Background: The recent expansion of genomic biobank research in the Arab region in the Middle East North Africa has raised complex ethical and regulatory issues. However, there is a lack of studies regarding the views of Arab researchers involved in such research. We aimed to assess the perceptions and attitudes of Arab researchers regarding these issues in biobank research. Methods: We developed a questionnaire to assess the perceptions and attitudes regarding genetic research of researchers from Egypt, Sudan, Morocco, and Jordan. The questionnaire requested demographic data, perceptions, and attitudes regarding the collection, storage, and use of biospecimens and data, the use of broad consent, data security, data sharing, and community engagement. We used multiple linear regressions to identify predictors of perceptions and attitudes. Results: We recruited 383 researchers. Researchers favored equally the use of broad and tiered consent (44.1% and 39.1%, respectively). Most respondents agreed with the importance of confidentiality protections to ensure data security (91.8%). However, lower percentages were seen regarding the importance of community engagement (64.5%), data sharing with national colleagues and international partners (60.9% and 41.1%, respectively), and biospecimen sharing with national colleagues and international partners (59.9% and 36.2%, respectively). Investigators were evenly split on whether the return of individual research results should depend on the availability or not of a medical intervention that can be offered to address the genetic anomaly (47.5% and 46.4%, respectively). Predictors of attitudes toward biospecimen research included serving on Research Ethics Committees, prior research ethics training, and affiliation with nonacademic institutions. Conclusions: We recommend further exploratory research with researchers regarding the importance of community engagement and to address their concerns about data sharing, with researchers within and outside their countries.

Biopreservation and Biobanking

Ischemia–reperfusion injuries are important issues after ovarian tissue transplantation (OTT). Our study examined the effects of N-acetylcysteine (NAC) and estradiol (E2) on mouse ovarian autografts. Mice (6–8 weeks) were divided into ovarian autograft as follows: Control: fresh OTT; Sham: cryopreserved/warmed OTT; NAC: cryopreserved/warmed OTT with NAC treatment; E2: cryopreserved/warmed OTT with E2 treatment; NAC+E2: cryopreserved/warmed OTT with the treatment of NAC and E2. In all groups, grafts were harvested on days 2, 7, and 28 after transplantation to evaluate histological parameters, inflammation relative to genes expression, and oxidative status. Histological analysis showed that NAC, E2, and a combination of NAC+E2 significantly increased the primordial, preantral, and antral follicular number. When NAC was used, it significantly reduced the expression of Tnf-α and Fgf-2, whereas it increased Il-1β, Il-6, and Vegf expression levels. The levels of Il-6, Fgf-2, and VEGF were dramatically increased in the E2-treated group. The combination of NAC and E2 significantly increased levels of Il-1β, Il-6, Fgf-2, and Vegf. NAC and E2 alone or in combination significantly increased total antioxidant capacity but did not affect the superoxide dismutase and glutathione peroxidase activities. In conclusion, after transplantation, NAC and E2 alone or in combination, could improve follicular development and angiogenesis as well as decline inflammation and ovarian oxidative damage.

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.

Background: Rheumatoid arthritis is a long-lasting inflammatory disease that usually involves joints, but it can also affect other organs, including the skin and lungs. In this case, it is important to maintain a balance between beneficial pro-inflammatory activity and harmful overactivation of the T helper cells (Th). We strive to investigate in this study the possibilities for the effect of mesenchymal stem cells (MSCs)-derived exosomes containing miR-146a/miR-155 on the lymphocyte population and function. Methods: Exosomes were isolated from overexpressed miR-146a/miR-155 MSCs for the purpose of this analysis. Splenocytes were isolated from collagen-induced arthritis (CIA) and control mice. It was important to consider the expressions of certain predominant autoimmune-response genes, including T-bet and interferon-γ (IFNγ), by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. It turned out to be a significant consideration with p < 0.05. Results: The results are expressed in percentages with respect to miR-146a/AntimiR-155 transduced MSC-derived exosomes treatment, which significantly decreased the mRNA expression level of IFNγ in healthy mice (p < 0.05). miR-146a transduced MSC-derived exosomes treatment significantly reduced the mRNA expression level of IFNγ in CIA mice (p < 0.05). It should be noted that the secretion of the pro-inflammatory factor IFNγ in CIA mice was inhibited in almost all groups (p < 0.05). Conclusion: Many research groups have mainly focused on strategies for reducing pro-inflammatory cytokines. This approach was recently suggested and investigated in our research team and suggested that manipulation of MSCs-derived exosomes could minimize pro-inflammatory cytokine production to strike a balance among Th subsets. These approaches tend to appear to achieve better results in the regulation of the immune system by the use of engineered exosomes derived from MSCs. By providing accurate information the reasonably practicable use of exosomes for cell-free therapy can be established.

This systematic review provides an overview of the history and current status of cryopreservation of fish sperm and a detailed evaluation of cryoprotocols using powdered milk. A literature search was performed in PubMed, Scopus, Web of Science, and SciELO databases. Twenty-nine articles were selected after excluding duplicate articles or articles that did not meet the eligibility criteria. Rhamdia quelen and Danio rerio were the most studied species. Slow freezing method, dry-shipper, freezing rate of −35.6°C/min, thawing in water bath (35.93°C ± 10°C), and 0.25 and 0.5 mL plastic straws were the main approaches evaluated. Methanol was the most used permeable cryoprotectant in combination with powdered milk, yielding the best results at 10% concentration. Motility rate was the main analysis performed after cryopreservation in virtually all studies, being subjectively evaluated by most authors. Powdered milk at 15% promoted the best results in the analyzed studies. For motility rate, the gains with the addition of powdered milk were observed in the orders Perciformes (Oreochromis mossambicus), Siluriformes (Pangasius pangasius, Pseudoplatystoma corruscans, and Pseudoplatystoma mataense), and Cypriniformes (Tor soro and Barbonymus gonionotus). For fertilization, gains were observed in the order Siluriformes (P. mataense) and Cypriniformes (T. soro). Sperm viability gains were observed in the orders Siluriformes (P. pangasius), Characiformes (Piaractus brachypomus), and Cypriniformes (B. gonionotus). The scientific evidences we present, in this study, may contribute and serve as a starting point for new and more refined studies to be developed in the field.

Objective: Recently, researchers have been focusing on characterizing the tongue coating microbiome from patients with digestive tract disease. However, to the best of our knowledge, the tongue coating collection methods have not been standardized until now. This article focuses on bridging this gap by exploring and validating the conditions suitable for the collection of tongue coating samples. Methods: One hundred forty-one healthy subjects were involved in the standardization of the tongue coating collection method. We conducted our standardization experiment by comparing different sampling tools, different preservation solutions, different scraping times, and different storage days with preservation at room temperature. The tongue coating samples from 59 normal individuals were analyzed using 16S ribosomal RNA (rRNA) gene-sequencing technology. The assessment of the quality of extracted DNA was used to verify our established method. We separated the 59 subjects into two groups (aged and younger), and the sequencing results were used to explore the age-related changes in microbiome. Results: Sterile oral swab B is suitable for the collection of tongue coating samples. To obtain a sufficient amount of DNA from a tongue coating sample, we recommend 30 times of tongue coating scraping. Normal saline, phosphate-buffered saline, and commercial preservation solution are all suitable for short-term sample storage (<1 hour). The commercial long-term preservation solution, which stores samples at room temperature (0 hour to 7 days) and can provide for fast commercial transportation, ensures the integrity of the sample DNA as well as the stability of the DNA quality. By using the established method, extracted DNA from all the 59 normal individuals' tongue coating samples passed an appropriate quality bar for microbiome studies. The average value of OD 260/280 is 1.72 ± 0.10; the average total DNA amount is 334.92 ng (±183.81 ng). The bacterial diversity of the tongue coating is increased and the bacterial community composition changes greatly in the NC group (aged normal subjects). Fusobacteriota is found as the dominant bacteria phyla in aged normal subjects with the 16S rRNA gene-sequencing technology. At the genus level, the relative abundance of Fusobacterium, Haemophilus, and Leptotrichia are significantly higher in aged individuals (all p < 0.05), and Neisseria, Streptococcus, and Porphyromonas are significantly higher in younger individuals (all p < 0.05). Conclusion: A participant-friendly tongue coating collection method for microbiome analyses can be established with good reliability and reproducibility. By taking advantage of our established method and 16S rRNA gene sequencing, significant differences were found in diversity and composition of tongue coating microbiota between aged and younger individuals, which contributes to a better understanding of the age-related composition of tongue coating microbiota.

Biopreservation and Biobanking

Biopreservation and Biobanking

Background: The measurement of nucleic acid quality, especially the analysis of integrity, is a key step for many downstream experiments in biomedical research and quality control of biomaterials. General gel electrophoresis is a traditional method for nucleic acid integrity analysis. Currently, more electrophoresis techniques are becoming standardized and automated operations with higher precision. In this study, we have evaluated the comparability and bias of the outcomes from three commercial assay systems. Methods: Seventy-two deoxyribonucleic acid (DNA) and 67 ribonucleic acid (RNA) samples were selected for methodological comparison among different systems. The DNA Quality Number (DQN) and RNA Quality Number (RQN) of BIOptic Qsep400, DNA Quality Score (DQS) and RNA Quality Score (RQS) of PerkinElmer Labchip GX Touch HT were separately compared with the DNA Integrity Number (DIN) and RNA Integrity Number (RIN^e) of the Agilent 4200 TapeStation according to Clinical and Laboratory Standards Institute (CLSI) guideline (EP09-A3). Results: The biases of the mean estimated between DQN and DIN, DQS and DIN both exceeded the acceptance criteria. The Passing–Bablok regression analysis between DQN and DIN, and the Deming regression analysis between DQS and DIN, showed the biases were both within the acceptance criteria, and the bias between DQN and DIN was smaller. For the comparisons of RQN and RIN^e, RQS and RIN^e, the regression analyses revealed the biases were both within the acceptance criteria. The bias of the mean estimated between RQS and RIN^e was outside of the acceptance criteria. Conclusions: There was a good comparability in nucleic acid integrity detection between BIOptic Qsep400 and PerkinElmer Labchip GX Touch HT with the Agilent 4200 TapeStation. However, the bias and linear correlations require more attention between systems.

Biopreservation and Biobanking

Biopreservation and Biobanking

Biopreservation and Biobanking

High-quality, well-annotated, healthy tissue specimens are crucial to the success of basic and translational research, but often difficult to procure. Postmortem (PM) tissue collections provide the opportunity to collect these healthy biospecimens. PM procurement programs led by biobanks can further contribute by providing researchers with rare biospecimens collected with short postmortem intervals (PMI) in controlled environments. To support biomedical and translational research, the Cornell Veterinary Biobank (CVB), an ISO 20387 accredited core resource at the Cornell University College of Veterinary Medicine, has performed PM tissue collections from research and privately owned animals since 2013. The CVB PM collection team, consisting of a board-certified veterinary pathologist, a licensed veterinary technician collection specialist, and a data capture specialist, performs rapid tissue collections during controlled warm necropsies, with an accepted PMI of ≤2 hours and a target PMI of ≤1 hour. A retrospective analysis of PM collections between 2013 and 2020 was completed, consisting of 4077 aliquots of 1582 biospecimens from 69 donors (48 canine, 16 feline, and 5 equine). An average of 22.93 biospecimens per donor were collected (range: 1–49). The average PMI for standard collections was 43.48 ± 2.30 minutes, starting on average 20.81 ± 1.61 minutes after time of death. Thus far, the CVB has a favorable utilization rate, with 414 aliquots (10.15%) from 350 specimens (20.12%) and 45 animals (65.22%) distributed to researchers. The success of the CVB PM tissue biobanking program, collecting high-quality biospecimens with short PMIs, was due to support from veterinary pathologists, the competence of CVB personnel, and the continuous evolution of methods within a quality management system. Improvement of PM tissue collection programs in biobanks, with standardized practices for all processes and specialized personnel, can enhance the quality and increase utilization of its biospecimens and associated data.

Background: Biobanks process, store, and supply biological materials for research. Preanalytical factors, especially storage time and temperature, must be controlled and standardized at all stages when handling biospecimen samples, especially because the literature reports highly contradictory optimal parameters. As large-sample studies are required to better understand the influence of time and temperature on cryopreserved samples' quality for genomic research, this study evaluated the integrity and quality of cryopreserved samples stored for up to 9 years at the biobank of Barretos Cancer Hospital, one of the largest biobanks in Latin America. Methods: We randomly selected 447 samples with tumor tissue paired with buffy coat or peripheral blood mononuclear cells (PBMCs) that were stored from 2008 to 2016. The genetic material quality was evaluated based on RNA integrity (RIN) and DNA integrity (DIN) ≥7, which indicated undegraded samples, and compared with storage time, which means that for DNA storage time, samples <8.1 and ≥8.1 years and for RNA <4.5 and ≥4.5 were used. Results: A total of 190 tumor tissues were eligible for DNA and RNA extraction. Those stored for 8 years had lower DIN (68%) than those stored for a shorter period (92%). A similar pattern, based on storage time (<8.1 and ≥8.1 years), was observed in the buffy coat (74% and 95%, respectively) and PBMCs (54% and 96%, respectively). For RNA extracted from tumor tissues, we observed lower RIN in samples stored for 4.5 years (17%) than in samples stored for a shorter period (45%). Buffy coat and PBMC samples stored at −30°C exhibited greater degradation (26%) than those stored at −80°C (1%). The DIN (p = 0.15) and RNA (p = 0.18) were unrelated to topography type. Conclusion: The temperature, particularly cryopreservation methodology, and storage time were the main factors that affected nucleic acid integrity, especially RNA, during cryopreservation of biospecimens.

Biopreservation and Biobanking

This review provides an update on the current state of cryopreservation studies coupled with ultrastructural observation. Research in these fields has evolved and advanced since its inception in the 1950s. Different techniques have different advantages, but the researcher's technical proficiency is also necessary to derive a sound conclusion. Sperm samples are the most widely studied specimen because they are less sensitive to freezing and have high fluidity in the membrane and low water content. Some studies have also investigated oocytes, embryos, larvae, and algae from aquatic species. Cryopreservation studies have formulated a method applicable to every species of interest to preserve their biodiversity and prevent extinction. However, the avoidance of cryoinjury because of intracellular ice formation is a species-specific challenge. More comprehensive studies on ultrastructural observation can assist in understanding the underlying mechanisms of failed cellular responses to cryopreservation. Thus, optimizing protocols and increasing the survival rates of thawed samples can improve current cryopreservation techniques. Nevertheless, investigations into the effects of freezing on organisms' ultrastructure remain limited, especially regarding aquatic organisms.

Purpose: Promoting neurogenesis is a promising strategy to treat neurodegenerative disorders. In the present study, we aimed to evaluate the effect of mastic gum resin from the Pistacia lentiscus var. Chia (Anacardiaceae family) in proliferation capacity and differentiation of embryonic mesenchymal stem cells into a neural lineage. Methods: For this purpose, mastic gum was applied as a neural inducer for stem cell differentiation into the neuronal lineage. Following treatment of embryonic stem cells (ESCs) with mastic gum, verification differentiation of the ESCs into the neuronal lineage, gene expression analysis, and immunocytochemistry staining approach were performed. Results: Gene expression analysis demonstrated that mastic gum increased the expression level of neuron markers in the ESCs-derived neuron-like cells. Moreover, our immunocytochemistry staining results of two important neural stem cell markers, including Nestin and microtubule-associated protein-2 (Map2) expression confirmed that mastic gum has the potential to promote neuronal differentiation in ESCs. Conclusion: In summary, the use of mastic gum to stimulate the differentiation of ESCs into a neural lineage can be considered as a good candidate in stem cell therapy.

The effect of antifreeze protein (AFP) as a cryoprotectant used in different concentrations of glycerol on post-thaw quality of epididymal sperm was investigated. Sperm were isolated from 50 testicles, obtained from 25 healthy mature goat bucks, with progressive motility >80%, and total morphological abnormalities <10% were pooled in each replication. The semen samples were diluted with Tris-citrate-fructose-soybean lecithin extender containing different concentration of AFP [0 μg/mL (A0), 5 μg/mL (A5), 10 μg/mL (A10)]. Each concentration of AFP was added in an extender containing either 7% (G7) or 5% (G5) glycerol. Post-thaw total and progressive motility were found to be higher (p < 0.05) in groups A5G5 and A5G7. Plasma membrane integrity, sperm acrosome integrity, DNA integrity, acrosome intact sperm, and mitochondrial membrane potential were found to be higher (p < 0.05) in groups A5G5 and A10G5. Sperm viability was found to be higher (p < 0.05) in group A5G5, while lipid peroxidation was recorded lower (p < 0.05) in groups A5G5 and A5G7. Regarding the apoptosis occurrence, the results demonstrate higher (p < 0.05) live post-thawed spermatozoa for groups containing 5 μg/mL AFP with 5% and 7% glycerol in addition to the lowest (p < 0.05) value for groups containing 0 μg/mL AFP with 5% and 7% glycerol. Based on these results, the present study concludes that the addition of 5 μg/mL AFP in combination with 5% glycerol in freezing extender improves the post-thaw quality, structure, and function parameters for buck spermatozoa.

Background: Colorectal cancer (CRC) is a common and lethal cancer worldwide. Extraction of high-quality RNA from CRC samples plays a key role in scientific research and translational medicine. Specimen collection and washing methods that do not compromise RNA quality or quantity are needed to ensure high quality specimens for gene expression analysis and other RNA-based downstream applications. We investigated the effect of tissue specimen collection and different preparation processes on the quality and quantity of RNA extracted from surgical CRC tissues. Materials and Methods: After surgical resection, tissues were harvested and prepared with various washing processes in a room adjacent to the operating room. One hundred fourteen tissues from 36 CRC patients were separately washed in either cold phosphate-buffered saline reagent (n = 34) or Dulbecco's modified Eagle's medium (DMEM; n = 34) for 2–3 minutes until the stool was removed, and unwashed specimens served as controls (n = 34). Six tissue specimens were washed and immersed in DMEM for up to 1 hour at 4°C. Before RNA extraction, all specimens were kept in the stabilizing reagent for 3 months at −80°C. RNA was extracted, and the concentration per milligram of tissue was measured. RNA quality was assessed using the RNA integrity number (RIN) value. Results: Different washing processes did not result in significant differences in RNA quantity or RIN values. In the six tissues that were washed and immersed in DMEM for 1 hour, RIN values significantly decreased. The quality of the extracted RNA from most specimens was excellent with the average RIN greater than 7. Conclusions: RNA is stable in specimens washed in different processes for short periods, but RIN values may decrease with prolonged wash times.

Aims: Bacterial contamination may occur in feces during collection and processing of semen. Bacteria not only compete for nutrients with spermatozoa but also produce toxic metabolites and endotoxins and affect sperm quality. The aim of the present study was to investigate the effect of antibiotic supplementation on the sperm quality of Indian red jungle fowl, estimation and isolation of bacterial species and their antibiotic sensitivity. Materials and Methods: Semen was collected and initially evaluated, diluted, and divided into six experimental extenders containing gentamicin (2.5 μg/mL), kanamycin (31.2 μg/mL), neomycin (62.5 mg/mL), penicillin (200 U/mL), and streptomycin (250 μg/mL), and a control having no antibiotics were cryopreserved and semen quality was evaluated at post-dilution, post-cooling, post-equilibration, and post-thawing stages (Experiment 1). A total aerobic bacterial count was carried out after culturing bacteria (Experiment 2) and subcultured for antibiotic sensitivity (Experiment 3). Results: It was shown that penicillin-containing extender improved semen quality (sperm motility, plasma membrane integrity, viability, and acrosomal integrity) compared with the control and other extenders having antibiotics. The bacteria isolated from semen were Escherichia coli, Staphylococcus spp., and Bacillus spp. Antibiotic sensitivity results revealed that E. coli shows high sensitivity toward neomycin, kanamycin, and penicillin. Staphylococcus spp. shows high sensitivity toward streptomycin, neomycin, and penicillin. Bacillus spp. shows high sensitivity toward kanamycin and penicillin. Conclusions: It was concluded that antibiotics added to semen extender did not cause any toxicity and maintained semen quality as that of untreated control samples, and penicillin was identified as most effective antibiotic. It is recommended that penicillin can be added to the semen extender for control of bacterial contamination without affecting the semen quality of Indian red jungle fowl.

Semen banking is an efficient method of artificial insemination for commercial breeders. However, the cryopreservation process induces severe damages to plasma membranes, which lead 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.

Introduction: Sample handling can influence biomarker measurement and introduce variability when combining data from multiple studies or study sites. To inform the development of blood collection protocols within a multisite cohort study, we directly quantified concentrations of 54 biomarkers in blood samples subjected to different handling conditions. Materials and Methods: We obtained serum, lithium heparin plasma, and EDTA plasma from 20 adult volunteers. Tubes of chilled whole blood were either centrifuged and processed within 2 hours of collection (the “reference standard”) or were stored with cool packs for 24 or 48 hours; centrifuged before and/or after this delay; or collected in tubes with/without gel separators. We used linear mixed models with random intercepts to estimate geometric mean concentrations and relative percent differences across the conditions. Results: Compared to the reference standard tubes, concentrations of many biomarkers changed after processing delays, but changes were often small. In serum, we observed large differences for B vitamers, glutamic acid (37% and 73% increases with 24- and 48-hour delays, respectively), glycine (12% and 23% increases), serine (16% and 27% increases), and acetoacetate (−19% and −26% decreases). Centrifugation timing and separator tube use did not affect concentrations of most biomarkers. Conclusion: Sample handling should be consistent across samples within an analysis. The length of processing delays should be recorded and accounted for when this is not feasible.

Background: Parallel to the rapid advancement of biological and information technologies, the role and forms of biobank research have been constantly changing. The ethical, legal, and social implications of consent in biobank research are in a state of flux. This study aimed to clarify current Japanese public preferences regarding the consent model and explore how public attitudes are determined. Methods: We conducted an online, population-based quantitative survey among Japanese residents aged between 20 and 69 years. Statistical analyses consisted of univariate and multivariate logistic regression. Results: Of the 1580 respondents, 60.9% preferred autonomy-based consent (specific or dynamic consent) and 23.9% preferred broad-type consent (opt-out or broad consent). Marital status, gender, and privacy concerns were significantly associated with the preference for a consent model. Conclusions: Our results demonstrated the public's current preference for autonomy-based consent, including dynamic consent. However, our findings also revealed that approximately half of the respondents considered broad consent as somewhat preferable.

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.

Objectives: This work investigates whether changes in a biospecimen's molecular composition from formaldehyde fixation drive changes in the mid infrared (MID-IR) spectrum. Our ultimate goal was to develop an analytical metrology that could be used to accurately determine the fixation time of a tissue sample as a surrogate to overall tissue quality. Methods: Multiple unstained formalin-fixed paraffin-embedded tissue samples were scanned with an MID-IR microscope to identify a molecular fingerprint of formaldehyde fixation. The fixation specific patterns were then mined to develop a predictive model. A multiple tissue experiment using greater than 100 samples was designed to train the algorithm and validate the accuracy of predicting fixation status. Results: We present data that formaldehyde crosslinking results in alterations to multiple bands of the MID-IR spectra. The impact was most dramatic in the Amide I band, which is sensitive to the conformational state of proteins. The spectroscopic fixation signature was used to train a machine-learning model that could predict fixation time of unknown tissues with an average accuracy of 1.4 hours. Results were validated by histological stain quality for bcl-2, FOXP3, and ki-67. Further, two-dimensional imaging was used to visualize the spatial dependence of fixation, as demonstrated by multiple features in the tissue's vibrational spectra. Conclusions: This work demonstrates that it is possible to predict the fixation status of tissues for which the preanalytics are unknown. This novel capability could help standardize clinical tissue diagnostics and ensure every patient gets the absolutely best treatment based on the highest quality tissue sample.

Biopreservation and Biobanking

Biopreservation and Biobanking

Biopreservation and Biobanking

Objective: Semen analysis is performed as one of the screening tests for infertility, including motility, morphology, and concentration observation. We aimed to investigate the expression rates of tumor necrosis factor-α (TNF-α) and heat shock protein (HSP)-70 as two opposite affectors of apoptosis in men with normal semen parameters and abnormal parameters to find the possible effect of this pathway on sperm parameters. We also aimed to investigate the apoptotic markers (DNA fragmentation and Caspase-3 expression) to observe the correlation of this pathway with apoptosis. Materials and Methods: A total of 32 men who applied for infertility evaluation were included in the study. Semen analysis was performed according to WHO criteria. Liquefaction time, appearance, volume, pH, viscosity, sperm concentration, total motility rate, sperm motility, and percentage of spermatozoa with normal morphology were determined. TNF-α, HSP-70, and Caspase-3 immunolocalization were scored histologically. A sperm chromatin dispersion test was used to observe DNA fragmentation. Results: There was no significant difference in TNF-α protein expression rate (mild level). The HSP-70 expression rate was lower, especially in the head region of normo. Caspase-3 was higher totally in non-normo. DNA fragmentation levels were similar in both the groups. Conclusion: From TNF-α protein expression at the mild level in both the groups, it may be hypothesized that the apoptotic pathway might not be triggered by the extrinsic pathway. We found a negative correlation between HSP-70 and Caspase-3 expressions, providing further evidence that HSP-70 works as an inhibitor to apoptosis. This, particularly on specific points, made us think the communication might begin in the anterior chamber, then flow through the cell body to the tail. HSP-70 expression was lower in normo than in non-normo, indicating the possible role of HSP-70 as an answer to any type of stressor in non-normozoospermic patients. Correspondingly, it may be concluded that HSP has an antiapoptotic effect, causing inhibition in the elimination of abnormal sperm cells impairing sperm parameters.

Preservation and transportation are essential for the clinical application of chimeric antigen receptor T (CAR-T) cells. This study aimed to optimize a cryopreservation solution for CAR-T cells and evaluate the antitumor efficiency of CAR-T cells using this optimized solution in vitro and in vivo. First, the stability of the cryopreservation solution for CAR-T infusion was detected by the L27 (3⁷) orthogonal experiment. Subsequently, osmolality and pH were analyzed for the preservation reagent. Additionally, apoptosis and CAR expression of CAR-T cells were measured by flow cytometry, and the cytotoxicity was determined by calcein-AM staining. The results showed that cryopreservation solutions used in this study demonstrated high chemical stability, which induced only 2% CAR-T cells apoptosis in optimal solutions, which were slightly lower than other commercial solutions. Moreover, the CAR expression was not significantly affected by preservation with these solutions. There were no significant differences in the cytotoxicity between fresh and thawed CAR-T cells cryopreserved in the cryopreservation solutions in vivo and in vitro. This study developed a new cryopreservation solution for CAR-T cells, and it was safe and also had negligible effects on the CAR-T cells antitumor activity.

Cell lines are valuable tools to safeguard genetic material from species threatened with extinction that is mainly due to human action. In this scenario, the puma constitutes a species whose population is being rapidly reduced in the ecosystems it inhabits. For the first time, we characterized puma skin-derived cell lines and assessed these cells after extended culture (experiment 1) and cryopreservation (experiment 2). Initially, we identified and characterized four dermal fibroblast lines using morphology, ultrastructure, and immunofluorescence assays. Moreover, we evaluated the effects of culture time (1st, 3rd, and 10th passages) and cryopreservation on their morphology, ultrastructure, viability, metabolism, proliferative activity, reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm), and apoptosis. The cells showed a typical spindle-shaped morphology with centrally located oval nuclei. The cells were identified as fibroblasts by staining for vimentin. In vitro culture after the 1st, 3rd, and 10th passages did not alter most of the evaluated parameters. Cells in the 3rd and 10th passages showed a reduction in ROS levels (p < 0.05). The ultrastructure revealed morphological damage in the prolongments, and nuclei of cells derived from the 3rd and 10th passages. Moreover, cryopreservation resulted in a reduction in ΔΨm compared with that of noncryopreserved cells, suggesting that the optimization of cryopreservation methods for puma fibroblasts is essential. In conclusion, we found that viable fibroblasts could be obtained from puma skin, with slight changes after the 10th passage in in vitro culture and cryopreservation. This is the first report on the development of cell lines derived from pumas.

This study investigated the cryoprotectant effects of dimethylformamide (DMF), ethylene glycol (EG), and dimethyl sulfoxide (DMSO) as substitutes for glycerol (GLY) in a soybean lecithin (SL)-based extender in the cryopreservation of buck sperm. In this study, the semen of three Saanen bucks was individually extended in SL supplemented with 5% GLY (control), DMF, EG, or DMSO. After this, the extended semen was cryopreserved and two straws from each group were thawed (37°C for 30 seconds), pooled, and analyzed for sperm motion parameters, plasma membrane integrity (PMI), acrosomal integrity (ACI), and high mitochondrial membrane potential (HMMP). Samples were analyzed after 15 minutes (T0) and after 2 hours of incubation at 37°C (T2). The results revealed higher values of motility (total and progressive) and sperm motion parameters for DMF than the other cryoprotectants (p < 0.0001). PMI and HMMP did not differ (p > 0.05) between GLY and DMF, but ACI was higher (p < 0.01) for DMF compared with GLY. Based on these results, DMF and GLY samples were used in heterologous in vitro fertilization assays by using bovine oocytes (n = 337) obtained from a slaughterhouse. No differences (p > 0.05) were observed between GLY and DMF for unfertilized (GLY: 38.8%; DMF: 25.33%), pronucleus (GLY: 25.68%; DMF: 27.92%), and cleavage rates (GLY: 35.52%; DMF: 46.75%). Based on these results, it is concluded that DMF preserves sperm motion characteristics and ACI better than GLY, EG, and DMSO, and it is the penetrating cryoprotectant of choice for the cryopreservation of buck sperm in SL extender.

This study investigated the effect of ascorbic acid (vitamin C) and proline amino acid alone or together on the quality and fertility of frozen/thawed honey bee spermatozoa. The experiments were designed to compare a single ascorbic acid, a single proline amino acid, and different combinations of ascorbic acid with proline amino acid on the cryopreservation of honey bee semen based on sperm motility, viability, intact membrane (hypo-osmotic swelling test), and fertility rates. Eight cryopreserved study groups comprised Control II with no supplement, along with groups with ascorbic acid (2 mg), proline 25 mM, proline 50 mM, proline 100 mM, and combination groups of both ascorbic acid (2 mg) and proline 25 mM, proline 50 mM, and lastly proline 100 mM groups, respectively. Using 50 mM proline in the tested groups had the greatest impact on sperm motility, viability, the percentage of spermatozoa with intact membrane, and fertility. The cryopreservation process caused a gradual decrease in motility, viability, intact membrane (p < 0.05), and fertility rates (p < 0.01) in all the tested research groups as against the fresh semen control group. Successful honey bee sperm cryopreservation and fertility are achievable when using an appropriate sperm freezing protocol and antioxidant. Proline amino acid as an antioxidant in semen extender had a more beneficial influence on sperm quality parameters and fertility. The success of cryopreservation with antioxidants is related to the chosen antioxidant in a dose-dependent manner.

Freezing of sperm is known as an important part of assisted reproduction. However, many studies have illustrated that cryopreservation negatively affects the quality and fertility rate of sperm. This study aimed to evaluate the effects of trehalose and pentoxifylline (PTX) in diluents on cooled and frozen-thawed Markhoz goat sperm. Preassessed samples were pooled and diluted with a basic diluent using trehalose and PTX. The cooled sperm showed significant improvement. The motion characteristics of cryopreserved sperm were evaluated based on computer-assisted system analysis. In this study, we investigated the viability, membrane integrity, malondialdehyde concentration, total abnormality, acrosome integrity, and seminal hyaluronidase enzyme. Also, the hypo-osmotic swelling test, mitochondrial activity, apoptotic features, caspase activity, chromatin dispersion test, active mitochondria, and reactive oxygen species (ROS) activity were assessed as complementary parameters. The data illustrate that the total motility, progressive motility, average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL), and the ratio of sperm chromatin dispersion, viable sperm were improved significantly (p < 0.05) using 3 mM PTX alone or 3 mM PTX plus 50 mM trehalose, while other characteristics indicate significant enhancement by 3 and 6 mM PTX and 50 and 70 mM trehalose alone or in combination, except amplitude of lateral head displacement (ALH), beat/cross frequency (BCF), and intracellular ROS-(O⁻), which demonstrate no significant difference among treatments. In conclusion, this study indicates that addition of 3 and 6 mM PTX alone or with 50 and 70 mM trehalose seems to reduce the damage caused by cooling and cryopreservation processes.

Background: A functional artificial ovary is a promising strategy to recover fertility and restore endocrine function in cancer patients. The aim of this study is to optimize the follicle isolation protocol for cryopreserved human ovarian tissues. Methods: Each of the cryopreserved human ovarian cortex pieces (OCPs) from 10 patients was cut into two equal parts and randomly distributed into two treatment groups. Group 1: OCPs digested with Tumor Dissociation Enzyme (TDE); Group 2: OCPs digested with Liberase Dispase High (DH). The efficiency of both groups were evaluated in terms of yield, viability, morphology, and a short-term in vitro culture (IVC) in alginate scaffolds. Results: The TDE can isolate more primordial follicles and smaller diameter of follicles than Liberase DH. The TDE also enabled the isolation of more bright red follicles, higher percent of viable follicles, more morphologically normal follicles, and lower oxidative stress levels compared with Liberase DH. After eight days of IVC, follicles in the TDE group had a higher growth rate from Day 0 to Day 8, and higher viability on Day 8 than the Liberase DH Group. Conclusion: The TDE can be considered an alternative to Liberase DH, enables the isolation of a higher number of healthy follicles from human OCPs, and improves follicle survival after IVC in contrast to Liberase DH.

Oxidative stress is a major contributory factor to cellular damage during semen cryopreservation and results in a decreased fertilizing capacity of cryopreserved bull sperm. The inclusion of exogenous antioxidants sometimes exerts deleterious effects on sperm quality. Thus, enhancing the endogenous production of antioxidants is a requirement. This study aimed to investigate the effect of milk type heated at different temperatures on the antioxidant potential of extenders, and the subsequent post-thaw quality parameters and in vivo fertility of buffalo bull semen. Cow (C) and buffalo whole milk (B) were used separately for semen extender preparation, heated at five different temperatures (T₁ = 90°C, T₂ = 100°C, T₃ = 110°C, T₄ = 120°C, T₅ = 130°C) for 10 minutes. Reactive sulfhydryl groups were measured in each subgroup by Ellman's reagents as CT₁ = 143.2 μM, CT₂ = 147.4 μM, CT₃ = 151.5 μM, CT₄ = 157.2 μM, CT₅ = 161.8 μM, BT₁ = 168.3 μM, BT₂ = 172.5 μM, BT₃ = 176.7 μM, BT₄ = 196.3 μM, and BT₅ = 205.7 μM. All semen samples were cryopreserved in milk-based extenders by using standard procedures. Post-thaw quality parameters including total and progressive motility, mitochondrial membrane potential, plasma membrane integrity, and acrosome integrity were found to be higher (p < 0.05) in the group (BT₃) containing buffalo milk heated at 110°C, whereas in the same group, lipid peroxidation was found to be lower (p < 0.05) as compared with other treatment groups and control group. In vivo fertility of cryopreserved buffalo sperm was compared among BT₃, CT₁ (conventionally used milk extender), and a Tris egg yolk extender group. The fertility rates [47% (54/114), 30% (33/108), and 36% (37/103)] were higher (p < 0.05) in BT₃ as compared with other groups. This study suggests that buffalo milk heated at 110°C has high antioxidant potential and improves post-thaw quality and in vivo fertility of cryopreserved buffalo bull semen.

The present study was performed to investigate the effects of supplementing flaxseed oil (FO) or vitamin E (VE) or their combination to an extender for Simmental bull semen cryopreservation. In experiment 1, different concentrations of FO (0, 10, 100, and 1000 ng/mL) and VE (0.05, 0.1, and 0.2 mg/mL) were added to the extenders. In experiment 2, FO, VE, and FO + VE were added and a control group was included. Sperm viability, motility, motion parameters, acrosome integrity and membrane integrity, endogenous antioxidant indices, reactive oxygen species, and malondialdehyde levels were evaluated after semen thawing. A higher percentage of viability, motion parameters, endogenous antioxidant indices, and membrane integrity was observed after supplementation with 10 ng/mL FO or 0.1 mg/mL VE compared with the control group (p < 0.05). Also, combined supplementation of 10 ng/mL FO +0.1 mg/mL VE further improved the quality of frozen–thawed sperm by regulating viability, motion parameters, membrane integrity, and endogenous antioxidant indices compared with the FO or VE alone (p < 0.05). These results indicated that FO (10 ng/mL) + VE (0.1 mg/mL) could further improve the protective effects on bull sperm post-thaw.

This study assessed the outcomes of nonsurgical embryo recovery (NSER) after superovulation (SOV) in five locally adapted Brazilian breeds of sheep and goats. The objective was to evaluate the feasibility and efficiency of using SOV combined with a less-invasive embryo collection technique for supplying the Brazilian animal gene bank with germplasm from specific genotypes of interest. Morada Nova (n = 20), Santa Inês (n = 20), and Somalis (n = 20) ewes received an intravaginal progesterone (330 mg) device for 9 days, while Canindé (n = 15) and Moxotó (n = 15) goats received an intravaginal medroxyprogesterone acetate (60 mg) device for 6 days. All females received 133 mg of porcine follicle-stimulating hormone (pFSH) administrated in six decreasing doses 12 hours apart, starting 60 hours before device removal, plus 37.5 μg of d-cloprostenol at the fifth and sixth pFSH dose. Donors in estrus were mated with fertile males. The corpora lutea (CL) number was assessed by ultrasonography 1 day before NSER. On day 6.5 or 7 after estrus, NSER was performed following hormonally induced cervical relaxation. A total of 97% of sheep and 90% of goats responded with estrus, and among those, 91% of sheep and 85% of goats presented a CL. In ewes, the numbers of CL were greater (p < 0.05) in the Santa Inês breed, while similar (p > 0.05) CL numbers were found among the goat breeds. All viable embryos were freezable (excellent and good quality) and the number per donor was 7.8 for sheep and 4.9 for goats. All parameters of NSER efficiency, embryo yield, and fertility post-NSER did not differ (p > 0.05) between breeds among each species. The SOV-NSER procedures applied for an embryo biobank supply of locally adapted Brazilian breeds of small ruminants were efficient regarding production of cryopreservable embryos, and preservation of donor fertility. Therefore, SOV followed by NSER is recommended for embryo biobank assembly in sheep and goats.

Background: Formalin-fixed, paraffin-embedded (FFPE) tissues are a valuable resource for clinical and basic science research. Paraffin blocks and the resulting unstained sections (USS) are often stored for years before being used. Previous studies have evaluated the effects of time, temperature, humidity, and inert gases on preservation of USS; however, no study has examined all four variables together. Methods: In the current work, we prospectively and blindly assessed time points from 0 to 24 months, room versus refrigerated temperature, and presence of a desiccant and/or nitrogen atmosphere on a variety of benign and malignant tissues from North America and Africa. End points included immunohistochemistry (IHC), in situ hybridization (ISH), extracted RNA and DNA quantity and quality, and messenger RNA performance in a novel, multiplexed digital gene expression profiling assay of both housekeeping and tumor-specific genes. Results: We found that using current methods of antigen retrieval, staining, and extraction, the end points of IHC, ISH, RNA, and DNA were well preserved under the various conditions tested, with implications that pre-embedding factors contribute to variability in subsequent tissue integrity. We also document that spectrophotometric estimations of nucleic acid concentrations were in general estimated to be higher than with fluorimetric methods, which may be pertinent to end assay development. We further describe a new multiplex assay, the PlexSet digital gene expression assay, suitable for evaluating RNA quality in FFPE tissues. Conclusion: Altogether, these results may provide helpful guidance with regard to approaches for long-term storage conditions for USS.

Cryopreservation of somatic tissue has been studied as a tool for the knowledge and conservation of endangered species, such as Antillean manatees. The use of vitrification protocols is an important step in the establishment of biological banks. To decrease the damage caused by this technique, a reduction in the concentration of cryoprotectants has been proposed. Therefore, we aimed to evaluate combinations and concentrations of intracellular cryoprotectants for the conservation of somatic tissues derived from Antillean manatees. Dulbecco's modified Eagle's medium, F-12 composed of 10% fetal bovine serum and 0.25 M sucrose, was supplemented with 3.0 M ethylene glycol (EG) plus 3.0 M dimethyl sulfoxide (DMSO), or 1.5 M EG plus 1.5 M DMSO or 3.0 M EG or 3.0 M DMSO, to produce four solutions for solid-surface vitrification. Noncryopreserved tissues were used as the controls. After warming, tissues derived from four Antillean manatees were evaluated for ultrastructure, histology, and in vitro culture. No differences were observed among the cryopreserved and noncryopreserved tissues in terms of ultrastructure. The dermis thickness of the cryopreserved fragments in solutions containing 3.0 M EG plus 3.0 M DMSO, 3.0 M EG, and 3.0 DMSO was similar to that of the control. Moreover, cryopreservation with 3.0 M EG plus 3.0 M DMSO maintained tissue proliferative capacity potential evaluated by quantification of nucleolar organizing regions. Nevertheless, none of the cryopreserved fragments were able to maintain the number of fibroblasts and the collagen percentage as compared with that of the noncryopreserved fragments. Also, none of the cryopreserved fragments in the different solutions were able to produce cells in vitro. In summary, even reducing the concentration of intracellular cryoprotectants as well as their association did not guarantee the maintenance of cells after in vitro culture. Further studies are needed to optimize the cryopreservation protocols in Antillean manatee somatic tissues.

Xenarthra—a superorder of placental mammals endemic to the Neotropics—is represented by armadillos, anteaters, and sloths. Considering their long history in the Americas, extant xenarthrans represent an important group for understanding the impact of past environmental changes on species diversification and serve key ecological functions as ecosystem engineers. Unfortunately, most wild xenarthran populations are at risk, due primarily to anthropogenic activities, necessitating urgent conservation efforts. Moreover, the paucity of information on some species has rendered population estimation and, consequently, conservation management challenging. In addition, relatively few groups are researching this superorder, perhaps because fieldwork with armadillos, anteaters, or sloths and their captive care are challenging tasks. Nevertheless, dedicated research and efforts to ensure the long-term conservation of these animals are deemed essential. In this context, cryobanks are a practical approach for breeding and maintaining genetic diversity in wildlife, and they are important tools for assisting and improving both ex situ and in situ conservation strategies. Therefore, cryopreservation of biological resources may be a promising strategy for conserving xenarthrans. Specifically, semen cryopreservation, which has already been applied in some species, may be the most effective strategy for this group. The present article provides an overview of ex situ conservation of xenarthrans, which will contribute to the development and implementation of additional strategies for protecting these unique mammals.

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 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 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.

Background: Two physicochemical effects occur during vitrification: nucleation and crystallization. Nucleation is a statistical occurrence by its nature. Thus, the more water molecules that are present the higher are the chances for nucleation to occur. Crystallization is a first-order transition where a water molecule is incorporated into ice crystals. Intracellular viscosity, which is the combination of water, salts, and cryoprotectants (CPs), affects both the nucleation and crystal growth rates. Ice velocity is inversely correlated with the viscosity and directly proportional to the function of the system's supercooling. However, little is known about the speed of ice crystals propagation in vitrification solutions containing different concentrations of CPs. Methods: This article describes the ice crystal propagation velocity while referring to vitrification. Ice crystal propagation velocity was measured in solutions containing different CP (dimethyl sulfoxide [DMSO], propylene glycol [PG], ethylene glycol [EG], and glycerol) concentrations at a supercooled temperature. The different CPs solutions were inserted into 0.25 mL straws and placed in different temperatures of an alcohol bath (T_c) at supercooling temperatures of −8°C to −10°C. Results: We found that ice crystal propagation is inversely correlated to CP concentrations. Interestingly, PG showed, with statistically significant results, lower ice crystals growth velocities up to concentrations of 30% (v/v), compared with DMSO, and EG at the same concentrations. The combination of EG with PG showed better results (0.25 mm/s) than EG with DMSO (0.39 mm/s) in terms of decreasing the ice crystals growth velocity. When the concentration was increased to 40% (v/v), EG showed the lowest ice crystal propagation velocity (0.09 mm/s), although not significantly different than PG and glycerol but significantly lower than DMSO (0.13 mm/s). Conclusion: These results suggest that current vitrification solutions are not optimized. Based on our results, we suggest that combining PG with EG has advantages over the combination of DMSO and EG, which might promote successful cell and tissue vitrification.

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.

The aim of this study was to investigate differences in the sperm response to a vitrification-warming process between ejaculated and epididymal dog spermatozoa, and to evaluate the efficacy of an animal protein-free extender for vitrification of both types of sperm cells. Vitrified-warmed spermatozoa from the epididymis showed greater (p < 0.001) progressive motility and total motility values than ejaculated spermatozoa, regardless of the diluent. The vitrification procedure returned better results for viability and intact acrosome when human tubal fluid (HTF^®) was used (25.10 ± 7.90 and 56.50 ± 6.7, respectively) compared with Tris-Citric acid-Glucose (TCG) (15.20 ± 4.70 and 43.70 ± 7.9, respectively) in ejaculated samples. Similarly, higher total motility (34.5 ± 4.5) was observed in HTF postwarmed samples compared with TCG-treated samples (19.52 ± 5.1). The interaction source (epididymis, ejaculated) × extender had a significant effect (p < 0.001) on the values of total motile spermatozoa after warming. HTF-based extender improved (p < 0.001) total motility values in epididymal samples, but not in ejaculated samples. In conclusion, epididymal spermatozoa show higher cryoresistance to the vitrification process than ejaculated spermatozoa in dogs. The use of HTF is adequate for both ejaculated and epididymal canine sperm vitrification.

Biobanking is becoming increasingly important as a key tool for precision medicine, but neither biobanking nor precision medicine itself have generally been integrated in medical curricula. However, most medical students will encounter these topics in their future careers as physicians or researchers. The European Union (EU)-funded project eduBRoTHER aims to close this gap of professional input. Since the academic year 2020/21, students at the Faculty of Medicine of Pilsen—Charles University and the Faculty of Medicine of the University of Regensburg have been offered an innovative core elective subject that focuses on biobanking and precision medicine issues, using the concept of blended learning.

Freshly isolated human hepatocytes are an important model for translational research, validation of experiments done in animals, and preclinical studies. Human hepatocyte isolation often cannot be carried out easily on demand in common research laboratories, and researchers often collaborate to share hepatocytes or outsource hepatocyte isolations. As a prerequisite for such a strategy, hepatocytes have to maintain their phenotypes after transport. Therefore, this study aimed to determine if overnight storage or shipment of hepatocytes affects their quality when viability, adherence, and cytochrome P450 (CYP) activities are considered. Hepatocytes were stored overnight or shipped to a collaborator in a cold storage solution on wet ice. On the next day, viability of hepatocytes was assessed before plating the cells to determine adherence. Hepatocytes were also cultured in a sandwich culture to determine CYP activities and inducibility. The results showed that although viability (79% ± 0.7% on isolation) was significantly decreased by overnight storage or shipment by 11% (p < 0.001) or 15% (p < 0.001), respectively, the viability of hepatocytes the next day at above 64% ± 2.2% remained sufficiently high for further experiments. In addition, hepatocytes stored for 18 or 24 hours were adherent the next day, and a high confluence of 81% ± 10% to 91% ± 4% was achieved after 48 hours in culture when hepatocytes were adhered on collagen-coated plates. Furthermore, CYP enzyme activities were inducible and not affected by variables such as fibrosis, age, type of operation, steatosis, and body mass index. However, our data would suggest that the type of cancer (primary/secondary), sex (male/female), hypertension, glutamic oxaloacetic transaminase activity, partial thromboplastin time, and size of perfused liver had significant effects (p < 0.05) on induction of some CYP enzymes. In conclusion, human hepatocyte isolation can be carried out at a centralized site and shared between multiple researchers, increasing flexibility and access to a representative human liver in vitro model.