An explanation for why it is difficult to form slush nitrogen from liquid nitrogen used previously for this purpose

Baker, Michael J.; Denton, Travis T.; Herr, C.

doi:10.1016/j.cryobiol.2012.10.007

Cited by 15 publications

(9 citation statements)

References 8 publications

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“…When tissue samples are frozen directly in liquid nitrogen, an insulating layer of nitrogen vapor forms around the sample that slows the freezing process (the Leidenfrost effect). 12 The Leidenfrost effect can be avoided by freezing tissue samples in an empty metal container that floats on the liquid nitrogen. This procedure also avoids the formation of large ice crystals in the tissue.…”

Section: Introductionmentioning

confidence: 99%

The Influence of Tissue Procurement Procedures on RNA Integrity, Gene Expression, and Morphology in Porcine and Human Liver Tissue

Kap

Sieuwerts

Kubista

et al. 2015

Biopreservation and Biobanking

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The advent of molecular characterization of tissues has brought an increasing emphasis on the quality of biospecimens, starting with the tissue procurement process. RNA levels are particularly affected by factors in the collection process, but the influence of different pre-analytical factors is not well understood. Here we present the influence of tissue specimen size, as well as the transport and freezing protocols, on RNA quality. Large, medium, and smaller porcine liver samples were stored either dry, on moist gauze, or in salt solution for various times, and then frozen in either liquid nitrogen or in pre-cooled isopentane. Large and small human liver samples were frozen in pre-cooled isopentane either immediately or after one hour at room temperature. The small samples were stored dry, on moist gauze, or in salt solution. RNA was isolated and RIN values were measured. The RNA for six standard reference genes from human liver was analyzed by RT-qPCR, and tissue morphology was assessed for artifacts of freezing. Experiments using porcine liver samples showed that RNA derived from smaller samples was more degraded after one hour of cold ischemia, and that cooled transport is preferable. Human liver samples showed significant RNA degradation after 1 h of cold ischemia, which was more pronounced in smaller samples. RNA integrity was not significantly influenced by the transport or freezing method, but changes in gene expression were observed in samples either transported on gauze or in salt solution. Based on observations in liver samples, smaller samples are more subject to gene expression variability introduced by post-excision sample handling than are larger samples. Small biopsies should be transported on ice and snap frozen as soon as possible after acquisition from the patient.

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Section: Introductionmentioning

confidence: 99%

The Influence of Tissue Procurement Procedures on RNA Integrity, Gene Expression, and Morphology in Porcine and Human Liver Tissue

Kap

Sieuwerts

Kubista

et al. 2015

Biopreservation and Biobanking

View full text Add to dashboard Cite

show abstract

“…However, enclosing specimens prevents direct contact between specimen and isopentane or liquid nitrogen vapor, which may have affected the speed of the freezing process. Freezing of samples in liquid nitrogen that are already placed in vials, are reported to be particularly adversely affected by the Leidenfrost effect (Baker et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

“…Freezing in liquid nitrogen is the easiest and fastest method for snap-freezing, but the freezing process itself is hampered by the Leidenfrost effect. This effect leads to the formation of a vapor layer around warm surfaces, which inhibits direct contact to liquid nitrogen or dry ice and therefore slows down the freezing process (Baker et al, 2013). As rapid freezing is considered to be crucial for the preservation of morphological and molecular features, most biobanking best practice guidelines and/or standard protocols recommend the use of a freezing medium for snap-freezing.…”

Section: Introductionmentioning

confidence: 99%

Example for process validation in biobanking: Fit for purpose testing of a cryopreservation method without isopentane

Wieser

Burger²,

Ertl³

et al. 2022

Front. Mol. Biosci.

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Background: The freezing process of tissue samples is crucial for the preservation of morphological and molecular features. Several biobanking guidelines describe freezing techniques for optimal outcomes. As the Vetbiobank standard freezing protocol does not comply with those recommendations in detail, a process validation was performed to demonstrate that samples are suitable for downstream applications. Here we give a formal example of a process validation in the biobanking setting, as required by the biobanking guideline ISO 20387 (2018).Methods: Three different freezing protocols, freezing in liquid nitrogen, freezing via isopentane precooled on dry ice and freezing via liquid nitrogen vapor, were assessed based on morphological integrity of mouse liver and muscle tissue samples. Samples were either frozen in cryotubes (without Optimal Cutting Temperature compound, OCT) or in cryomolds (with OCT). The protocol providing the best results was validated for reproducibility and robustness in terms of defined acceptance criteria for morphological evaluability, A260/A280 ratio, and RNA integrity number values (RIN). In addition, performance tests were run by gene expression analyzes of selected, tissue specific biomarkers to confirm that processed samples are fit for purpose.Results: From the three applied freezing protocols, freezing in liquid nitrogen generated best results. Reproducibility acceptance criteria were met for both, morphological integrity and RNA quality. The freezing method was robust for the tested tissue types and the application of OCT, with exception of liver tissue, where it led to a significant decrease of the RIN value. Gene expression analyzes showed good comparability of results regardless of the applied freezing method.Conclusion: Freezing of tissue samples in liquid nitrogen provides samples of adequate quality for subsequent RNA investigations. A negative impact of OCT on the RIN value of liver samples was observed, which was independent from the applied freezing protocol and showed no impact on subsequent gene expression analysis.

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“…Among the many plausible conditions to influence pregnancy rates after ART, the number of retrieved oocytes, the number of available embryos and the number of transferred TQEs seem to be the most important factors of reproductive prognosis. [16][17][18][19][20] However, there are few studies evaluating the relationship between pregnancy rate and live birth rate and the number and morphological quality of the embryos concomitantly. In women over 40 years, Opsahl et al 21 found that patients who had less than 4 embryos presented lower chances of pregnancy than those who formed !…”

Section: Discussionmentioning

confidence: 99%

Association between Number of Formed Embryos, Embryo Morphology and Clinical Pregnancy Rate after Intracytoplasmic Sperm Injection

Luz

Giorgi

Neto

et al. 2016

Rev Bras Ginecol Obstet

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Introduction Infertility has a high prevalence in the general population, affecting $ 5 to 15% of couples in reproductive age. The assisted reproduction techniques (ART) include in vitro manipulation of gametes and embryos and are an important treatment indicated to these couples. It is well accepted that the implantation rate is positively influenced by the morphology of transferred embryos. However, we question if, apart from the assessment of embryo morphology, the number of produced embryos per cycle is also related to pregnancy rates in the first fresh transfer cycle. Purpose To evaluate the clinical pregnancy rate according to the number of formed embryos and the transfer of top quality embryos (TQEs). Methods In a retrospective cohort study, between January 2011 and December 2012, we evaluated women who underwent intracytoplasmic sperm injection (ICSI), aged < 40 years, and with at least 1 formed embryo fresh transferred in cleavage stage. These women were stratified into 3 groups according to the number of formed embryos (1 embryo, 2-3 and ! 4 embryos). Each group was divided into 2 subgroups according to the presence or not of at least 1 transferred TQE (1 with TQE; 1 without TQE; 2-3 with TQE, 2-3 without TQE; ! 4 with TQE; ! 4 without TQE). The clinical pregnancy rates were compared in each subgroup based on the presence or absence of at least one transferred TQE. Results During the study period, 636 women had at least one embryo to be transferred in the first fresh cycle (17.8% had 1 formed embryo [32.7% with TQE versus 67.3% without TQE], 42.1% of women had 2-3 formed embryos [55.6% with TQE versus 44.4% without TQE], and 40.1% of patients had ! 4 formed embryos [73.7% with TQE versus 26.3% without TQE]). The clinical pregnancy rate was significantly higher in the subgroup with ! 4 formed embryos with at least 1 transfered TQE (45.2%) compared with the subgroup without TQE (28.4%).

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An explanation for why it is difficult to form slush nitrogen from liquid nitrogen used previously for this purpose

Cited by 15 publications

References 8 publications

The Influence of Tissue Procurement Procedures on RNA Integrity, Gene Expression, and Morphology in Porcine and Human Liver Tissue

The Influence of Tissue Procurement Procedures on RNA Integrity, Gene Expression, and Morphology in Porcine and Human Liver Tissue

Example for process validation in biobanking: Fit for purpose testing of a cryopreservation method without isopentane

Association between Number of Formed Embryos, Embryo Morphology and Clinical Pregnancy Rate after Intracytoplasmic Sperm Injection

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