Vapor Phase Of Liquid Nitrogen Research Articles

In Vivo Potency of Hematopoietic Stem Cells after Long-Term Storage at -80C in DMSO+HES Cryprotectant Mixture: Comparing Engraftment Kinetics and Efficacy Data in First and Second Autologous Stem Cell Transplantation in Myeloma

Background: The use of a cryoprotectant combining both intracellular and extracellular protectants (DMSO+HES) permits large volume hematopoietic stem cell (HSC) cryopreservation in mechanical freezers at -80C without the development of clumping after thawing (Stiff et al, Blood 1983. PMID: 6187518) and was recently utilized for allograft cryopreservation during the COVID-19 pandemic. However, there are competing methods for cryopreservation. Many centers are utilizing vapor phase liquid nitrogen (VPLN) storage protocols with DMSO alone instead. While HSC stored with DMSO alone in VPLN have shown to remain viable after long-term storage (Makhani et al, TCT 2022. PMID:3524877), this has not yet been proven for DMSO+HES stored HSCs at -80C. Here we report on second autologous hematopoietic stem cell transplants (AHSCT2) for relapsed multiple myeloma (RRMM) performed > 2 years after cell storage began at - 80C to determine if there were differences between engraftment and outcomes between initial and long-term storage in DMSO+HES at -80C. Methods: We searched our own institution's records from 1990-2023 to find patients who received two separate AHSCT for MM after only one cell collection prior to first AHSCT (AHSCT1). Prior allogeneic transplants patients were excluded. The primary outcome was neutrophil and platelet engraftment times. Secondary outcomes were progression free survival (PFS) and overall survival (OS). We reviewed patient characteristics, disease state prior to both transplants, engraftment kinetics for both, and survival outcomes. Outcomes were analyzed by Kaplan Meier (KM) and longrank test where applicable, and median PFS (mPFS) and median OS (mOS) were determined by reverse KM. Differences in engraftment were compared using the Kruskal-Wallis test. Results: 35 patients met inclusion criteria, of whom 57% of patients were male. Median age at AHSCT1 was 56.3 years and at AHSCT2 was 60.1 years. Median follow up was 8 years from second transplant and 12.3 years from stem cell collection. Median time from stem cell collection to AHSCT1 was 11 days (range 3-39 days) and to AHSCT2 was 4.6 years (range 2.2-15.8 years). Prior to AHSCT1, 65% of patients were in PR, 20% in VGPR, and 14% in CR. Prior to AHSCT2, 44% were in PR, 38% in VGPR, and 18% in CR. 80% and 83% of patients were conditioned with melphalan alone in AHSCT1 and 2 respectively. Median CD34+ doses were 5.1x10^6/kg and 4.2x10^6/kg (p=0.028) for AHSCT1 and 2 respectively. There was no statistical difference between the engraftment kinetics of the first or second transplant with a median ANC engraftment time of 11 days for both groups (p=0.88), and median platelet engraftment of 17.0 and 17.5 days (p=0.55) for AHSCT1 and AHSCT2 respectively. Between categories of cells stored between 2-5 years and >5 years, there was no statistical difference in ANC (p=0.85) or platelet engraftment (p=0.29). mPFS from AHSCT1 for the overall study was 3.7 years and for AHSCT2 was 3.6 years. The mOS was 4.8 years from AHSCT2. There was a statistical difference in 2-year PFS between ASHCT1 and 2 at 86% and 62% (p=0.0001) respectively. The mPFS differed by disease state prior to AHSCT2 dramatically; those in CR had mPFS of 7.8 years, those in VGPR 4.3 years, and those in PR 1.3 years. Discussion: Our results suggest that HSC stored long-term at -80°C using the DMSO + HES combination does not adversely affect product quality even for cells stored up to 15.8 years. Furthermore, our method is simpler, more cost-effective and poses no risk to the working environment when compared to current freezing method where VPLN storage is mainly used. This is particularly important to expand access to cryopreserved HSC for centers and countries where resources are more limited. These data also add evidence to the efficacy of AHSCT2 as we saw a 2-year PFS of 63% for these patients, which compares favorably to 50% 1-year PFS in AHSCT2 noted in the large CIBMTR retrospective by Dhakal (Leukemia 2021, PMID 32747684). In addition, some patients benefit greatly, best seen in those achieving a CR prior to AHSCT2 who here had a mPFS of 7.8 years. This is an important selection criteria we now evaluate when considering AHSCT2 in RRMM. AHSCT2 is cost effective and compared to recently approved CAR-T and bispecifics is likely a more economical approach in select patients relapsing after long remission following AHSCT1 who achieve deep responses to salvage therapy.

Cord Blood–Derived Endothelial Progenitor Cells Promote In Vivo Regeneration of Human Hematopoietic Bone Marrow

Victims of acute radiation exposure are susceptible to hematopoietic toxicity due to bone marrow damage and loss of mature blood elements. Here, we evaluated cord blood-derived endothelial progenitor cells (CB-EPCs) as a potential cellular therapy for mitigation of hematologic acute radiation syndrome. CB-EPCs express endothelial cell markers and maintain their growth characteristics beyond 10+ passages without diminishing their doubling capacity. Further, CB-EPCs can be cryopreserved in vapor-phase liquid nitrogen and easily recovered for propagation, making them an attractive nonimmunogenic cellular therapy for off-the-shelf use. Importantly, we show CB-EPCs have the capacity to potently expand adult human bone marrow hematopoietic progenitor cells both in vitro and in vivo. To demonstrate the role of CB-EPCs in promoting in vivo human immune reconstitution after irradiation, we employed a novel humanized mouse model established by transplant of CD34+ bone marrow cells from 9 unique adult organ donors into immunocompromised NSG-SGM3 mice. The response of the humanized immune system to ionizing irradiation was then tested by exposure to 1 Gy followed by subcutaneous treatment of CB-EPCs, Food and Drug Administration-approved growth factor pegfilgrastim (0.3 mg/kg), or saline. At day 7, total human bone marrow was decreased by 80% in irradiated controls. However, treatment with either growth factor pegfilgrastim or CB-EPCs increased recovery of total human bone marrow by 2.5-fold compared with saline. Notably, CB-EPCs also increased recovery of both human CD34+ progenitors by 5-fold and colony-forming capacity by 3-fold versus saline. Additionally, CB-EPCs promoted recovery of endogenous bone marrow endothelial cells as observed by both increased vessel area and length compared with saline. These findings indicate the feasibility of using humanized mice engrafted with adult bone marrow for radiation research and the development of CB-EPCs as an off-the-shelf cellular therapy for mitigation of hematologic acute radiation syndrome.

Streamlined Methods for Processing and Cryopreservation of Cell Therapy Products Using Automated Systems.

Streamlined procedures for processing and cryopreservation of cell therapies using good laboratory practices are integral to biomanufacturing process development and clinical applications. The protocol herein begins with the preparation of human cell types cultured as adherent (i.e., mesenchymal stromal cells, MSCs) or suspension cells (i.e., peripheral blood mononuclear cells, PBMCs) to comprehensively demonstrate procedures that are applicable to commonly used primary cell cultures. Cell processing steps consist of preparing high yields of cells for cryopreservation using instruments routinely used in cell manufacturing, including the Finia® Fill and Finish System and a controlled-rate freezer. The final steps comprise the storage of cells at subzero temperatures in liquid nitrogen vapor phase followed by the analysis of cell phenotypes before and after processing and cryopreservation, along with cell quality metrics for validation. Additionally, the protocol includes important considerations for the implementation of quality control measures for equipment operation and cell handling, as well as Good Laboratory Practices for cell manufacturing, which are essential for the translational use of cell therapies. Key features • The protocol applies to small- or large-scale manufacturing of cell therapy products. • It includes streamlined procedures for processing and cryopreservation of cells cultured as adherent cells (MSCs) and suspension cells (PBMCs). • Provides temperature control and rapid partitioning of sample in cryopreservation solution to maintain high viability of a range of cell types throughout the procedures. • This protocol employs the Finia® Fill and Finish System and a controlled-rate freezer. Graphical overview.

Field performance of cryopreserved seed-derived tomato plants and post-thaw survival of viral-infected meristems

The effectiveness of different cryopreservation techniques of tomato meristems isolated from viral-infected plants ‘Irishka’ cultivar was determined. The pieces of stem were protected with dimethyl sulfoxide and propylene glycol and cooled in vapour phase of liquid nitrogen (–170 °C). For the vitrification and droplet-vitrification protocols, the meristems were treated with loading solution and dehydrated with different plant vitrification solutions (PVS1 modified, PVS2, 88 % PVS3, PVSN). The samples were placed to sterilized aluminum foil pieces, in 1.2 ml cryovials or in 50 µl aluminum pans for differential scanning calorimetry and were directly immersed into liquid nitrogen. Acсording to the dehydration technique, the meristems were dehydrated with sterile airflow for 120 min. The post-thaw survival rates of meristems (from 34.2 to 78.5 %) were observerved only for 50 µl aluminum pans and airflow dehydration. We determined the productivity of plants, obtaned from cryopreserved seeds (‘Seven’, ‘Potiron Ecarlate’ and ‘Druzhba’ cultivars). We observed increasing in total and marketable yields for the plants grown from the cryopreserved seeds for all the cultivars. Total number of diseased plants decreased by 33 % for ‘Seven’, for ‘Potiron Ecarlate’ it did by 6.7 %, for that of ‘Druzhba’ the total percentage of sick and healthy plants did not differ after seeds cryopreservation.

Cellular Therapy Product Viability during Long Term Cryopreserved Storage

Introduction Cryopreservation of human cellular therapy products (CTPs) is integral for modern blood and marrow transplant (BMT) programs. However, there is limited data available regarding CTP stability during long-term storage, particularly beyond ten years. Our policy assigns a 20-year expiration to cryopreserved HPC(A) and HPC(M) products. To support the 20-year expiration period, a validation program was developed and assessed. Methods Cryopreserved samples and products stored in vapor phase liquid nitrogen tanks were tested using two sample groups. HPC(A) or HPC(M) specimens from deceased patient samples stored over 15 years were the initial group tested. A second group was added from an ongoing Prospective Stability Program (PSP). Post-thaw viability testing was performed by flow cytometry per our validated procedure based on the ISHAGE gating strategy, producing a percent viability for CD34+ cells (CD34v) and CD3+ cells (CD3v). Acceptance criteria was defined as ≥ 75% viability for both cell populations. Simple linear regression was used to predict the 75% threshold. After initial analysis, data from the PSP was included for an expanded analysis. This provided an additional 14 CD34v and 13 CD3v results, ranging in age from < 1 week (e.g., soon after cryopreservation) to 8 years; all PSP results are from cryovial specimens. Results Twenty-six collections were identified for testing, collected 15 to 31 years prior. The 25 HPC(A) specimens ranged in age from 15 to 26 years. The single HPC(M) collection was 31 years old (yo). Initially, 11 cryovials, 12 sample bags and 3 product bags from 26 different patients were tested. All but two specimens <20yo were cryovials; one product and one sample bag were also <20yo. All but three specimens >20yo were sample bags; the remainder were products. A second product from the HPC(M) collection was tested four months after the initial test for an additional HPC(M) data point. All specimens < 20yo and both HPC(M) specimens met the acceptance criteria. Of the 13 HPC(A) over 20yo, nine met the 75% CD34v acceptance criteria (range = 66 - 95%) and four met the 75% CD3v acceptance criteria (range = 42 - 80%). Notably, two HPC(A) only days over 20yo failed to meet CD3v, with results of 52% and 74%; CD34v was 79% and 90%, respectively. However, several 21-22yo met both acceptance criteria. Both HPC(M) results of the 31yo product met acceptance criteria; CD34v mean ± SD = 95% ± 0.4 and CD3v mean ± SD = 86% ± 3.9. Linear regression analysis of the initial HPC(A) dataset predicts CD34v would reach 75% after more than 30 years and CD3v would reach 75% after 21 years. Similar evaluation of the expanded dataset (which includes stability program samples up to 8 years old as well as both results from the retested HPC(A) and the HPC(M)) predicts reaching 75% CD34v at almost 48 years and CD3v at about 24 years. The accompanying CD34v and CD3v graphs show the expanded dataset and include 95% confidence interval bands for the linear regression analysis. Conclusions CD34+ cells appear to maintain viability well during cryopreserved storage. CD3+ cell viability is maintained adequately but less robustly compared to CD34+ cells and may begin to decline more dramatically after 20 years of cryopreserved storage. The linear regression analysis supports our current 20-year expiration date. We plan to expand the dataset as the PSP specimens continue to age. We are evaluating viable cell recovery to ensure viability is not artificially high due to cell loss. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Multivariate Evaluation of DNA Quality Differences in Different Preanalytical Procedures in Mouse Livers.

Successful histogenetic research relies on proper handling of tissue samples to maximize DNA quality. As the largest gland in the body, the liver is particularly sensitive to sample mishandling owing to its enzymatic and transcriptional activity. However, the impact of preanalytical procedures on the quality of extracted liver DNA remains poorly understood. In this study, we assessed the impact of extraction methods, duration of ex vivo liver ischemia, liver storage time, and temperature on extracted DNA quality. Comprehensive parameters such as DNA yields, purity, DNA integrity number, the percentage of double-stranded DNA (%dsDNA), and PCR amplification of the GAPDH gene fragment were assessed to identify the quality of extracted DNA. Our results revealed that these preanalytical processes had little effect on DIN values and PCR efficiency of GAPDH gene fragments for each sample, whereas the DNA yields, purity, and %dsDNAs varied widely across different processes. For liver DNA extraction, RNase is necessary to isolate "pure" DNA, and the presence of RNase could significantly increase the %dsDNA. In addition, significant increases in the yields, purity, and %dsDNA of extracted DNA were observed in the TissueLyser-processed livers compared with the mortar and pestle or shear cell disruption methods. Further investigation revealed that livers experiencing longer periods of ex vivo ischemia resulted in significantly compromised DNA yields, and to obtain sufficient DNA, the ex vivo liver ischemia should be limited to within 30 minutes. Moreover, compared with storage of livers at -80°C, storage of livers in the vapor phase of liquid nitrogen yielded a higher quality of the extracted DNA. Our findings exhibited significant implications for liver-derived DNA quality assessment and management.

Single-cell RNA-seq of out-of-thaw mesenchymal stromal cells shows tissue-of-origin differences and inter-donor cell-cycle variations

BackgroundHuman Mesenchymal stromal cells (hMSCs) from various tissue sources are widely investigated in clinical trials. These MSCs are often administered to patients immediately after thawing the cryopreserved product (out-of-thaw), yet little is known about the single-cell transcriptomic landscape and tissue-specific differences of out-of-thaw human MSCs.Methods13 hMSC samples derived from 10 “healthy” donors were used to assess donor variability and tissue-of-origin differences in single-cell gene expression profiles. hMSCs derived and expanded from the bone marrow (BM) or cord tissue (CT) underwent controlled-rate freezing for 24 h. Cells were then transferred to the vapor phase of liquid nitrogen for cryopreservation. hMSCs cryopreserved for at least one week, were characterized immediately after thawing using a droplet-based single-cell RNA sequencing method. Data analysis was performed with SC3 and SEURAT pipelines followed by gene ontology analysis.ResultsscRNA-seq analysis of the hMSCs revealed two major clusters of donor profiles, which differ in immune-signaling, cell surface properties, abundance of cell-cycle related transcripts, and metabolic pathways of interest. Within-sample transcriptomic heterogeneity is low. We identified numerous differentially expressed genes (DEGs) that are associated with various cellular functions, such as cytokine signaling, cell proliferation, cell adhesion, cholesterol/steroid biosynthesis, and regulation of apoptosis. Gene-set enrichment analyses indicated different functional pathways in BM vs. CT hMSCs. In addition, MSC-batches showed significant variations in cell cycle status, suggesting different proliferative vs. immunomodulatory potential. Several potential transcript-markers for tissue source differences were identified for further investigation in future studies. In functional assays, both BM and CT MSCs suppressed macrophage TNFα secretion upon interferon stimulation. However, differences between donors, tissue-of-origin, and cell cycle are evident in both TNF suppression and cytokine secretion.ConclusionsThis study shows that donor differences in hMSC transcriptome are minor relative to the intrinsic differences in tissue-of-origin. hMSCs with different transcriptomic profiles showed potential differences in functional characteristics. These findings contribute to our understanding of tissue origin-based differences in out-of-thaw therapeutic hMSC products and assist in the identification of cells with immune-regulatory or survival potential from a heterogeneous MSC population. Our results form the basis of future studies in correlating single-cell transcriptomic markers with immunomodulatory functions.

Cell Viability Assessment Using Fluorescence Vital Dyes and Confocal Microscopy in Evaluating Freezing and Thawing Protocols Used in Cryopreservation of Allogeneic Venous Grafts.

The authors present their contribution to the improvement of methods suitable for the detection of the freezing and thawing damage of cells of cryopreserved venous grafts used for lower limb revascularization procedures. They studied the post-thaw viability of cells of the wall of cryopreserved venous grafts (CVG) immediately after thawing and after 24 and 48 h culture at +37 °C in two groups of six CVG selected randomly for slow thawing in the refrigerator and rapid thawing in a water bath at +37 °C. The grafts were collected from multi-organ and tissue brain-dead donors, cryopreserved, and stored in a liquid nitrogen vapor phase for five years. The viability was assessed from tissue slices obtained by perpendicular and longitudinal cuts of the thawed graft samples using in situ staining with fluorescence vital dyes. The mean and median immediate post-thaw viability values above 70% were found in using both thawing protocols and both types of cutting. The statistically significant decline in viability after the 48-h culture was observed only when using the slow thawing protocol and perpendicular cutting. The possible explanation might be the “solution effect damage” during slow thawing, which caused a gentle reduction in the graft cellularity. The possible influence of this phenomenon on the immunogenicity of CVG should be the subject of further investigations.

Effects of Warm Ischemia Time, Cryopreservation, and Grinding Methods on RNA Quality of Mouse Kidney Tissues.

Background: High-quality RNA extraction from tissue samples is of key importance for scientific research and translational medicine. Tissue collection and preparation may affect RNA quality. In this study, we investigated effects of warm ischemia time, cryopreservation, and grinding methods on RNA quality. Methods: Total RNA was extracted from mouse kidney tissues with warm ischemia times of 0, 30, 60, 90, and 120 minutes. Half of the tissues were used to extract RNA immediately, while the others were cryopreserved in the vapor phase of liquid nitrogen for 6 months before RNA extraction. A mortar, homogenizer, and tissue lyser were used to grind tissues. RNA was extracted by TRIzol, and RNA integrity was assessed by the RNA integrity number (RIN) value. Results: For fresh tissues and frozen tissues with warm ischemia time within 60 minutes, RIN values were above 7.0 and remained above 6.0 with warm ischemia time within 120 minutes. For the same warm ischemia time, RIN values of frozen tissues were slightly lower than those of fresh tissues. No significant RIN value alterations were observed among grinding methods, but for RNA extraction efficiency, a mortar was much less efficient than the homogenizer or tissue lyser. For frozen tissues, RNA tended to degrade within 8 minutes at room temperature. Conclusions: Mouse kidney tissues with a warm ischemia time within 120 minutes are suitable for general RNA-related research. For tissues with a warm ischemia time within 60 minutes, cryopreservation may not affect RNA quality. The duration of frozen tissues held at room temperature before grinding affects the integrity of RNA, while grinding methods do not affect RNA integrity.

CRISPR Mediated ELANE Single-Allele Knock-out Restores Proliferation and Myeloid Differentiation of Neutropenia Patient Derived BM HSCs

Background: Mutations in ELANE are a cause of cyclic and severe congenital neutropenia (SCN), predominantly autosomal dominant disorders. ELANE encodes neutrophil elastase (NE), a tissue specific serine protease. Neutropenia occurs because mutant NE impairs survival and maturation of myeloid cells. More than 130 disease causing mutations in ELANE have been identified and cell permeable inhibitors of NE can correct the defect in cell survival and maturation in cellular models. (Makaryan et al. J Leukoc Biol. 2017;102:1143). CRISPR/Cas9 knock-out (KO) of ELANE in patients' hematopoietic stem cells (HSCs) corrects myeloid cell differentiation, (Nasri et al Haematologica 2020:105:598), but this approach targets both alleles, eliminates normal NE and may impair innate immunity. Hypothesis: Selective, single allele editing is a preferred strategy for correcting ELANE associated neutropenia and other autosomal dominant disorders. Methods: Emendo Biotherapeutics developed CRISPR-associated allele specific KO based on single nucleotide polymorphisms (SNPs) located in the vicinity of ELANE. Three SNPs cover ~80% of the population. In the current study, editing of the mutated allele was achieved by targeting SNP rs1683564 located downstream to the 3'UTR of ELANE and mediating a biallelic break in intron 4, with excision of the 3'UTR, destabilized the mutated allele transcript. For editing we utilized RNPs assembled from Emendo's proprietary nuclease, OMNI-50, a guide targeting the SNP and a guide targeting a region in intron 4. OMNI-50 has low off-target activity and high allele specific editing when targeting either the reference or the alternative form of the SNPs. The efficacy of this composition was tested on normal and SCN patient-derived HSCs harboring the relevant SNP. (Figure 1) A patient's bone marrow CD34+ cells with the S126L mutation and rs1683564 were enriched using RosetteSep™ Progenitor Cell Enrichment Cocktail (Stemcell Technologies), purified by density gradient centrifugation using Lymphoprep (Stemcell Technologies). The purified CD34+ cells were expanded 4 days in StemSpan™ SFEM II media, supplemented with StemSpan™ CD34+ Expansion Supplement. Expanded CD34+ cells were further purified using StemSep™ Human CD34 Positive Cocktail and purity verified by FACS analysis using CD34 and CD45 antibodies. Purified CD34+ cells were cryopreserved using serum free CryoStor® CS10 media (Stemcell Technologies) and stored in Liquid Nitrogen Vapor Phase. Normal human blood CD34+ progenitor cells, harboring rs1683564 were similarly treated as the control. Allele specific excision was determined by ddPCR. To determine excision efficiency for the patient and control, we amplified two regions in ELANE, Exon 1 and Exon 5 (excised region), using two different probes labeled with FAM and HEX, respectively. We used OMNI-50 nuclease (Emendo Bio, Ness Ziona, Israel), electroporation (Lonza 4D nucleofector) and StemCell media (Stemcell Technologies, Vancouver, BC) to modify, grow and differentiate the CD34+ cells. We assessed myeloid cell proliferation and differentiation using daily cell counts, cytospins stained with Diff-Quik, and CD14, CD66b, CD15, CD16, CD11b labeling and flow cytometry. Results: Unedited patient cells demonstrated significant abnormalities in proliferation and differentiation. Cytospins from unedited patient CD34+ cells at 14 days showed block of myeloid differentiation and 4-fold greater monocytes compared to control, consistent with the hematopoietic defect in SCN patients. Single allele ELANE KO significantly improved these cellular abnormalities: total cell proliferation increased 41% and CD14/CD66b and CD15/CD11b positive cells increased by 107% and 65.5%, respectively. Studies are in progress for additional patients harboring different ELANE mutations. Conclusions: Single allele KO may resolve concerns about unwanted effects of total elimination of the normal gene products with gene editing for autosomal dominant diseases and be an effective strategy for treating ELANE associate neutropenia. Disclosures Sabo: Emendo BioTherapeutics: Research Funding. Makaryan:Emendo BioTherapeutics: Research Funding. Poulsen:Emendo BioTherapeutics: Research Funding. Povodovski:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Dicken:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Herman:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Emmanuel:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Dale:X4 Pharmaceuticals: Research Funding; Emendo BioTherapeutics: Consultancy; X4 Pharmaceuticals: Honoraria.

The Effect of Different Thawing Rates on Cryopreserved Human Iliac Arteries Allograft's Structural Damage and Mechanical Properties.

Introduction The rate of thawing of cryopreserved human iliac arteries allografts (CHIAA) directly affects the severeness of structural changes that occur during this process. Method The experiment was performed on ten CHIAA. The 10% dimethylsulphoxide in 6% hydroxyethyl starch solution was used as the cryoprotectant; all CHIAA were cooled at a controlled rate and stored in the vapor phase of liquid nitrogen (-194°C). Two thawing protocols were tested: (1) placing the CHIAA in a water bath at 37°C, and (2) the CHIAA were thawed in a controlled environment at 5°C. All samples underwent analysis under a scanning electron microscope. Testing of the mechanical properties of the CHIAA was evaluated on a custom-built single axis strain testing machine. Longitudinal and circumferential samples were prepared from each tested CHIAA. Results Ultrastructural analysis revealed that all five CHIAA thawed during the thawing protocol 1 which showed significantly more damage to the subendothelial structures when compared to the samples thawed in protocol 2. Mechanical properties: Thawing protocol 1—longitudinal UTS 2, 53 ± 0, 47 MPa at relative strain 1, 27 ± 0, 12 and circumferential UTS 1, 94 ± 0, 27 MPa at relative strain 1, 33 ± 0, 09. Thawing protocol 2—longitudinal ultimate tensile strain (UTS) 2, 42 ± 0, 34 MPa at relative strain 1, 32 ± 0, 09 and circumferential UTS 1, 98 ± 0, 26 MPa at relative strain 1, 29 ± 0, 07. Comparing UTS showed no statistical difference between thawing methods. Conclusion Despite the significant differences in structural changes of presented thawing protocols, the ultimate tensile strain showed no statistical difference between thawing methods.

Compared DNA and RNA quality of breast cancer biobanking samples after long-term storage protocols in \u2212 80\xa0\xb0C and liquid nitrogen

Molecular investigations are crucial for further developments in precision medicine. RNA sequencing, alone or in combination with further omic-analyses, resulted in new therapeutic strategies. In this context, biobanks represent infrastructures to store tissue samples and body fluids in combination with clinical data to promote research for new predictive and prognostic biomarkers as well as therapeutic candidate molecules. Until today, the optimal storage conditions are a matter of debate especially with view to the storage temperature. In this unique approach we compared parallel samples from the same tumour, one half stored at − 80 °C and one half in the vapor phase of liquid nitrogen, with almost identical pre-analytical conditions. We demonstrated that RNA isolated from breast cancer samples revealed significantly higher RINe-values after 10 years of storage in the vapor phase of liquid nitrogen compared to storage at − 80 °C. In contrast, no significant difference was found regarding the DIN-values after DNA isolation. Morphological changes of the nucleus and cytoplasm, especially in the samples stored at − 80 °C, gave insights to degenerative effects, most possibly due to the storage protocol and its respective peculiarities. In addition, our results indicate that exact point-to point documentation beginning at the sample preparation is mandatory.

Cryopreservation of Seeds and Seed Embryos in Orthodox-, Intermediate-, and Recalcitrant-Seeded Species.

Seeds are one of the preferable and most used sources of germplasm for the ex situ preservation of plant genetic resources. They are generally stored dry at -20°C in seed banks following international standards. However, some seeds do not tolerate drying and/or storage at -20°C, or present short lifespans at these conditions. For them cryopreservation is indicated for long-term preservation. When seeds tolerate desiccation (i.e., orthodox seeds), they can be dried at about 32±3% relative humidity at 18°C and stored in the vapor phase of liquid nitrogen. This is the method followed in the Millennium Seed Bank of the Royal Botanic Gardens, Kew, for wild species with short lifespans in the standard conditions of seed banks. When seeds do not tolerate desiccation (i.e., recalcitrant seeds) or their tolerance to desiccation and/or -20°C storage is limited (i.e., intermediate seeds), drying and cooling procedures must be adjusted, and often, cryoprotection is also required. Some methods are detailed for diverse species of temperate and tropical origin.

Conservation protocols for Ensete glaucum, a crop wild relative of banana, using plant tissue culture and cryopreservation techniques on seeds and zygotic embryos

Ensets (Ensete spp., family Musaceae), are multipurpose plants with great versatility and potential as a food security crop, although they are cultivated only in Ethiopia and few other regions of Africa. Currently due to loss of habitat, intensification of agriculture, genetic diversity of both wild and domestic ensets is at high risk of erosion. Ensete glaucum (Roxb.) Cheesman, a less-known and under-utilized crop wild relative of banana, is found as natural strands in North-eastern regions of India. No report exists on seed conservation or cryopreservation of E. glaucum and the present work was undertaken to fill this knowledge gap. Mature fruits of two seeded accessions of E. glaucum (MZU-DBT-T15 and HSB-8) were collected from plants in natural populations from two geographically distinct North-eastern region of India. Seed morphology, moisture content (MC) of seed and embryo, viability and germination of excised embryos, desiccation and cryosensitivity were studied. Fresh seed (31% MC) viability measured through in vitro germination of excised embryos was not significantly different in both accessions (87–89%). Seeds could tolerate desiccation up to 5–10% MC without significant loss in germination potential (83–84%) and safely stored up to 1 year at room temperature (RT), without loss of viability. Air dehydrated seeds (10 ± 2% MC) and embryos (12 ± 2% MC) were cryopreserved (in liquid and vapour phase of liquid nitrogen). High post-thaw recovery was achieved in both tissues i.e. seeds (80.2 ± 2.2%) and embryos (83.1 ± 2.6%) by in vitro culture of excised embryos on MS medium + 2 µM gibberellic acid + 1 µM 6-benzyl amino purine (BAP) + 1 µM ascorbic acid. The seedlings converted into multiple shoots (15–20 shoots/explant) within 2 months of culture on medium with MS salts + 10 µM BAP + 1 µM indole-3-acetic acid (IAA) + 1 µM ascorbic acid. This study is the first successful report of cryopreservation of E. glaucum using simple air dehydration. Using the method devised, seeds and zygotic embryos of the two accessions of E. glaucum are safely conserved in the Cryogenebank, while in vitro plantlets are maintained in the In Vitro Genebank of ICAR-NBPGR, New Delhi, India. This study is the first successful report of cryopreservation of Ensete glaucum using seeds and zygotic embryos

Feasibility of direct venous inoculation of the radiation-attenuated Plasmodium falciparum whole sporozoite vaccine in children and infants in Siaya, western Kenya

PfSPZ Vaccine, composed of radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum sporozoites, is administered by direct venous inoculation (DVI) for maximal efficacy against malaria. A critical issue for advancing vaccines that are administered intravenously is the ability to efficiently administer them across multiple age groups. As part of a pediatric safety, immunogenicity, and efficacy trial in western Kenya, we evaluated the feasibility and tolerability of DVI, including ease of venous access, injection time, and crying during the procedure across age groups. Part 1 was an age de-escalation, dose escalation trial in children aged 13 months-5 years and infants aged 5–12 months; part 2 was a vaccine efficacy trial including only infants, using the most skilled injectors from part 1. Injectors could use a vein viewer, if needed. A total of 1222 injections (target 0.5 mL) were initiated by DVI in 511 participants (36 were 5–9-year-olds, 65 were 13–59-month-olds, and 410 infants). The complete volume was injected in 1185/1222 (97.0%) vaccinations, 1083/1185 (91.4%) achieved with the first DVI. 474/511 (92.8%) participants received only complete injections, 27/511 (5.3%) received at least one partial injection (<0.5 mL), and in 10/511 (2.0%) venous access was not obtained. The rate of complete injections by single DVI for infants improved from 77.1% in part 1 to 92.8% in part 2. No crying occurred in 51/59 (86.4%) vaccinations in 5–9-year-olds, 25/86 (29.1%) vaccinations in 13–59-month-olds and 172/1067 (16.1%) vaccinations in infants. Mean administration time ranged from 2.6 to 4.6 minutes and was longer for younger age groups.These data show that vaccination by DVI was feasible and well tolerated in infants and children in this rural hospital in western Kenya, when performed by skilled injectors. We also report that shipping and storage in liquid nitrogen vapor phase was simple and efficient. (Clinicaltrials.gov NCT02687373).

Cryopreservation of Heart Valves: A Deceptive Illusion

Objective: Frozen cryopreservation (FC) with storage in the vapour phase of liquid nitrogen remains the standard bio-banking technique for preservation of allogeneic heart valves. Since its introdu...

Impact of long-term storage and freeze-thawing on eight circulating microRNAs in plasma samples

Sample collection, processing, storage and isolation methods constitute pre-analytic factors that can influence the quality of samples used in research and clinical practice. With regard to biobanking practices, a critical point in the sample’s life chain is storage, particularly long-term storage. Since most studies examine the influence of different temperatures (4°C, room temperature) or delays in sample processing on sample quality, there is only little information on the effects of long-term storage at ultra-low (vapor phase of liquid nitrogen) temperatures on biomarker levels. Among these biomarkers, circulating miRNAs hold great potential for diagnosis or prognosis for a variety of diseases, like cancer, infections and chronic diseases, and are thus of high interest in several scientific questions. We therefore investigated the influence of long-term storage on levels of eight circulating miRNAs (miR-103a-3p, miR-191-5p, miR-124-3p, miR-30c-5p, miR-451a, miR-23a-3p, miR-93-5p, miR-24-3p, and miR-33b-5p) from 10 participants from the population-based cohort study KORA. Sample collection took place during the baseline survey S4 and the follow-up surveys F4 and FF4, over a time period spanning from 1999 to 2014. The influence of freeze-thaw (f/t) cycles on miRNA stability was also investigated using samples from volunteers (n = 6). Obtained plasma samples were profiled using Exiqon’s miRCURYTM real-time PCR profiling system, and repeated measures ANOVA was used to check for storage or f/t effects. Our results show that detected levels of most of the studied miRNAs showed no statistically significant changes due to storage at ultra-low temperatures for up to 17 years; miR-451a levels were altered due to contamination during sampling. Freeze-thawing of one to four cycles showed an effect only on miR-30c-5p. Our results highlight the robustness of this set of circulating miRNAs for decades of storage at ultra-low temperatures and several freeze-thaw cycles, which makes our findings increasingly relevant for research conducted with biobanked samples.

Comparison of Different Thawing Protocols in Human Cryopreserved Venous Grafts

The aim of our study was to assess the impact of different thawing protocols on morphological changes arising in cryopreserved human saphenous vein grafts. The study was performed in 12 saphenous vein grafts harvested in brain death donors. Storage in the vapor phase of liquid nitrogen for 3 or 5years followed. Two thawing protocols were tested: slow thawing in a refrigerator at temperature +4°C for 2hr and rapid thawing-in a water bath at +37°C. Grafts were processed for scanning electron microscopy. Comparisons of continuous parameters under study between experimental groups were performed using the t-test (age, cold ischemia time, exposure to cryoprotectant, time of storage, total thawing time, mean thawing rate, morphology scoring of thawed HSVG) and the median test (HSVG length). Categorical parameters (sex and blood group) were formally tested using the chi-square test. All samples were evaluated according to morphological changes and scored in terms of morphologically intact endothelium, confluent endothelium with structural inhomogeneity, disruption of the intercellular contacts, separation of the endothelial cells, complete loss of the endothelium, and damage of the subendothelial layers. There is no statistically significant difference between the sample sets at the significance level of 0.05. There was no association with donors' age, sex, and time of storage. Human cryopreserved saphenous vein grafts in our experimental work showed no difference in terms of structural deterioration of the endothelial surface and basal membrane depending on different thawing protocols used.

Sustained Hematopoietic Recovery after Salvage Autologous Stem Cell Transplants after Long Term Storage of Cryopreserved Hematopoietic Progenitor Stem Cells (HPSC)

Sustained Hematopoietic Recovery after Salvage Autologous Stem Cell Transplants after Long Term Storage of Cryopreserved Hematopoietic Progenitor Stem Cells (HPSC)

Effects of supplementation of Tris-egg yolk extender with royal jelly on chilled and frozen-thawed ram semen characteristics

This study was conducted to examine the effect of supplementation of Tris-egg yolk extender with lyophilized royal jelly (RJ) on chilled and frozen-thawed ram semen parameters. Ejaculates were collected by artificial vagina from 4 mature rams, twice a week for 4 weeks. Only samples with motility of ≥70% were included, pooled and divided into four equal parts and then diluted in extenders with various concentrations of RJ (0, 1, 3 and 5%, vol/vol) to a final concentration of 200 × 106 sperm/mL and was incubated at 37 °C for 30 min and were subsequently evaluated. After equilibration of extended semen for 2 h at 4 °C, some semen samples were packed in 0.25 mL plastic straws. Then, the straws were frozen in the liquid nitrogen vapor phase for 15 min and stored at −196 °C in liquid nitrogen. The frozen straws were thawed in warm water (37 °C) for 30 s and evaluated; whereas, other semen samples were stored in the refrigerator (4 °C) up to 7 days. The chilled samples were kept in water bath (37 °C) for 5 min and then were evaluated. After dilution, the lowest and highest sperm total abnormality was recorded in 3 and 5% RJ supplemented groups, respectively (P < 0.05). The chilled sperm total motility and membrane integrity were significantly (P < 0.05) higher in 3% than those in 0% and 5% RJ supplemented groups. The chilled sperm progressive motility and viability was significantly (P < 0.05) higher in 1 and 3% than those in 0 and 5% RJ supplemented groups. The frozen-thawed sperm total motility, progressive motility, membrane integrity and viability were significantly higher in 3% RJ supplemented group (P < 0.05). In conclusion, supplementation of Tris-egg yolk extender with 3% lyophilized RJ had a protective effect on chilled and cryopreserved ram spermatozoa.