Assessment of formalin preserved and fresh frozen quadriceps tendon graft-suture constructs for load to failure testing: a biomechanical cadaveric study.

PURPOSE: Fresh-frozen specimen availability and cost may be a barrier for initiation of biomechanical studies where soft tissue is used in a construct with other medical devices. The impact of soft tissue preservation method on the outcomes of biomechanical studies in the specific case of graft-suture constructs is relatively unexplored. This study aimed to observe peak loads and failure modes in biomechanical testing of fresh-frozen (FF) versus formalin embalmed (FE) quadriceps tendon (QT) graft-suture constructs for soft tissue fixation in ACLR and assess suitability of FE QT graft constructs for load-to-fail testing. METHODS: Twenty QT grafts were harvested from human cadaver specimens. Ten grafts came from fresh-frozen donors and 10 from embalmed donors. All grafts were prepared with the modified Prusik knot using a braided composite suture and subjected to tensile loading. Comparisons between the biomechanical properties of the graft-suture constructs were made with unpaired t tests with α = 0.05. RESULTS: FE and FF constructs displayed similar peak loads and failure modes. FF constructs had greater elongation after pre-tensioning than FE (7.3 vs. 5.5 mm, p = 0.02) and greater elongation after cyclic loading than FE constructs (17.5 vs. 10.5 mm, p = 0.01). Hysteresis was greater for FF constructs at the 50th, 100th, 150th, and 200th cycle (p = 0.02, p = 0.07, p < 0.001, p = 0.004, respectively). FE constructs were stiffer than fresh-frozen (103 vs. 84 N/mm, p < 0.001). CONCLUSION: FE constructs were significantly stiffer but displayed similar peak load and failure mode to FF which was reflective of the strength of the suture material. FE grafts can offer an alternative to FF grafts in graft-suture constructs for biomechanical studies where load at failure and knot security and strength is of main interest.

Cryopreservation and assessment of genetic fidelity Acorus calamus Linn., an endangered medicinal plant.

BACKGROUND: Acorus calamus Linn. is a medicinally valuable monocot plant belonging to the family Acoraceae. Over-exploitation and unscientific approach towards harvesting to fulfill an ever-increasing demand have placed it in the endangered list of species. OBJECTIVE: To develop vitrification-based cryopreservation protocols for A. calamus shoot tips, using conventional vitrification and V cryo-plate. MATERIALS AND METHODS: Shoot tips (2 mm in size) were cryopreserved with the above techniques by optimizing various parameters such as preculture duration, sucrose concentration in the preculture medium, and PVS2 dehydration time. Regenerated plantlets obtained post-cryopreservation were evaluated by random amplified polymorphic DNA (RAPD) to test their genetic fidelity. RESULTS: The highest regrowth of 88.3% after PVS2 exposure of 60 min was achieved with V cryo-plate as compared to 75% after 90 min of PVS2 exposure using conventional vitrification. After cryopreservation, shoot tips developed into complete plantlets in 28 days on regrowth medium (0.5 mg/L BAP, 0.3 mg/L GA3, and 0.3 mg/L ascorbic acid). RAPD analysis revealed 100% monomorphism in all cryo-storage derived regenerants and in vitro donor (120-days-old) plants. CONCLUSION: Shoot tips of A. calamus that were cryopreserved had 88.3% regrowth using V cryo-plate technique and the regerants retained genetic fidelity. https://doi.org/10.54680/fr24210110412.

Assessment of Faecal Microbiota Transplant Stability in Deep-Freeze Conditions: A 12-Month Ex Vivo Viability Analysis.

BACKGROUND: Faecal microbiota transplantation (FMT) is an established treatment for Clostridioides difficile infection and is under investigation for other conditions. The availability of suitable donors and the logistics of fresh stool preparation present challenges, making frozen, biobanked stools an attractive alternative. AIMS: This study aimed to evaluate the long-term viability of bacterial populations in faecal samples stored at -80°C for up to 12 months, supporting the feasibility of using frozen grafts for FMT. METHODS: Fifteen faecal samples from nine healthy donors were processed, mixed with cryoprotectants and stored at -80°C. Samples were assessed at baseline and after 3, 6 and 12 months using quantitative culturing methods to determine the concentration of live bacteria. RESULTS: Quantitative analysis showed no significant decrease in bacterial viability over the 12-month period for both aerobic and anaerobic cultures (p = 0.09). At all timepoints, the coefficients of variability in colony-forming unit (CFU) counts were greater between samples (102 ± 21% and 100 ± 13% for aerobic and anaerobic cultures, respectively) than the variability between measurements of the same sample (30 ± 22% and 30 ± 19%). CONCLUSIONS: The study confirmed that faecal microbiota can be preserved with high viability in deep-freeze storage for up to a year, making allogenic FMT from biobanked samples a viable and safer option for patients. However, a multidonor approach may be beneficial to mitigate the risk of viability loss in any single donor sample.

Assessment and comparison of viability assays for cellular products.

BACKGROUND AIMS: Accurate assessment of cell viability is crucial in cellular product manufacturing, yet selecting the appropriate viability assay presents challenges due to various factors. This study compares and evaluates different viability assays on fresh and cryopreserved cellular products, including peripheral blood stem cell (PBSC) and peripheral blood mononuclear cell (PBMC) apheresis products, purified PBMCs and cultured chimeric antigen receptor and T-cell receptor-engineered T-cell products. METHODS: Viability assays, including manual Trypan Blue exclusion, flow cytometry-based assays using 7-aminoactinomycin D (7-AAD) or propidium iodide (PI) direct staining or cell surface marker staining in conjunction with 7-AAD, Cellometer (Nexcelom Bioscience LLC, Lawrence, MA, USA) Acridine Orange/PI staining and Vi-CELL BLU Cell Viability Analyzer (Beckman Coulter, Inc, Brea, CA, USA), were evaluated. A viability standard was established using live and dead cell mixtures to assess the accuracy of these assays. Furthermore, precision assessment was conducted to determine the reproducibility of the viability assays. Additionally, the viability of individual cell populations from cryopreserved PBSC and PBMC apheresis products was examined. RESULTS: All methods provided accurate viability measurements and generated consistent and reproducible viability data. The assessed viability assays were demonstrated to be reliable alternatives when evaluating the viability of fresh cellular products. However, cryopreserved products exhibited variability among the tested assays. Additionally, analyzing the viability of each subset of the cryopreserved PBSC and PBMC apheresis products revealed that T cells and granulocytes were more susceptible to the freeze-thaw process, showing decreased viability. CONCLUSIONS: The study demonstrates the importance of careful assay selection, validation and standardization, particularly for assessing the viability of cryopreserved products. Given the complexity of cellular products, choosing a fit-for-purpose viability assay is essential.

Short-duration cold exposure decreases fasting-induced glucose intolerance but has no effect on resting energy expenditure.

The aim of the present study was to investigate whether brief cold exposure can reverse fasting-induced glucose intolerance and insulin resistance, and improve resting energy expenditure (REE). Twelve young non-obese women were randomly assigned to undergo the following conditions: 2 days of fasting with two 10-min whole-body cold-water immersions on separate days (FAST-COLD), 2 days of fasting without cold-water immersions (FAST), 2 days of usual diet with two 10-min whole-body cold-water immersions on separate days (COLD), or 2 days of usual diet without cold-water immersions (CON) in a randomised crossover fashion. Changes in REE and substrate utilisation, and glucose tolerance and insulin sensitivity from the oral glucose tolerance test were examined. The results showed that FAST-COLD and FAST trials increased (P < 0.05) REE and decreased (P < 0.05) respiratory quotient, but these variables did not differ significantly between the FAST-COLD and FAST trials. The glucose and insulin area under the curves (AUCs) were higher (P < 0.05) in the FAST-COLD and FAST trials than in the CON and COLD trials, and these AUCs were lower (P < 0.05) in the FAST-COLD than in the FAST trial. Matsuda index was lower in the FAST trial than in the CON trial (P < 0.05), and tended to be greater after the FAST-COLD trial than after the FAST trial (P = 0.060). In conclusion, cold exposure had no effect on REE but decreased fasting-induced glucose intolerance which was accompanied by a maintained insulin sensitivity.

Assessment of the stallion sperm acrosome in two different extenders: Spermac stain in comparison with PNA/PSA/PI triple-staining.

Since the stallion acrosome is very small compared to other species and cannot be properly assessed without additional staining, several labelling techniques were developed to facilitate its assessment. The aim of this study was to compare the Spermac stain (Minitüb GmbH) and a PNA/PSA/PI triple-staining detected by flow cytometry with regard to method agreement for detecting non-intact acrosomes within two different extenders. For this purpose, eighteen stallion ejaculates were split in half and diluted with the semen extenders EquiPlus or Gent (Minitüb GmbH) to a final concentration of 50 × 106 sperm/mL, respectively. Subsequently, 126 semen samples were stained with both methods between 4 and 240 h (mean: 63.8 ± 48.9 h) after semen collection. Calculated Intraclass correlation coefficients revealed excellent correlations between both methods for EquiPlus (r = .77, p < .001) and fair correlations for Gent (r = .49, p < .001). Interestingly, flow cytometry detected more non-intact acrosomes in EquiPlus than in Gent (p < .001), whereas the Spermac stain showed no differences (p = .902) between extenders. The poorer method agreement in Gent could be caused by egg yolk artefacts, which made interpretation difficult, so flow cytometry might be preferred. The differences in detected non-intact acrosomes between extenders highlighted the importance of establishing adapted laboratory protocols for different extender types in order to generate comparable results.

Comparative Assessment of Survival and Clinical Outcome Between Two Commercial Vitrification Kits with Different Warming Protocols After Blastocyst Culture: Potential Perspectives Toward Simplified Warming Procedures.

This study investigates whether there is an effect on laboratory results and clinical outcome using commercial kits with similar vitrification but different warming procedures for blastocysts vitrified on day 5 or day 6. A single-center retrospective cohort study was performed between 2011 and 2020. A change from a stage-specific kit (Kit 1) to a universal kit (Kit 2) was undertaken in 2017. A total of 1845 untested blastocysts were warmed for single vitrified-warmed blastocyst transfers (SVBT). Eight hundred and twenty-five blastocysts were vitrified with Kit 1 and 1020 with Kit 2. Blastocyst survival was not different (96.1% versus 97.3%). Seven hundred seventy-seven SVBT were performed from Kit 1 and 981 from Kit 2. Overall clinical pregnancy and live birth rates were not different (35.4% versus 34.1% and 30.9% versus 30.5% for Kit 1 and 2, respectively). Subgroup analysis for live birth rates in relation to the day of blastocyst vitrification showed no differences (36.1% and 36.1% for day 5 and 25.4% and 23.5% for day 6 blastocysts, respectively). For both kits, the mean gestational age was not different (38.8 ± 2.5 weeks versus 38.8 ± 2.0 weeks) with a singleton birth weight of 3413 ± 571 g and 3410 ± 528 g for Kit 1 and Kit 2, respectively. Differences in warming procedures do not affect laboratory performance or clinical outcome after blastocyst vitrification. The plasticity of a human blastocyst may allow for further investigation on simplification of blastocyst warming procedures.

Fish sperm cryopreservation using biodegradable containers: New low-cost and environment-friendly methodology.

In brief: The containers used in cell cryopreservation are essential to maintain cell integrity and viability after thawing. This paper reveals the methodology of using biodegradable containers for fish sperm cryopreservation. Cryopreserved sperm in biodegradable containers showed high fertility capability. Biodegradable capsules could be alternative containers to plastic straws for sperm cryopreservation. Abstract: Containers used to cryopreserve sperm are made with non-biodegradable plastic compounds, having a high monetary and environmental cost. Therefore, the development of biodegradable alternative containers for cell cryopreservation is necessary. Thus, this study aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as low-cost and biodegradable alternative containers for sperm cryopreservation. Sperm from 12South American silver catfish Rhamdia quelen were individually cryopreserved in plastic straws 0.25 mL (as control), hard-gelatin, and hard-HPMC capsules. The quality of post-thaw sperm cryopreserved in the different containers was checked by measuring spermatozoa membrane integrity, kinetic parameters, mitochondrial activity, fertilization, hatching, and normal larvae rates. The samples cryopreserved in straws showed a higher percentage of membrane integrity (68%) than those frozen in hard-gelatin (40%) and hard-HPMC capsules (40%). However, we did not observe differences between the samples stored in straws and hard capsules for the rest of the tested sperm parameters. Thus, based on the high sperm fertility capability, both capsules were efficient as cryopreservation containers for maintaining sperm functionality.

Assessment of semen quality and anti-oxidative enzyme activity between bovine sex-sorted and non-sex-sorted frozen-thawed semen.

In the current study, the difference between the sex-sorted and non-sex-sorted frozen semen of Holstein Friesian breed cattle was investigated. Significant variation (p < .05) was found in the semen quality parameters such as motility; vitality; acrosome integrity rate; the anti-oxidative enzyme activity including GSH (glutathione); SOD (superoxide dismutase); CAT (catalase); GSH-Px (glutathione peroxidase) and the rate of fertilization. The results showed that the sperm acrosome integrity and motility of the non-sorted sperm were higher compared to sex-sorted sperm (p < .05). The linearity index and mean coefficient analysis revealed that the percentage of 'grade a' in sex-sorted sperm were significantly (p < .05) lower than non-sorted sperm. Interestingly, low SOD level and high CAT level was found in the non-sexed semen than in the sexed semen (p < .05). Furthermore, the GSH and GSH-Px activity in the sexed semen was found lower than the non-sexed semen (p < .05). In conclusion, sperm motility characteristics were lower in sex-sorted semen than in non-sex-sorted semen. This might be related to the complex process of sexed semen production, which could reduce sperm motility and movement characteristics, acrosomal integrity, CAT, SOD, GSH and GSH-Px, and finally lead to the decline in the fertilization rate.

Freeze-all embryos during treatment with assisted reproduction: Health economic aspects.

Assisted reproductive technologies are evolving, with the most recent example being the introduction of the freeze-all policy during which a fresh embryo transfer does not take place and all embryos of good quality are cryopreserved to be used in future frozen embryo transfers. As the freeze-all policy is becoming more prevalent, it is important to review the economic aspects of this approach, along with considerations of efficacy and safety, and the role of emerging freeze-all-specific ovarian stimulation strategies. Based on the available evidence, the freeze-all policy presents distinct clinical advantages, particularly for high responders. Available health economic evaluations are limited. Two good-quality cost-effectiveness analyses based on randomized controlled trials suggest that the freeze-all strategy is unlikely to be cost-effective in non-polycystic ovarian syndrome (non-PCOS), normally responding patients. However, the cost-effectiveness of the freeze-all strategy in different populations of patients and in different settings has not been evaluated, nor has the clinical and economic efficacy of modern freeze-all-specific ovarian stimulation protocols that are likely to simplify treatment and make it more affordable for patients. Economic evaluations that incorporate good practice health technology assessment (HTA) methods are needed to compare freeze-all with conventional embryo transfer strategies. Furthermore, future research should address the unique limitation of traditional HTA methods in valuing a life conceived through fertility treatment.

Quality Assessment of Cryopreserved Human Biological Samples from the Biobank of Barretos Cancer Hospital.

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.

Transcriptome assessment in 'Red Globe' grapevine zygotic embryos during the cooling and warming phase of the cryopreservation procedure.

Cryopreservation has the potential for long-term germplasm storage. The metabolic pathways and gene regulation involved in cryopreservation procedures are still not well documented. Hence, the genetic expression profile was evaluated using RNA-Seq in zygotic embryos of grapevines subjected to cryopreservation by vitrification. Sequencing was performed on the Illumina NextSeq 500. The average alignment of reads was 96% against the reference genome. The expression profiles showed 229 genes differentially expressed (186 repressed and 46 induced). The main biological processes showing upregulated enrichment were related to nucleosome assembly, while downregulated processes were related to organ growth. The most highly repressed processes were associated with the organization of the cell wall and membrane components. The unnamed protein product and 17.3 kDa class II heat shock protein (HSP) were highly induced, while ATPase subunit 1 and expansin-A1 were repressed. The response to the cooling and warming process during cryopreservation probably indicates that the changes occurring in transcription may be related to epigenetics. In addition, the cell exhibits an increase in the reserve of nutrients while seeking to survive modestly using available energy and pausing the plant's development. Additionally, energy containment occurred to cope with the stress caused by the treatment where deactivation of components of the cell membrane was observed, possibly due to changes in fluidity caused by alterations in temperature.

Improved cryopreservation media formulation reduces costs of maintenance while preserving function of genetically modified insect cells.

Insect cell lines are an invaluable resource that facilitate various fundamental and applied research programs. Genetically engineered insect cell lines, in particular, serve as a platform through which the function of heterologously expressed proteins can be studied. However, a barrier to more widespread utilization and distribution of insect cell lines, genetically modified or not, is the technical and operational challenge associated with traditional cryopreservation methods, including their dependence on the use of liquid nitrogen facilities, animal or human serum products, and relatively high concentrations of permeating cryoprotectants (e.g., DMSO). Recent innovations in cryopreservation technologies have produced reagents with improved abilities to effectively preserve mammalian cell lines for long periods in regular laboratory deep freezers without using serum products, but their effectiveness in preserving genetically engineered insect cell lines has not yet been evaluated. In this study, we engineered Sf9 cells to express a dopamine receptor and used them as a model for evaluating the efficacy of a novel cryopreservation medium product, C80EZ®-INSECT, in not only preserving cell viability and proliferation efficiency but also maintaining the insect cell line's "functionality" after storage at -80°C. We found that the engineered Sf9 cells frozen using C80EZ®-INSECT with 5% DMSO alone and stored at -80°C for 6 mo displayed higher viability and growth rates than cells frozen using traditional fetal bovine serum (FBS)-based cryopreservation media with 10% DMSO that were stored at -80°C or in liquid nitrogen for the same period of time. We also found that after 6 mo of storage at -80°C or in liquid nitrogen the cells retained a responsiveness to dopamine comparable to that of the initial cell line, regardless of the cryopreservation reagent used. These results suggest that, due to the unique characteristics of C80EZ®-INSECT in preventing ice recrystallization and reducing ice crystal size and cellular apoptosis during cryostorage procedures, it is an effective cryopreservation reagent for genetically engineered Sf9 cells, and it practically eliminates the need for liquid nitrogen-based storage facilities and FBS-based cryopreservation formulations, as well as reduces the use of permeating cryoprotectants.

Oocyte quality assessment in marine invertebrates: a novel approach by fluorescence spectroscopy.

BACKGROUND: The assessment of oocyte quality is, nowadays, a major challenge in aquaculture, oocyte cryopreservation, and environmental science. Oocyte quality is a determining factor in fertilization and embryo development; however, there is still a lack of rapid and sensitive cellular markers for its assessment. Currently, its estimation is predominantly based on morphological analysis, which is subjective and does not consistently reflect the developmental competence of the oocytes. Despite several recent studies investigating molecular markers related to oocyte quality, methods currently available for their determination pose various technical challenges and limitations. In this study, we developed a novel approach based on fluorescence spectroscopy to assess different intrinsic physiological parameters that can be employed to evaluate egg quality in marine invertebrates that are widely used as animal models such as sea urchins and mussels. RESULTS: Different physiological parameters, such as viability, mitochondrial activity, intracellular ROS levels, plasma membrane lipid peroxidation, and intracellular pH, for egg quality evaluation have been successfully assessed in sea urchins and mussels by using specific fluorescent dyes and detecting the fluorescent signals in eggs through fluorescence spectroscopy. CONCLUSIONS: Based on our findings, we propose these physiological markers as useful predictors of egg quality in marine invertebrates; they can be estimated rapidly, selectively, and sensitively by employing this novel approach, which, due to the speed of analysis, the low cost, and easy use can be considered a powerful analytical tool for the egg quality assessment.

Quantitative assessment of intracellular/extracellular dimethyl sulfoxide concentrations during freezing with low-temperature confocal Raman micro-spectroscopy.

Although cryopreservation plays an indispensable role in the clinical application of cell therapy, the research on the osmotic behavior of cells during freezing is still at the level of theoretical models, and quantitative experimental data are still lacking. Therefore, the Raman spectra of dimethyl sulfoxide (DMSO) solutions with different standard concentrations (5%-80% v/v) were recorded experimentally to establish a quantitative evaluation method with the intensity ratio of different labeled peaks to the hydrogen bonding peak (as the internal standard) of water molecules in relation to different DMSO concentrations. By using this method, the characteristics of quantitative changes in intra- and extracellular concentrations under three different freezing methods were explored, including direct freezing, ice seeding freezing and vitrification. It was found that the intracellular concentration (@ -50 °C) after the ice seeding (@ -7 °C) freezing (1 °C min-1) method could reach 41.6%-49.2%, significantly higher than that of the direct freezing method (1 °C min-1 to -50 °C) of 32.4%-39.1%. Moreover, it is worth noting that the quantitative values of concentrations (@ -50 °C) of the ice seeding freezing are more consistent with the primary saturation curve of the DMSO solution. Thus, for the first time, it was revealed from the experimental data that the fundamental reason for the improvement of cell survival after ice seeding operation was pre-dehydration, higher concentration and smaller osmotic pressure difference between the inside and outside of the cell. These results also confirmed the validity of the famous two-factor hypothesis and more work will be carried out in depth.

Real-time computer assisted measurement of oocyte and embryo volume for assessment of transport parameters.

Cryopreservation of gametes has revolutionized both animal agriculture and human reproductive medicine. Although many new technologies have tremendously improved the cryopreservation of oocytes and embryos, osmotic stress encountered during the equilibration process can cause their loss of function. Rational cryoprotective agent (CPA) equilibration strategies can be used to minimize this stress but require trained personnel to monitor the process in individual oocytes or embryos or require the use of suboptimal average transport parameter values in mathematically guided protocols. To enable individually optimized equilibration of CPAs in individual cells, here we establish experimental and computational techniques to track the osmotic behavior of individual bovine oocytes and embryos during CPA equilibration in real time. We designed a microfluidic device to provide a controlled flow of CPA and modified standard image analysis techniques to estimate real-time cell volume changes. In particular, we used a level-set method to define a boundary within a contour plot which could automate the image analysis process. A colour based level set algorithm coupled with contour smoothing not only provided the best fit but also reduced the segmentation time to well under a second per image. The accuracy of the automated method was comparable to human segmented images for both oocytes and embryos. This technology should enable both rapid evaluation of key biophysical parameters in oocytes and embryos undergoing CPA equilibration and the development of real-time feedback-control of CPA equilibration, enabling individual oocyte- and embryo-specific optimal protocols.