Biopreservation and Biobanking

The recently published fifth edition of the International Society for Biological and Environmental Repositories (ISBER) Best Practices signifies a pivotal milestone in navigating the complexities of repository management. Repositories operate within a constantly evolving landscape influenced by the changing fields of biospecimen science, technology, legal requirements, and ethical considerations. This dynamic is further amplified by unprecedented local and global challenges, such as pandemics, conflicts, and supply chain disruptions. Creating this new edition required a comprehensive approach capable of delivering a focused and coherent resource reflecting the broad horizon of its diverse users. The innovative approach used the existing phased development process and integrated the canvassing of opinions, formal evaluation, and real-time collaboration platforms. Merging these techniques enabled efficient collection and effective distillation of the latest in biobanking practices, enhancing the value of the fifth edition for repositories of specimens and associated data. The expanded document is a testament to the collective efforts of many dedicated individuals who have built upon the foundations of prior editions.

The present study analyzes the effects of different disaccharide concentrations and two thawing temperatures on the characteristics of ultrarapid frozen (URF) bovine sperm, compared with conventional slow-frozen (CF) sperm. For URF sperm, samples were diluted in media comprising 2% bovine serum albumin (BSA) and various nonpermeable cryoprotectants. Five groups were compared: control (without cryoprotectant), sucrose 0.15 M, sucrose 0.3 M, trehalose 0.15 M, and trehalose 0.3 M. In addition, the influence of warming temperatures, 37°C and 65°C, was analyzed. The aspect of different diluents (by drops) immersed in liquid nitrogen was also evaluated. Sperm quality was assessed by measuring motility, viability, acrosome status, and membrane lipid peroxidation (LPO). Moreover, the cryoresistance rate (CR) was determined. The drops immersed in liquid nitrogen showed that crystallization occurred, but not vitrification. CF sperm exhibited significantly higher scores for total motility (TM) and progressive motility (PM), viability, and acrosome integrity, in contrast with URF samples. Cryoprotectants for URF sperm showed a significant (p ≤ 0.05) influence on the TM and PM, viability, acrosome integrity, and CR, but not on LPO. Sperm viability was reduced after ultrarapid freezing, and the control samples were observed to have significantly lower values than those treated with disaccharides. Samples supplemented with 0.3 M sucrose exhibited higher LPO when they were thawed at 37°C. In short, a limited number of spermatozoa were able to maintain their motility and other functional attributes after ultrarapid freezing, but disaccharides showed a moderate protective effect. Samples with trehalose and sucrose at 0.15 and 0.3 M, respectively, showed higher sperm quality than samples containing only BSA. In sum, the function of spermatozoa was moderately maintained when disaccharides were used for ultrarapid freezing, although motility was significantly reduced. In addition, thawing temperatures did not modify the sperm values, suggesting that the easier procedure, that is, 37°C for 30 seconds, can be used.

This research examined the antioxidant and cryoprotective effects of melatonin (ME) and caffeine (CAF) supplementation in freezing medium on the cryosurvival of Peruvian Paso horse sperm using a two-step accelerating cooling rate. Twenty ejaculates from four adult and fertile stallions were recovered, initially diluted with INRA-96^®, and finally frozen with INRA-Freeze^® with either no supplementation (as control), 1 μM ME, or 2 mM CAF using a two-ramp freezing system content inside a cryogenic-box and liquid nitrogen vapors. The sperm kinematic parameters and integrity of the plasma and acrosomal membranes of fresh semen and cryopreserved samples were evaluated using the CASA system (SCA-Evolution^® 2018) and PI/fluorescein isothiocyanate-conjugated peanut (Arachis hypogaea) agglutinin double fluorescent test, respectively. The oxidative stress of post-thaw sperm samples was also assessed using the CellRox Deep Red fluorescence test. The results showed that curvilinear velocity and average-path velocity were greater (p < 0.05) after freezing with CAF than the control group. In addition, there were significance differences (p < 0.01) between stallions (1–4) in post-thaw kinematic parameters regardless of ME or CAF addition. Both ME and CAF improved (p < 0.05) the proportion of sperm with intact plasma membranes and intact acrosomes. Nevertheless, neither CAF nor ME improved the oxidative stress after the cryopreservation process.

This study investigated the effect of silymarin on human sperm quality during cryopreservation. Samples were collected from 20 normospermic individuals, and each sample was divided into different concentrations of silymarin comprising the following groups: (0, 20, 100, 500, and 1000 μg/mL silymarin). Sperm quality parameters, such as plasma membrane integrity, mitochondrial membrane potential, acrosomal membrane integrity, and caspase 3 were estimated. Silymarin concentrations of 100–500 μg/mL significantly increased motility, plasma membrane integrity, and mitochondrial activity compared with the frozen control group. Acrosomal integrity was increased in the 1000 μg/mL silymarin group. Moreover, 20 and 100 μg/mL concentrations significantly decreased the percentage of caspase 3. The addition of silymarin antioxidant to the frozen medium reduced damage in the sperm after freezing and thawing. This is the first study that showed silymarin can be useful in cryopreservation of human sperm.

Oocyte vitrification is widely used for female fertility preservation. However, the efficacy of this procedure may depend on the women's age. The aim of the study was to compare the morphology, viability of cryopreserved oocytes, and their fertilization outcomes (fertilization, blastulation rate, level of embryo chromosomal aneuploidy—preimplantation genetic testing for aneuploidy [PGT-A]) in women of different reproductive ages. The studied oocytes were divided into groups depending on the age of patients: up to 30 years (group 1), 30–35 years (group 2), 36–40 years (group 3), and older than 40 years (group 4). It has been shown that in women of older reproductive age, the number of oocytes with polymorphism of endo- and extracytoplasmic structures was higher compared with younger patients. This could reflect on their cryosurvival rate, which was the highest in group 1 (98.1%) and the lowest was in group 4 (47.4%). With increasing age, the fertilization rate of cryopreserved oocytes and subsequent blastulation was decreased. However, the number of embryos with an aneuploid chromosome set number was increased. The chromosome set number euploidy rate of the embryos obtained from cryopreserved oocytes of advanced age women (group 4) did not differ from the fresh group with the same age (31.2% vs. 24.4%, p > 0.05), but the number of euploid embryos per patient was less than one (0.8 ± 0.1). Therefore, the decision to cryopreserve the oocytes of a patient of older reproductive age should be made individually for each situation, taking into account the prospects of obtaining full-fledged embryos and the chances of pregnancy.

The aim of this study was to evaluate the effect of both pure rainbow trout seminal plasma (RTSP) supplementation and RTSP-cysteine combination on cryopreservation success and post-thaw incubation resilience of ram semen in the nonbreeding season. For this purpose, different doses of RTSP (0%, 1%, 10%, and 15%) with or without cysteine supplementation were used for experiments. Ejaculates chosen for experiments were pooled and then divided into eight equal volumes for grouping (Control-ControlC, RTSP1-RTSP1C, RTSP10-RTSP10C, and RTSP15-RTSP15C). After cryopreservation, frozen-thawed semen samples were incubated for 5 hours at 37°C for determination of post-thaw incubation resistance. Motility, HOST, TUNEL, Rh123-PI, and CTC tests were performed at 0 hour and 3rd and 5th hours of post-thaw incubation to evaluate the efficacy of all experimental groups. The RTSP10 and RTSP10C groups were noted to provide the best protection on motility, plasma membrane integrity, DNA integrity, and mitochondrial function of cryopreserved ram semen. On the other hand, the best protection against cryo-capacitation was observed in RTSP15 and RTSP15C groups. The addition of cysteine was found to be effective when the higher (15%) or lower (1%) doses of RTSP were used, as well as for no use of RTSP.

Semen banking is an efficient method of artificial insemination for commercial breeders. However, the cryopreservation process induces severe damages to plasma membranes, which leads to reduced fertility potential of thawed sperm. The replacement of membrane lipids with oxidized membrane lipids repairs the cell membrane and improves its stability. The aim of this study was to investigate the effects of glycerophospholipid (GPL) nanomicelles on the cryosurvival of thawed rooster semen. Semen samples were collected from six 29-week Ross broiler breeder roosters, then mixed and divided into five equal parts. The samples were diluted with the Beltsville extender containing different concentrations of GPL according to the following groups: 0 (GPL-0), 0.1% (GPL-0.1), 0.5% (GPL-0.5), 1% (GPL-1), and 1.5% (GPL-1.5), then diluted semen was gradually cooled to 4°C during 3 hours and stored in liquid nitrogen. The optimum concentration of GPL was determined based on the quality parameters of thawed sperm. Our results showed sperm exposed to GPL-1 had significantly increased motion parameters and mitochondrial activity. The percentages of viability and membrane integrity were significantly higher in the GPL-1, and GPL-1.5 groups compared with the other groups (p < 0.05). Moreover, the lowest rate of apoptosis and lipid peroxidation were observed in the GPL-1 and GPL-1.5 groups in comparison with the frozen control group. Our findings indicated that membrane lipid replacement with GPL nanomicelles (1% and 1.5%) could substitute for damaged lipids in membranes and protect sperm cells against cryoinjury.

In recent years, cells provided by cell banks and medical facilities have been used for cell therapy, regenerative therapy, and fundamental research. Cryopreservation is an effective means of maintaining stable cell quality over a long period of time. The slow freezing method is most suitable for processing many human cells isolated simultaneously from organs and tissues, but it is necessary to develop a freezing solution for this method. In this study, we report the successful development of a dimethyl sulfoxide (DMSO)-free freezing medium for differentiated neuronal cells. Neuronal differentiation results in the differentiation of undifferentiated SK-N-SH cells into neuronal cells. A basic freezing medium (BFM) was prepared using Dulbecco's modified Eagle's medium, 1 M maltose, and 1% sericin as the essential ingredients, supplemented with 5%–40% propylene glycol (PG). Each BFM supplemented with 5%–40% PG was evaluated in undifferentiated cells. After thawing, BFM supplemented with 10% and 20% PG were 83% and 88% viable, respectively. There was no significant difference between the 10% and 20% PG groups. However, a significant difference was observed when the concentration of PG in the BFM decreased by 5% (5% PG vs. 10% PG; p = 0.0026). Each DMSO-free BFM was evaluated using differentiated neuronal cells. There was no significant difference between the 10% PG BFM and stem-CB-free groups. Viability was significantly different in the 10% glycerol BFM (4.8%) and 10% PG BFM (45%) (p = 0.028). The differentiated cells with 10% PG BFM showed higher adherence to culture dishes than those with 10% glycerol BFM. These results show that BFM containing PG was effective in differentiating neuronal cells. DMSO affects the central nervous system at low concentrations. This report indicates that DMSO is unsuitable for neuronal cells with multipotent differentiation potential. Therefore, it is essential for cell banking and transplantation medicine services to select appropriate cell freezing media.