Months Of Cryopreservation Research Articles

Impact of cryoprotective agents on human gut microbes and invitro stabilized artificial gut microbiota communities.

The availability of microbial biobanks for the storage of individual gut microbiota members or their derived and artificially assembled consortia has become fundamental for invitro investigation of the molecular mechanisms behind microbe-microbe and/or microbe-host interactions. However, to preserve bacterial viability, adequate storage and processing technologies are required. In this study, the effects on cell viability of seven different combinations of cryoprotective agents were evaluated by flow cytometry for 53 bacterial species representing key members of the human gut microbiota after one and 3 months of cryopreservation at -80°C. The obtained results highlighted that no universal cryoprotectant was identified capable of guaranteeing effective recovery of intact cells after cryopreservation for all tested bacteria. However, the presence of inulin or skimmed milk provided high levels of viability protection during cryoexposure. These results were further corroborated by cryopreserving 10 artificial gut microbiota produced through invitro continuous fermentation system technology. Indeed, in this case, the inclusion of inulin or skimmed milk resulted in a high recovery of viable cells, while also allowing consistent and reliable preservation of the artificial gut microbiota biodiversity. Overall, these results suggest that, although the efficacy of various cryoprotective agents is species-specific, some cryoprotectants based on glycerol and the addition of inulin or skimmed milk are preferable to retain viability and biodiversity for both single bacterial species and artificial gut microbiota.

Effects of Long-Term Cryopreservation on the Transcriptomes of Giant Grouper Sperm.

The giant grouper fish (Epinephelus lanceolatus), one of the largest and rarest groupers, is a fast-growing economic fish. Grouper sperm is often used for cross-breeding with other fish and therefore sperm cryopreservation is important. However, freezing damage cannot be avoided. Herein, we performed a transcriptome analysis to compare fresh and frozen sperm of the giant grouper with frozen storage times of 0, 23, 49, and 61 months. In total, 1911 differentially expressed genes (DEGs), including 91 in El-0-vs-El-23 (40 upregulated and 51 downregulated), 251 in El-0-vs-El-49 (152 upregulated and 69 downregulated), and 1569 in El-0-vs-El-61 (984 upregulated and 585 downregulated), were obtained in the giant grouper sperm. DEGs were significantly increased at 61 months of cryopreservation (p < 0.05). GO and KEGG enrichment analyses of the DEGs revealed significant enrichment in the pilus assembly, metabolic process, MAPK signaling pathway, apoptosis, and P53 signaling pathway. Time-series expression profiling of the DEGs showed that consistently upregulated modules were also significantly enriched in signaling pathways associated with apoptosis. Four genes, scarb1, odf3, exoc8, and atp5f1d, were associated with mitochondria and flagella in a weighted correlation network analysis. These genes may play an important role in the response to sperm freezing. The experimental results show that long-term cryopreservation results in freezing damage to the giant grouper sperm. This study provides rich data for studies of the mechanism underlying frozen fish sperm damage as well as a technical reference and evaluation index for the long-term cryopreservation of fish sperm.

Drug selection pressure and fitness cost for artemether-resistant Plasmodium berghei ANKA parasites in vivo

The clinical use of artemisinin-based combination therapies is threatened by increasing failure rates due to the emergence and spread of multiple drug resistance genes in most human Plasmodium strains. The aim of this study was to generate artemether-resistant (AMR) parasites from Plasmodium berghei ANKA (AMS), and determine their fitness cost. Artemether resistance was generated by increasing drug pressure doses gradually for 9 months. Effective doses (ED50 and ED90) were determined using the 4-day suppressive test, and the indices of resistance (I) at 50% and 90% (I50 and I90) were determined using the ratio of either ED50 or ED90 of AMR to AMS, respectively. The stability of the AMR parasites was evaluated by: five drug-free passages (5DFPs), 3 months of cryopreservation (CP), and drug-free serial passages (DFSPs) for 4 months. Analysis of variance was used to compare differences in growth rates between AMR and AMS with 95% confidence intervals. ED50 and ED90 of AMS were 0.61 and 3.43 mg/kg/day respectively. I50 and I90 after 20 cycles of artemether selection pressure were 19.67 and 21.45, respectively; 5DFP values were 39.16 and 15.27, respectively; 3-month CP values were 29.36 and 10.79, respectively; and DFSP values were 31.34 and 12.29, respectively. The mean parasitaemia value of AMR (24.70% ± 3.60) relative to AMS (37.66% ± 3.68) at Day 7 post infection after DFSPs revealed a fitness cost of 34.41%. A moderately stable AMRP. berghei line was generated. Known and unknown mutations may be involved in modulating artemether resistance, and therefore molecular investigations are recommended.

Establishment and characterization of a skeletal myoblast cell line of grass carp (Ctenopharyngodon idellus).

Skeletal muscle myoblastic cell lines can provide a valuable new in vitro model for the exploration of the mechanisms that control skeletal muscle development and its associated molecular regulation. In this study, the skeletal muscle tissues of grass carp were digested with trypsin and collagenase I to obtain the primary myoblast cell culture. Myoblast cells were obtained by differential adherence purification and further analyzed by cryopreservation and resuscitation, chromosome analysis, immunohistochemistry, and immunofluorescence. A continuous grass carp myoblast cell line (named CIM) was established from grass carp (Ctenopharyngodon idellus) muscle and has been subcultured > 100 passages in a year and more. The CIM cells revived at 79.78-95.06% viability after 1-6 months of cryopreservation, and shared a population doubling time of 27.24 h. The number of modal chromosomes of CIM cells was 48, and the mitochondrial 12S rRNA sequence of the CIM cell line shared 99% identity with those of grass carp registered in GenBank. No microorganisms (bacteria, fungi, or mycoplasma) were detected during the whole study. The cell type of CIM cells was proven to be myoblast by immunohistochemistry of specific myogenic protein markers, including CD34, desmin, MyoD, and MyHC, as well as relative expression of key genes. And the myogenic rate and fusion index of this cell line after 10 days of induced differentiation were 8.96 ~ 9.42% and 3-24%, respectively. The telomerase activity and transfection efficiency of CIM cell line were 0.027 IU/mgprot and 23 ~ 24%, respectively. These results suggest that a myoblast cell line named CIM with normal biological function has been successfully established, which may provide a valuable tool for related in vitro studies.

Recovering Spermatogenesis By Protected Cryopreservation Using Metformin and Transplanting Spermatogonial Stem Cells Into Testis in an Azoospermia Mouse Model.

Cryopreservation and transplantation of spermatogonial stem cells (SSCs) may serve as a new method to restore male fertility in patients undergoing chemotherapy or radiotherapy. However, SSCs may be damaged during cryopreservation due to the production of reactive oxygen species (ROS). Therefore, different antioxidants have been used as protective agents. Studies have shown that metformin (MET) has antioxidant activity. The aim of this study was to assess the antioxidant and antiapoptotic effects of MET in frozen-thawed SSCs. In addition, the effect of MET on the proliferation and differentiation of SSCs was evaluated. To this end, SSCs were isolated from mouse pups aged 3-6 days old, cultured, identified by flow cytometry (ID4, INTEGRIN β1+), and finally evaluated for survival and ROS rate. SSCs were transplanted after busulfan and cadmium treatment. Cryopreserved SSCs with and without MET were transplanted after 1 month of cryopreservation. Eight weeks after transplantation, the recipient testes were evaluated for the expression of apoptosis (BAX, BCL2), proliferation (PLZF), and differentiation (SCP3, TP1, TP2, PRM1) markers using immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction. The findings revealed that the survival rate of SSCs was higher in the 500 μm/mL MET group compared to the other groups (50 and 5000 μm/mL). MET significantly decreased the intracellular ROS production. Transplantation of SSCs increased the expression level of proliferation (PLZF) and differentiation (SCP3, TP1, TP2, PRM1) markers compared to azoospermia group, and their levels were significantly higher in the MET group compared to the cryopreservation group containing basic freezing medium (p < 0.05). MET increased the survival rate of SSCs, proliferation, and differentiation and decreased the ROS production and the apoptosis rate. Cryopreservation by MET seems to be effective in treating infertility.

Enhancing the Cryopreservation System of Larch Embryogenic Culture by Optimizing Pre-Culture, Osmoprotectants, and Rapid Thawing

Cryopreservation is considered the safe and efficient strategy for the long-term conservation of embryogenic cultures. The objective of this study was to cryopreserve the embryogenic tissues of hybrid larch to overcome the result raised by rapid growth rates of conifer embryogenic cultures necessitating frequent sub-culturing. We systematically evaluated several parameters, including the pre-culture method (liquid or solid), osmoprotectant type (DMSO, sucrose, or PEG6000), duration of cryoprotection (1–3 h), and thawing temperature (4 °C, 25 °C, or 40 °C). After one month of cryopreservation, we assessed the regeneration efficiency and maturation ability of both cryo-preserved and non-cryopreserved tissues. Our optimized protocol involves pre-culturing embryonic tissue on the solid medium with 0.4 M sorbitol for 48 h, followed by treatment with 10% DMSO, 0.4 M sucrose, and 15% PEG6000 for 1 h on ice, and immersion in liquid nitrogen with rapid thawing at 40 °C. Notably, the use of solid media during pre-culturing was crucial to enhancing the success rate of cryopreservation. Using protocol optimization, we achieved high embryogenic tissue survival rates of over 80% without affecting the ability of somatic embryogenesis. This work provides a comprehensive set of steps for routine cryopreservation of embryogenic tissues for long-term conservation in hybrid larch, along with sample protocols for cryopreservation of larch. The results demonstrate that vitrification is a reliable method for preserving embryogenic tissues of hybrid larch with broader implications for the cryopreservation of other plant species. Further optimization and standardization of protocols across different species would ensure the preservation of genetic diversity and facilitate future research in plant biotechnology that benefits human health, food security, and environmental sustainability.

MP79-08 EFFECT OF HORMONE THERAPY ON SEMEN ANALYSIS PARAMETERS IN ADOLESCENT TRANSGENDER FEMALES

You have accessJournal of UrologyCME1 Apr 2023MP79-08 EFFECT OF HORMONE THERAPY ON SEMEN ANALYSIS PARAMETERS IN ADOLESCENT TRANSGENDER FEMALES Sai Allu, Sreeya Komaravolu, Mark Sigman, Gabriella Avellino, and Sarah Moore Sai AlluSai Allu More articles by this author , Sreeya KomaravoluSreeya Komaravolu More articles by this author , Mark SigmanMark Sigman More articles by this author , Gabriella AvellinoGabriella Avellino More articles by this author , and Sarah MooreSarah Moore More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003356.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Feminizing transgender patients represent an important urological patient population with unique healthcare needs. Although not every patient chooses to cryopreserve, everyone should be given the option. Hormone therapy (HT) is often started prior to proceeding with surgical orchiectomy. Research has suggested that transgender females on HT have reduced semen analysis (SA) parameters compared to cisgender patients, including reduced sperm concentration and motility. [1,2] There is limited information regarding fertility preservation by transgender patients on HT. The goal of this study is to report cryopreservation outcomes by adolescent feminizing transgender patients who are currently on HT. METHODS: We retrospectively reviewed charts of donors with our Men’s Health Center for direct donations to the sperm bank between 01/2011 to 07/2022. Inclusion criteria included adolescent age (16–24 years old) and the use of HT within three months of cryopreservation. Patient data collected included patient demographics, type of hormone therapy, and SA parameters. RESULTS: Five transgender patients were found who were on HT within three months prior to their semen cryopreservation and analysis. Their median age was 18 (range 16–22). Three patients were on spironolactone, one patient was on estradiol, and one patient was on a combination of both medications. All four patients on single-medication HT had normal concentration and motility on their semen analysis, as defined by the WHO Laboratory Manual for the Examination and Process of Human Semen (Table 1). Three of four patients on single HT had normal volume. The patient on combination HT demonstrated lowered overall SA parameters. CONCLUSIONS: This is the largest cohort of adolescent feminizing transgender patients on hormone therapy in which fertility preservation has been described. Semen cryopreservation could be a viable method for fertility preservation among this patient population who have already initiated hormonal therapies, specifically spironolactone or estradiol. Patients on single HT appear to have improved semen analysis parameters compared to patients on combination HT. Future studies are needed to further characterize fertility preservation with specific information on duration and dosage of HT, as well as outcomes of cryopreservation. Source of Funding: N/A © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1144 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sai Allu More articles by this author Sreeya Komaravolu More articles by this author Mark Sigman More articles by this author Gabriella Avellino More articles by this author Sarah Moore More articles by this author Expand All Advertisement PDF downloadLoading ...

Cryostorage of white cachama (Piaractus orinoquensis) sperm: Effects on cellular, biochemical and ultrastructural parameters

To date, little attention has been paid to identifying the effects of long-term cryopreservation on sperm quality for Piaractus orinoquensis. The object of this study was therefore to evaluate the effect of long-term cryopreservation (24 h, 1, 6 and 12 months) on sperm motility, viability, DNA integrity, ATP content, total antioxidant capacity (TAC), morphology and sperm ultrastructure in this species. Milt samples from six males were cryopreserved in a medium containing final concentrations of 7.5% Me2SO, 4.1% glucose and 9.0% egg yolk. The samples were frozen in liquid nitrogen (LN) vapor and stored in LN for periods of 24 h and 1, 6 and 12 months. After thawing, both the motility rate and the viability decreased significantly compared with fresh sperm; however, these parameters did not differ among the four cryopreservation times. The DNA integrity and ATP content decreased significantly after 6 months of cryopreservation. There were no significant differences in TAC values between fresh and cryopreserved sperm. The total sperm abnormalities in cryopreserved samples were about 5-fold higher than in fresh sperm; short tail was the most common defect occurring after cryostorage. The ultrastructural analysis reveals that P. orinoquensis spermatozoa consist of an ovoid head without acrosome, a cylindrical mid-piece, and a single flagellum. The nuclear fossa is located at the base of the nucleus and contains the centriolar complex. There are 1–2 ring-shaped mitochondria located in the mid-piece. The flagellum shows a 9 + 2 organization of microtubules in the axoneme. Post-thaw spermatozoa presented damage such as swelling and rupture of the plasma membrane, mitochondrial damage, loss of the electron-dense chromatin of the nucleus, and degeneration in the middle region.

EFFECTIVENESS OF CRYOPRESERVED CHICKEN EMBRYO FIBROBLAST CELLS FOR PROPAGATION OF DUCK ENTERITIS VIRUS AND FOWLPOX VIRUS

Primary chicken embryo fibroblast (CEF) cultures are excellent model systems for in-vitro virus propagation. However, the cumbersome preparation process of CEF makes cryopreservation highly essential. Cryopreservation in Liquid Nitrogen liquid phase (-196 °C) or vapour phase (-156 °C) are frequently used methods. In the present study, efficiency of cryopreserved primary CEF cells for the propagation of Duck plague virus and Fowl pox virus was evaluated. The cells were preserved for different time periods and different cooling methodologies were employed in order to assess the effects exerted by these variations. The efficiency of the cells was compared based on cell viability/ revival rate, ability to form monolayer and TCID50 of the propagated viruses. Cell count revealed viability of 75.60% and 68% post 2 weeks and 6 months of cryopreservation respectively following step freezing method. On the other hand, cells preserved using insulated cryo-containers exhibited 89% and 80% viability post 2 weeks and 6 months of cryopreservation respectively. Development of complete monolayer could be seen within 24 hours in all the batches irrespective of the cooling method used and the cryopreservation storage time. Infection with DPV and FPV yielded respective cytopathic effect from 48 hours onwards. CPE observed in the freshly prepared CEF and in those revived 2 weeks and 6 months post freezing, were similar in terms of typical characteristics and duration of appearance. From the findings of the present study it is evident that cryopreservation of CEF cells is a convenient and practical approach for virus propagation. Cryopreservation of primary cells offers a definite advantage over repeated culture preparation which is labour intensive, time consuming and dependent on the availability of embryonated eggs.

Peptide-DNA origami as a cryoprotectant for cell preservation.

Cryopreservation of cells is essential for the conservation and cold chain of bioproducts and cell-based medicines. Here, we demonstrate that self-assembled DNA origami nanostructures have a substantial ability to protect cells undergoing freeze-thaw cycles; thereby, they can be used as cryoprotectant agents, because their nanoscale morphology and ice-philicity are tailored. In particular, a single-layered DNA origami nanopatch functionalized with antifreezing threonine peptides enabled the viability of HSC-3 cells to reach 56% after 1 month of cryopreservation, surpassing dimethyl sulfoxide, which produced 38% viability. It also exhibited minimal dependence on the cryopreservation period and freezing conditions. We attribute this outcome to the fact that the peptide-functionalized DNA nanopatches exert multisite actions for the retardation of ice growth in both intra- and extracellular regions and the protection of cell membranes during cryopreservation. This discovery is expected to deepen our fundamental understanding of cell survival under freezing environment and affect current cryopreservation technologies.

Suitability of salivary leucocytes to assess DNA repair ability in human biomonitoring studies by the challenge-comet assay

The challenge-comet assay is a simple but effective approach that provides a quantitative and functional determination of DNA repair ability, and allows to monitor the kinetics of repair process. Peripheral blood mononuclear cells (PBMC) are the cells most frequently employed in human biomonitoring studies using the challenge-comet assay, but having a validated alternative of non-invasive biomatrix would be highly convenient for certain population groups and circumstances. The objective of this study was to validate the use of salivary leucocytes in the challenge-comet assay. Leucocytes were isolated from saliva samples and challenged (either in fresh or after cryopreservation) with three genotoxic agents acting by different action mechanisms: bleomycin, methyl methanesulfonate, and ultraviolet radiation. Comet assay was performed just after treatment and at other three additional time points, in order to study repair kinetics. The results obtained demonstrated that saliva leucocytes were as suitable as PBMC for assessing DNA damage of different nature that was efficiently repaired over the evaluated time points, even after 5 months of cryopreservation (after a 24 h stimulation with PHA). Furthermore, a new parameter to determine the efficacy of the repair process, independent of the initial amount of damage induced, is proposed, and recommendations to perform the challenge-comet assay with salivary leucocytes depending on the type of DNA repair to be assessed are suggested. Validation studies are needed to verify whether the method is reproducible and results reliable and comparable among laboratories and studies.

Time Dependent Impact of Reactive Oxidants on Seminal Attributes, Mitochondrial Membrane Potential, Lipid Peroxidation and Capacitation-Like Changes of Karan-Fries (KF) Bulls During Cryopreservation

BACKGROUND: Cryopreservation of semen is a valuable technique; however, it is also known to be detrimental to the structure of spermatozoa and fertility due to cryo-injury and subsequent generation of reactive oxidants. OBJECTIVE:To determine the time-dependent impact of reactive oxidants on seminal attributes, mitochondrial membrane potential (MMP), lipid peroxidation status (LPO) and early capacitation like changes. MATERIALS AND METHODS:Semen samples were collected by artificial vagina technique from six Karan-Fries (KF) bulls and subsequently examined at 0 h (before cryopreservation) and at 24 hours, 15 days and 2-months of storage for various seminal attributes, MMP (Δψm) , and early capacitation-like changes. Simultaneously, LPO (TBARS) was determined in fresh and post-thaw seminal plasma. RESULTS: A sharp decrease (P&lt;0.01) in semen quality was observed only after 24 h of cryopreservation except for viability and acrosomal integrity. Sperm viability and acrosome integrity reduced significantly up to 2 months of cryopreservation. The lipid peroxidation status was found to be lower in fresh seminal plasma (2.63±0.22 vs. 3.51±0.34 units/mL) as compared to post-thaw. Furthermore, the active Δψm of fresh semen showed a significant (P &lt;0.01) decrease after 24 hours (77.92±0.387 vs. 54.52±0.28%) of cryopreservation, while there was a non- significant decrease in active MMP after 15 d and 2-months (53.68±0.138 and 52.76±0.16%). Moreover, significant (P &lt;0.01) early capacitation-like changes were found in post-thaw spermatozoa (25.72±0.12 vs. 9.1±0.19%) as compared to fresh ejaculate. CONCLUSION: Spermatozoa incur the majority of damages during the early phase of cryopreservation, however the damage associated by different stressors cannot be neglected.

PB2187: VIABILITY AND PROLIFERATIVE POTENTIAL OF AUTOGRAFT HEMATOPOIETIC STEM CELLS

Background: Autograft cellularity is a fundamental condition for the timing of hematopoiesis recovery in patients with multiple myeloma (MM) after autologous hematopoietic stem cell transplantation (AutoSCT). Taking the number of CD34+ cells in a volume of 2x10/6/kg of the patient’s weight as a suboptimal value that provides fast and reliable graft engraftment, it remains unclear whether this number will be sufficient for subsequent transplantation, if tandem AutoHSCT assumes an interval of 6 months, and repeated - not earlier 36 months. Aims: Determination of the preservation of colony-forming (CFU) and CD34+ cells in the autograft of MM patients depending on the terms of their cryopreservation. Methods: A retrospective analysis of the results of the study of 127 samples of the product of leukocytapheresis was performed. The prepared cell suspension was placed in disposable CryoMacs Freesing bags and mixed with dimethyl sulfoxide to its final concentration of 7.5%. Freezing was carried out in a Cryo 560-16 Planer RLC programmed freezer. Containers with cell suspension were stored in liquid nitrogen. To assess the effect of the duration of the cryopreservation period on CFU, the numerical values of each sample were converted into percentages: the primary CFU values from each sample were taken as 100%, and the CFU values after the cryopreservation period were compared with them. Results: Depending on the terms of cryostorage, 3 groups were formed. Group 1: 83 samples, shelf life from 1 to 5.9 months. Group 2: 34 samples, shelf life from 6 to 10 months. Group 3: 10 samples, shelf life over 10 months. A trend towards a decrease in the number of colonies was revealed as the duration of the cryopreservation period increased. Thus, the number of CFU-totals decreased from 66% recorded after 1–5.9 months of storage, to 60% after 6–10 months and to 44% of the original number when stored for more than 10 months; p=0.131. The decrease in the number of colony-forming cells of granulomonocytopoiesis was distinct in cell suspension samples stored for more than 10 months, but also did not reach significant values: 64%, 58% and 37%, respectively; p=0.451. On the contrary, the decrease in the number of burst-forming cells of erythrocytopoiesis (BFU-E) after 10 months of cryostorage was significantly lower than at earlier periods of cell suspension thawing. So if the content of BFU-E after 1–5.9 and 6–10 months of storage was 65% and 57% of the initial amount, respectively, then after 10 months it was only 26%; p=0.01. When analyzing the preservation of CD34+ cells, a tendency was also found to decrease in their percentage as the duration of cryopreservation increased, but the difference did not reach a significant value: 77% during thawing after 1–5.9 months of cryopreservation, 49% after 6–10 months and 39% in case of cell suspension thawing after 10 months of storage; p=0.465. Despite the fact that in some patients with the maximum terms of cell suspension cryopreservation, the need for a larger number of doses of donor erythrocytes was recorded and there were longer terms for platelet recovery than in patients in the other two groups, the median values of the analyzed parameters did not differ. Summary/Conclusion: When planning the first and subsequent, repeated, AutoHSCT in MM patients, it is advisable to prepare an autograft with a greater than suboptimal cellularity due to a possible decrease in the number of CFU during long-term cryostorage of the cell suspension.

Isolation and Cryopreservation of Toxoplasma gondii Isolates from Cats and Chickens from Selected Households in the Thika Region, Kenya

There is a shortage of information in Africa regarding the genotypic and phenotypic characteristics of the Toxoplasma gondii circulating in domestic cats (Felis catus) and the intermediate hosts such as chicken. The situation is compounded by a lack of collection of well-stored isolates. The present study was aimed at creating a cryobank of T. gondii bradyzoites, tachyzoites, and oocysts. The parasites were isolated from cats and chickens kept in households in the Thika region, Kenya. Eight (8) cat fecal samples positive for T. gondii oocysts and 38 chicken brain tissue cysts (bradyzoites) were obtained and used for propagation in mice before cryopreservation. For each sample from the cats, (two donors) BALB/c mice were infected orally with 1 x 104/ml of oocysts. From each chicken sample, (two donors) BALB/c mice were infected intraperitoneally with 20-30 tissue cysts. On the third day after infection, tachyzoites were harvested from the peritoneal cavity of one donor mouse. The other two infected mice were further monitored for eight weeks, euthanized and the brain tissue harvested for toxoplasma cysts which were purified and cryopreserved. From the mice infected with oocysts from the cats’ samples, 2 (25%) tachyzoites but a higher 8 (100%) isolation was obtained from brain tissue cysts. On the other hand, from chicken samples generated 18 (47.3%) tachyzoites and 38 (100%), tissue cysts were obtained. The isolated oocysts (from cats), tachyzoites, and tissue cysts (from mice) were cryopreserved using 15% glycerol as cryoprotectant and stored in liquid nitrogen (-196oC). After 6 months of cryopreservation, the viability of the isolates was tested using Trypan blue dye exclusion on a manual hemocytometer. Viability (99.5% - 96%) of the cryopreserved samples was maintained for the three toxoplasma stages and there was no significant change (p&gt;0.05) in the viability of the parasites before and after cryopreservation. The cryobank will serve as a repository for subsequent studies on molecular and phenotypic characterization of T. gondii isolates from Kenya.

Endophytic fungi of Brunfelsia uniflora: isolation, cryopreservation, and determination of enzymatic and antioxidant activity.

Brunfelsia uniflora (Pohl.) D. Don (Solanaceae), commonly known as manacá-de-cheiro, is widely distributed in Brazil and used by local indigenous peoples as an antirheumatic, antisyphilitic, depurative, emetic, vermifuge, and purgative agent. Several studies have examined the biological activities and phytochemical profile of Brunfelsia; however, few have focused on the diversity of endophytic microorganisms that colonize members of the genus. This study aimed to isolate and cryopreserve endophytic fungi from B. uniflora and determine their cellulase, laccase, and antioxidant activities. Endophytic fungi were isolated from B. uniflora stems, cultured on wheat grains, immersed in a 150gL-1 aqueous sucrose solution, and cryopreserved at -80°C for 1 and 6months. Cellulase activity was determined by a qualitative test using carboxymethylcellulose medium and laccase activity by a quantitative test based on the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate). Prior to antioxidant activity assays, fungi were grown in malt extract broth for production of mycelial biomass. A methanolic extract was prepared for evaluation of DPPH· scavenging activity, FRAP activity, and total phenolic content. A total of 46 endophytic fungal isolates were obtained from B. uniflora stems and classified into 24 groups according to morphological similarities. B. uniflora was shown to harbor different genera of ascomycete fungi as endophytic organisms. Mycelial viability was observed after 1 and 6months of cryopreservation at - 80°C. Fungi exhibited cellulase and laccase activities. Isolate CE23 had the highest laccase activity after 7days of cultivation. Twelve isolates were found to have low total phenolic contents and DPPH· and FRAP activities.

Deciphering dose and time dependent effect of supplementing SNP and L-NAME in extender on progressive motility of Murrah bull spermatozoa

Male germ cells have the potential to generate reactive oxygen species (ROS) during different stages of differentiation, laboratory manipulation and during their interaction with dissolved oxygen in extender. These ROS and its subset reactive nitrogen species (RNS) perform a concentration specific bimodal role, favouring oxidative eustress at physiological concentrations. The present study was aimed to investigate the dose and time dependent effect of the most potent RNS, i.e. nitric oxide (NO) compounds, specifically SNP (Sodium nitroprusside) and L-NAME (N-Nitro-L-Arginine Methyl Ester) supplementation in extender on progressive motility. The semen samples were divided into 8 aliquots comprising of various concentrations of SNP (Control, 0.1, 1 and 10 μmol/L) and L-NAME (Control, 1, 10 and 100 μmol/L) and refrigerated for 0, 6, 24 and 48 h. The progressive motility (%) improved significantly particularly with the addition of 1 μM SNP and 10 μM L-NAME as compared to control during all time intervals. Subsequently semen sample was cryopreserved with optimum level of SNP (1μmol/L) and L-NAME (10 μmol/L) from these results by adding it in extender and assessed for progressive motility before cryopreservation and after 24 h, 1 month and 2 months of cryopreservation. It is concluded that the addition of SNP and L NAME in cryopreservation media at 1 μM and 10 μM respectively improved the sperm motility significantly. On further evaluation a significant increase in progressive motility was observed for L-NAME treated extender at 24 h and SNP treated extender at 1 and 2 month interval in comparison to control.

Seed Cryopreservation, Germination, and Micropropagation of Eastern Turkeybeard, Xerophyllum asphodeloides (L.) Nutt.: A Threatened Species from the Southeastern United States.

Xerophyllum asphodeloides (Xerophyllaceae), known as eastern turkeybeard, is an herbaceous perennial found in eastern North America. Due to decline and destruction of its habitat, several states rank X. asphodeloides as “Imperiled” to “Critically Imperiled”. Protocols for seed cryopreservation, in vitro germination, sustainable shoot micropropagation, shoot establishment in soil, and seed germination are presented. Seeds from two tested sources were viable after 20 months of cryopreservation. Germination of isolated embryos in vitro was necessary to overcome strong seed dormancy. Shoot multiplication and elongation occurred on ½ MS medium without PGRs. Shoots rooted in vitro without PGRs or with 0.5 mg/L NAA or after NAA rooting powder treatment and placement in potting mix. When planted in wet, peaty soil mixes, shoots grew for two months and then declined. When planted in a drier planting mix containing aged bark, most plants continued growth. In the field, plant survival was 73% after three growing seasons. Safeguarding this species both ex situ and in situ is possible and offers a successful approach to conservation. Whole seeds germinated after double dormancy was overcome by incubation under warm moist conditions for 12 weeks followed by 12 weeks cold at 4 °C and then warm.

Therapeutic Efficacy of Cryopreserved, Allogeneic Extracellular Vesicles for Treatment of Acute Myocardial Infarction.

Extracellular vesicles (EV) that are derived from endothelial progenitor cells (EPC) have been determined to be a novel therapy for acute myocardial infarction, with a promise for immediate "off-the-shelf" delivery. Early experience suggests delivery of EVs from allogeneic sources is safe. Yet, clinical translation of this therapy requires assurances of both EV stability following cryopreservation and absence of an adverse immunologic response to EVs from allogeneic donors. Thus, more bioactivity studies on allogeneic EVs after cold storage are necessary to establish quality standards for its widespread clinical use. Thus, in this study, we aimed to demonstrate the safety and efficacy in delivering cryopreserved EVs in allogeneic recipients as a therapy for acute myocardial infarction.In this present study, we have analyzed the cardioprotective effects of allogeneic EPC-derived EVs after storage at -80°C for 2 months, using a shear-thinning gel (STG) as an in vivo delivery vehicle. EV size, proteome, and nucleic acid cargo were observed to remain steady through extended cryopreservation via nanoparticle tracking analysis, mass spectrometry, and nanodrop analysis, respectively. Fresh and previously frozen EVs in STG were delivered intramyocardially in a rat model of myocardial infarction (MI), with both showing improvements in contractility, angiogenesis, and scar thickness in comparison to phosphate-buffered saline (PBS) and STG controls at 4 weeks post-MI. Pathologic analyses and flow cytometry revealed minimal inflammatory and immune upregulation upon exposure of tissue to EVs pooled from allogeneic donor cells.Allogeneic EPC-EVs have been known to elicit minimal immune activity and retain therapeutic efficacy after at least 2 months of cryopreservation in a post-MI model.

Cryopreservation of Cyanobacteria and Eukaryotic Microalgae Using Exopolysaccharide Extracted from a Glacier Bacterium.

Exopolysaccharide (EPS) has been known to be a good cryoprotective agent for bacteria, but it has not been tested for cyanobacteria and eukaryotic microalgae. In this study, we used EPS extracted from a glacier bacterium as a cryoprotective agent for the cryopreservation of three unicellular cyanobacteria and two eukaryotic microalgae. Different concentrations of EPS (10%, 15%, and 20%) were tested, and the highest concentration (20%) of EPS yielded the best growth recovery for the algal strains we tested. We also compared EPS with 5% dimethyl sulfoxide (DMSO) and 10% glycerol for the cryopreservation recovery. The growth recovery for the microalgal strains after nine months of cryopreservation was better than 5% DMSO, a well-known cryoprotectant for microalgae. A poor recovery was recorded for all the tested strains with 10% glycerol as a cryoprotective agent. The patterns of growth recovery for most of these strains were similar after 5 days, 15 days, and 9 months of cryopreservation. Unlike common cryopreservants such as DMSO or methanol, which are hazardous materials, EPS is safe to handle. We demonstrate that the EPS from a psychrotrophic bacterium helped in the long-term cryopreservation of cyanobacteria and microalgae, and it has the potential to be used as natural cryoprotective agent for other cells.

Comparative sensitivity of three new cell lines developed from gill, liver and brain tissues of goldfish, Carassius auratus (L.) to cyprinid herpesvirus-2 (CyHV-2)

Cyprinid herpesvirus 2 (CyHV-2) is the etiological agent of Goldfish herpesviral haematopoietic necrosis (GHVHN) in goldfish. In this study, three new cell lines including Fantail goldfish gill (FtGG), Fantail goldfish liver (FtGL) and Fantail goldfish brain (FtGB) had been established and characterized from the gill, liver and brain tissue of C. auratus respectively. Cell lines were optimally grown at 28 °C in Leibovitz-15 (L-15) medium supplemented with 10 % fetal bovine serum (FBS). The PDT during exponential growth of FtGG, FtGL and FtGB cells were determined to be 41.47 h, 63.43 h and 79.79 h respectively. Karyotyping analysis of cell lines remained diploid (2n = 100). The revival rate was 82 %, 72 % and 70 % in FtGG, FtGL and FtGB cells respectively after 6 months of cryopreservation. All the three cells showed similar cytopathic effect (CPE) between 3–5 days post-infection (dpi) with CyHV-2 and complete destruction of the monolayer was observed at 8–10 dpi. The viral titers of CyHV-2 in FtGG, FtGL and FtGB reached 107.375±0.35 TCID50 ml−1, 104·55±0.070 TCID50 ml−1 and 106.45±0.070 TCID50 ml−1 respectively. These newly established cell lines will be a useful diagnostic tool for viral diseases in this fish species and also for the isolation and study of goldfish viruses in future.