Long-term storage in liquid nitrogen does not affect cell viability in cardiac valve allografts

We describe a method for the possible cryopreservation of embryogenic callus of Piceaabies and Pinustaeda at −196 °C and the regeneration of somatic embryos from thawed cells of subcultured embryonal–suspensor masses. Piceaabies and Pinustaeda were frozen without cryoprotective agent, in the presence of dimethyl sulfoxide (10%), or in a mixture of polyethylene glycol, glucose, and dimethylsulfoxide (10, 8, and 10% w/v, respectively). Cell masses placed in plastic vials or aluminum envelopes were frozen at 1 °C/min to −30 °C and then immersed for 10 min in liquid nitrogen. Cells were thawed rapidly and placed on modified MS subculture medium. Six to seven somatic embryos per gram of fresh weight were regenerated from each piece of frozen cell mass as compared with 12–13 embryos per gram from unfrozen cells. Post-thaw cell growth was inhibited initially by up to 5 weeks. Inhibition was reversed after the third 10-day subculture. Results suggest that the long-term storage of embryogenic cell lines in liquid nitrogen may be feasible for tree improvement programs in circumstances where testing of progeny may take several years.

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Microbial occurrence in liquid nitrogen storage tanks: a challenge for cryobanking?

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11531-4 ◽

2021 ◽

Author(s):

Felizitas Bajerski ◽

Manuela Nagel ◽

Joerg Overmann

Keyword(s):

Risk Assessment ◽

Quality Management ◽

Liquid Nitrogen ◽

Potential Risk ◽

Biological Materials ◽

Cross Contamination ◽

Storage Tanks ◽

Long Term Storage ◽

Term Storage

Abstract Modern biobanks maintain valuable living materials for medical diagnostics, reproduction medicine, and conservation purposes. To guarantee high quality during long-term storage and to avoid metabolic activities, cryostorage is often conducted in the N2 vapour phase or in liquid nitrogen (LN) at temperatures below − 150 °C. One potential risk of cryostorage is microbial cross contamination in the LN storage tanks. The current review summarises data on the occurrence of microorganisms that may compromise the safety and quality of biological materials during long-term storage. We assess the potential for the microbial contamination of LN in storage tanks holding different biological materials based on the detection by culture-based and molecular approaches. The samples themselves, the LN, the human microbiome, and the surrounding environment are possible routes of contamination and can cause cross contaminations via the LN phase. In general, the results showed that LN is typically not the source of major contaminations and only a few studies provided evidence for a risk of microbial cross contamination. So far, culture-based and culture-independent techniques detected only low amounts of microbial cells, indicating that cross contamination may occur at a very low frequency. To further minimise the potential risk of microbial cross contaminations, we recommend reducing the formation of ice crystals in cryotanks that can entrap environmental microorganisms and using sealed or second sample packing. A short survey demonstrated the awareness for microbial contaminations of storage containers among different culture collections. Although most participants consider the risk of cross contaminations in LN storage tanks as low, they prevent potential contaminations by using sealed devices and − 150 °C freezers. It is concluded that the overall risk for cross contaminations in biobanks is relatively low when following standard operating procedures (SOPs). We evaluated the potential sources in detail and summarised our results in a risk assessment spreadsheet which can be used for the quality management of biobanks. Key points • Identification of potential contaminants and their sources in LN storage tanks. • Recommendations to reduce this risk of LN storage tank contamination. • Development of a risk assessment spreadsheet to support quality management.

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Development of cryopreservation methods of seed of Nepeta cataria

Bulletin of the Karaganda University. “Biology, medicine, geography Series” ◽

10.31489/2021bmg2/37-42 ◽

2021 ◽

Vol 102 (2) ◽

pp. 37-42

Author(s):

Margarita Ishmuratova ◽

◽

Damirzhan Baigarayev ◽

Saltanat Tleukenova ◽

Elena Gavrilkova ◽

...

Keyword(s):

Liquid Nitrogen ◽

Room Temperature ◽

Seed Viability ◽

Positive Temperature ◽

Seed Rate ◽

Nepeta Cataria ◽

Long Term Storage ◽

Medical Plant ◽

Term Storage

This article presents the summarized data on cryopreservation of seeds of the medical plant Nepeta cataria. Cryopreservation is a highly promising method for saving of seed materials, allowing to organize long-term storage without viability loss. The purpose of present work is to optimize conditions of cryopreservation of seed materials of Nepeta cataria. Assessment of seed survival rate in the storage showed a linear decrease in seed viability and energy of germination. After 30 months of storage at the low positive temperature (+5 ºC) in paper pack seed rate decreased to 12.0 % and energy of germination to 11.2 %; after 4 years of storage seeds lost viability. During conduction of research the type of container, condition of thawing, optimal moisture of seeds and cryoprotectants are optimized. The optimal container for cryopreservation in liquid nitrogen was plastic cryo tubes; defrosting at room temperature. The best seed rate is found at moisture 3 %; the best cryoprotectant was glucose, the optimal concentration was 15 %. The result of the research is used for creation of the long-term storage medicinal cultures’ seed bank in the liquid nitrogen.

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Cryopreservation of Shoot Tips and Pollen of Potato

Plant Breeding and Seed Science ◽

10.1515/plass-2017-0025 ◽

2017 ◽

Vol 76 (1) ◽

pp. 75-80

Author(s):

Paulina Smyda-Dajmund

Keyword(s):

Liquid Nitrogen ◽

Shoot Tips ◽

Popular Method ◽

Long Term Storage ◽

Term Storage ◽

Direct Immersion

Abstract Cryopreservation is a frequently used method of long-term storage of potato meristems and pollen in liquid nitrogen (LN) in temperature of -196°C. This technique allows for theoretically unlimited storage of potato material. The most popular method of potato shoot tips preservation is cryopreservation by the solidification of liquids without crystallization (vitrification).The best method of pollen conservation is its direct immersion in LN. The successful regeneration after vitrification is genotype-dependent, which require optimization of protocol.

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

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab40 ◽

2020 ◽

Vol 32 (2) ◽

pp. 146

Author(s):

L. Palazzese ◽

D. A. Anzalone ◽

P. Toschi ◽

P. Loi

Keyword(s):

Liquid Nitrogen ◽

Rapid Freezing ◽

Cell Stage ◽

Food Industries ◽

Long Term Storage ◽

Freeze Dried ◽

Cheap Alternative ◽

Group 2 ◽

Term Storage

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.

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LONG-TERM STORAGE OF HAZELNUT EMBRYONIC AXES IN LIQUID NITROGEN

Acta Horticulturae ◽

10.17660/actahortic.2001.556.24 ◽

2001 ◽

pp. 177-180 ◽

Cited By ~ 1

Author(s):

Barbara Reed ◽

Kim Hummer

Keyword(s):

Liquid Nitrogen ◽

Embryonic Axes ◽

Long Term Storage ◽

Term Storage

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

Šumarski list ◽

10.31298/sl.143.7-8.3 ◽

2019 ◽

Vol 143 (7-8) ◽

pp. 323-324

Author(s):

Daniela Pilarska ◽

Andreas Linde ◽

Gernot Hoch ◽

Manana Kereselidze

Keyword(s):

Liquid Nitrogen ◽

Lymantria Dispar ◽

Spore Viability ◽

Microsporidian Species ◽

Long Term Storage ◽

Gipsy Moth ◽

Term Storage

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.

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