMSO concentration. The Weibull probabilistic model fitting in this study is an approach that can be applied by other researchers for effective cryopreservation variability estimation and analysis.

Concepts: Concentration, Liquid, Probability theory, Solution, Dimethyl sulfoxide, Cryobiology, Cryoprotectant, Dimethyl sulfide

25

In swine spermatozoa, the damage caused by cryopreservation is more severe than other species, provoking reduced potential for fertilization. Adjustments in the freezing extender composition may be an important alternative to increase its efficiency. The objective of this study was to test the efficiency of different cryoprotectant solutions during cryopreservation of swine semen with a controlled cooling curve. Three cryoprotectant solutions (5% dimethylformamide, 3% glycerol and the combination of these two cryoprotectants) were used in association with three base media (powdered coconut water, lactose and trehalose), constituting nine different treatments. The semen was frozen using a controlled-rate freezer (TK-3000). After thawing, semen was evaluated for total sperm motility, vigor, morphology, plasma membrane integrity and acrosome integrity. Cryopreservation with the controlled curve using an automated system showed satisfactory results, guaranteeing practicality and repeatability for the process of freezing swine sperm. With this curve, the solutions of lactose, trehalose and powdered coconut water associated with glycerol, as well as the solution of coconut water containing dimethylformamide, presented higher quality of sperm compared to the other solutions. Powdered coconut water associated with dimethylformamide appears as a new solution for swine sperm cryopreservation. The freezing controlled curve used in this study allowed standardization of the cryopreservation technique.

Concepts: Sperm, Spermatozoon, Semen, Liquid, Freezing, Cryobiology, Cryopreservation, Cryoprotectant

25

We recently demonstrated that the hollow fibre vitrification (HFV) method (Matsunari et al. 2012) could effectively be applied to the cryopreservation of embryos from diverse species. In this study, we applied the HFV method to the cryopreservation of highly cryosensitive specimens, such as in vitro matured (IVM)/IVF-derived porcine zona-free morulae and blastomeres isolated from those morulae, as well as IVM/IVF-derived cattle embryos at early cleavage stages. Porcine parthenogenetic morulae (d-4) derived from IVM oocytes were treated with 0.25% pronase to remove zona pellucidae. The resulting blastomeres were isolated from the zona-free morulae by a decompaction treatment followed by gentle pipetting. Bovine IVM-IVF embryos at the 2 to 4 cell (d-1), 8 to 16 cell (d-3), and morula stages (d-5) were then subjected to vitrification. The HFV procedure was performed as described previously using 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5M trehalose as cryoprotectants. Four to twenty embryos, or all of the blastomeres isolated from a single morula, were individually loaded into a cellulose acetate hollow fibre (25mm long, 185μm φ, 15μm membrane thickness) and vitrified. Survival of the vitrified embryos was assessed by in vitro development to blastocysts. Blastomeres recovered after vitrification were aggregated in micro-wells to examine their ability to form blastocysts. The HFV method was demonstrated to be effective for cryopreserving zona-free in vitro-produced porcine morulae and the blastomeres isolated from them (Table 1), as well as bovine IVM-IVF embryos at early cleavage stages. These data demonstrate that the HFV method is effective for highly cryosensitive specimens, such as IVM/IVF-derived porcine zona-free morulae and blastomeres isolated from those morulae, and IVM/IVF-derived cattle embryos at early cleavage stages. These achievements may expand the technological options in the production of cloned and genetically modified pigs that are useful for biomedical research.

Concepts: Developmental biology, Embryology, Zygote, Ethylene glycol, Cryobiology, Morula, Blastomere, Cryoprotectant

25

The production of long shelf-life highly concentrated dried probiotic/starter cultures is of paramount importance for the food industry. The aim of the present study was to evaluate the protective effect of glucose, lactose, trehalose, and skim milk applied alone or combined upon the survival of potentially probiotic Lactobacillus rhamnosus CTC1679, Lactobacillus casei/paracasei CTC1677 and L. casei/paracasei CTC1678 during freeze-drying and after 39 weeks of storage at 4 and 22 °C. Immediately after freeze-drying, the percentage of survivors was very high (≥94%) and only slight differences were observed among strains and cryoprotectants. In contrast, during storage, survival in the dried state depended on the cryoprotectant, temperature and strain. For all the protectants assayed, the stability of the cultures was remarkably higher when stored under refrigeration (4 °C). Under these conditions, skim milk alone or supplemented with trehalose or lactose showed the best performance (reductions ≤0.9 log units after 39 weeks of storage). The lowest survival was observed during non-refrigerated storage and with glucose and glucose plus milk; no viable cells left at the end of the storage period. Thus, freeze-drying in the presence of appropriate cryoprotectants allows the production of long shelf-life highly concentrated dried cultures ready for incorporation in high numbers into food products as starter/potential probiotic cultures.

Concepts: Milk, Food, Probiotic, Food preservation, Freeze drying, Trehalose, Lactobacillus rhamnosus, Cryoprotectant

10

We previously demonstrated that whole hair follicles could be cryopreserved to maintain their stem-cells differentation potential. In the present study, we demonstrated that cryopreserved mouse whisker hair follicles maintain their hair growth potential. DMSO better cryopreserved mouse whisker follicles compared to glycerol. Cryopreserved hair follicles also maintained the hair follicle-associated-pluripotent (HAP) stem cells, evidenced by P75NTR expression. Subcutaneous transplantation of DMSO-cryopreserved hair follicles in nude mice resulted in extensive hair fiber growth over 8 weeks, indicating the functional recovery of hair shaft growth of cryopreserved hair follicles.

Concepts: Present, Gene, Mouse, Hair, Hair follicle, Cryobiology, Facial hair, Cryoprotectant