Storage In Liquid Nitrogen Research Articles

Development of a common PVS2 vitrification method for cryopreservation of several fruit and vegetable crops

Many cryopreservation techniques are currently available, and it is common for new modifications to be developed for individual crops or specific genotypes. In this study, results of variations of the PVS2 cryopreservation protocol are compared to provide evidence for the suitability of a standard form of this technique for cryopreservation of a range of fruit, berry crops, and potato. Shoot cultures of Malus, Solanum, Lonicera, and Berberis were tested with variations of cold acclimation, pretreatment media, and PVS2 exposure times. A general protocol with some modifications was produced that was suitable for all four genera. The regenerative capacity of shoot tips after cryopreservation by this method exceeded a mean of 50% for Malus, Solanum, Lonicera, and Berberis, which is sufficient for setting storage in a cryobank. After liquid nitrogen storage, the shoot cultures that survived had a healthy appearance and developed rapidly. For each species tested, the only optimization required was the preparation of donor plants by cold acclimation and pretreatment. The choice of one common method simplifies the methodology for conducting experiments and storing a range of germplasm. The use of the PVS2 vitrification method with a 0.3-M sucrose pretreatment is multiuse and can be recommended as the most effective method for the cryopreservation of shoot tips from many plant species.

Abstract 4827: Establishing a platform for the generation of organoids from diverse tumor types as part of the NCI patient-derived models (PDM) initiave

Abstract Cancer Organoids are discrete multicellular structures that recapitulate tumor microanatomy (1). These reagents can be generated by extended culture of partially or fully dissociated tumor samples in three-dimensional matrices. By maintaining tumor and accessory cells in an appropriate context, they provide a biosimilar platform for studying disease pathogenesis and cellular pharmacology (2). Similarly, cancer organoid culture is useful for propagating slow growing tumors or those requiring heterotypic cell-cell interactions. Here, preliminary data will be presented regarding generation of organoids from diverse tumor types as part of the NCI patient-derived models (PDM) initiative. This initiative aims to develop a national repository of patient-derived cancer models (PDMs) consisting of clinically annotated patient-derived xenografts (PDXs) and patient-derived tumor cell cultures (PDCs) prepared from primary and metastatic tumors (3). A standardized panel of different organoid media formulations was constructed to optimize culture conditions for disease subsets. Using this approach, organoids were generated for colon, prostate, pancreatic, breast, melanoma, NSCLC, and bladder tumors. Although some samples were refractory to organoid generation, in several instances, samples that failed to generate 2D cultures thrived as organoids. A further finding was that direct implantation of organoid cultures was an efficient means of generating xenografts. Indeed, work will be presented detailing the exact number of organoids required to establish xenograft tumors. Protocols were developed for routine culture, passaging and long-term storage in liquid nitrogen. Similarly, organoids were amenable to characterization by FACS analysis, ICC/IHC and qRT-PCR to evaluate metrics such as tumor type, histological similarity with patient tumor, cell viability, percentage stroma and whether mouse cells persist in PDX-derived organoids. In summary, growth, expansion, analysis and storage of tumor organoids is feasible for a wide range of tumor types. Importantly, for certain samples, generation of cancer organoids appears to be a useful intermediary step for subsequent PDX model and 2D culture generation. Funded by NCI Contract No. HHSN261200800001E.

Effect of the Duration of Liquid Nitrogen Storage on the Regrowth of Blackberry Cryopreserved by Droplet Vitrification

Summary In vitro shoot tips of the blackberry cultivar ‘Čačanska Bestrna’ were cryopreserved using the droplet vitrification technique. Upon loading (30 min) in a solution of 1.9 M glycerol and 0.5 M sucrose, the explants were dehydrated for 40 min on ice with the PVS A3 vitrification solution (glycerol 37.5%, dimethyl sulfoxide 15%, ethylene glycol 15% and sucrose 22.5%) and for 40 min at room temperature with the PVS3 solution (glycerol 50% and sucrose 50%). They were subsequently frozen in individual microdroplets of vitrification solution, by direct immersion in liquid nitrogen (LN), and kept therein for 2, 4, 8 and 24 h. The explant rewarming was performed in an unloading solution (0.8 M sucrose) for 30 min at room temperature. The duration of LN exposure did not exert significant effects on the survival and regrowth of explants in both types of vitrification solutions. The survival and regrowth of cryopreserved shoot tips dehydrated with PVS3 solution ranged between 90–95% and 80–90%, respectively. However, dehydration with PVS A3 resulted in a lower survival rate (80–90%) and a considerably lower regrowth rate (55–65%) of explants. Monitoring the shoots regenerated in the in vitro culture revealed their normal capacity for multiplication and rooting in comparison with the controls, which fully confirms the purpose of cryopreservation in the long-term preservation of plant material.

Manufacture of Autologous CD34+ Selected Grafts in the NIAID-Sponsored HALT-MS and SCOT Multicenter Clinical Trials for Autoimmune Diseases

To ensure comparable grafts for autologous hematopoietic cell transplantation (HCT) in the National Institute of Allergy and Infectious Diseases–sponsored Investigational New Drug protocols for multiple sclerosis (HALT-MS) and systemic sclerosis (SCOT), a Drug Master File approach to control manufacture was implemented, including a common Master Production Batch Record and site-specific standard operating procedures with “Critical Elements.” We assessed comparability of flow cytometry and controlled rate cryopreservation among sites and stability of cryopreserved grafts using hematopoietic progenitor cells (HPCs) from healthy donors. Hematopoietic Progenitor Cells, Apheresis-CD34+ Enriched, for Autologous Use (Auto-CD34+HPC) graft specifications included ≥70% viable CD34+ cells before cryopreservation. For the 2 protocols, 110 apheresis collections were performed; 121 lots of Auto-CD34+HPC were cryopreserved, and 107 of these (88.4%) met release criteria. Grafts were infused at a median of 25 days (range, 17 to 68) post-apheresis for HALT-MS (n = 24), and 25 days (range, 14 to 78) for SCOT (n = 33). Subjects received precryopreservation doses of a median 5.1 × 106 viable CD34+ cells/kg (range, 3.9 to 12.8) for HALT-MS and 5.6 × 106 viable CD34+ cells/kg (range, 2.6 to 10.2) for SCOT. Recovery of granulocytes occurred at a median of 11 days (range, 9 to 15) post-HCT for HALT-MS and 10 days (range, 8 to 12) for SCOT, independent of CD34+ cell dose. Subjects received their last platelet transfusion at a median of 9 days (range, 6 to 16) for HALT-MS and 8 days (range, 6 to 23) for SCOT; higher CD34+/kg doses were associated with faster platelet recovery. Stability testing of cryopreserved healthy donor CD34+ HPCs over 6 months of vapor phase liquid nitrogen storage demonstrated consistent 69% to 73% recovery of viable CD34+ cells. Manufacturing of Auto-CD34+HPC for the HALT-MS and SCOT protocols was comparable across all sites and supportive for timely recovery of granulocytes and platelets.

Is it possible to detect PEth 16:0/18:1 and PEth 18:1/18:1 in red blood cells after 20 years of storage in liquid nitrogen?

Phosphatidylethanol (PEth), as measured in freshly drawn blood samples, could be a promising new biomarker for habitual alcohol consumption, but it is still unknown whether PEth can also be determined from blood samples having been stored frozen for a longer period. PEth 16:0/18:1 and PEth 18:1/18:1 were determined by LC-MS/MS from red blood cells (RBC) derived from blood samples of (I) 20 healthy volunteers (after 1month of storage at -80°C) and (II) 232 participants of the population-based European Prospective Investigation into Cancer and Nutrition (EPIC)-Heidelberg study (after 20years of storage at -196°C). Analyses involved liquid-liquid extraction from 100μl aliquots with phosphatidylpropanol (PProp 18:1/18:1) as the internal standard. Extracts were subjected to a 10-min LC gradient separation, using multiple reaction monitoring of deprotonated molecules for quantification. After 1month of storage at -80°C, PEth was detectable in all samples at mean concentrations of 393.6±12.4ng/ml (PEth 16:0/18:1) and 43.3±1.1ng/ml (PEth 18:1/18:1). In samples stored for 20years at -196°C, PEth was detectable in 23.7% of all samples at mean concentrations of 412.2±655.5ng/ml (PEth 16:0/18:1) and 38.0±74.8ng/ml (PEth 18:1/18:1). PEth can be determined reliably from samples of moderate habitual alcohol consumers after 1month of storage at -80°C. Our data suggest that PEth is generally also detectable in samples after 20years of storage at -196°C. Further studies are needed to assess the still unknown impact of storage duration and temperature on different PEth specimen concentrations.

The effect of low- and high-power microwave irradiation on in vitro grown Sequoia plants and their recovery after cryostorage.

Two distinct microwave power levels and techniques have been studied in two cases: low-power microwave (LPM) irradiation on in vitro Sequoia plants and high-power microwave (HPM) exposure on recovery rates of cryostored (-196°C) Sequoia shoot apices. Experimental variants for LPM exposure included: (a) in vitro plants grown in regular conditions (at 24±1°C during a 16-h light photoperiod with a light intensity of 39.06 μEm-2 s-1 photosynthetically active radiation), (b) in vitro plants grown in the anechoic chamber with controlled environment without microwave irradiation, and (c) in vitro plants grown in the anechoic chamber with LPM irradiation for various times (5, 15, 30, 40 days). In comparison to control plants, significant differences in shoot multiplication and growth parameters (length of shoots and roots) were observed after 40 days of LPM exposure. An opposite effect was achieved regarding the content of total soluble proteins, which decreased with increasing exposure time to LPM. HPM irradiation was tested as a novel rewarming method following storage in liquid nitrogen. To our knowledge, this is the first report using this type of rewarming method. Although, shoot tips subjected to HPM exposure showed 28% recovery following cryostorage compared to 44% for shoot tips rewarmed in liquid medium at 22±1°C, we consider that the method represent a basis and can be further improved. The results lead to the overall conclusion that LPM had a stimulating effect on growth and multiplication of in vitro Sequoia plants, while the HPM used for rewarming of cryopreserved apices was not effective to achieve high rates of regrowth after liquid nitrogen exposure.

Recovery of bovine Babesia spp. after long-term cryostorage and comparison of bovine donor erythrocytes and serum

Cultured Babesia bovis and Babesia bigemina were recovered from liquid nitrogen storage nearly 30 years after they were cryopreserved. Four cattle were compared as donors of erythrocytes and serum for microaerophilous stationary phase (MASP) cultures for recovery of B. bigemina. Erythrocytes and serum from only one (#913) of the four animals supported growth of B. bigemina. Two B. bigemina (frozen in 1986 and 1987) and two B. bovis (both frozen in 1986) cryostocks were recovered from liquid nitrogen storage and all four recovered and thrived in #913 erythrocytes and serum. In the third passage after recovery, B. bovis cultures were cryopreserved. Six months later they were successfully recovered using #913 erythrocytes and serum. This study shows that B. bovis and B. bigemina stored nearly 30 years in liquid nitrogen can be successfully recovered in the MASP system. This study also confirms previous observations that selection of a suitable bovine donor of erythrocytes and serum is critical to the success of the culture.

Long-term cryopreservation of decellularised oesophagi for tissue engineering clinical application.

Oesophageal tissue engineering is a therapeutic alternative when oesophageal replacement is required. Decellularised scaffolds are ideal as they are derived from tissue-specific extracellular matrix and are non-immunogenic. However, appropriate preservation may significantly affect scaffold behaviour. Here we aim to prove that an effective method for short- and long-term preservation can be applied to tissue engineered products allowing their translation to clinical application. Rabbit oesophagi were decellularised using the detergent-enzymatic treatment (DET), a combination of deionised water, sodium deoxycholate and DNase-I. Samples were stored in phosphate-buffered saline solution at 4°C (4°C) or slow cooled in medium with 10% Me2SO at -1°C/min followed by storage in liquid nitrogen (SCM). Structural and functional analyses were performed prior to and after 2 and 4 weeks and 3 and 6 months of storage under each condition. Efficient decellularisation was achieved after 2 cycles of DET as determined with histology and DNA quantification, with preservation of the ECM. Only the SCM method, commonly used for cell storage, maintained the architecture and biomechanical properties of the scaffold up to 6 months. On the contrary, 4°C method was effective for short-term storage but led to a progressive distortion and degradation of the tissue architecture at the following time points. Efficient storage allows a timely use of decellularised oesophagi, essential for clinical translation. Here we describe that slow cooling with cryoprotectant solution in liquid nitrogen vapour leads to reliable long-term storage of decellularised oesophageal scaffolds for tissue engineering purposes.

Cryopreserved storage of clonal germplasm in the USDA National Plant Germplasm System

The US Department of Agriculture-Agricultural Research Service (USDA-ARS), National Plant Germplasm System (NPGS) plant collections are a critical source of genetic diversity for breeding and selection of improved crops, including vegetatively propagated plants. Information on these collections is readily accessible to breeders and researchers on the internet from the Germplasm Resources Information Network (GRIN). The clonal collections are at risk for loss due in part to their genetic diversity that makes growing them in one location a challenge, but also because it is difficult to have duplicate collections without incurring great expense. The development of cryopreservation techniques during the last two decades provides a low maintenance form of security backup for these collections. National plant collections for vegetatively propagated crop plants and their wild relatives are maintained by the USDA-ARS, NPGS at 15 sites across the country. These sites include various combinations of field, greenhouse, screenhouse, and in vitro collections. Cryopreserved backup collections in liquid nitrogen storage were instituted in the 1990s, increased greatly in the 2000s with the advent of new techniques, and are continuing today. Collections of dormant buds of temperate trees, shoot tips of in vitro cultures of many crops, and embryonic axes of some large seeded or recalcitrant seeded plants are all part of the clonal backup storage system.

Subculture, cryopreservation and recovery of osteoclasts

To explore the feasibility of passage, cryopreservation, and recovery of osteoclasts in order to develop new techniques facilitating osteoclast research. Passage of osteoclasts: adult male SD rat(SPF grade, weight of 250 g) was sacrificed and the abdominal aorta was exposed for blood draw. Monocytes isolated from peripheral circulation was treated with RANKL and M-CSF for 2 weeks. After formation of osteoclasts, they were trypsinized with pipetting, centrifuged, re-suspended with α-MEM containing RANKL and M-CSF, and cultured in 6 well-plates and 35 mm culture dishes. Freezing of osteoclasts: trypsinized osteoclasts were centrifuged and resuspended with DMSO, FBS, α-MEM (1:2:7), and were stored in liquid nitrogen(-196 °C). Recovery of osteoclasts: frozen osteoclasts were taken out of liquid nitrogen tank and thawed quickly at 37 °C in water bath. After wash with PBS, the cells were resuspended with α-MEM containing RANKL and M-CSF, and were cultured in 6 well dishes and 35 mm culture dishes. Meanwhile, cells were checked with inverted phase contrast microscope and observed in the live cell station for real time imaging. TRAP staining was performed 3 days after plating. Trypsinization together with pipetting and shaking can detach the adherent osteoclasts, and the resuspended cells can be used for passage and storage in liquid nitrogen. The passaged cells became fully attached to the culture dishes in 2 hours, and the multinucleated feature could be clearly seen. The osteoclasts recovered from liquid nitrogen could completely spread out for 2 to 3 hours so that the multinucleated cells were clearly seen. These cells were still TRAP positive. Although osteoclasts strongly adhere to the bottom of culture dishes, a large majority of the osteoclasts can be detached after appropriate digestion with trypsin, pipetting and shaking. These cells can be used for passage and cryopreservation. After recovering from liquid nitrogen, these cells still preserve the viability and the feature of osteoclasts. The results provide a new and powerful tool for future study of osteoclast biology.

Expansion and cryopreservation of porcine and human corneal endothelial cells

Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me2SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me2SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine.

Effects of long-term liquid nitrogen storage on the post-thaw motility of cryopreserved equine semen

Effects of long-term liquid nitrogen storage on the post-thaw motility of cryopreserved equine semen

Propagation, micropropagation and cryopreservation of Buxus hyrcana Pojark., an endangered ornamental shrub

Buxus hyrcana Pojark. is an important ornamental shrub, widely used for landscape and gardens. This ornamental shrub is a critically endangered species due to invasion of some pathogens. Rooting of this species is slow using propagation technique such as cutting. This study was carried out in three sections: 1) the effect of indole-3-butiyric acid (IBA) and ɑ-naphthalene acetic acid (NAA) on rooting of semi-hardwood cuttings in greenhouse condition; and 2) the effect of 6-benzylaminopurine (BAP), IBA and NAA on micropropagation in tissue culture condition and 3) cryopreservation of shoot tip by encapsulation-dehydration technique. For the first experiment, the basal region of the cutting was treated with various IBA and NAA solutions: 0 (free of IBA), 500, 1000, 2000 and 3000mgl−1. The 1000mgl−1 IBA along with 1000mgl−1 NAA induced the largest number of root (8.700/plantlet). For the second experiment, shoot tip dissected from 2-years-old pot-grown plants was cultured on Murashige and Skoog (MS) medium supplemented with 50 combinations of BAP, IBA and NAA at concentrations of 0.00, 0.50, 1.00, 1.50 and 2.00mgl−1. Here, we present a new protocol for sterilization of shoot tip. This shoot tip sterilization protocol minimized bacterial and fungal contamination. Multiple shoot formation (6.433/plantlet) was obtained through the use of 1.00mgl−1 BAP. Also, the optimum plant growth regulators (PGRs) combination for the largest number of root (7.633/plantlet) was 0.50mgl−1 BAP plus 1.00mgl−1 NAA. Plantlets were hardened-off and transferred to soil for further growth. About 95% of the acclimatized plantlets were established successfully. Regenerated plantlets were morphologically identical. For the third experiment, B. hyrcan shoot tip was cryopreserved by direct immersion in liquid nitrogen (LN) using encapsulation-dehydration method. After rapid rewarming, encapsulated and non-encapsulated shoot tips were cultured on MS medium enriched with 0.00, 0.50, 1.00, 1.50 and 2.00mgl−1 of BAP, IBA and NAA for rapid germination and growth. The highest percentage of re-growing the cryopreserved shoot tip was 60% for those grown on medium containing 0.50mgl−1 BAP along with 1.50mgl−1 NAA. No survival was observed for non-encapsulated shoot tip after storage in LN and through cultivation on regeneration medium.

Long term storage in liquid nitrogen leads to only minor phenotypic and gene expression changes in the mammary carcinoma model cell line BT474

Background/AimCancer cell lines are indispensible surrogate models in cancer research, as they can be used off-the-shelf, expanded to the desired extent, easily modified and exchanged between research groups for affirmation, reproduction or follow-up experiments.As malignant cells are prone to genomic instability, phenotypical changes may occur after certain passages in culture. Thus, cell lines have to be regularly authenticated to ensure data quality. In between experiments these cell lines are often stored in liquid nitrogen for extended time periods.Although freezing of cells is a necessary evil, little research is performed on how long-term storage affects cancer cell lines. Therefore, this study investigated the effects of a 28-year long liquid nitrogen storage period on BT474 cells with regard to phenotypical changes, differences in cell-surface receptor expression as well as cytokine and gene expressional variations.MethodsTwo batches of BT474 cells, one frozen in 1986, the other directly purchased from ATCC were investigated by light microscopy, cell growth analysis, flow cytometry and cytokine as well as whole-transcriptome expression profiling.ResultsThe cell lines were morphologically indifferent and showed similar growth rates and similar cell-surface receptor expression. Transcriptome analysis revealed significant differences in only 26 of 40,716 investigated RefSeq transcripts with 4 of them being up-regulated and 22 down-regulated.ConclusionThis study demonstrates that even after very long periods of storage in liquid nitrogen, cancer cell lines display only minimal changes in their gene expression profiles. However, also such minor changes should be carefully assessed before continuation of experiments, especially if phenotypic alterations can be additionally observed.

Effect of cryopreservation on germination of seeds and zygotic embryos of Calamus shendurunii an endemic rattan of Western Ghats

<p><em>Calamus shendurunii </em>is an endemic rattan of Western Ghats having restricted distribution and limited population. As a prerequisite to device an appropriate method for <em>ex situ </em>conservation of the species, desiccation and cryopreservation of seeds and zygotic embryo has been studied. Seeds extracted from ripened fruits possessed 35 per cent moisture content and exhibited 97 per cent germination. Desiccation to 28 per cent moisture content reduced the germination to 77 per cent. Desiccation below 14 per cent moisture content caused complete loss of seed germinability. Seeds stored under ambient conditions (28±2oC/60% RH) for more than seven days reduced germination to less than 40 per cent. Thus, conventional storage is not effective for their <em>ex situ </em>conservation. As an alternative method, excised zygotic embryos were subjected to desiccation and storage in liquid nitrogen. The embryos tolerated desiccation down to 5 per cent exhibiting 60 to 90 per cent germination upon culture into MS medium. Desiccated embryos subjected to liquid nitrogen exposure showed post freeze recovery and germination (80-90%) equal to that of desiccated control samples. Thus the study proved the extreme recalcitrance of <em>C. shendurunii </em>seeds and embryo cryopreservation as an alternative method of their <em>ex situ </em>conservation in gene banks.</p>

Allograft Heart Valves: Current Aspects and Future Applications.

Human heart valve allografts continue to represent almost perfect substitutes for heart valves. They have optimal hemodynamic characteristics and are highly resistant to infections. The first clinical use of allograft heart valves was as homovitals being transplanted after antibiotic incubation without any preservation. Since 1968, relatively standardized frozen cryopreservation (SFC) has been employed, including storage in vapor-phase liquid nitrogen. Disadvantages, particularly in pediatric patients, are limited availability due to organ scarcity, inability to grow, degeneration, immune response, and long-term failure. However, in contrast to alternative prosthetic or bioprosthetic heart valve replacements, they represent the best pediatric and juvenile replacement options for the pulmonary valve. Application of multiphoton imaging analysis for three-dimensional visualization of elastin and collagen by induction of autofluorescence without chemical fixation, embedding, and staining has revealed partial destruction of elastic and collagenous matrix in SFC valves. As the overall amount of collagen and elastin remains unchanged, the observed destruction is attributed to freezing-induced extracellular matrix damages due to ice crystal formation during SFC. The objective of this review is an assessment of current allograft preservation methods and the potential of novel preservation techniques to avoid ice formation with accompanied better long-term function.

Tools for the ex situ conservation of the threatened species, Cycladenia humilis var. jonesii.

Ex situ conservation is critical for hedging against the loss of plant diversity. For those species (exceptional species) that cannot be conserved long-term in standard seed banks, alternative methods are required, often involving in vitro culture and cryopreservation, or storage in liquid nitrogen. Cycladenia humilis var. jonesii is a federally threatened perennial native to Utah and Arizona. It is classified as an exceptional species, because it produces few seeds, and, thus, in vitro propagation and cryopreservation were investigated as tools for its propagation and preservation. Shoot-propagating cultures were established from both seedling and wild-collected shoots, but cultures from both sources displayed an extreme form of the physiological disorder, hyperhydricity. This phenotype could be at least partially normalized by the use of vented closures, as well as by using agar, rather than gellan gum, in the medium. The hyperhydric (HH) phenotype had a lower dry weight, more branching, minimal leaf development and more poorly developed vascular tissue than the more normal (MN) phenotype. Only more normalized shoots could be rooted and the resulting plants acclimatized. Both HH and MN shoots also provided shoot tips capable of surviving cryopreservation using the droplet vitrification method. These in vitro and cryopreservation methods provide tools that can be used for propagating plants of C. humilis var. jonesii for research and restoration, as well as for supplying shoot tips for the ex situ conservation of this species. The two distinct phenotypes also provide a useful system for studying factors involved in the HH response of this dryland species in vitro.

A new simple and reliable vitrification device based on Hollow Fiber Vitrification (HFV) method evaluated using IVP bovine embryos

A new vitrification device based on hollow fiber vitrification (HFV) was constructed using a glass capillary, which lead to simplified construction process and increased practicality of the device. The hollow fiber was attached to heat-pulled tip of the glass capillary using forceps. A protective sheath fitted on the capillary provided protection for the cellulose triacetate hollow fiber with loaded embryos and allowed safe storage in liquid nitrogen for long periods of time (2-12 month), transfer between tanks with liquid nitrogen and transportation within these tanks. No embryos were lost in the process. The device was tested using seven-dayold and eight-day-old IVP bovine blastocysts and expanded blastocysts as a model. Obtained survival (90% at 24 h post warming) and hatching rates (62% at 72 h post warming) of day 7 blastocysts and expanded blastocysts were comparable to those gained using various vitrification carriers. Vitrified embryos did not show an increase in the number of cells with damaged membrane or a decrease in total cell number per embryos in comparison to their non-vitrified counterparts. Day 7 and 8 expanded blastocysts did not differ significantly in terms of survival at 24 (97.01 vs. 97.50%) and 48 h post warming (95.52 vs. 95%), but showed significantly higher survival and hatching rates than day 7 and 8 blastocysts. These results indicated that high and repeatable survival rates can be obtained by selection of IVP bovine embryos at the developmental stage of expanded blastocyst for HFV. Further modification of the method may be required to achieve high and stable results with different developmental stages of IVP bovine embryo. The vitrification device presented in the current article may contribute to wider application of HFV method in livestock production.

Appendix D: Irvine Scientific® Vitrification System.

This chapter will describe the use of the Irvine Scientific® vitrification and warming solutions (Vit Kits®) along with a detailed protocol for the correct use of the CryoTip® vitrification device for human embryos and oocytes. Successful pregnancies have been reported after carrying out rapid vitrification methods of oocytes, cleavage stage embryos, and blastocysts using the CryoTip® (Popwell et al. Fertil Steril, 101:e20, 2014; Kuwayama et al. Reprod Biomed 11:608-614, 2005; Kuwayama et al. Fertil Steril 84:S187, 2005; Kuwayama et al. Vitrification of human embryos using the CryoTip TM method. Reprod Biomed 11:608-614). Compared to other vitrification carrier devices, the CryoTip® is considered a closed carrier for vitrification, thereby eliminating the theoretical risk of disease transmission through contaminated liquid nitrogen during cooling and storage (Bielanski et al. Cryobiology, 40:110-116, 2000). The CryoTip® is cleared by the FDA and has CE mark approval as a closed device to carry gametes or embryos in a specialized medium during cryopreservation procedures and subsequent long-term storage in liquid nitrogen. The CryoTip® has been shown to be a safe and reliable vitrification device, and when compared to other open system vitrification devices, it has provided similar results (Martino et al. Reprod Biol Endocrinol 11:27, 2013, Valbuena et al. Fertil Steril 97:209-217, 2012; Kuwayama et al. Reprod Biomed 11:608-614, 2005). The CryoTip® has also been shown to be suitable for use as a vitrification device for the cryopreservation of small volumes of sperm (Tanaka et al. Fertil Steril 90: S292, 2008).

Cryopreservation of Coffea arabica L. Zygotic Embryos by Vitrification

As a consequence of the difficulty in conventional coffee seed storage, biotechnological alternatives such as cryopreservation have been investigated. The objective of this study was to develop a protocol for the cryopreservation of Coffea arabica L. (cv. ‘Catuaí Vermelho’ - IAC 144) zygotic embryos by vitrification. For the cryopreservation study, the embryos were immersed in Plant Vitrification Solution 2 at different times (0, 10, 25, 50, 100, and 250 min) and two temperatures (0 and 25 °C). Subsequently, the best thawing time was determined in a water bath (1, 3, 5 minutes or directly in Recovery Solution). An anatomical study was conducted on non-stored and stored embryos, with or without the use of Plant Vitrification Solution 2. The immersion in cryoprotectant solution for 100 min at 0 °C allows embryo cryopreservation. Embryos can be directly thawed in Recovery Solution after storage in liquid nitrogen. It was observed that Plant Vitrification Solution 2 reduced internal water content in the cells, allowing subsequent embryo growth resumption.