Storage In Liquid Nitrogen Research Articles

Evaluation of the TMRW vapor phase cryostorage platform using reproductive specimens and in vitro extended human embryo culture

To assess the impact of shipment and storage of sperm, oocytes, and blastocysts in vapor phase nitrogen compared with static storage in liquid phase nitrogen. Prospective cohort-matched study. Multiple invitro fertilization laboratories in an invitro fertilization network. Fifty-eight human embryos, 32 human oocytes, 15 units of bovine semen. Vapor vs. liquid nitrogen. The postwarming survival of oocytes, sperm, and blastocysts, and the developmental potential of blastocysts during invitro extended culture. Custom-designed labware, for use with the TMRW platform, enables continuous temperature monitoring during shipment and/or storage in the vapor phase robotic storage system. The highest temperature recorded for specimens shipped to a domestic laboratory was -180.2 °C with a mean ± SD of -190.4 ± 0.5 °C during shipment and -181.1 ± 0.6 °C during storage. Likewise, specimens shipped internationally had a high of -180.2 °C with a mean ± SD of -193.5 ± 0.6 °C during shipment and -181.2 ± 0.7 °C during storage. Results from the extended culture assays have revealed no deleterious effect of shipment and storage in nitrogen vapor. The viability of mammalian gametes and embryos was equivalent between the vapor phase and liquid phase storage. The evaluated system did not have any deleterious effects on the postwarming survival of sperm, oocytes, and blastocysts. The postwarming developmental potential of human blastocysts during invitro extended culture was unaffected by storage and handling in the vapor phase nitrogen TMRW platform when compared with static liquid phase nitrogen storage. Our results suggest that the vapor phase cryostorage platform is a safe system to handle and store reproductive specimens for human assisted reproductive technology.

Establishment and characterization of a cell line (RBME-6) of Rhipicephalus (Boophilus) microplus from Brazil

Tick cell lines have already proved to be a useful tool for obtaining more information about possible vector species and the factors governing their ability to transmit a pathogen. Here, we established and characterized a cell line (RBME-6) derived from embryos of Rhipicephalus microplus from Brazil. Primary tick cell cultures were prepared in L-15B medium supplemented with 20% fetal bovine serum and 10% tryptose phosphate broth. The cell monolayers were subcultured when they reached a density of approximately 8 × 10 5 cells/mL (95% viability). Only after the sixth subculture were cells thawed from storage in liquid nitrogen successfully. Cytological analyses were performed using live phase contrast microscopy and cytocentrifuge smears stained with Giemsa, while periodic acid-Schiff and bromophenol blue staining techniques were used to detect total polysaccharides and total protein, respectively . No DNA from Anaplasma spp., Anaplasma marginale, Babesia spp., Bartonella spp., Coxiella spp., Ehrlichia canis, Rickettsia spp. or Mycoplasma spp. was detected in the cells through PCR assays. In addition, we performed chromosomal characterization of the tick cell line and confirmed the R. microplus origin of the cell line through conventional PCR and sequencing of a fragment of the mitochondrial 16S rRNA gene. In conclusion, we established and characterized a new cell line from a Brazilian population of R. microplus, which may form a useful tool for studying several aspects of ticks and tick-borne pathogens.

Autologous Stem Cells as Backup for Patients Undergoing Allogeneic Related Transplant for Thalassemia Major.

Abstract 4539Autologous backup of harvested stem cells from patients with Beta thalassemia major, undergoing allogeneic HLA matched sibling transplantation, in order to rescue the patient in the event of graft failure, has been practiced in some transplant centers in the world. From February 2007 we instituted a program of harvesting and cryopreserving autologous bone marrow for all patients undergoing transplant for thalassemia at our center and this abstract is a report on 62 patients for whom this was done over the past 26 months till June 2009. Since most of the patients were below 10 years a bone marrow harvest was performed under general anesthesia. The harvest was depleted of red cells by gravity sedimentation using hydroxyethyl starch, plasma depleted and cryopreserved using 10% dimethyl sulfoxide and stored at -18-degC in the vapor phase of a liquid nitrogen storage system. The mean volume of the product before freezing was 180 ml. The median CD34 cell dose was 8.35 × 106 cells/kg (range 0.6 to 24.6 × 106 cells/kg).During this 26 month period there was one early rejection, after initial engraftment, without autologous recovery with the patient being critically ill and febrile. Autologous stem cells were re-infused on day + 90 when the ANC was 0. However there was progressive deterioration in respiratory function and the patient died on day+92 with disseminated pulmonary aspergillosis. It is extremely difficult to decide when to re-infuse autologous stem cells in a patient who is sick post transplant and may not engraft. There was only one other rejection in this cohort of 62 patients but this patient had spontaneous recovery of autologous hematopoiesis and did not require intervention.The total cost of harvesting autologous stem cells under anesthesia and Cryopreservation, at this center is approximately $ 1600 and this represents 10% of the total cost of an allogeneic stem cell transplant. In this cohort of 62 patients, cryopreserved stem cells were used in only one patient without preventing mortality. The increased cost, manpower and time required for this procedure needs further evaluation and also the criteria on which autologous stem cells are to be re-infused needs to be established. Disclosures:No relevant conflicts of interest to declare.

Universal Engraftment after Allogeneic Hematopoietic Cell Transplantation Using Cryopreserved CD34-Selected Grafts

As a result of the COVID-19 pandemic, most centers performing allogeneic hematopoietic cell transplantation (allo-HCT) have switched to the use of cryopreserved grafts. Previous investigators have suggested that cryopreserved allografts may heighten risk of nonengraftment. To date, no study has investigated the effect of cryopreservation of CD34-selected hematopoietic progenitor cells (CD34+ HPCs) used as the sole graft source. In this study, we sought to evaluate outcomes after unrelated donor or matched sibling allo-HCT with cryopreserved CD34+ HPCs. This was a single-center analysis of adult patients with hematologic malignancies who underwent allo-HCT with cryopreserved CD34-selected allo-HCT grafts between January 2010 and June 2017. All patients received ablative conditioning and antirejection prophylaxis with rabbit antithymocyte globulin. G-CSF-mobilized leukapheresis products underwent CD34 selection using the CliniMACS Reagent System. Cells were then cryopreserved in DMSO (final concentration 7.5%) to -90 °C using a controlled-rate freezing system before being transferred to vapor-phase liquid nitrogen storage. In internal validation, this method has shown 92% mean CD34+ cell viability and 99.7% mean CD34+ cell recovery. Engraftment was defined as the first of 3 consecutive days of an absolute neutrophil count of ≥0.5. Platelet recovery was recorded as the first of 7 consecutive days with a platelet count ≥20 K/μL without transfusion. Kaplan-Meier methodology was used to estimate overall survival (OS) and relapse-free survival (RFS), and cumulative incidence functions were used to estimate rates of relapse, nonrelapse mortality (NRM), and acute graft-versus-host disease (GVHD). A total of 64 patients received a cryopreserved CD34-selected graft. The median CD34+ cell count before cryopreservation was 6.6×106/kg (range, 1.4 to 16.1×106/kg), and the median CD3+ cell count was 2.0×103/kg (range, 0 to 21.1×106/kg). All patients were engrafted, at a median of 11 days post-HCT (range, 8 to 14 days). One patient had poor graft function in the setting of cytomegalovirus viremia, necessitating a CD34-selected boost on day +57. The median time to platelet recovery was 16 days (range, 13 to 99 days). The estimated 2-year OS was 70% (95% confidence interval [CI], 58% to 83%) with cryopreserved grafts versus 62% (95% CI, 57% to 67%) with fresh grafts (hazard ratio [HR], 0.86; 95% CI, 0.54 to 1.35; P=.5). The estimated 2-year RFS in the 2 groups was 59% (95% CI, 48% to 74%) versus 56% (95% CI, 51% to 61%; HR, 1.01; 95% CI, 0.68 to 1.51; P > .9). The cumulative incidence of relapse at 2 years was 29% (95% CI, 17% to 41%) versus 23% (95% CI, 19% to 27%; P=.16), and the cumulative incidence of NRM at 2 years was 17% (95% CI, 9% to 28%) versus 23% (95% CI, 19% to 28%; P=.24). The cumulative incidence of grade II-IV acute GVHD by day +100 was 16% with cryopreserved grafts (95% CI, 8% to 26%) and 16% (95% CI, 13% to 20%; P=.97) with fresh grafts. Moderate to severe chronic GVHD by day +365 occurred in only 1 recipient of a cryopreserved graft (2%). Our data show that in patients with hematologic malignancies who received cryopreserved allogeneic CD34+ HPCs, engraftment, GVHD, and survival outcomes were consistent with those seen in recipients of fresh allogeneic CD34+ HPC grafts at our center. Our laboratory validation and clinical experience demonstrate the safety of our cryopreservation procedure for CD34-selected allografts.

Temporal-dependent effects of DNA degradation on frozen tissues archived at −80°C

Abstract Frozen tissues, associated with natural history and biological collections, historically have been archived at temperatures between −20°C and −80°C. More recently, the availability of liquid nitrogen systems has enabled the storage of tissue samples (biobanking) at temperatures as low as −196°C. Currently, it is not known how the degree of coldness (e.g., −80°C or −196°C) or longevity (time in storage) impacts preservation of tissue samples. To examine the effects of long-term storage (−80°C and −196°C) on DNA degradation, tissue samples (muscle and liver) archived for 30, 20, 10, or 1 years were obtained from the Natural Science Research Laboratory at Texas Tech University. The integrity of DNA (measured as molecular weight and fragment length) extracted from samples was determined using automated DNA isolation methods followed by microfluidic distribution measurement. DNA distributions were compared using measures of central tendency, a regression-based molecular mass profile, and as a latent variable in a structural equation model. Muscle samples consistently outperformed liver samples in terms of quality of DNA yield. Also, muscle samples exhibited a significant linear relationship with time in which older samples were more degraded than were recent samples. The signal for a temporal effect on DNA was strongest when considering a latent variable of DNA quality based on mode and kurtosis; 37% of the variation in the latent variable was explained by variation in units of time. More recent time points tended to be more similar, but the temporal effect on the latent variable remained strong even when the oldest samples were removed from the analysis. In contrast, integrity of DNA from liver samples did not have a significant linear relationship with time; however, in some years they exhibited non-normally distributed DNA quality metrics that may have reflected sensitivity of liver tissue to degradation during specimen preparation, DNA extraction, or archive parameters. Results indicated that tissue type and temporal effects influenced rates of DNA degradation, with the latter emphasizing the long-term value of biobanking at the coldest temperatures possible (liquid nitrogen storage) to mitigate degradation of biological samples of ever-increasing scientific value.

Cryopreservation method for Drosophila melanogaster embryos

The development of a widely adopted cryopreservation method remains a major challenge in Drosophila research. Here we report a robust and easily implemented cryopreservation protocol of Drosophila melanogaster embryos. We present innovations for embryo permeabilization, cryoprotectant agent loading, and rewarming. We show that the protocol is broadly applicable, successfully implemented in 25 distinct strains from different sources. We demonstrate that for most strains, >50% embryos hatch and >25% of the resulting larvae develop into adults after cryopreservation. We determine that survival can be significantly improved by outcrossing to mitigate the effect of genetic background for strains with low survival after cryopreservation. We show that flies retain normal sex ratio, fertility, and original mutation after successive cryopreservation of 5 generations and 6-month storage in liquid nitrogen. Lastly, we find that non-specialists are able to use this protocol to obtain consistent results, demonstrating potential for wide adoption.

Comparison of two methods for prolong storage of decellularized mouse whole testis for tissue engineering application: An experimental study.

BackgroundBiological scaffolds are derived by the decellularization of tissues or organs. Various biological scaffolds, such as scaffolds for the liver, lung, esophagus, dermis, and human testicles, have been produced. Their application in tissue engineering has created the need for cryopreservation processes to store these scaffolds.ObjectiveThe aim was to compare the two methods for prolong storage testicular scaffolds.Materials and MethodsIn this experimental study, 20 male NMRI mice (8 wk) were sacrificed and their testes were removed and treated with 0.5% sodium dodecyl sulfate followed by Triton X-100 0.5%. The efficiency of decellularization was determined by histology and DNA quantification. Testicular scaffolds were stored in phosphate-buffered saline solution at 4°C or cryopreserved by programmed slow freezing followed by storage in liquid nitrogen. Masson's trichrome staining, Alcian blue staining and immunohistochemistry, collagen assay, and glycosaminoglycan assay were done prior to and after six months of storage under each condition.ResultsHematoxylin-eosin staining showed no remnant cells after the completion of decellularization. DNA content analysis indicated that approximately 98% of the DNA was removed from the tissue (p = 0.02). Histological evaluation confirmed the preservation of extracellular matrix components in the fresh and frozen-thawed scaffolds. Extracellular matrix components were decreased by 4°C-stored scaffolds. Cytotoxicity tests with mouse embryonic fibroblast showed that the scaffolds were biocompatible and did not have a harmful effect on the proliferation of mouse embryonic fibroblast cells.ConclusionOur results demonstrated the superiority of the slow freezing method for prolong storage of testicular scaffolds.

Cryopreservation of human cerebral microvascular endothelial cells and astrocytes in suspension and monolayers.

The blood–brain barrier (BBB) keeps pathogens and toxins out of the brain but also impedes the entry of pharmaceuticals. Human cerebral microvascular endothelial cells (hCMECs) and astrocytes are the main functional cell components of the BBB. Although available commercially as cryopreserved cells in suspension, improvements in their cryopreservation and distribution as cryopreserved monolayers could enhance BBB in vitro studies. Here, we examined the response to slow cooling and storage in liquid nitrogen of immortalized hCMEC/D3 cells and human primary astrocytes in suspension and in monolayers. HCMEC/D3 cells in suspension cryopreserved in 5% dimethyl sulfoxide (DMSO) and 95% fetal bovine serum or in 5% DMSO and 6% hydroxyethyl starch (HES) showed post-thaw membrane integrities above 90%, similar to unfrozen control. Cryopreservation did not affect the time-dependent ability of hCMEC/D3 cells to form tubes on Matrigel. Primary astrocytes in suspension cryopreserved in the presence of 5% DMSO and 6% HES had improved viability over those cryopreserved in 10% DMSO. Monolayers of single cultures or co-cultures of hCMEC/D3 cells and astrocytes on fibronectin-coated Rinzl coverslips retained membrane integrities and metabolic function, after freezing in 5% DMSO, 6% HES, and 2% chondroitin sulfate, that were comparable to those of unfrozen controls even after overnight incubation. Rinzl is better than glass or Thermanox as an underlying solid substrate for cryopreserving hCMEC/D3 monolayers. Cryopreserved hCMEC/D3 monolayers expressed the junction proteins ZO-1 and claudin-5 similar to their unfrozen counterparts. Hence, we describe improved cryopreservation protocols for hCMEC/D3 cells and astrocytes in suspension, and a novel protocol for the cryopreservation of monolayers of hCMEC/D3 cells and astrocytes as single cultures or co-cultures that could expand their distribution for research on disease modeling, drug screening, and targeted therapy pertaining to the BBB.

Cryopreservation and post-thaw genetic integrity of Viola stagnina Kit., an endangered species of wet habitats – A useful tool in ex situ conservation

The use of cryopreservation techniques for plant species confined to wet habitats is difficult to standardize due to low tolerance for dehydration of plant tissue. The study aimed to develop a procedure for the long-term storage in liquid nitrogen (LN) of Viola stagnina, a rare and endangered species of moist habitats whose European range is constantly decreasing. Adventitious shoot apices of different sizes (small, medium and large) were cryopreserved using encapsulation/dehydration, encapsulation/vitrification (in PVS2 or PVS3 solutions) and droplet/vitrification methods. Encapsulation/dehydration was the most efficient technique, and 47.7 % regrowth was obtained with an average rate of shoot regeneration of 2.4 %. Less than 27.6 % of cryopreserved shoot apices were able to produce shoots with an efficiency of 0.35 or less based on vitrification procedures. In post-thaw recovery culture, adventitious shoots were produced indirectly (via callus). The genetic variation among recovered shoots after different cryopreservation methods was low (Dice coefficient not exceeded 0.09). Some clones were genetically uniform with the mother plant estimated by ISSR molecular markers. The genome size of all post-thaw recovered regenerants, except one individual with a multiplicated genome, was uniform with the initial plant.This paper presents the first report on cryopreservation of V. stagnina with a developed protocol that can be used in ex situ conservation of the species.

Cryopreservation and low-temperature storage of seeds of Tillandsia species (Bromeliaceae) with ornamental potential.

Tillandsia (Bromeliaceae) species have high endemism, and due to their strong ornamental potential, predatory extraction is threatening the extinction or drastic population reduction of many of them. In light of this scenario, it is necessary to find strategies for the conservation of these endangered species. The objective of this study was to evaluate two seed preservation strategies (freezing at - 5°C and cryopreservation at - 196°C) for 20 Tillandsia species occurring in the state of Bahia. We initially evaluated the morphometry, thousand-seed weight, and water content, followed by tests of germination and desiccation. After selecting the best result of the germination test (Germitest paper and incubation at 30°C) and desiccation (3h on silica gel), we established conservation tests utilizing two temperatures (freezing at - 5°C and liquid nitrogen at - 196°C), with storage times of 1, 7, 30, 180 and 450days. Analysis of variance indicated that the 20 species had different behaviors when submitted to the two temperatures and different storage times. After 450days there was a reduction in the germination percentage and germination speed index (GSI) of all the species studied when the seeds were preserved in the freezer. The storage in liquid nitrogen was efficient for the preservation of Tillandsia seeds when dried to a moisture content of approximately 7%. Our results support the establishment of a cryobank for Tillandsia to conserve these endemic species.

Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo

Age-related macular degeneration (AMD) is the primary cause of blindness in adults over 60 years of age, and clinical trials are currently assessing the therapeutic potential of retinal pigmented epithelial (RPE) cell monolayers on implantable scaffolds to treat this disease. However, challenges related to the culture, long-term storage, and long-distance transport of such implants currently limit the widespread use of adherent RPE cells as therapeutics. Here we report a xeno-free protocol to cryopreserve a confluent monolayer of clinical-grade, human embryonic stem cell-derived RPE cells on a parylene scaffold (REPS) that yields viable, polarized, and functional RPE cells post-thaw. Thawed cells exhibit ≥ 95% viability, have morphology, pigmentation, and gene expression characteristic of mature RPE cells, and secrete the neuroprotective protein, pigment epithelium-derived factor (PEDF). Stability under liquid nitrogen (LN2) storage has been confirmed through one year. REPS were administered immediately post-thaw into the subretinal space of a mammalian model, the Royal College of Surgeons (RCS)/nude rat. Implanted REPS were assessed at 30, 60, and 90 days post-implantation, and thawed cells demonstrate survival as an intact monolayer on the parylene scaffold. Furthermore, immunoreactivity for the maturation marker, RPE65, significantly increased over the post-implantation period in vivo, and cells demonstrated functional attributes similar to non-cryopreserved controls. The capacity to cryopreserve adherent cellular therapeutics permits extended storage and stable transport to surgical sites, enabling broad distribution for the treatment of prevalent diseases such as AMD.

The new sheep ovarian cell culture and the prospect its application in virology

The new sheep ovarian cell culture named as SO was derived and its reproduction has been successful carried out using available growth mediums such as mixture of modified Eagle’s medium, lactalbumin hydrolysate and bovine serum. The monolayer was formed at a seeding concentration of 100-120 thousand cells/ml in 3-5 days. The proliferation index was 2.81-3.24. Fibroblast-like cells predominated at early generations of the culture. During high passages, domination of epithelial-like cells was observed. While passaging, the modal class of 54 chromosomes was retained but the number of chromosome dispersion increased and the number of cells with a diploid set decreased. The cell viability was 82-75% during long-term storage in liquid nitrogen. The monolayer was retained for at least two weeks without changing the medium at 37°C. The culture produced estradiol and progesterone. Cell culture was highly sensitive to bovine herpesvirus 1, parainfluenza-3, nodular dermatitis and pustular stomatitis (Orf) viruses, but had low sensitivity to Orthopneumovirus. It didn’t allow reproduction of pestivirus – causative agent of bovine viral diarrhea – mucosal disease.

The impact of five years storage/biobanking at -80°C on mouse spermatozoa fertility, physiology, and function.

We previously demonstrated how mouse spermatozoa can be efficiently stored for two years in a -80°C freezer, maintaining their ability to fertilize mouse eggs. The main objective here was to evaluate the effects of five years at -80°C compared to liquid nitrogen storage (LN2 , control condition) on mouse sperm viability, physiological parameters, and fertilization capacity. Three different strains were used: C57BL/6N, C57BL/6J and CD1. Flow cytometry experiments were performed to analyze sperm viability (SYBR-14 + Propidium Iodide +Hoechst33342), the intracellular calcium concentration (Fluo 3-AM), the membrane lipid disorder (Merocyanine 540), and the mitochondrial activity (MitoTracker Red) in live spermatozoa. The in vitro fertilization (IVF) was used to evaluate the sperm fertilizing ability. Flow cytometry analysis showed that the percentage of live cells are reduced in B6N and B6J, but not in CD1 mice. However, in the live population no differences in terms of intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity were reported when comparing both biobanking methods. Spermatozoa stored at -80°C for 5years successfully fertilized the eggs and developed mouse embryo normally both in culture and in vivo, generating live pups with no differences compared to control samples stored in LN2 . Long-term mouse sperm storage at -80°C (five years) could be considered an ideal alternative to the most common LN2 approach, giving economical and logistic advantages. Moreover, the precise information originated from the flow cytometry analysis stands up this technique as an optimal strategy to evaluate the sperm quality and ranking. It is demonstrated here the possibility to store mouse spermatozoa for up to five years in a -80°C freezer with no significant differences compared to the storage in LN2 in terms of fertilizing ability, sperm viability, intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity.

Cryopreserved skin epithelial cell sheet combined with acellular amniotic membrane as an off-the-shelf scaffold for urethral regeneration

BackgroundAutologous tissue transplantation for urethral repair is often limited and causes donor site complications. Here, a cryopreserved rabbit skin epithelial cell sheet (SEC) combined with an acellular amniotic membrane (AM) was used to repair rabbit urethral defects. MethodsAbdominal skin was collected from 4-week-old New Zealand rabbits, and primary epithelial cells were extracted and cultured to form a cell sheet. Fresh SEC-AMs were constructed and cryopreserved. A cryopreservation system including optimized medium, two-pump perfusion, a programmed freezer and liquid nitrogen storage was established. Cell viability, mechanical strength, electron microscopy, and histological staining were performed in vitro after 1 month. Next, the sheets were transplanted subcutaneously for 2 weeks, and the graft was used to repair the rabbit urethral defect. Urinary function was measured and samples were collected for histological staining after 1 month. ResultsWe confirmed that cryopreservation damage of SECs was reduced by composition with acellular AMs in terms of high cell activity. The SEC mechanical strength was also enhanced by AMs, which was convenient for the operation. In in vivo experiments, we transplanted sheets into the groin area for two weeks and found that cryopreservation reduced inflammatory cell infiltration and significantly improved vascular density. In the urethral repair experiment, the near-normal passive urine flow rate, smooth mucosa of the gross specimen, intact epithelialization and abundant neovascularization were confirmed in the cryopreserved-SEC-AM group compared with the other groups. ConclusionsCryopreserved SEC-AMs demonstrated similar outcomes of rabbit urethral defect repair as fresh SEC-AMs, showing good clinical application prospects.

Optimisation and evaluation of cryopreservation method for Aurantiochytrium limacinum

Marine microorganisms are gaining increased global importance owing to their richness in various bioactive compounds. The thraustochytrid Aurantiochytrium limacinum produces high amounts of the omega-3 fatty acid, DHA, and thus is of biotechnological importance. Aurantiochytrium limacinum cultures are usually maintained by continuous sub-culturing or plating, which is labour and resource-intensive. Having a cryopreservation technique will be useful for the preservation of A. limacinum cultures. Hence, we explored and developed a cryopreservation technique to preserve A. limacinum. Cryopreservation was done at two different temperatures by using a mechanical freezer (− 80 °C) and liquid nitrogen (− 196 °C). In the mechanical freezer, the cells were preserved in glycerol, while in liquid nitrogen four widely used cryoprotectants namely ethylene glycol, glycerol, methanol and dimethyl sulfoxide were used. The cells stored in a mechanical freezer had a relatively constant growth rate up to 12 months of storage. For liquid nitrogen storage, glycerol, methanol and dimethyl sulfoxide showed promising cryoprotectant activity with viability as high as 80% even after 12 months of storage. However, ethylene glycol did not exhibit any cryoprotective effect evidenced by marginal viability. Post-thaw assessment is crucial for judging the success of cryopreservation protocol and therefore, the study also looked into the functionality of cells on revival. On reviving no significant difference in the fatty acid profile and cell morphology among treatments was observed. The cryopreservation technique thus developed for preserving A. limacinum will help save human efforts and time and also conserve the organism’s genetic integrity.

Cryopreservation of mesenchymal stem cells derived from dental pulp: a systematic review.

ObjectivesThe aim of the present systematic review was to investigate the cryopreservation process of dental pulp mesenchymal stromal cells and whether cryopreservation is effective in promoting cell viability and recovery.Materials and MethodsThis systematic review was developed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement and the research question was determined using the population, exposure, comparison, and outcomes strategy. Electronic searches were conducted in the PubMed, Cochrane Library, Science Direct, LILACS, and SciELO databases and in the gray literature (dissertations and thesis databases and Google Scholar) for relevant articles published up to March 2019. Clinical trial studies performed with dental pulp of human permanent or primary teeth, containing concrete information regarding the cryopreservation stages, and with cryopreservation performed for a period of at least 1 week were included in this study.ResultsThe search strategy resulted in the retrieval of 185 publications. After the application of the eligibility criteria, 21 articles were selected for a qualitative analysis.ConclusionsThe cryopreservation process must be carried out in 6 stages: tooth disinfection, pulp extraction, cell isolation, cell proliferation, cryopreservation, and thawing. In addition, it can be inferred that the use of dimethyl sulfoxide, programmable freezing, and storage in liquid nitrogen are associated with a high rate of cell viability after thawing and a high rate of cell proliferation in both primary and permanent teeth.

The effect of glutathione antioxidant addition in maturation medium on the morphology of Garut sheep (Ovis aries) oocytes after vitrification

The study regarding the addition of glutathione antioxidants in the maturation medium in Garut sheep oocytes has been done. The purpose of this study was to evaluate the addition of glutathione antioxidants on oocyte morphology at post-vitrification. A total of 129 oocytes were matured in vitro using TCM-199 medium combined with glutathione antioxidants with a concentration of 0 mM (KK); 0.5 mM (KP1); 1 mM (KP2); and 1.5 mM (KP3). Oocytes were then vitrified using a combination of 15 % ethylene glycol and 15 % dimethyl sulfoxide as cryoprotectants. Oocyte evaluation was carried out after 7 days of storage in liquid nitrogen, including oocyte maturity and oocyte morphology. The percentages of mature oocytes are 53.13 % (0 mM), 55.88 % (0.5 mM ), 59.38 % (1 mM ), and 67.74 % (1.5 mM ) and the percentages of normal oocytes are 56.25 % (0 mM ), 64.71 % (0.5 mM ), 71.88 % (1 mM ) and 87.10 % (1.5 mM ). The results of ANOVA tests showed that the data were not significantly different between groups (P > 0.05), but the percentage tends to increase with the increase of antioxidants concentration. The addition of glutathione antioxidants in the maturation medium seems to help maintain oocyte morphology until post-vitrification, although not significant.

The effect of glutathione antioxidant addition in slow freezing medium on oocyte viability of Garut sheep (Ovis aries)

Research has been conducted on the addition of glutathione antioxidants in slow freezing medium on the oocyte viability of garut sheep (Ovis aries) after-cryopreservation. The study aimed to evaluate the effect of adding glutathione antioxidants in slow freezing medium with a concentration of 0.5 mM; 1 mM; 1.5 mM on the oocyte viability of garut sheep. Cryopreservation of 136 oocytes was carried out using cryoprotectant, such as 10 % ethylene glycol and 0.1 M sucrose. Viability of oocytes was evaluated using Hoechst and propidium iodide dyes, after 7 days of storage in liquid nitrogen (-196 °C). Based on the results of the study, the percentage of viable oocytes after-cryopreservation was 68.40 % (0 mM), 69.76 % (0.5 mM ), 75.55 % (1 mM ) and 83.08 % (1.5 mM ). Based on ANOVA statistical test, there were no significant differences between treatment groups (P > 0.05), but results showed an increase pattern with the addition of glutathione antioxidant concentrations. In conclusion the results showed that the addition of glutathione antioxidants in the slow freezing medium did not affect the oocyte viability of Garut sheep after cryopreservation.

The effect of addition alpha tocopherol antioxidant on slow freezing medium to the quality of Garut sheep oocyte (Ovis aries)

This study has evaluated the ability of alpha tocopherol antioxidant in maintaining the quality of Garut sheep oocytes post-cryopreservation using slow freezing method. Total of 226 oocytes with two different qualities (A and B) are matured in vitro in TCM-199 medium added with 150 µM alpha tocopherol. Afterwards, the oocytes were cryopreserved in the slow freezing medium with the addition of 0 µM (KK), 100 µM (KP1), 150 µM (KP2) and 200 µM (KP3) alpha tocopherol. Combination of cryoprotectant 10 % ethylene glycol (EG) and 0.1 M sucrose were also added to the medium slow freezing. Oocyte evaluation was carried out after 7 days of storage in liquid nitrogen (-196 °C), including oocyte morphology and oocyte viability using Hoechst and Propidium Iodide (PI) dyes. Based on the results of the study, the percentage of normal oocyte post-cryopreservation are 0 µM (67.86 %), 100 µM (68.42 %), 150 µM (74.58 %) and 200 µM (61.11 %). The percentage of viable oocyte post-cryopreservation are 0 µM (76.79 %), 100 µM (80.70 %), 150 µM (86.44 %), and 200 µM (70.37 %). The results shows that the addition of alpha tocopherol did not have an effect to the quality of oocytes post-cryopreservation, but there was a tendency for quality improvement with increasing doses of alpha tocopherol. In this study, addition of 150 µM alpha tocopherol into slow freezing medium is the best dose for maintaining the quality of Garut sheep oocytes until post-cryopreservation, although not significant (P > 0.05).

Cryopreservation of Potato Shoot Tips for Long-Term Storage.

Cryopreservation is currently the only method which allows long-term conservation of living clonal plant material in the vapor or liquid phase of nitrogen (at -140 to -196°C) allowing tissue to be viable for decades or perhaps centuries. Specifically, for species with recalcitrant seeds or requiring constant vegetative propagation, it is the method of choice for the long-term conservation of its genetic resources. The protocol described here is a modification of a previously developed plant vitrification solution 2 (PVS2)-droplet vitrification method of potato shoot tips, adapted from Musa species. Utilizing this protocol, the International Potato Center (CIP) has successfully stored in the cryobank more than 3000 cultivated potato accessions, belonging to seven species and nine different taxa[16], originating principally from ten countries in South and Central America. As part of CIP's quality management system, all vegetative material placed in cryo is routinely subsampled, thawed, and assessed to confirm that whole plantlets can be produced after storage in liquid nitrogen. Complete plant recovery rates of thawed shoot tips range from 20% to 100% (average rate: 60%). This chapter describes the complete set of steps from the routine procedure of cryopreserving potato shoot tips for long-term conservation.