Long-term Storage In Liquid Nitrogen Research Articles

Evaluating shoot-tip regrowth of 25 Rubus L species and hybrids after 15 to 20 years of cryopreserved storage

Rubus L. species are pan-global in their distribution and used as food throughout the world. Their fruits, collectively called brambles, come in a variety of colors from black, through various shades of red and yellow to white. The USDA-ARS, National Plant Germplasm System maintains >2250 accession seed- and screenhouse-based Rubus collection in Corvallis, OR and the USDA-ARS, National Laboratory for Genetic Resources Preservation in Fort Collins, CO has cryopreserved and maintains 200 accessions in liquid nitrogen (LN). We investigated regrowth of 25 selected Rubus accessions cryopreserved by droplet vitrification and stored cryogenically for 15 to 20 years, to determine the need for possible repropagation and reprocessing. The initial regrowth of the stored accessions after 24 h in LN ranged from 50 to 100%. After long-term LN storage, overall regrowth was significantly lower than the initial regrowth (on the average for the 25 accessions by 8.7%). However, except for one accession with recovery of 23% (R. laciniatus PI 553651), the regrowth of the other 24 accessions ranged from 40 to 100%, with 16 accessions at >73% regrowth. High initial viability was important for recovering large numbers of living shoots after long-term storage. Extending regrowth of five accessions from six to 12 wk did not significantly increase the regrowth, suggesting that a six-wk cultivation is sufficient for regrowth assessment. Knowledge of changes in viability over time in cryopreserved collections is vital information for curators to determine what repropagation intervals are needed for specific germplasm collections. These studies also provide large-scale information on changes that might be needed in the cryopreservation protocol for the next storage cycle.

Poxvirus sensitivity of a novel diploid sheep embryonic heart cell line.

Lumpy skin disease virus (LSDV), camelpox virus (CPV), and orf virus (ORFV) are members of the family Poxviridae. These viruses are usually isolated or produced in embryonated eggs or primary cells because continuous cell lines are less sensitive to infection. Disadvantages of the use of eggs or primary cells include limited availability, potential endogenous contaminants, and a limited ability to perform multiple passages. In this study, we developed a diploid cell culture from sheep embryonic hearts (EHs) and demonstrated its high proliferative and long-term storage capacities. In addition, we demonstrated its sensitivity to representatives of three genera of the family Poxviridae: Capripoxvirus (LSDV), Orthopoxvirus (CPV), and Parapoxvirus (ORFV). The cell culture had a doubling time of 24 h and reached 40 passages with satisfactory yield. This is comparable to that observed in primary lamb testis (LT) cells at passage 5 (P5). After infection, each poxvirus titer was 7.0-7.6 log TCID50/mL for up to five passages and approximately 6.8, 6.4, and 5.6 for the three viruses at P6-P25, P30, and P40, respectively. The sensitivity of sheep EH cells to poxvirus infection did not decrease after long-term storage in liquid nitrogen and was higher than that of primary LT cells, which are used for capripoxvirus and parapoxvirus detection and growth, and Vero cells, which are used for orthopoxvirus detection and growth. Thus, EH diploid cells are useful for poxvirus isolation and production without embryonated eggs or primary cells.

Evaluation of long-term cryostorage on sperm function and fertility of brown-marbled grouper (Epinephelus fuscoguttatus)

Cryopreservation of fish semen is a valuable technique for reproductive management in aquaculture. The aim of this study was to evaluate the effect of long-term storage in liquid nitrogen on post-thaw sperm functional parameters of brown-marbled grouper (Epinephelus fuscoguttatus). Samples were cryopreserved in a simple freezing medium (10 % DMSO + 0.3 M Glucose), sperm quality parameters were assessed at different storage periods (one month, one year, 2 years and 3 years). Our results indicated that cryostorage periods had a significant effect on the quality of frozen-thawed spermatozoa. Compared with fresh semen, cryopreserved sample was characterized by an overall decline in motility, viability, mitochondrial membrane potential (MMP), ATP content and increased level of lipid peroxidation with the storage time going on. For the analyzed quality parameters, there were no significant differences in storage time cryopreserved over one month. After 3 years of storage, frozen sperm still showed relatively high total motility, viability and MMP (all over 80 %), yielding acceptable fertilizing capacity close to that of fresh sperm. Therefore, it can be concluded that brown-marbled grouper sperm could be effectively cryopreserved for at least 3 years. The results of this study would be helpful to optimize gamete management in routine hatchery practices.

Establishment of an in vitro model from the vulnerable fish species Coregonus maraena (maraena whitefish): Optimization of growth conditions and characterization of the cell line.

In this study, a cell line of the fish species Coregonus maraena was produced for the first time. C.maraena is an endangered species, and studies indicate that this fish species will be affected by further population declines due to climate change. This cell line, designated CMAfin1, has been maintained in Leibovitz L-15 supplemented with 10% fetal bovine serum over 3 years. Both subculturing and storage (short-term storage at -80°C and long-term storage in liquid nitrogen) was successful. Cell morphology and growth rate were consistent from passage 10 onwards. Immunocytochemical examination of cellular proteins and matrix components confirmed the mechanical stability of the cells. Actin, fibronectin, vinculin, vimentin, and tubulin are present in the cells and form a network. In addition, the transport of molecules is ensured by the necessary proteins. Gene expression analysis showed a shift in the expressions of stem cell markers between younger and higher passages. While SOX2 and IGF1 were more highly expressed in the seventh passage, SOX9 and IGF2 expressions were significantly increased in higher passages. Therefore, the stable cell culture CMAfin1 can be used for applied analysis to further understand the cell physiology of C.maranea.

Detection and Isolation of Rickettsia tillamookensis (Rickettsiales: Rickettsiaceae) From Ixodes pacificus (Acari: Ixodidae) From Multiple Regions of California.

The western black-legged tick (Ixodes pacificus) is the most frequently identified human-biting tick species in the western United States and the principal vector of at least three recognized bacterial pathogens of humans. A potentially pathogenic Rickettsia species, first described in 1978 and recently characterized as a novel transitional group agent designated as Rickettsia tillamookensis, also exists among populations of I. pacificus, although the distribution and frequency of this agent are poorly known. We evaluated DNA extracts from 348 host-seeking I. pacificus nymphs collected from 9 locations in five California counties, and from 916 I. pacificus adults collected from 24 locations in 13 counties, by using a real-time PCR designed specifically to detect DNA of R. tillamookensis. DNA of R. tillamookensis was detected in 10 (2.9%) nymphs (95% CI: 1.6-5.2%) and 17 (1.9%) adults (95% CI: 1.2-3.0%) from 11 counties of northern California. Although site-specific infection rates varied greatly, frequencies of infection remained consistently low when aggregated by stage, sex, habitat type, or geographical region. Four novel isolates of R. tillamookensis were cultivated in Vero E6 cells from individual adult ticks collected from Alameda, Nevada, and Yolo counties. Four historical isolates, serotyped previously as 'Tillamook-like' strains over 40 yr ago, were revived from long-term storage in liquid nitrogen and confirmed subsequently by molecular methods as isolates of R. tillamookensis. The potential public health impact of R. tillamookensis requires further investigation.

Integration of Engineered "Spark-Cell" Spheroids for Optical Pacing of Cardiac Tissue.

Optogenetic methods for pacing of cardiac tissue can be realized by direct genetic modification of the cardiomyocytes to express light-sensitive actuators, such as channelrhodopsin-2, ChR2, or by introduction of light-sensitized non-myocytes that couple to the cardiac cells and yield responsiveness to optical pacing. In this study, we engineer three-dimensional “spark cells” spheroids, composed of ChR2-expressing human embryonic kidney cells (from 100 to 100,000 cells per spheroid), and characterize their morphology as function of cell density and time. These “spark-cell” spheroids are then deployed to demonstrate site-specific optical pacing of human stem-cell-derived cardiomyocytes (hiPSC-CMs) in 96-well format using non-localized light application and all-optical electrophysiology with voltage and calcium small-molecule dyes or genetically encoded sensors. We show that the spheroids can be handled using liquid pipetting and can confer optical responsiveness of cardiac tissue earlier than direct viral or liposomal genetic modification of the cardiomyocytes, with 24% providing reliable stimulation of the iPSC-CMs within 6 h and >80% within 24 h. Moreover, our data show that the spheroids can be frozen in liquid nitrogen for long-term storage and transportation, after which they can be deployed as a reagent on site for optical cardiac pacing. In all cases, optical stimulation was achieved at relatively low light levels (<0.15 mW/mm2) when 5 ms or longer pulses were used. Our results demonstrate a scalable, cost-effective method with a cryopreservable reagent to achieve contactless optical stimulation of cardiac cell constructs without genetically modifying the myocytes, that can be integrated in a robotics-amenable workflow for high-throughput drug testing.

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.

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.

Flow cytometric analysis of cell lineage and immune activation markers using minimal amounts of human whole blood—Field method for remote settings

Remote laboratory settings – such as those where studies on neglected tropical diseases are performed – often lack specialized equipment required for flow cytometric analysis of immune cell subsets, which complicates evaluations on a single cell level using peripheral blood. Our aim was to establish a method to use whole blood for phenotypic characterization of T-cells for specific markers including CD3, CD4, HLA-DR, CD38, CCR5, CD27, CD45RA, CD25, and FoxP3. This method uses 100 μL whole blood which is stained for extracellular markers, lysed, and cryopreserved at −20 °C at a field laboratory before transferring to liquid nitrogen for long-term storage and transportation. Cells can then be transported to a central laboratory for flow cytometry analysis. The method was initially established using samples from healthy donors; expression levels after cryopreservation were comparable to fresh whole blood samples from the same individuals. Moreover, data sets were also comparable to those which were stored in liquid nitrogen for up to one year. The method was then transferred to field studies in a remote area of Ghana which was used to observe its practicality and robustness in limited resource settings. Collectively, the low amount of whole blood (such as that taken from a finger prick), lack of any specialized equipment, and ease of use make this method suitable for utilization in remote field locations.

40 Comparison of slow and rapid freezing in freeze-dried ram spermatozoa

Semen lyophilisation is an interesting technique that might be a cheap alternative to long-term storage in liquid nitrogen. The first significant result of this method was achieved by Wakayama and Yanagimachi in the 1998 and demonstrated, for the first time, the birth of healthy mouse pups from epididymal freeze-dried (mouse) spermatozoa. The authors follow the lyophilisation technique, commonly used in the pharmaceutical and food industries, namely, deep freezing, which requires direct immersion of the semen sample into liquid nitrogen before vacuum drying. In this work, we focused on the freezing phase to improve and make the technique more reliable. We compared two protocols: 1) rapid freezing, where the semen is plunged directly into liquid nitrogen (LN group), and 2) slow freezing, where the sample is frozen with a freezing rate of 1°C min−1 until −50°C (SL group). Then, both frozen samples were lyophilized. Subsequently, after an interval ranging between 1 and 3 months, dry spermatozoa from LN and SL groups were used for intracytoplasmic sperm injection (ICSI), and the embryo development was evaluated at 24h (2-cell stage) and 7 days (expanded blastocyst) post-ICSI. Moreover, acrosome integrity was evaluated with Pisum sativum agglutinin (PSA) staining on part of the semen, immediately after freezing. The LN-group semen showed the acrosome completely melted, whereas the SL group showed better integrity of the acrosome, which was comparable to that of the normal frozen (vital) spermatozoa. At 24h post-ICSI the number of cleaved embryos in the SL group was higher than in the LN group (42/100 (42%) vs. 19/75 (25.3%), SL and LN, respectively; P=0.0253). The blastocyst rate 7 days after ICSI in the SL group was higher (7/100 (7%) than that in the LN group (2/75 (2.7%); P=0.0238). Our data show that lyophilisation can be conveniently achieved in ram spermatozoa without liquid nitrogen, thus simplifying the procedure. These data support the idea that lyophilisation might be a valuable and cheaper alternative to liquid nitrogen for long-term storage of ram semen.

Spore viability of microsporidian species isolated from gipsy moth larvae (Lymantria dispar) after long-term storage in liquid nitrogen

U radu se prikazuju rezultati preživljenja mikrosporidija izoliranih iz gubara (Lymantria dispar) nakon dugotrajne pohrane u tekućem dušiku. Infektivnost osam mikrosporidijskih L. dispar izolata testirano je na ličinkama gubara: Vairimorpha disparis, Nosema lymantriae, Nosema portugal, Nosema sp. (Poljska), Nosema sp. (Ebergassing), Nosema sp. (Njemačka), Nosema sp. (Schweinfurt) and Nosema sp. (Veslec). Preživljenje spora u tekućem dušiku detaljno je praćeno kod N. portugal i Nosema sp. (Ebergassing) koje su tako čuvane skoro 19 godina i aplicirane oralnom infekcijom i ponaosob na svaku pojedinu ličinku gubara. Ostalih 6 izolata aplicirano je površinskom kontaminacijom hranjivog supstrata, također oralnim infekcijskim putem. Od 8 mikrosporidijskih izolata apliciranih površinskom kontaminacijom samo kod 4 izolata, Vairimorpha disparis, Nosema lymantriae, Nosema sp. (Ebergassing) i Nosema sp. (Poljska), došlo je do uspješne infekcije ličinki gubara. Dok je kod svih testiranih ličinki (100%) uspješno realizirana infekcija sporama prve tri mikrosporidije, tek 21,1% tretiranih ličinki uspješno je inficirano sporama Nosema sp. (Poljska). Na kraju inkubacijskog razdoblja, kod svih ličinki inficiranih sa sva 4 izolata disekcijom je utvrđen visok stupanj zaraze; tkiva su bila ispunjena sporama. Mikroskopska pretraga ličinki L. dispar individualno tretiranih sporama Nosema portugal uz tri različite doze (100, 1000 i 10000 spora) pokazala je da su spore N. portugal izgubile infektivnost nakon 19 godina pohrane u tekućem dušiku; niti jedna od testiranih ličinka nije bila zaražena. Naprotiv, spore Nosema sp. (Ebergassing) zadržale su infektivnost nakon istog razdoblja pohrane u tekućem dušiku od 18,75 godina. Niti jedna od testiranih ličinki nije zaražena nakon oralnog unosa od 100 spora. Oralna aplikacija od 1000 spora po ličniki rezultirala je ukupnom uspješnom infekcijom 4,1% ličinki, a aplikacija od 10000 spora s 68,8% inficiranih ličinki. U svim uspješnim slučajevima uspješno zaraženih ličinki, na kraju inkubacijskog razdoblja infekcija je bila dobro razvijena, a masno tijelo prepuno spora. Istraživanje je potvrdilo da je pohrana mikrosporidija Nosema i Vairimorpha vrsta i domaćina iz reda leptira prikladna opcija za dugotrajno čuvanje izolata. Spore su preživjele i do 18 i pola godina, iako je tijekom eksperimenta zamijećen i značajan pad njihove infektivnosti. U pojedinim slučajevima spore su odumrle već nakon 7 godina pohrane u tekućem dušiku. Preporuka je da prilikom skladištenja i pohrane mikrosporidijskih izolata u tekućem dušiku svakih 5 godina repozitorij obnavlja svježim izolatima. Materijal koji se dulje vrijeme skladišti u tekućem dušiku nije prikladan i ne bi se smio koristiti u infektološkim testovima.

Changes of pollen viability of ornamental plants after long-term preservation in a cryopreservation pollen bank

This study determined the changes in pollen viability of 102 species/cultivars of ornamental plants (affiliated to 32 genera of 14 families) following long-term liquid nitrogen storage in a cryopreservation pollen bank. The goal was to provide information on the safety and stability of pollen cryopreservation technology. Fresh pollen at the time of storage was used as the control, and the study examined the pollen viability of ornamental plants cryopreserved for 8, 9, or 10 years. The results show that pollen of the 102 species/cultivars in the cryopreservation pollen bank retained viability ranging from 1% to 58%, After long-term storage there were changes in viability: 11.76% (12 species/cultivars) had increased viability, 16.67% (17 species/cultivars) had stable viability, and the viability of 71.57% (73 species/cultivars) showed a decreasing trend.

Implementation of Citrus shoot tip cryopreservation in the USDA-ARS National Plant Germplasm System

The USDA-ARS National Plant Germplasm System (NPGS) maintains a citrus genetic resource collection of 1522 accessions of Citrus cultivars and crop wild relatives. Of these, 540 are maintained as pathogen-free duplicate clones in a screenhouse at the National Clonal Germplasm Repository for Citrus and Dates (NCGRCD) in Riverside, California. An additional 815 accessions of citrus and citrus relatives are maintained in greenhouses or orchards in Riverside until sanitation for pathogens can be completed. This paper describes progress in backing up the 540 pathogen-tested accessions using cryopreservation techniques and methods to accelerate processing and viability testing. Vegetative budwood was harvested from trees in the screenhouse and sent to NLGRP. At least 170 shoot tips from each accession were excised from surface-sterilized budwood, and then cryopreserved using a PVS2 droplet-vitrification technique. Viability was assessed by micrografting 10 thawed shoot tips onto in vitro grown 'Carrizo' seedling rootstock. The remaining shoot tips are held in long-term liquid nitrogen storage. A total of 451 of the pathogen-tested citrus accessions were cryoprocessed: 354 accessions had regrowth levels of 40% or greater, 17 had 0% regrowth, 47 had viability levels between 10 and 30%, and 33 had endogenous contaminants. Technical staff familiar with shoot-tip cryoprocessing could be fully trained for specific applications of citrus in about 2 months and thereafter processed a single accession of at least 170 shoot tips in about 16 h. This large-scale effort has revealed that shoot tip cryopreservation can be successfully scaled-up to secure the NPGS Citrus collection.

Sperm cryopreservation protocols for the large-scale fertilization of Japanese eel using a combination of large-volume straws and low sperm dilution ratio

The aim of this study was to develop sperm cryopreservation protocols for large-scale fertilization in Japanese eel (Anguilla japonica), by testing the feasibility of combining large-volume straws and a low sperm dilution ratio. Experiments were conducted using both 0.25-ml straws and 2.5- and 5-ml straws (macrotubes) for sperm cryopreservation. Cooling rates in macrotubes were measured at three internal positions and at different heights above the surface of liquid nitrogen (LN). There was no significant difference in the cooling rate among internal positions in the straws. The cooling rate could be adjusted to the appropriate range for Japanese eel sperm (6.3 °C–8.6 °C min−1) by positioning the 2.5- and 5.0-ml straw 0.5–5.0 cm above the surface of LN. We succeeded in scaling up the storage volume from 0.25 to 4.5 ml (18-fold) without decreasing the motility using a 5.0-ml straw. The number of storable motile spermatozoa was the highest at a range of dilution ratios (sperm to extender, v/v) from 1:1 to 1:5. Although frozen-thawed sperm showed a significant decrease in the proportion of motile sperm to egg compared with than fresh sperm, there was no significant difference in the fertilization rate, and fertilized eggs with cryopreserved sperm did not affect the hatching rate or survival rate at 7 days after hatching. Using a segmented linear analysis, we found that 10,180 ± 3077 motile spermatozoa per egg are required to maximize fertilization success. We estimated that spermatozoa stored in a 2.5- and 5.0-ml straw can fertilize approximately 184,185 and 331,532 eggs, respectively. Furthermore, our investigation of the effect of long-term storage in LN on the sperm quality showed that the storage period does not affect the post-thaw motility rate even after 2 years of storage. Our results demonstrate that cryopreservation techniques for Japanese eel sperm using 2.5- and 5.0-ml straws could be used for large-scale fertilization programs.

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.

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

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).

Patient-Derived Tumor Xenograft Models of Breast Cancer.

The need for model systems that more accurately predict patient outcome has led to a renewed interest and a rapid development of orthotopic transplantation models designed to grow, expand, and study patient-derived human breast tumor tissue in mice. After implanting a human breast tumor piece into a mouse mammary fat pad and allowing the tumor to grow in vivo, the tumor tissue can be either harvested and immediately implanted into mice or can be stored as tissue pieces in liquid nitrogen for surgical implantation at a later time. Here, we describe the process of surgically implanting patient-derived breast tumor tissue into the mammary gland of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice and harvesting tumor tissue for long-term storage in liquid nitrogen.

Cord Blood Unit Cryopreservation: Positioning Segments for Potency Assessment

Introduction: Banked, unrelated umbilical cord blood units (CBU) are a rich source of hematopoietic stem cells for hematopoietic reconstitution. Potency assays on attached segments have been used for CBU release from the bank to the transplant center. CBUs have historically been manufactured and cryopreserved in bags with three segments (made from the tubing used to fill the bag) arranged across the top of the cassette. Prior to cryopreservation, the CBU is overwrapped, placed in a metal cassette and frozen in a control-rate freezer and submerged under liquid nitrogen for long-term storage. After cryopreservation, segments can be removed from the cassette, without disturbing the bag, for HLA typing, potency assays, or other testing. Last year, in an effort to increase the number of segments available for testing, we changed the configuration of the cryobag to create a fourth segment. To fit in the cassette, the segments were arranged with two segments across the top and two segments bent at 90 degrees to fit between the 20% and 80% portions of the bag. When performing our annual stability testing, we realized that the two distal segments, positioned between the 20% and 80% compartments yielded lower potency results than the segments positioned across the top of the cassette. We investigated this finding as described below.Method: Three large fresh CBUs were processed separately on a Sepax cell processor (Biosafe, Geneva, Switzerland) and evenly divided into two cryopreservation bags. For each CBU, one bag was created with three segments and the other bag with four segments. The cryopreservative, dimethylsulfoxide in dextran, was added at a final concentration of 10% to both bags prior to cryopreservation. Segments were positioned either across the top (3 segments) or in the bent configuration (4 segments) for each of the paired CBUs. All units were cryopreserved and stored in a Thermogenesis Bioarchive. After two weeks in storage, the CBUs were tested. The bags and segments were assayed for ALDHbr, viable CD34, CD45, glycophorin A and viability (7-AAD) along with colony forming units (CFUs), the potency assay used by our bank.Results: The segments configured across the top of the bag correlated with results from the cryobag for ALDHbr (as a percent of viable CD45+, Table 1), CD34+ (as a percent of viable CD45+) and CFU. In contrast, segments placed between the 20% and 80% compartments were significantly lower than the bag for both ALDHbr (as a percent of viable CD45+) and CFU, although CD34+ (as a percent of viable CD45+) was equivalent.Conclusions: Segments attached to CBUs can predict potency of the CBU by various parameters. However, proper placement of the segments within the cryopreservation cassette must be performed to ensure that potency measured on the segments reflects potency measured on the cord blood unit. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cryopreservation of gametophytic thalli of Ulva prolifera (Ulvales, Chlorophyta) from Korea

Cryopreservation is needed to ensure long-term storage without any genetic drift and contamination. To conserve Ulva species, gametophytic thalli of Ulva prolifera (Chlorophyta) were first cryopreserved at −196 °C using the two-step cooling method with cooling rate of 1 °C min−1 to −40 °C. Three cryoprotectants (dimethyl sulfoxide (DMSO), glycerol, and proline) at five concentrations (5, 10, 15, 20, and 25 %) and mixed cryoprotectants were assessed to determine the optimal solution. The mixture of cryoprotectants with the highest viability was 10 % glycerol in combination with 5 % DMSO and 5 % proline, with a viability of 89.2 %. The cryoprotectant treated singly with the highest viability was 20 % glycerol with a viability of 91.6 %. However, there was no statistically significant difference between 20 % glycerol and the mixed treatment. An evaluation of long-term storage in liquid nitrogen showed very high viability of 91.2–92.1 % with no statistically significant effect from storage time up to 120 days. The specimens developed normally in culture after cryopreservation for 120 days with the rate of gametogenesis reaching 95.7 % on the fourth day of culture. The released gametes developed normally into gametothalli. In conclusion, the use of glycerol as a cryoprotectant was very effective for cryopreservation of the gametothalli of U. prolifera. With 20 % glycerol, viability was very high as 91.6 %. The results suggest that gametothalli of U. prolifera can be cryopreserved completely by a treatment with 15–25 % glycerol singly as well as by a mixture of cryoprotectants with usual efficiency as a cryoprotectant.