Cryopreservation is a method adopted for storage of autologous skulls. Herein, this current research sought to explore the effects of different cryoprotectants on the biological characteristics of rat calvarial osteoblasts after cryopreservation. Neonatal Sprague-Dawley rats were selected and their skull tissues were isolated. The skull tissues were allocated into the refrigerating-3M, refrigerating-6M, M199-3M, M199-6M, povidone iodine-3M, and povidone iodine-6M groups according to the usage of cryoprotectants and treatment time (month) and the fresh group. Osteoblasts were isolated from skull tissues in each group through digestion. The histomorphology of the skull was evaluated by H&E staining and cell morphology was observed by microscopy. The viability, proliferation, apoptosis, and osteogenic activity of osteoblasts were assessed by trypan blue staining, MTT, flow cytometry, and alkaline phosphatase (ALP) staining. The skull histomorphology and osteoblast morphology were similar between the fresh and refrigerating groups. Osteoblast viability was weakened after cryopreservation. The longer the refrigeration time, the lower the number of living cells and the higher the apoptosis rate. However, cryopreservation using different cryoprotectants did not evidently affect osteoblast proliferation and ALP activity. Different cryoprotectants show no apparent effect on the osteogenic activity of rat calvarial osteoblasts after cryopreservation.

Primordial germ cells (PGCs) constitute an important cell lineage that directly impacts genetic dissemination and species conservation through the creation of cryobanks. In order to advance the field of animal genetic cryopreservation, this work aimed to recover intact PGCs cryopreserved in embryonic tissues during the segmentation phase for subsequent in vitro maintenance, using the yellow-tailed tetra (Astyanax altiparanae) as a model organism. For this, a total of 202 embryos were distributed in two experiments. In the first experiment, embryos in the segmentation phase were dissociated, and isolated PGCs were maintained in vitro. They were visualized using gfp-Pm-ddx4 3\'UTR labeling. The second experiment aimed to vitrify PGCs using 3 cryoprotective agents or CPAs (dimethyl sulfoxide, ethylene glycol, and 1,2 propanediol) at 3 molarities (2, 3, and 4 M). The toxicity, somatic cell viability, and recovery of intact PGCs were evaluated. After cryopreservation and thawing, 2 M ethylene glycol produced intact PGCs and somatic cells (44 ± 11.52 % and 42.35 ± 0.33 %, respectively) post-thaw. The recovery of PGCs from frozen embryonic tissues was not possible without the use of CPAs. Thus, the vitrification of PGCs from an important developmental model and Neotropical species such as A. altiparanae was achieved, and the process of isolating and maintaining PGCs in a culture medium was successful. Therefore, to ensure the maintenance of genetic diversity, PGCs obtained during embryonic development in the segmentation phase between 25 and 28 somites were stored through vitrification for future applications in the reconstitution of species through germinal chimerism.

BACKGROUND: Buffalo spermatozoa have a distinct membrane structure that makes them more vulnerable to cryopreservation, resulting in lower-quality post-thawed sperm. This decreases the success rate of artificial insemination in buffaloes. Understanding and addressing these specific vulnerabilities are essential for improving reproductive techniques in buffalo populations. The properties of cryopreserved buffalo bull semen were examined in this study regarding the impact of adding autologous platelet-rich plasma (PRP) to OptiXcell® or Tris egg yolk-based extenders. Ten buffalo bulls were used to collect semen. Each bull\'s ejaculate was separated into two main equal amounts, each of which was then diluted with either OptiXcell® or Tris egg yolk-based extender, supplemented with various PRP concentrations (5%, 10%, and 15%), and the control (0%), before being cryopreserved according to established protocols. Following equilibration and thawing, the quality and functionality of the sperm were evaluated, along with the antioxidant enzyme activities (GSH and TAC), malondialdehyde (MDA) content, and in vivo fertilization rate of the thawed semen.
RESULTS: All PRP concentrations in both extenders, particularly 10% PRP, improved the quality and functionality of the sperm in both equilibrated and frozen-thawed semen. Additionally, the antioxidant enzyme activities in both extenders were higher in the PRP-supplemented groups compared to the control group in thawed semen (P < 0.05). All post-thaw sperm quality, antioxidant enzyme activities, and functionality aside from DNA integrity were higher (P < 0.05) in the PRP-supplemented OptiXcell® than in the PRP-supplemented Tris egg yolk-based extender. The fertility of cryopreserved semen in the extenders supplemented with 10% and 15% PRP increased (P < 0.05) significantly more than that of the control extenders, with 10% PRP being the optimum concentration in OptiXcell® (80%) compared to that of Tris egg yolk-based extender (66.67%) and control of two extenders (53.33% and 46.67%, respectively).
CONCLUSIONS: Even though autologous PRP-supplemented extenders have a protective impact on equilibrated and cryopreserved semen, 10% PRP-supplemented OptiXcell® extenders are more effective at preserving post-thaw semen quality, functionality, and antioxidant capacity, which increases the in vivo fertility of buffalo bulls.

Antifreeze proteins (AFPs) are natural biomolecules found in cold-adapted organisms that lower the freezing point of water, allowing survival in icy conditions. These proteins have the potential to improve cryopreservation techniques by enhancing the quality of genetic material postthaw. Deschampsia antarctica, a freezing-tolerant plant, possesses AFPs and is a promising candidate for cryopreservation applications. In this study, we investigated the cryoprotective properties of AFPs from D. antarctica extracts on Atlantic salmon spermatozoa. Apoplastic extracts were used to determine ice recrystallization inhibition (IRI), thermal hysteresis (TH) activities and ice crystal morphology. Spermatozoa were cryopreserved using a standard cryoprotectant medium (C+) and three alternative media supplemented with apoplastic extracts. Flow cytometry was employed to measure plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) postthaw. Results showed that a low concentration of AFPs (0.05 mg/mL) provided significant IRI activity. Apoplastic extracts from D. antarctica demonstrated a cryoprotective effect on salmon spermatozoa, with PMI comparable to the standard medium. Moreover, samples treated with apoplastic extracts exhibited a higher percentage of cells with high MMP. These findings represent the first and preliminary report that suggests that AFPs derived from apoplastic extracts of D. antarctica have the potential to serve as cryoprotectants and could allow the development of novel freezing media.

The objective of this study was to evaluate the effects of different concentrations of antifreeze protein (AFP) extracted from the larva of the beetle, Tenebrio molitor (TmAFP), on vitrification of in vitro-produced bovine embryos. In vitro-produced blastocysts were divided into three experimental groups and vitrified using a cryotop. TmAFP was added to the equilibrium solution (ES) and vitrification solution (VS) at a concentration of 0 ng/mL (control), 500 ng/mL (500TmAFP), or 1000 ng/mL (1000TmAFP). Vitrification was carried out by first placing the blastocysts in ES for 2 minutes (7.5% ethylene glycol [EG] and 7.5% dimethyl sulfoxide [DMSO]). The blastocysts were then transferred to VS (15% EG and 15% DMSO) and promptly deposited on a cryotop stem and submerged in liquid nitrogen. Warming was carried out in three steps with decreasing sucrose concentrations. After warming, the blast cells were cultured for 24 hours for subsequent survival analysis and ultrastructural evaluation. There was a significant difference in the survival rate and expansion in the 500TmAFP group compared with the other groups. The ultrastructural analysis revealed intracellular lesions in all vitrified embryos; however, the embryos of the 500TmAFP and 1000TmAFP groups showed fewer cytoplasmic lesions compared with the control group. Taken together, addition of TmAFP can mitigate cellular changes that involve organelles and cellular components essential for proper functioning and improve the viability of warmed and vitrified in vitro-produced bovine embryos.

Semen cryopreservation is a widely used procedure for fertility preservation, despite some level of cryodamage that may occur in spermatozoa after thawing. However, there is some evidence that lactobacilli, one of the bacteria found in semen, might benefit sperm quality.
This study aims to determine whether the addition of Lactobacillus plantarum secretions to sperm freezing medium has an impact on sperm motility, morphology, and DNA fragmentation.
This is a prospective auto-controlled study. It was conducted on 30 raw semen samples from 30 infertile men attending a fertility center for semen analysis. Before freezing, all the samples were analyzed for motility, morphology, and DNA fragmentation percentages. Each sample was then divided equally into three aliquots. Cryopreservation was performed on each aliquot using one of the following three media: without Lactobacillus plantarum secretions (control group) or with 107 or 108 colony-forming units/mL Lactobacillus plantarum secretions. Sperm motility, morphology, and DNA integrity were evaluated after the cryopreservation media were added and after semen thawing.
The results of this study indicated that after thawing, no statistically significant decrease in progressive motility and non-progressive percentages were detected in the sperm freezing medium supplemented with 108 colony-forming units/mL Lactobacillus plantarum secretions than the fresh raw semen. Moreover, multivariate linear regression model analyses showed that the progressive motility (p = 0.02), non-progressive motility (p = 0.016), and non-motile spermatozoa (p = 0.012) percentages were significantly decreased in the freezing medium (without Lactobacillus plantarum secretions) compared to the fresh raw semen.
To the best of our knowledge, this is the first study showing that Lactobacillus plantarum secretions had a cryoprotective effect on sperm motility when added to the sperm freezing medium. Furthermore, Lactobacillus plantarum secretions were found to protect sperm DNA integrity more effectively than the freezing medium without Lactobacillus plantarum secretions in non-normozoospermia group. Cryopreservation procedures must therefore be optimized to minimize any iatrogenically induced sperm DNA damage, given the correlation between sperm DNA damage and increased mutation loads in progeny.

We designed a highly adhesive cryoprotectant-gel composite (CGC), based on regular liquid-form cryoprotectant base (CB), aiming to protect cartilage tissue during frozen osteoarticular autograft reconstruction for high-grade sarcoma around the joint. This study aimed to evaluate its effectiveness in rat and porcine distal femur models.
Fresh articular cartilage samples harvested from distal rat and porcine femurs were divided into 4 test groups: untreated control group, liquid nitrogen (LN) freezing group, LN freezing group pretreated with CB (CB group), and LN freezing group pretreated with CGC (CGC group). Microscopic and macroscopic evaluation of cartilage condition, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, and apoptotic protein analysis of chondrocytes were performed to confirm our results.
In the rat model, CGC could prevent articular cartilage from roughness and preserve more proteoglycans when compared with the LN freezing and CB groups. Western blot analysis showed CGC could prevent cartilage from LN-induced apoptosis supported by caspase-3/8 apoptotic signaling cascade. Macroscopically, we observed CGC could reduce both articular clefting and loss of articular luminance after freezing in the porcine model. In both models, CGC could reduce articular chondrocytes from degeneration. Fewer TUNEL-positive apoptotic and more viable chondrocytes in cartilage tissue were observed in the CGC group in our animal models.
Our study proved that CGC could effectively prevent cartilage surface and chondrocytes from cryoinjury after LN freezing. Freezing articular cartilage surrounded with high concentration of CGC can be a better alternative to preserve articular cartilage during limb salvage surgery for malignant bone tumor.

Shellfish aquaculture needs the development of new tools for the improvement of good practices avoiding the reliance on natural spat collection to increase production efficiently. The aim of this work was to improve the cryopreservation protocol for Mytilus galloprovincialis larvae described in Paredes et al. (in: Wolkers, Oldenhof (eds) Cryopreservation and freeze-drying protocol, methods in molecular biology, Humana Press, 2021, pp 2180, https://doi.org/10.1007/978-1-0716-0783-1_18 ). Moreover, the capability of producing adult mussels from cryopreserved 72 h-old D-larvae and potential long-term effects of cryopreservation through progenies were evaluated. The selection of 72-h old D-larvae for cryopreservation yielded 75% of recovery, higher than 50% from trochophores. The best combination was 10% Ethylene-Glycol + 0.4 M Trehalose in Filtered Sea Water (FSW) with cooling at - 1 °C/min and a water bath at 35 °C for thawing. Sucrose (SUC) solutions did not improve larval recovery (p > 0.05). At settlement, 5.26% of cryopreserved F1 larvae survived and over 70% settled. F2 cryopreservation produced 0.15% survival of spat and settlement varied from 35 to 50%. The delay of shell size showed on cryopreserved larvae declined throughout larval rearing without significant differences with controls from settlement point (p > 0.05). Long-term experiments showed that it is possible to obtain adult mussels from cryopreserved larvae and this tool does not compromise the quality of following progenies, neither for cryopreservation nor post-thawing development of them.

The influence of four common cryoprotectants (dimethyl sulfoxide, glycerol, ethylene glycol and dimethylformamide) on monolayers of four common phospholipids (DPPC, DOPC, POPC and POPE) have been studied using Langmuir isotherms and monolayer insertion experiments. The cryoprotectant concentrations were chosen to be directly relevant to cryoprotection. We show that DMSO causes an expansion of the DPPC area per lipid (in contrast to previous work at higher concentrations). However, it caused compression for POPC, and had little effect for POPE or DOPC. As most previous studies have involved only DPPC, this highlights the importance of studying different lipid types as these may have a significant effect on the interactions. We show that both ethylene glycol and glycerol cause a small expansion of the monolayer at fixed pressure, implying that they insert into the headgroup regions, regardless of lipid species, and consistent with their ability to penetrate membranes. By contrast, dimethylformamide causes monolayer compression for all lipid species, implying it dehydrates the lipid head groups. Membrane insertion experiments at physiological values of lateral pressure highlight that DPPC is the most difficult lipid to penetrate, implying that the penetrating action of cryoprotectants may only occur for unsaturated phospholipids. Thus, extrapolations of results based solely on the DPPC need to be made with care.

There is scarcity of breast cancer tissues derived from women of African origin available for patient - derived xenograft and organoid models.
We aim to create a versatile protocol for processing mastectomy and cryopreservation of breast cancer tissue.
An immediate collection of breast cancer tissue from mastectomy was bathed in 4 °C HBSS and immediately transferred to 4 °C RPMI1640 containing HEPES, 10% FBS, Streptomycin and Penicillin. Tissues were processed over ice yielding nine samples of cold ischemic time (20-45 min) stored at 3 min interval. Cut samples were transferred into cryovials containing 4 °C cryoprotectant agent (90% FBS +10% Me2SO) before snap -freezing in liquid Nitrogen vapour and final short-term storage in -80 °C Freezer. The histomorphology, tissue and molecular viability were assessed.
The cold ischemic times had no detrimental effect to the nine samples despite being processed in a resource poor setting, hence providing a reproducible and reliable protocol.