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Results for “cryopreservation”

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At least 19 recordsLinked to original sources

PubMed2026

Cryopreservation-induced proteomic alterations in Pêga donkey (Equus asinus) spermatozoa.

Semen cryopreservation is a key tool for assisted reproduction and genetic conservation, but its efficiency remains limited in donkeys, compromising post-thaw sperm quality. This study aimed to characterize the proteomic profile of Pêga donkey spermatozoa and to investigate molecular mechanisms associated with cryopreservation-induced impairment of sperm function. Semen samples were collected from Pêga jacks and evaluated for sperm motility and vigor before and after cryopreservation. Quantitative proteomic analysis was performed by LC-MS/MS, followed by bioinformatic characterization of differentially abundant proteins. Cryopreservation markedly reduced sperm motility in all animals, whereas sperm vigor showed only a non-significant tendency toward reduction, suggesting sublethal cryoinjury primarily affecting flagellar efficiency. Proteomic profiling identified 554 proteins, of which 98 were differentially abundant between in natura and cryopreserved spermatozoa. Functional enrichment analyses showed that these proteins were mainly associated with energy metabolism, mitochondrial oxidative phosphorylation, glycolysis, cytoskeletal organization, signal transduction, proteostasis, and oxidative stress response. Notably, proteins involved in ATP production, mitochondrial function, and axonemal organization were significantly altered, supporting a mechanistic link between metabolic dysfunction, flagellar structural disorganization, and reduced post-thaw motility. Overall, cryopreservation induced coordinated and compartment-specific remodeling of the donkey sperm proteome, particularly affecting pathways essential for motility and functional competence. These findings provide new molecular insights into the cryobiological vulnerability of donkey spermatozoa and establish a mechanistic basis for the development of more effective, biology-driven cryopreservation strategies for this species.

Animals↗

PubMed2026

Research note: Efficient preservation of genome-edited chicken germplasm via N-methylacetamide-based semen cryopreservation.

The rapid development of genome-edited chicken lines for agricultural and biomedical use requires effective methods for long-term preservation. In birds, cryopreservation of oocytes and embryos is challenging due to the structure of the egg. This makes semen cryopreservation one of the most practical alternatives, however, this method has not been validated in genome-edited chickens. This study evaluated the feasibility of cryopreserving semen from genome-edited chickens using an N-methylacetamide (N-MA)-based protocol. Two genome-edited chicken lines were used in this study which are a DAZL-GFP germ cell reporter line and a RAG1 knockout line. Semen was stored for either one week or one month prior to assessing fertility and hatchability. The results demonstrated that cryopreserved semen from both genome-edited chicken lines maintained their fertilizing capacity, with no significant differences in fertility or hatchability compared to cryopreserved wild-type groups at either storage duration. Hatchability remained above 80% across all groups, indicating that semen cryopreservation did not significantly affect embryo development after successful fertilization. Furthermore, SYBR-14/PI staining revealed no significant differences in sperm viability between genome-edited and control groups, although both showed a significant decrease in viability compared to fresh semen. These findings demonstrate that N-MA-based semen cryopreservation provides a reliable and practical method for the preservation of genome-edited chicken lines.

Chicken↗

PubMed2025

CryoSCAPE: Scalable immune profiling using cryopreserved whole blood for multi-omic single cell and functional assays.

BACKGROUND: The field of single cell technologies has rapidly advanced our comprehension of the human immune system, offering unprecedented insights into cellular heterogeneity and immune function. While cryopreserved peripheral blood mononuclear cell (PBMC) samples enable deep characterization of immune cells, challenges in clinical isolation and preservation limit their application in underserved communities with limited access to research facilities. We present CryoSCAPE (Cryopreservation for Scalable Cellular And Proteomic Exploration), a scalable method for immune studies of human PBMC with multi-omic single cell assays using direct cryopreservation of whole blood. RESULTS: Comparative analyses of matched human PBMC from cryopreserved whole blood and density gradient isolation demonstrate the efficacy of this methodology in capturing cell proportions and molecular features. The method was then optimized and verified for high sample throughput using fixed single cell RNA sequencing and liquid handling automation with a single batch of 60 cryopreserved whole blood samples. Additionally, cryopreserved whole blood was demonstrated to be compatible with functional assays, enabling this sample preservation method for clinical research. CONCLUSIONS: The CryoSCAPE method, optimized for scalability and cost-effectiveness, allows for high-throughput single cell RNA sequencing and functional assays while minimizing sample handling challenges. Utilization of this method in the clinic has the potential to democratize access to single-cell assays and enhance our understanding of immune function across diverse populations.

Humans↗

PubMed2026

Protective effects of seminal exosomes on cryopreserved sperm via inhibiting oxidative damage.

This study aimed to explore the protective effect of seminal plasma exosomes (SPEs) on human sperm structure and function during cryopreservation and its potential mechanism. The samples were divided into two groups: the control group was treated solely with sperm cryoprotectant before freezing, while the exosome group was supplemented with SPEs. After cryopreservation and thawing, sperm progressive motility, normal morphological rate, and survival rate were evaluated. Furthermore, PKH67 labeling experiments were performed, and oxidative stress markers as well as energy metabolism indicators in sperm were detected. Subsequent mechanism exploration was conducted via proteomic analysis and protein validation assays. This work reveals that adding SPEs at a concentration of 1 or 2 mg/ml effectively improves sperm progressive motility after cryopreservation. After supplementing with SPEs, sperm glucose levels are reduced and mitochondrial membrane potential is enhanced. Simultaneously, SPEs alleviate oxidative stress by decreasing reactive oxygen species (ROS) and DNA fragment index (DFI) while increasing superoxide dismutase (SOD) activity. Functional annotation of proteomics reveals that 14 of the differentially expressed proteins (DEPs) are associated with sperm motility. Enriched metabolic pathways related to sperm motility and sperm protein validation experiments indicate that the expression of MAPK, p-MAPK, and p-JNK proteins in sperm is higher in the Exosome group than in the Control group. This study provides important theoretical support for the application of SPEs in mitigating cryopreservation damage to sperm by enhancing antioxidant capacity. The specific mechanism may be mediated by the MAPK/p-JNK pathway.

Male↗

PubMed1977

Morphological transformation of early passage golden Syrian hamster embryo cells derived from cryopreserved primary cultures as a reliable in vitro bioassay for identifying diverse carcinogens.

Cryopreserved primary cultures of golden Syrian hamster embryo cells were used as the source of target and feeder cells for establishing an in vitro carcinogenesis bioassay. The primary culture giving the best overall response in a pretest before freezing gave positive results in 20 consecutive experiments when retested with 3-methylcholanthrene after cryopreservation, indicating that pretested cryopreserved cultures can serve as a source of susceptible target cells in an in vitro carcinogenesis bioassay. Similarly prepared and cryopreserved cultures served satisfactorily as feeder cells. Susceptible positive cultures were used to test a large number of carcinogenic and non-carcinogenic chemicals in this system. The results showed a very high positive correlation (90.8%) between morphological transformation and the reported carcinogenic activity of the chemicals. Transformation was not observed when cells were tested with a few carcinogens that may not be metabolized to their active forms by early passage hamster embryo cells. N-2-acetylaminofluorene transformed cells only when tested in the presence of hamster liver microsomes. No false positive results were obtained when non-carcinogens were bioassayed, nor was spontaneous transformation observed in control cultures treated with medium alone, 0.2% dimethylsulfoxide or other solvents. Cultures derived from morphologically transformed colonies arising after treatment of cells with several known carcinogens were tumorigenic in vivo, confirming the correlation of morphological transformation with tumorigenicity and the validity of altered morphology as an in vitro criterion for carcinogenicity in vivo.

Animals↗

PubMed1977

Semen cryopreservation: an update.

Cryopreservation of semen has a place in reproductive medicine. It is done best using liquid nitrogen as the refrigerant and the pre-freeze semen should be of good quality. High quality spermatozoa survive the freezing-thawing process and ordinarily result in good babies. Abnormal sperm generally do not survive the freezing-thawing process, which, consequently, results in a more viable union and outcome from the germ cells. Cryopreservation of semen can be used to preserve semen before medical or surgical sterilization, to augment the sperm count in patients with certain types of oligospermia and to manage the childless couple by donor semen artificial insemination in those instances in which the husband is infertile. Cryopreserved semen produces babies and perhaps thousands of humans have been the result of conception that resulted from cryopreserved spermatozoa.

Female↗

PubMedDate not supplied

Ultrastructural alterations and phagocytic function of cryopreserved platelets.

Fresh human platelets and platelets cryopreserved in 4% dimethylsulfoxide were examined ultrastructurally before and after incubation in a suspension of latex particles. Cryopreserved platelets had fewer discoid forms than fresh platelets. The cryopreserved platelets had many sphered platelets containing an increased number of vacuoles; the sphered platelets were more electron-lucent margination and pallor of organelles. Phagocytosis of latex into vacuoles was markedly impaired in the cryopreserved platelets. The morphologic alterations suggested that the freezing, thawing, and washing procedures reduced the functional activity of platelets.

Blood Platelets↗

PubMed1978

Experimental cryopreservation and autotransplantation of parathyroid glands: technique and demonstration of function.

A canine model for cryopreservation and autotransplantation of parathyroid glands was developed as a prototype for cryopreservation of human parathyroid glands; Each of 18 dogs had two parathyroids removed, sectioned, and cooled at a controlled rate to -80 degrees in media containing 10% dimethylsulfoxide and 10% autologous serum. After storage for two months at -196 degrees the tissue was thawed and implanted in muscle; the remaining two parathyroid glands were removed from the neck. Function was assessed by monitoring serum calcium levels and measuring parathyroid hormone levels in venous effluent from the graft beds. Autograft function was demonstrated in ten of 18 dogs; graft failure occurred in four dogs. In the remaining four dogs, function could not be evaluated because of accessory parathyroid tissue. Parathyroid tissue cryopreserved for nine months was documented to function in six of seven dogs. Histologic study of the cryopreserved, autografted tissue showed normal parathyroid architecture.

Animals↗

PubMed1979

Autotransplantation of cryopreserved parathyroid tissue in man.

Human cryopreserved parathyroid autografts have been performed in six patients following reoperative parathyroid surgery. All patients were rendered hypoparathyroid by their most recent reoperation. Parathyroid tissue was successfully autotransplanted after as long as eighteen months of cryopreservation. Viability and expected in vivo function of cryopreserved parathyroid tissue may be predicted by in vitro testing of parathyroid hormone secretion in response to varying ambient calcium concentration. Parathyroid cryopreservation with subsequent autotransplantation is a practical solution to the problem of permanent hypoparathyroidism that may follow multiple surgical procedures for persistent hyperparathyroidism.

Calcium↗

PubMed1978

Advantages of cryopreserved lymphocytes for sequential evaluation of human immune competence. II. Mixed lymphocyte cultures and mononuclear cell subpopulations.

Peripheral blood lymphocytes were obtained from normal volunteer donors over a 2-week period. One-way or two-way mixed lymphocyte reactions, percentages of T-cells and B-cells, and percentages of monocytes were quantitated on both fresh and cryopreserved lymphocytes. These comparisons revealed that the measurement of mixed leukocyte reactions, fractions of E-rosettes and EA rosettes, and the fraction of esterase-staining cells were not altered by cryopreservation and storage at -196 degrees C up to 1 year. Cryopreserved reference lymphocytes provided a stable standard for use as stimulating cells in mixed lymphocyte cultures and could be irradiated or treated with mitomycin C prior to being frozen, without alteration of their function as stimulator cells. Cryopreserved cells thus appeared to be utilizable in the sequential measurement of the immune competence of humans.

B-Lymphocytes↗

PubMed1978

Successful engraftment of cryopreserved autologous bone marrow in patients with malignant lymphoma.

In an effort to evaluate the possible utility of cryopreserved autologous bone marrow infusions in man, 22 patients with malignant lymphoma resistant to conventional chemotherapy were treated with high-dose chemotherapy. This was followed in 12 patients by an infusion of their cryopreserved autologous bone marrow; 10 patients received chemotherapy alone and serve as controls. Following chemotherapy, severe leukopenia (less than 100 leukocytes/mm3) lasted 6-10 (median 8) days in patients receiving cryopreserved marrow, compared to 10-29 (median 16) days in controls (p less than 0.001). Recovery to 1000 leukocytes/mm3 occurred 10-18 (median 13) days after chemotherapy in autograft recipients but was delayed until 12-38 (median 23) days after chemotherapy in controls (p less than 0.001). Autografted patients also recovered granulocyte and platelet function significantly faster and had significantly fewer febrile days after chemotherapy than did controls. Cryopreserved autologous bone marrow infusions can hasten hemopoietic recovery in man after high-dose chemotherapy; this earlier reconstitution may be of clinical benefit to the patient.

Adolescent↗

PubMed1978

Maintenance of viable arterial allografts by cryopreservation.

An experimental study, designed to investigate the feasibility of maintaining fresh, viable arterial allografts by cryopreservation, is presented. Cryopreserved femoral arterial allografts were shown to be equal in patency rate when compared with fresh controls. The cryopreserved allografts were shown to be superior to fresh allografts in the parameters of intimal integrity, absence of host rejection, and reduced surface thrombogenicity. This experimental study supports the thesis that cryopreservation in liquid nitrogen, utilizing 15% dimethyl sulfoxide (DMSO) and methylprednisolone as a cryoprotectant solution, is an excellent method of preserving and storing fresh allograft arteries for use as a small vessel replacement.

Animals↗

PubMed2026

Mitochondrial uncoupler BAM15 attenuates cryopreservation-induced damage in human sperm by stabilizing mitochondrial homeostasis†.

Human sperm cryopreservation is essential for sperm banking and assisted reproduction, yet freeze-thaw stress promotes oxidative injury that reduces motility and damages the acrosome and nuclear DNA. Here, we tested whether the mitochondrial uncoupler BAM15 improves post-thaw human sperm quality and examined mechanisms linked to mitochondrial homeostasis. Ejaculates were cryopreserved using a standard protocol supplemented with graded concentrations of BAM15. After thawing, total and progressive motility and viability were assessed. Flow cytometry quantified the DNA fragmentation index and the proportion of high DNA stainability cells. Mitochondrial membrane potential, intracellular reactive oxygen species, and lipid peroxidation were measured to evaluate mitochondrial function and oxidative status. Ultrastructural preservation of the acrosome, plasma membrane, midpiece mitochondria, and flagellar axoneme was examined by transmission electron microscopy. Compared with untreated controls, BAM15 increased total and progressive motility and improved viability. BAM15 reduced DNA fragmentation and decreased high DNA stainability, indicating enhanced genomic integrity. Consistently, BAM15 improved mitochondrial membrane potential while suppressing intracellular reactive oxygen species and lipid peroxidation, supporting attenuation of freeze-thaw oxidative damage. Transmission electron microscopy further revealed more continuous acrosomal and plasma membranes, fewer swollen or vacuolated midpiece mitochondria, and improved preservation of axonemal architecture. Collectively, these findings identify BAM15 as a promising cryopreservation supplement that stabilizes mitochondrial homeostasis and improves the functional and structural quality of human sperm after thawing.

Humans↗

PubMed1977

Cryopreservation of blood mononuclear leukocytes and stem cells suspended in a large fluid volume. A preclinical model for a blood stem cell bank.

It was the purpose of this study to establish and evaluate a freezing-and-thawing method for preservation of hemopoietic stem cells from the peripheral blood. Blood leukocytes collected by means of an IBM Blood-Cell-Separator were frozen in plastic bags using 10% DMSO and controlled cooling rates. Thawing was performed rapidly, and DMSO was diluted and removed prior to the in-vitro and in-vivo assays. The mean recovery of mononuclear cells collected from 82 leukaphereses was 86%. To assess the recovery of cryopreserved hemopoietic stem cells, the soft agar culture method adapted for the dog was used. There was no significant difference in the CFUc recovery per 1 X 10(6) mononuclear cells or in per leukapheresis after different cryopreservation times (1--6 and 7--27 months). To evaluate the hemopoietic repopulation capability of cryopreserved blood stem cells, leukapheresis-derived leukocytes were transfused into 1200 R whole body x-irradiated dogs. The hemopoietic repopulation pattern at day 10 after transfusion of comparable numbers of fresh or frozen leukocytes was not significantly different, as measured in bone marrow smears and sections and by granulocyte concentration in the peripheral blood.

Animals↗

PubMedDate not supplied

Evaluation of platelet cryopreservation techniques by isolated kidney perfusion.

The isolated perfused rabbit kidney, a model system used previously to assess platelet function, was adapted for evaluation of human platelet cryopreservation techniques. A new, simple, efficient device for controlling cooling rates before, during, and after freezing was used. Platelet concentrates frozen with 5 per cent dimethyl sulfoxide (DMSO) under different conditions were the most effective of those tried in maintaining the hemostatic function and vascular integrity of perfused kidneys. Our studies indicate that the isolated, perfused rabbit kidney can be used to evaluate platelet cryopreservation techniques and is potentially adaptable for studies of organ cryopreservation.

Animals↗

PubMedDate not supplied

Platelet preservation V. Survival, serotonin uptake velocity, and response to hypotonic stress of fresh and cryopreserved human platelets.

The survival in vivo of 51Cr-labeled autologous human platelets, the serotonin uptake velocity, and the response to hypotonic stress were determined both before and after cryopreservation. Changes in platelet survival corresponded to changes in serotonin uptake velocity and response to hypotonic stress. No correlation was observed between the values of the survivla in vivo, the serotonin uptake velocity, and the response to hypotonic stress within the separate suspensions of fresh and cryopreserved platelets. There was no indication that hypercoagulability was induced by infusion of small amounts of cryopreserved platelets.

Blood Platelets↗

PubMed1977

Standardization of the chromium-51 release, cell-mediated cytotoxicity assay: cryopreservation of mouse effector and target cells.

Utilizing controlled cryopreservation techniques, we were able to standardize the 51Cr release cytotoxicity assay and thereby ensured reliable comparisons between results obtained on different days. Optimal conditions for freezing of both effector and target cells were quite similar. Dimethyl sulfoxide (DMSO) at a concentration of 7.5-10.0% was employed as the cryoprotective agent and cells were frozen at the rate of -1 degrees C/minute. The handling procedures for the cells before and after freezing were important. Factors affecting recovery of functional reactivity were related to toxicity of DMSO for the cells, the osmotic stress placed upon the cells as the DMSO was being removed after thawing, the handling temperature of the freshly thawed cells, and the susceptibility of cells to mechanical damage immediately after thawing. The recovery of lymphocytes after freezing was about 70%; the recovery of cytotoxicity was around 85%. Syngeneic cytotoxic reactivity induced by inoculation with the Moloney strain of murine sarcoma virus was cryopreserved, as were allogeneic cytotoxicity and natural cytotoxic reactivity. Multiple tests employing effector cells from the same frozen pool gave reproducible results; the standard error of the mean percent cytotoxicity was less than 1.5%. Cryopreserved target cells gave decreased day-to-day variability in susceptibility to lysis, since the same population of cells could be employed in each assay. These results demonstrated conclusively that we can now have a constant source of effector cells and target cells, which can be used from assay to assay as an internal standard.

Animals↗

PubMed1978

Human parathyroid cryopreservation: in vitro testing of function by parathyroid hormone release.

The functional viability of cryopreserved human parathyroid tissue was assessed by determining suppressibility of parathyroid hormone release by evaluation of ambient calcium concentration. Parathyroid hormone release from dispersed human parathyroid cells prepared from both fresh tissue and tissue cryopreserved for up to 200 days was suppressed 0-90% in response to four-fold increases in calcium concentration. In the tissue that demonstrated suppression precryopreservation, the suppression curve was similar in form postcryopreservation. The ability to retain functional integrity within human parathyroid cells by cryopreservation, allows preservation for periods of time probably sufficient to determine the presence of the aparathyroid state, and allows for subsequent successful parathyroid autotransplantation. This technique has particular applicability to patients reoperated upon for persistent hyperparathyroidism where the remaining amount of normal parathyroid tissue is obscure or unknown.

Calcium↗