Long-term Storage In Liquid Nitrogen Research Articles

Lymphocyte Cultures and FISH Analysis after Long-term Storage of Chernobyl Cleanup Worker Samples

The aim was to establish optimal culture conditions for lymphocytes after long-term liquid nitrogen storage and to determine the frequency of translocations after fluorescence in situ hybridization (FISH) painting of samples from Estonian Chernobyl cleanup workers with average recorded doses of about 130 mSv. Lymphocyte samples from more than 2000 cleanup workers and controls have been stored at STUK in liquid nitrogen during 1995-1997. In the present study, 150 cell samples from exposed cleanup workers and population controls were thawed during 2004-2005 and cultured with routine methods. The most critical factors for successful cultures were: a) adequate freezing procedure of the samples, b) careful washing of cells to remove DMSO, c) cells kept in foetal serum after washing before cultures were set up, d) minimum cell density of 0.4 to 0.6 x 106 per ml in culture medium. More than 90% of the cultures were successful, i.e. the mitotic index and metaphase quality were adequate for FISH analysis. Chromosome aberrations from FISH painted slides were scored from 37 Estonian unexposed controls and 101 cleanup workers. One thousand metaphases were analysed per person. In preliminary analyses, the FISH translocation frequencies in the control group and the cleanup worker group were relatively similar. However, slightly higher translocation frequencies were observed among workers with recorded doses of more than 200 mSv. Results from regression analyses considering translocation yields with respect to age, exposure to ionizing radiation and smoking will be presented.

Microbial flora of spermatophores from black tiger shrimp(Penaeus monodon) declines over long-term cryostorage

Microbial contamination in sperm banks is of concern to sperm bank managers to prevent the perceived risk of disease transmission during cryostorage. The objective of this study was to examine the bacterial profile of cryopreserved spermatophores during long-term storage in liquid nitrogen in order to assess the risk of microbial flora during cryostorage. Spermatophores (N=216) collected from males (N=108) were equilibrated in calcium-free saline containing 5% dimethyl sulfoxide for 30 min and frozen at a cooling rate of −2 °C min−1 prior to storage in liquid nitrogen tanks. Cryopreserved spermatophores were sampled for bacterial profiles after 1 h and 30, 60, 90, 120, 150, 180 and 210 days using Plate count Agar, Thiosulphate Citrate Bile Salts Agar and Pseudomonas Isolation Agar. The incubation temperature was 37 °C for 24–48 h. Long-term storage of spermatophores in liquid nitrogen resulted in the reduction of the bacterial numbers. The number of total heterotrophic bacteria and total Vibrio decreased significantly (P<0.05) to 2.1±0.4×104 CFU/g (95.5% reduction) and 18.7±4.1×103 CFU/g (93.8% reduction), respectively, after 30 days in storage, while those of total Pseudomonas declined significantly (P<0.05) to 1.11±5.9×105 CFU/g (62.8% reduction) within 1 h of storage. Average sperm viability of cells from fresh spermatophores (96.4±1.8%) was not significantly different (P>0.05) from those of cryopreserved spermatophores held in liquid nitrogen for 1 h, 30 or 60 days (94.2±2.1, 89.8±3.5 and 86.7±3.9%, respectively, however it decreased significantly (P<0.05) after 90 days of storage. There were 23 bacterial species identified from fresh and cryopreserved spermatophores. The bacterial species least able to survive cold storage were Proteus mirabilis, Enterobacter spp., Vibrio spp., Kocuria varians, Bacillus cereus, B. amyloliquefaciens, B. circulans, Pseudomonas aeruginosa and Burkholderia cepacia because they were not detected from cryopreserved spermatophores after 90 or 120 days. The normal flora remaining at the end of the experiment (day 210) were Staphylococcus non aureus, Micrococcus spp., Proteus spp., Ochrobactrum anthropi, Corynebacterium spp., B. licheniformis and Pseudomonas spp. Cryopreservation of P. monodon spermatophores nearly eliminated the occurrence of pathogenic bacteria (Vibrio spp. and Ps. aeruginosa) culturable at 37 °C during long-term storage in liquid nitrogen.

Cryovial with partial membrane sealing can prevent liquid nitrogen penetration in submerged storage

Cryopreservation is one of the fundamental techniques in life science. To preserve the viability of cells and tissues, many researchers use plastic cryogenic vials and immerse them into liquid nitrogen for long-term storage. However, the non-sterile liquid nitrogen usually infiltrates into the vials and may cause a high rate of microbial contamination, and even some explosive incidents upon retrieval. To prevent these drawbacks while retaining the benefit of constant ultra-low temperature in submerged liquid nitrogen, we used a heat-sealable membrane to cover the upper portion of vials. After heat-sealing, the vials were completely free of liquid nitrogen penetration in the submerging test. Moreover, the sealing process did not affect the cell viability. This modified protocol provides an easy and efficient tool to ensure the integrity of biospecimens in long-term storage without interfering with existing cryobox storage systems.

Recovery of human sperm motility and ability to interact with the human zona pellucida after more than 28 years of storage in liquid nitrogen

Six donor semen samples were evaluated after 28 years cryopreservation in liquid nitrogen. The results showed that the samples retained good postthaw motility recovery and normal levels of binding to the human zona pellucida and that four of the five samples tested also gave normal levels of zona-induced acrosome reaction. In conclusion, human sperm can survive very long-term storage, which is pertinent information for clinicians referring boys and young men for sperm banking before chemotherapy.

A Universal Strategy for Proteomic Studies of SUMO and Other Ubiquitin-like Modifiers

Post-translational modification by the conjugation of small ubiquitin-like modifiers is an essential mechanism to affect protein function. Currently, only a limited number of substrates are known for most of these modifiers, thus limiting our knowledge of their role and relevance for cellular physiology. Here, we report the development of a universal strategy for proteomic studies of ubiquitin-like modifiers. This strategy involves the development of stable transfected cell lines expressing a double-tagged modifier under the control of a tightly negatively regulated promoter, the induction of the expression and conjugation of the tagged modifier to cellular proteins, the tandem affinity purification of the pool of proteins covalently modified by the tagged modifier, and the identification of the modified proteins by LC and MS. By applying this methodology to the proteomic analysis of SUMO-1 and SUMO-3, we determined that SUMO-1 and SUMO-3 are stable proteins exhibiting half-lives of over 20 h, demonstrated that sumoylation with both SUMO-1 and SUMO-3 is greatly stimulated by MG-132 and heat shock treatment, demonstrated the preferential usage of either SUMO-1 or SUMO-3 for some known SUMO substrates, and identified 122 putative SUMO substrates of which only 27 appeared to be modified by both SUMO-1 and SUMO-3. This limited overlapping in the subset of proteins modified by SUMO-1 and SUMO-3 supports that the SUMO paralogues are likely to be functionally distinct. Three of the novel putative SUMO substrates identified, namely the polypyrimidine tract-binding protein-associated splicing factor PSF, the structural microtubular component alpha-tubulin, and the GTP-binding nuclear protein Ran, were confirmed as authentic SUMO substrates. The application of this universal strategy to the identification of the pool of cellular substrates modified by other ubiquitin-like modifiers will dramatically increase our knowledge of the biological role of the different ubiquitin-like conjugations systems in the cell.

Cancers, environnements et modes de vie

Dr Riboli describes how, following his medical studies, he became interested in epidemiology, first in Milan, then in Boston, and since 1983 at the International Agency for Research on Cancer. He then describes how epidemiological studies have indicated that different dietary patterns, anthropometric characteristics and physical activity are related to the wide variations in cancer risk throughout the world. Western diet and lifestyle are generally associated with high incidence of cancers of the colorectum, breast, prostate and endometrium but with lower incidence of cancer of the stomach, oesophagus, liver and cervix uteri, whereas in most developing countries of Asia and Latin America the pattern of cancer incidence is almost the opposite. In 1992 Dr Riboli set up the European Prospective Investigation into Cancer and Nutrition (EPIC), a multi-centre prospective cohort study designed to investigate the relationship between nutrition and cancer, with the potential for studying other diseases as well. More than half a million study subjects have been recruited in 23 centres located in 10 European countries, to be followed up for cancer incidence and cause-specific mortality for several decades. Information was collected from the study participants through a non-dietary questionnaire on lifestyle variables and through a dietary questionnaire addressing usual diet. Anthropometric measurements were performed and blood samples taken, from which plasma, serum, red cells and leukocyte fractions were separated and aliquoted for long-term storage in liquid nitrogen, with a view to future genetic and biochemical studies.

Somatic embryo culture for propagation, artificial seed production, and conservation of sawara cypress (Chamaecyparis pisifera Sieb. et Zucc.)

Somatic embryogenesis in Chamaecyparis pisifera Sieb. et Zucc. was initiated from immature seeds collected from the end of June to early July. Mass propagation through adventitious shoot bud production from somatic embryo culture on Woody Plant (WP) medium and artificial seed production using sodium alginate was achieved. A high bud forming index value (25.8) was obtained on medium supplemented with 1μM 6-benzylaminopurine. The conversion rates from artificial seeds under aseptic and nonaseptic conditions were 60%–100% and 10%–12%, respectively. For germplasm conservation, somatic embryos and embryogenic cells were successfully stored at 4°C (medium-term storage) and in liquid nitrogen for long-term storage.

Function of cryopreserved horse semen is improved by optimized thawing rates

The use of cryopreserved semen for artificial insemination in equine breeding programs is increasing because of such benefits as reduction of injuries inherent in natural breeding. In the present study, semen straws from 1 ejaculate from each of 3 stallions predetermined to be poor freezers were thawed after long-term storage in liquid nitrogen (−196°C) at 3 different thaw rates (37°C for 30 seconds, 60°C for 8 seconds, 75°C for 7 seconds). Sperm samples were tested in triplicate for each stallion for motility in caffeine to detect all potentially motile sperm and sperm viability and capacitation status. The fast thaw rate significantly increased the percent of progressively motile sperm compared with the slow ( P = .0113) and moderate ( P = .0157) thaw rates. The fast thaw rate also improved sperm viability, as measured by the dual stain SYBR-14 and PI ( P < .05). Similar improvements were achieved with semen from 3 stallions with average postthaw semen quality. Finally, the chlortetracycline (CTC) stain found that the fast thaw rate reduced the number of sperm undergoing premature acrosome reactions ( P = .0329). In summary, thawing frozen equine sperm at 75°C for 7 seconds apparently caused less membrane damage on thawing resulting in enhanced postthaw motility and viability and reduced premature capacitation.

Survival of Chlorophyllous and Nonchlorophyllous Fern Spores through Exposure to Liquid Nitrogen

Survival of Chlorophyllous and Nonchlorophyllous Fern Spores through Exposure to Liquid Nitrogen

Skin substitutes from cultured cells and collagen-GAG polymers.

Engineering skin substitutes provides a potential source of advanced therapies for the treatment of acute and chronic wounds. Cultured skin substitutes (CSS) consisting of human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates have been designed and tested in preclinical and clinical studies. Cell culture techniques follow general principles of primary culture and cryopreservation in liquid nitrogen for long-term storage. Biopolymer substrates are fabricated from xenogeneic (bovine) collagen and glycosaminoglycan that are lyophilised for storage until use. At maturity in air-exposed culture, CSS develop an epidermal barrier that is not statistically different from native human skin, as measured by surface electrical capacitance. Preclinical studies in athymic mice show rapid healing, expression of cytokines and regulation of pigmentation. Clinical studies in burn patients demonstrate a qualitative outcome with autologous skin that is not different from 1:4 meshed, split-thickness autograft skin, and with a quantitative advantage over autograft skin in the ratio of healed skin to biopsy areas. Chronic wounds resulting from diabetes or venous stasis have been closed successfully with allogeneic CSS prepared from cryopreserved skin cells. These results define the therapeutic benefits of cultured skin substitutes prepared with skin cells from the patient or from cadaver donors. Future directions include genetic modification of transplanted cells to improve wound healing transiently or to deliver gene products systemically.

Recovery and Longevity of Cryopreserved Dormant Apple Buds

Clonally propagated crops, unlike seed-propagated crops, require intense and costly maintenance, generally in ex situ field gene banks. Consequently, large germplasm collections of tree species especially, are difficult to conserve in a well-replicated fashion and are vulnerable to damage from environmental stresses. Accordingly, long-term storage in liquid nitrogen presents a viable conservation alternative. To assess effectiveness of one approach to cryopreservation, dormant buds from 64 apple (Malus ×domestica Borkh. and other Malus spp.) accessions were collected and preserved in liquid nitrogen using a dormant-vegetative-bud method. Buds were retrieved from liquid nitrogen storage, rehydrated, and grafted onto rootstocks to determine survival. Mean recovery was 76% for 40 cold-hardy accessions, 66% for 20 moderately cold-hardy accessions, and 24% for four cold-tender accessions (range: 16% to 100%). Only four accessions had ≤25% recovery while 54 accessions had ≤50% recovery and 35 accessions had ≤75% recovery. No significant decline in recovery of these accessions by bud grafting occurred after 4 years of liquid nitrogen storage.

A new method of preservation of axenic amoebae of the genera Naegleria and Acanthamoeba

A new method of preservation of axenic strains of amoebae of the Naegleria and Acanthamoeba genera is described. The trophozoites are frozen in a solution with 2 cryoprotec-tors: fetal calf serum at 90% and dimethylsulphoxide at 10%. For 4 h and with a slow rate of cooling up to − 80 °C, one step is sufficient to reduce the temperature before long-term storage in liquid nitrogen. The ampules are quickly thawed at 40 °C and the medium is changed 4 h later. This technique gives confluent cells in 5 days and is quicker and more reliable than the methods previously described.

Successful long-term cryopreservation of highly purified canine islets.

This study evaluated the ability of cryopreservation to store large quantities of canine islets for transplantation studies. Islets were isolated by automated screen methods and purified by Euro-Ficoll gradients. After overnight 37 degrees C incubation, islets were equilibrated with 2 M dimethylsulfoxide, cryopreserved at a cooling rate of 0.25 degree C/min and subjected to long-term storage in liquid nitrogen. Six months later, some islets were thawed at a rate of 180 degrees C/min. The viability of cryothawed islets was determined in vitro by comparing islet count, total insulin content, morphology and perifusion studies in both control and cryothawed islets, and in vivo by transplantation into diabetic nude mice. The percentages of recovery of both islet count and total insulin content were 74.17 +/- 4.46 and 70.66 +/- 14.08, respectively. Islet morphology after cryothaw by light and electron microscopy revealed structurally intact islets with varying degrees of granulation. The results of perifusion indicated that there was no significant difference (p > 0.1) in both total stimulated insulin secretion and stimulation index response to high-dose glucose plus 3-isobutyl-1-methylxanthine and carbachol of isolated islets as compared to those of cryothawed islets. Transplantation into nude mice proved that grafted islets can successfully reverse diabetes. In conclusion, these findings indicate that the majority of purified canine islets can survive the frozen-thawed insult while maintaining their secretory function and permitting mass storage of canine islets for further transplant studies.

Smooth Muscle Function in Short- and Long-term Stored Cryopreserved Veins

There is considerable interest in banking of allograft veins for vascular recon struction procedures in patients for whom there are no suitable autologous or prosthetic vessels available. A major obstacle to vein banking has been posed by the lack of long-term storage studies and by a prior report indicating that veins deteriorated rapidly during liquid nitrogen storage. This study was, there fore, initiated to determine the effects of long-term liquid nitrogen storage on tissue viability. Viability was assessed by measurement of smooth muscle func tion in fresh, short-term (one to twenty-eight days), and long-term (&gt; two years) cryopreserved canine saphenous veins. There were no significant differences in the median effective doses of norepinephrine, serotonin, and KCL for genera tion of isometric force by vein rings from short- and long-term stored groups. Similarly, the maximum forces generated by long-term-stored cryopreserved veins in response to each reagent were the same as in short-term-stored veins. These results demonstrate that vein viability is maintained in cryopreserved banked veins.

Cryopreservation and long‐term liquid nitrogen storage of peripheral blood mononuclear cells for flow cytometry analysis effects on cell subset proportions and fluorescence intensity

The effect of cryopreservation and long-term liquid nitrogen storage on peripheral blood mononuclear cell (PBMC) subsets was prospectively analyzed using monoclonal antibodies and flow cytometry. Brief cryopreservation did not significantly alter the proportion of positively stained cells for CD3+, CD4+, CD8+, CD14+, CD16+, and CD19+ cells. A small but statistically significant increase in the proportion of positive cells was observed for HLA-DR+ and HLe-1+ cells. Brief cryopreservation was associated with a decrease in the mean fluorescence intensity (MFI) values for CD3+, CD4+, and CD8+ cells; an increase in MFI values for CD14+ and HLA-DR+ cells; and no change for CD16+, CD19+, and HLe-1+ cells. There was no significant change in the proportion of CD3+, CD4+, or CD16+ cells during 20 months of storage in liquid nitrogen. Small but statistically significant decreases in the proportion of CD8+ and CD19+ cells were observed over the same interval, and the proportion of CD14+ cells (monocytes) was highly variable. Chronologic changes in fluorescence intensity during long-term storage were observed for all cell subsets except CD16+ and CD19+ cells. Cryopreservation is a valuable technique for long-term storage of viable cells. For many laboratory applications, the small changes noted in the present study will have no practical importance. However, for clinical and epidemiological investigations encompassing large numbers of samples, statistical techniques to adjust for small changes during storage should be considered.

Long-term Storage in Liquid Nitrogen of an Embryogenic Cell Strain of Digitalis lanata

Summary Cells of the cardenolide-forming embryogenic strain VII of Digitalis lanata were stored in liquid nitrogen for about three years without loss of viability. The cells were pregrown in a nutrient solution containing 3% mannitol, then equilibrated with a mixture of 10% sucrose and 10% glycerol as cryoprotectors, slowly cooled (0.5 °C/min) till -100 °C in sealed glass ampoules, which were transferred to and stored in liquid nitrogen. For recultivation the ampoules were warmed rapidly in a water bath. The cells were grown for one week on a solidified medium and then cultivated as suspension. The cells used for cryopreservation and the recultivated cells showed the same growth rate, DNA content, rate of the glucosylation of added digitoxin and embryogenic capacity. The embryoid structures formed had the same ability for cardenolide biosynthesis and plant regeneration.

Vital-freezing of human muscle cultures for storage and reculture.

The vital-freezing of human muscle cultures enabling long-term storage in liquid nitrogen and future reculturing is described. Although human muscle is more fastidious in tissue culture than most cells of other origin, it was successfully frozen and re-established in culture. Cultures re-established after storage in liquid nitrogen differentiated and matured in the same way as primary human muscle cultures, and had the same histochemical and electron microscopic characteristics.

Survival of spermatozoa in a human sperm bank. Effects of long-term storage in liquid nitrogen

Survival of spermatozoa in a human sperm bank. Effects of long-term storage in liquid nitrogen

Viability of forty-two neoplasms after long-term storage in liquid nitrogen at −195 °C

Forty-two neoplasms stored at −195 °C for periods up to 6 years have remained transplantable and lethal to their inoculated hosts. A bioassay which estimates the surviving cell population based on the cell kinetics of exponentially growing neoplasms in vivo demonstrated an initial loss of viable cells due to the freezing procedures and no further losses due to storage. The response of 14 drug-induced resistant neoplasms to chemotherapeutic agents has remained unaltered after long-term storage.

53. The viability of cell cultures following long-term storage in liquid nitrogen: Arthur E. Greene, Balu Athreya, Herndon B. Lehr, and Lewis L. Coriell (South Jersey Medical Research Foundation, Camden, New Jersey, and Harrison Department of Surgical Research, University of Pennsylvania, Philadelphia, Pennsylvania)

53. The viability of cell cultures following long-term storage in liquid nitrogen: Arthur E. Greene, Balu Athreya, Herndon B. Lehr, and Lewis L. Coriell (South Jersey Medical Research Foundation, Camden, New Jersey, and Harrison Department of Surgical Research, University of Pennsylvania, Philadelphia, Pennsylvania)