Cryopreservation Period Research Articles

Optimisation of cryopreservation conditions, including storage duration and revival methods, for the viability of human primary cells

BackgroundCryopreservation is a crucial procedure for safeguarding cells or other biological constructs, showcasing considerable potential for applications in tissue engineering and regenerative medicine.AimsThis study aimed to evaluate the effectiveness of different cryopreservation conditions on human cells viability.MethodsA set of cryopreserved data from Department of Tissue Engineering and Regenerative Medicine (DTERM) cell bank were analyse for cells attachment after 24 h being revived. The revived cells were analysed based on different cryopreservation conditions which includes cell types (skin keratinocytes and fibroblasts, respiratory epithelial, bone marrow mesenchymal stem cell (MSC); cryo mediums (FBS + 10% DMSO; commercial medium); storage durations (0 to > 24 months) and locations (tank 1–2; box 1–5), and revival methods (direct; indirect methods). Human dermal fibroblasts (HDF) were then cultured, cryopreserved in different cryo mediums (HPL + 10% DMSO; FBS + 10% DMSO; Cryostor) and stored for 1 and 3 months. The HDFs were revived using either direct or indirect method and cell number, viability and protein expression analysis were compared.ResultsIn the analysis cell cryopreserved data; fibroblast cells; FBS + 10% DMSO cryo medium; storage duration of 0–6 months; direct cell revival; storage in vapor phase of cryo tank; had the highest number of vials with optimal cell attachment after 24 h revived. HDFs cryopreserved in FBS + 10% DMSO for 1 and 3 months with both revival methods, showed optimal live cell numbers and viability above 80%, higher than other cryo medium groups. Morphologically, the fibroblasts were able to retain their phenotype with positive expression of Ki67 and Col-1. HDFs cryopreserved in FBS + 10% DMSO at 3 months showed significantly higher expression of Ki67 (97.3% ± 4.62) with the indirect revival method, while Col-1 expression (100%) was significantly higher at both 1 and 3 months compared to other groups.ConclusionIn conclusion, fibroblasts were able to retain their characteristics after various cryopreservation conditions with a slight decrease in viability that may be due to the thermal-cycling effect. However, further investigation on the longer cryopreservation periods should be conducted for other types of cells and cryo mediums to achieve optimal cryopreservation outcomes.

Ultrasound-assisted extraction of bioactive compounds from Moringa oleifera leaves for beef patties preservation: Antioxidant and inhibitory activities, half-life, and sensory attributes.

This study aims to examine employing ultrasound-assisted extraction of bioactive components from Moringa oleifera leaves and apply them in beef patties preservation, as well as antioxidant and inhibitory activities and sensory qualities. The study included studying the chemical content and minerals of the M. oleifera leaves, preparation of aqueous and alcoholic extracts using an ultrasound device, then exploring the extraction yield. The results proved that the extraction yield by ultrasound using ethanol at 80% was the highest, reaching 19.22%. The total phenols in the ultrasonic extract with moringa leaves aqueous extract (AMEUS) amounted to 120,755 mg/mL. Since the AMEUS exhibited the highest value of 68.308 mg/mL calic acid - eight phenolic compounds discovered by HPLC - the total content of flavonoids was also calculated. The inhibitory and antioxidant effects of moringa leaf extracts are well documented. We monitored the changes in chemical indicators, such as the value of peroxide and thiobarbituric acid, as well as the percentage of free fatty acids and physical characteristics, such as water-carrying capacity, pH, and pigments, for storage periods 0, 4, 8, and 12 days after adding AMEUS to beef patties at a concentration of 0.5%. The patties were kept under refrigeration at 4 ± 1°C during this time. The values of peroxide number, thiobarbituric acid, free fatty acid, and metmyoglobin pigment were decreased in the beef patties treated with the AMEUS. However, they increased continuously during the cryopreservation period, and there was a significant increase in water-holding capacity (WHC) when the beef patties were treated with AMEUS. The results also showed that adding AMEUS to beef patties improved their qualitative characteristics.

P-156 Ultra-fast warming of blastocysts yields better survival rates for D5 non-PGT and similar survival rates for D5/D6 PGT blastocysts

Abstract Study question Can an ultra-fast single-step warming of vitrified blastocysts yield similar survival rates and post-warming development compared to the standard multi-step rehydration protocol? Summary answer Ultra-fast warming, shows significantly better rates of survival and post-warming development in vitro for non-PGT blastocysts and similar rates for PGT blastocysts. What is known already Excellent survival rates have been reported since the introduction of human blastocyst vitrification. Typically, most warming procedures involve three steps with decreasing concentrations of non-penetrating cryoprotectants. Recently, few studies have evaluated the effectiveness of simplifying the procedure to a single step for rehydration, while maintaining comparable survival and pregnancy rates. Adopting simpler procedures can optimize laboratory protocols, workflow, and safety, supporting further advancements in Assisted Reproduction Technologies (ART). Study design, size, duration This validation study ultra-fast warmed 246 vitrified blastocysts [129 non-PGT day5 (cryopreservation period 23/01/2017-24/04/2018 VIT-kit, Fujifilm), 57 PGT day5 (25/01/2023-31/03/2023 VIT-KitNX, Fujifilm), 60 PGTday6 (03/02/2023-22/08/2023 Vit-KitNX, Fujifilm)] and compared survival rates with 379 blastocysts [240 non-PGT day5, 97 PGT day5, 42 PGT day6] vitrified in the same time-periods and warmed for clinical application using the standard multi-step protocol. Ultra-fast warmed blastocysts were cultured for 24h in a time-lapse incubator and subjected to a viability staining. Participants/materials, setting, methods Blastocysts, originating from 111 patients that were donated to validation studies after the legal storage period(n = 129) or deemed non-transferable after PGT(n = 117), were ultra-fast warmed by immersing the straw in a 37 °C thawing solution (1M trehalose; Vit-kitwarmNX; Fujifilm) for 1 minute. Survival rates [total (≥50% intact) and fully (100% intact) ], transfer rates (2h post-warming, ≥50% intact), re-expansion time and the number of damaged cells (Live/Dead kit L-3224, Invitrogen) were analysed. Fisher’s Exact Test (p < 0.05). Main results and the role of chance Overall, the immediate post-warming survival rates (≥50% intact) were comparable between ultra-fast and multi-step warming, 98.4% vs 96.8% respectively (p = 0.3041). However, the fully intact survival rate immediately post-warming was significantly higher with ultra-fast warming (85.4% vs 76.3%; p = 0.0058) as well as the percentage of blastocysts considered transferable (96.7% vs 91.2%; p = 0.0076). Sub-group analysis demonstrated significantly better results for non-PGT blastocysts following ultra-fast warming. Higher immediate survival post-warming (100% vs 96.6%; p = 0.036) and transfer rates (100% vs 90%; p = 0.0002) were achieved. Similar results were observed for PGT blastocysts on both day 5 and day 6, with no significant differences in total, fully intact, and transfer rates observed between the two warming procedures [PGT Day5: 96.5%, 80.7%, and 93.0% vs. 97.9%(p = 0.6270), 69.1%(p = 0.1334), and 93.8%(p = 1.0000)] [PGT Day6: 96.7%, 68.3%, and 93.3% vs. 95.2%(p = 1.0000), 64.3%(p = 0.6761), and 92.8%(p = 1.0000)]. Ultra-fast warmed blastocysts showed a total re-expansion rate of 93.3% in 3.3h±2.7h on average. Viability staining demonstrated very few cells damaged. Zero cell damage was observed in 31.5% of the blastocysts, 59.0% had 1-10 cells damaged, 9.5% >10 cells damaged, and only 4 blastocysts were completely degenerated. Limitations, reasons for caution In order to limit the amount of embryos needed in this validation study, ultra-fast warming outcome parameters were compared with retrospective clinical results of blastocysts vitrified in the same time periods. Ultra-fast warming is a single-step process where precise temperature control(37 °C) of the warming medium is crucial for success. Wider implications of the findings Minimizing handling steps leads to an overall risk reduction in potential exposure to suboptimal warming conditions and operator/handling-related stress. This optimization brings huge efficiency to the busy work schedule that is characteristic for an IVF lab without dropping cryopreservation quality indicators. Trial registration number Not applicable

Selecting serum-free hepatocyte cryopreservation stage and storage temperature for the application of an “off-the-shelf” bioartificial liver system

The bioartificial liver (BAL) system can potentially rescue acute liver failure (ALF) patients by providing partial liver function until a suitable donor liver can be found or the native liver has self-regenerated. In this study, we established a suitable cryopreservation process for the development of an off-the-shelf BAL system. The viability of hepatocyte spheroids cryopreserved in liquid nitrogen was comparable to that of fresh primary hepatocyte spheroids. When hepatocyte spheroids were subjected to cryopreservation in a deep freezer, no statistically significant differences were observed in ammonia removal rate or urea secretion rate based on the cryopreservation period. However, the functional activity of the liver post-cryopreservation in a deep freezer was significantly lower than that observed following liquid nitrogen cryopreservation. Moreover, cryopreserving spheroid hydrogel beads in a deep freezer resulted in a significant decrease (approximately 30%) in both ammonia removal and urea secretion rates compared to the group cryopreserved in liquid nitrogen. The viabilities of spheroid hydrogel beads filled into the bioreactor of a BAL system were similar across all four groups. However, upon operating the BAL system for 24 h, the liver function activity was significantly higher in the group comprising hydrogel beads generated after thawing hepatocyte spheroids cryopreserved in liquid nitrogen. Consequently, the manufacturing of beads after the cryopreservation of hepatocyte spheroids is deemed the most suitable method, considering efficiency, economic feasibility, and liver function activity, for producing a BAL system.

Effect of post-vitrification cryopreservation duration on singleton birth-weight in frozen-thawed blastocysts transfer cycles.

This study aimed to explore the effect of cryopreservation duration after blastocyst vitrification on the singleton birth-weight of newborns to assess the safety of long-term preservation of frozen-thawed blastocyst transfer (FBT) cycles. This was a retrospective observational study conducted at the Gynecological Endocrinology and Assisted Reproduction Center of the Peking Union Medical College Hospital. Patients who gave birth to singletons between January 2006 and December 2021 after undergoing FBT cycles were included. Five groups were formed according to the duration of cryopreservation of embryos at FBT: Group I included 274 patients with a storage time < 3 months. Group II included 607 patients with a storage time of 3-6 months. Group III included 322 patients with a storage time of 6-12 months. Group IV included 190 patients with a storage time of 12-24 months. Group V included 118 patients with a storage time of > 24 months. Neonatal outcomes were compared among the groups. Multivariate linear regression analysis was performed to evaluate birth-weights and other birth-related outcomes. A total of 1,511 patients were included in the analysis. The longest cryopreservation period was 12 years. The birth-weights of neonates in the five groups were 3344.1 ± 529.3, 3326.1 ± 565.7, 3260.3 ± 584.1, 3349.9 ± 582.7, and 3296.7 ± 491.9 g, respectively (P > 0.05). The incidences of preterm birth, very preterm birth, low birth-weight, and very low birth-weight were similar in all groups (P > 0.05). The large-for-gestational-age and small-for-gestational-age rates did not differ significantly among the groups (P > 0.05). After adjusting for confounding factors that may affect neonatal outcomes, a trend for an increased risk of low birth-weight with prolonged cryopreservation was observed. However, cryopreservation duration and neonatal birth-weight were not significantly correlated (P > 0.05). The duration of cryopreservation after blastocyst vitrification with an open device for more than 2 years had no significant effect on the birth-weight of FBT singletons; however, attention should be paid to a possible increase in the risk of low birth-weight.

Investigating the cryoprotective efficacy of fructans in mammalian cell systems via a structure-functional perspective

Fructans have long been known with their role in protecting organisms against various stress factors due to their ability to induce controlled dehydration and support membrane stability. Considering the vital importance of such features in cryo-technologies, this study aimed to explore the cryoprotective efficacy of fructans in mammalian cell systems where structurally different fructan polymers were examined on in vitro cell models derived from organs such as the liver, frequently used in transplantation, osteoblast, and cord cells, commonly employed in cell banking, as well as human seminal fluids that are of vital importance in assisted reproductive technology. To gain insights into the fructan/membrane interplay, structural differences were linked to rheological properties as well as to lipid membrane interactions where both fluorescein leakage from unilamellar liposomes and membrane integrity of osteoblast cells were monitored. High survival rates obtained with human endothelial, osteoblast and liver cells for up to two months clearly showed that fructans could be considered as effective non-permeating cryoprotectants, especially for extended periods of cryopreservation. In trials with human seminal fluid, short chained levan in combination with human serum albumin and glycerol proved very effective in preserving semen samples across multiple patients without any morphological abnormalities.

Abstract 13 Long-Term Stability of Dus Cord Blood Units After &amp;gt;29 Years of Cryopreservation: Follow-Up Data from the José Carreras Cord Blood Bank

Abstract Introduction The José Carreras Cord Blood Bank (CBB) located in Düsseldorf stores 21215 active cryopreserved cord blood units (CBUs) and has released 1476 CBUs for allogeneic transplantation. The success of transplantations, but also the generation of cells for regenerative medicine for e.g. iPSC reprogramming, significantly depends on the quality of the cryopreserved CBUs after thaw. Objectives Therefore, typical parameters like cell count, recovery and viability are analysed from thawed CBUs yearly by a stability monitoring program according to the FACT standards. The longest cryopreservation period in liquid nitrogen tested to date is 338 months (&amp;gt;29 years) for unseparated units and 300 months (25 years) for volume-reduced units that are licensed by the Paul-Ehrlich Institute. Methods A retrospective analysis of unseparated CBUs processed from 1993-1997, CBUs processed from 1998-2005 by manual volume-reduction and CBUs processed since 2005 by automated volume-reduction applying the Sepax100 system was performed. In consideration of the distinct processing methods, storage time and existing follow-up data of released transplants, this approach should demonstrate the high CBU quality and proof the long-term engrafting capability of CBUs cryopreserved in liquid nitrogen. Results According to the CBB stability program means of TNC count (16.32±2.61 x 108), TNC recovery after thaw (106.8±13.91%, above 100% due to earlier applied cell-count method), TNC viability (88.91±5.01%), CD34+7AAD- viability (90.73±3.69%), CD45+7AAD- viability (69.09±5.38%) and CFC count (7.10±2.45 x 106) were determined after &amp;gt;29 years of cryopreservation for unseparated CBUs. In volume-reduced CBUs the mean TNC count (9.37±2.14 x 108), TNC recovery (97.78±3.70%), TNC viability (84.22±10.02%), CD34+7AAD- viability (88.11±5.16%), CD45+7AAD- viability (73.67±8.31%) and CFC count (1.37±0.59 x 106) were determined after 25 years of cryopreservation. Discussion In addition, these relevant parameters were retrospectively analyzed for released transplants in correlation to the storage time. The follow-up data of recipients from CBUs demonstrate an earlier engraftment of volume-reduced CBUs as compared to unseparated CBUs in accordance to an increased TNC count over time. With regard to applied processing methods, engraftment time and patient age (children and adults) this retrospective analysis confirms the high quality of old CBUs stored in the liquid phase at -196°C.

O-038 Is a 10-year retention period sufficient after elective oocyte vitrification (EOV)?

Abstract Study question In Belgium, by law, a retention period of 10-years is standard. Is this period sufficient for women to return to the clinic and use their vitrified oocytes? Summary answer A standard cryopreservation period of 10-years seems sufficient since half of the women returned to the fertility clinic and 96% used the vitrified oocytes. What is known already The main driver for elective oocyte cryopreservation is to buy more time to find a suitable partner. The question remains whether 10 years of cryopreservation is sufficient for women to accomplish their goal of finding a partner and procreate with or without using their vitrified oocytes. The return rates and the utilisation rates of the vitrified oocytes reported in the literature are low. Most women do not use their vitrified oocytes. Little is known about women's reproductive pathways after EOV and the destination of the expired surplus vitrified oocytes. Study design, size, duration Computerized clinical data were retrieved from a pioneer cohort of women who underwent EOV in our centre for age-related reasons more than 10 years ago (between 2009 and 2012). Hence, the legal cryopreservation period of their oocytes expired between 2019 and 2022. We documented reproductive choices for those who returned to the centre and we investigated the intended destination of the expired vitrified oocytes for those who did not return. Participants/materials, setting, methods Data were collected from clinical charts of women who vitrified oocytes. According to Belgian law, unused vitrified oocytes expire when reaching the age limit of 48 years for use or by reaching the standard cryopreservation period of 10 years. We evaluated whether women eventually started treatment, assessed their relational status, the utilisation rate of the vitrified oocytes and the intended destination of the vitrified oocytes as stated in their informed consents. Main results and the role of chance 117 women vitrified their oocytes at least 10 years ago. Women's oocyte cryopreservation period expired because they were either beyond the age limit for use (n = 82) or because the legal oocyte cryopreservation period of 10 years was reached (n = 35). Five women requested transport of their oocytes for treatment in a centre abroad. Fifty-two out of 117 women (44.4%) returned for assisted reproduction. Eventually, 96% (50/52) of them used their vitrified oocytes for treatment and 21/50 had a child or ongoing pregnancy. Upon return to the clinic, 22 women were single and 27 had found a partner, two women returned with a co-parent and one woman had a female partner. Furthermore, 7/117 women returned to the clinic but eventually refrained from treatment. Fifty-one women never returned to the centre. Only five women asked to prolong the cryopreservation period for medical reasons. The others not using their vitrified oocytes, did not initiate further action concerning the cryopreservation period, use or destination of their stored oocytes. Hence, the destination of the cryopreserved oocytes was according to the women's choice stated in the informed consent. The majority donated their oocytes for research (60%), others (31%) opted to destroy the oocytes after 10 years of cryopreservation. Limitations, reasons for caution It was not possible to map out the reproductive pathway of women who did not return to the centre and whose vitrified oocytes expired for use. Follow-up of reproductive choices and outcomes in women who did not return for treatment is required for a comprehensive appraisal of EOV. Wider implications of the findings The vast majority of the women who returned for treatment used their vitrified oocytes. Those not returning did not initiate further action concerning the use or destination of their vitrified oocytes. These results add to our knowledge on the utilisation rate after EOV, essential to improve counselling of future EOV-candidates. Trial registration number not applicable

Pregnancy and Delivery of a Healthy Baby Boy from a Patient Embryo Cryopreserved for Over 18 Years: A Case Report

Cryopreservation offers semipermanent embryo storage. Although there have been various reports on effects of cryopreservation, there are few regarding preservation for 10 years or more. Here, we describe the delivery of a healthy child to a mother 51 years old, using an embryo cryopreserved for 18.3 years. The patient had four cleaved embryos cryopreserved when she was 32 years old. After more than 18 years, a Day-3, 10-cell stage embryo was transferred during a hormone replacement therapy cycle. The patient became pregnant and gave birth to a healthy boy (3,046 g). This case demonstrates the potential for live births at advanced maternal age by cryopreserving embryos at a young maternal age. To our knowledge, this is the longest cryopreservation period reported for a birth involving transfer of the mother’s own frozen embryo.

Peptide-DNA origami as a cryoprotectant for cell preservation.

Cryopreservation of cells is essential for the conservation and cold chain of bioproducts and cell-based medicines. Here, we demonstrate that self-assembled DNA origami nanostructures have a substantial ability to protect cells undergoing freeze-thaw cycles; thereby, they can be used as cryoprotectant agents, because their nanoscale morphology and ice-philicity are tailored. In particular, a single-layered DNA origami nanopatch functionalized with antifreezing threonine peptides enabled the viability of HSC-3 cells to reach 56% after 1 month of cryopreservation, surpassing dimethyl sulfoxide, which produced 38% viability. It also exhibited minimal dependence on the cryopreservation period and freezing conditions. We attribute this outcome to the fact that the peptide-functionalized DNA nanopatches exert multisite actions for the retardation of ice growth in both intra- and extracellular regions and the protection of cell membranes during cryopreservation. This discovery is expected to deepen our fundamental understanding of cell survival under freezing environment and affect current cryopreservation technologies.

Abscisic acid induces the expression of AsKIN during the recovery period of garlic cryopreservation.

Abscisic acid induced the expression of AsKIN during the recovery period of garlic cryopreservation. AsKIN was identified as a gene involved in cold and osmotic stress resistance. Cryopreservation has been proven to be effective in removing viruses from garlic. However, oxidative damage in cryopreservation has a significant impact on the survival after preservation. Abscisic acid (ABA) has been shown to reduce oxidative stress and promote the survival after cryopreservation. However, it is not clear which genes play important roles in this process. In this study, we added ABA to the dehydration step and analyzed the transcriptomic divergences between the ABA-treated group and the control group in three cryogenic steps (dehydration, unloading and recovery). By short time-series expression miner (STEM) analysis and weighted gene co-expression network analysis (WGCNA), the recovery step was identified as the period of significant changes in gene expression levels in cryopreservation. The addition of ABA promoted the upregulated expression of microtubule-related genes in the recovery step. We further identified AsKIN as a hub gene in the recovery step and verified its function. The results showed that overexpression of AsKIN enhanced the tolerance of Arabidopsis to oxidative stress in cryopreservation, influenced the expression of genes in response to cold and osmotic stress and promoted plant growth after stress. The AsKIN gene is likely to be involved in the plant response to cold stress and osmotic stress. These results reveal the molecular mechanisms of ABA in cryopreservation and elucidate the potential biological functions of the kinesin-14 subfamily.

PB2187: VIABILITY AND PROLIFERATIVE POTENTIAL OF AUTOGRAFT HEMATOPOIETIC STEM CELLS

Background: Autograft cellularity is a fundamental condition for the timing of hematopoiesis recovery in patients with multiple myeloma (MM) after autologous hematopoietic stem cell transplantation (AutoSCT). Taking the number of CD34+ cells in a volume of 2x10/6/kg of the patient’s weight as a suboptimal value that provides fast and reliable graft engraftment, it remains unclear whether this number will be sufficient for subsequent transplantation, if tandem AutoHSCT assumes an interval of 6 months, and repeated - not earlier 36 months. Aims: Determination of the preservation of colony-forming (CFU) and CD34+ cells in the autograft of MM patients depending on the terms of their cryopreservation. Methods: A retrospective analysis of the results of the study of 127 samples of the product of leukocytapheresis was performed. The prepared cell suspension was placed in disposable CryoMacs Freesing bags and mixed with dimethyl sulfoxide to its final concentration of 7.5%. Freezing was carried out in a Cryo 560-16 Planer RLC programmed freezer. Containers with cell suspension were stored in liquid nitrogen. To assess the effect of the duration of the cryopreservation period on CFU, the numerical values of each sample were converted into percentages: the primary CFU values from each sample were taken as 100%, and the CFU values after the cryopreservation period were compared with them. Results: Depending on the terms of cryostorage, 3 groups were formed. Group 1: 83 samples, shelf life from 1 to 5.9 months. Group 2: 34 samples, shelf life from 6 to 10 months. Group 3: 10 samples, shelf life over 10 months. A trend towards a decrease in the number of colonies was revealed as the duration of the cryopreservation period increased. Thus, the number of CFU-totals decreased from 66% recorded after 1–5.9 months of storage, to 60% after 6–10 months and to 44% of the original number when stored for more than 10 months; p=0.131. The decrease in the number of colony-forming cells of granulomonocytopoiesis was distinct in cell suspension samples stored for more than 10 months, but also did not reach significant values: 64%, 58% and 37%, respectively; p=0.451. On the contrary, the decrease in the number of burst-forming cells of erythrocytopoiesis (BFU-E) after 10 months of cryostorage was significantly lower than at earlier periods of cell suspension thawing. So if the content of BFU-E after 1–5.9 and 6–10 months of storage was 65% and 57% of the initial amount, respectively, then after 10 months it was only 26%; p=0.01. When analyzing the preservation of CD34+ cells, a tendency was also found to decrease in their percentage as the duration of cryopreservation increased, but the difference did not reach a significant value: 77% during thawing after 1–5.9 months of cryopreservation, 49% after 6–10 months and 39% in case of cell suspension thawing after 10 months of storage; p=0.465. Despite the fact that in some patients with the maximum terms of cell suspension cryopreservation, the need for a larger number of doses of donor erythrocytes was recorded and there were longer terms for platelet recovery than in patients in the other two groups, the median values of the analyzed parameters did not differ. Summary/Conclusion: When planning the first and subsequent, repeated, AutoHSCT in MM patients, it is advisable to prepare an autograft with a greater than suboptimal cellularity due to a possible decrease in the number of CFU during long-term cryostorage of the cell suspension.

β-Estradiol 17-acetate enhances the in vitro vitality of endothelial cells isolated from the brain of patients subjected to neurosurgery.

In the current landscape of endothelial cell isolation for building in vitro models of the blood-brain barrier, our work moves towards reproducing the features of the neurovascular unit to achieve glial compliance through an innovative biomimetic coating technology for brain chronic implants. We hypothesized that the autologous origin of human brain microvascular endothelial cells (hBMECs) is the first requirement for the suitable coating to prevent the glial inflammatory response triggered by foreign neuroprosthetics. Therefore, this study established a new procedure to preserve the in vitro viability of hBMECs isolated from gray and white matter specimens taken from neurosurgery patients. Culturing adult hBMECs is generally considered a challenging task due to the difficult survival ex vivo and progressive reduction in proliferation of these cells. The addition of 10 nM β-estradiol 17-acetate to the hBMEC culture medium was found to be an essential and discriminating factor promoting adhesion and proliferation both after isolation and thawing, supporting the well-known protective role played by estrogens on microvessels. In particular, β-estradiol 17-acetate was critical for both freshly isolated and thawed female-derived hBMECs, while it was not necessary for freshly isolated male-derived hBMECs; however, it did counteract the decay in the viability of the latter after thawing. The tumor-free hBMECs were thus cultured for up to 2 months and their growth efficiency was assessed before and after two periods of cryopreservation. Despite the thermal stress, the hBMECs remained viable and suitable for re-freezing and storage for several months. This approach increasing in vitro viability of hBMECs opens new perspectives for the use of cryopreserved autologous hBMECs as biomimetic therapeutic tools, offering the potential to avoid additional surgical sampling for each patient.

Long-term cryopreservation of Lentinus crinitus strains by wheat grain technique

Lentinus crinitus (Basidiomycota: Polyporales) is a saprophytic fungus with biotechnological importance described more than 20 years ago. However, there are few studies on the long-term preservation of this basidiomycete. Cryopreservation is a long-term storage technique that reduces the metabolic activity of microorganisms, but its success depends on the adjustment of the freezing process, the cryoprotectants, and the protective substrates for each species. This study aimed to assess the mycelial viability and genetic stability of L. crinitus strains cryopreserved at −86 °C for two years by the wheat grain technique using different cryoprotectants and freezing methods. Three strains of L. crinitus (U9–1, U13–5, and U15–12) were subjected to different concentrations and types of cryoprotectants (dimethyl sulfoxide, glycerol, glucose, and sucrose), freezing methods such as immediate freezing from 25 to −86 °C and progressing freezing from 25 to −86 °C in a freezing container with isopropyl alcohol to control the rate of cell freezing at −1 °C min−1, protective substrate (wheat grain and 2% malt extract agar), and cryopreservation period (1, 6, 12, and 24 months). After thawing, samples were evaluated for mycelial viability, time to mycelial recovery, mycelial stability, and genetic stability of the fungus. All techniques achieved effective cryopreservation at −86 °C, mainly with the wheat grain technique. All cryoprotectants (3.5% glycerol, 1.5% dimethyl sulfoxide, 25% sucrose, and 5% glucose), freezing methods (immediate and gradual), and protective substrate (wheat grain and malt extract agar) were effective for cryopreservation of the three L.crinitus strains in an ultra-low temperature freezer for two years. Mycelial viability, mycelial stability, and genetic stability of the fungus were not affected after two-year cryopreservation, evidencing the robustness of the long-term cryopreservation technique and the fungus.

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation.

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible source for regenerative clinical applications. Different protocols have been used to obtain adipose-derived stem cells. This article describes different steps of an improved time-saving protocol to obtain a more significant amount of ADSC, showing how to cryopreserve and thaw ADSC to obtain viable cells for culture expansion. One hundred milliliters of lipoaspirate were collected, using a 26 cm three-hole and 3 mm caliber syringe liposuction, from the abdominal area of nine patients who subsequently underwent elective abdominoplasty. The stem cells isolation was carried out with a series of washes with Dulbecco's Phosphate Buffered Saline (DPBS) solution supplemented with calcium and the use of collagenase. Stromal Vascular Fraction (SVF) cells were cryopreserved, and their viability was checked by immunophenotyping. The SVF cellular yield was 15.7 x 105 cells/mL, ranging between 6.1-26.2 cells/mL. Adherent SVF cells reached confluence after an average of 7.5 (±4.5) days, with an average cellular yield of 12.3 (± 5.7) x 105 cells/mL. The viability of thawed SVF after 8 months, 1 year, and 2 years ranged between 23.06%-72.34% with an average of 47.7% (±24.64) with the lowest viability correlating with cases of two-year freezing. The use of DPBS solution supplemented with calcium and bag resting times for fat precipitation with a shorter time of collagenase digestion resulted in an increased stem cell final cellular yield. The detailed procedure for obtaining high yields of viable stem cells was more efficient regarding time and cellular yield than the techniques from previous studies. Even after a long period of cryopreservation, viable ADSC cells were found in the SVF.

Characteristics of mouse embryonic fibroblasts by cryopreservation period for tissue engineering

Characteristics of mouse embryonic fibroblasts by cryopreservation period for tissue engineering

The cytokinesis-block micronucleus assay for cryopreserved whole blood

Purpose The cytokinesis-block micronucleus (MN) assay is a widely used technique in basic radiobiology research, human biomonitoring studies and in vitro radiosensitivity testing. Fresh whole blood cultures are commonly used for these purposes, but immediate processing of fresh samples can be logistically challenging. Therefore, we aimed at establishing a protocol for the MN assay on cryopreserved whole blood, followed by a thorough evaluation of the reliability of this assay for use in radiosensitivity assessment in patients. Materials and methods Whole blood samples of 20 healthy donors and 4 patients with a primary immunodeficiency disease (PID) were collected to compare the results obtained with the MN assay performed on fresh versus cryopreserved whole blood samples. MN yields were scored after irradiation with 220 kV X-rays (dose rate 3 Gy/min), with doses ranging from 0.5–2 Gy. Results The application of the MN assay on cryopreserved blood samples was successful in all analyzed samples. The radiation-induced MN and NDI scores in fresh and cryopreserved blood cultures were found to be similar. Acceptable inter-individual and intra-individual variabilities in MN yields were observed. Repeated analysis of cryopreserved blood cultures originating from the same blood sample, thawed at different time points, revealed that MN values remain stable for cryopreservation periods up to one year. Finally, radiosensitive patients were successfully identified using the MN assay on cryopreserved samples. Conclusions To our knowledge, this study is the first report of the successful use of cryopreserved whole blood samples for application of the MN assay. The data presented here demonstrate that the MN assay performed on cryopreserved whole blood is reliable for radiosensitivity testing. Our results also support its wider use in epidemiological, biomonitoring and genotoxicity studies. The presented method of cryopreservation of blood samples might also benefit other assays.

Inactivated Nevus Tissue with High Hydrostatic Pressure Treatment Used as a Dermal Substitute after a 28-Day Cryopreservation Period.

Background Giant congenital melanocytic nevi (GCMN) treatment remains controversial. While surgical resection is the best option for complete removal, skin shortage to reconstruct the skin defect remains an issue. We report a novel treatment using a high hydrostatic pressurization (HHP) technique and a cryopreservation procedure. However, cryopreservation may inhibit revascularization of implanted nevus tissue and cultured epidermal autograft (CEA) take. We aimed to investigate the influence of the cryopreservation procedure on the HHP-treated dermis specimen and CEA take on cryopreserved tissue. Methods Nevus tissue harvested from a patient with GCMN was inactivated with HHP of 200 MPa and then cryopreserved at -30°C for 28 days. The cryopreserved specimen was compared with fresh (HHP-treated without cryopreservation) tissue and with untreated (without HHP treatment) tissue to evaluate the extracellular matrix, basal membranes, and capillaries. Cultured epidermis (CE) take on the cryopreserved tissue was evaluated following implantation of the cryopreserved nevus tissue with CE into the subcutis of nude mice. Results No difference was observed between cryopreserved and fresh tissue in terms of collagen or elastic fibers, dermal capillaries, or basement membranes at the epidermal-dermal junction. In 4 of 6 samples (67%), applied CE took on the nevus tissues and regenerated the epidermis in the cryopreserved group compared with 5 of 6 samples (83%) in the fresh group. Conclusion Cryopreservation at -30°C for 28 days did not result in significant damage to inactivated nevus tissue, and applied CE on the cryopreserved nevus tissues took and regenerated the epidermis. Inactivated nevus tissue with HHP can be used as a dermal substitute after 28-day cryopreservation.

Total nucleated cell counts are driving clinician's choice rather than cryopreservation period: Lesson for cord blood banks

According to the increase in both the number of cryopreserved cord blood (CB) units and the cryopreservation period for each CB unit in the largest public CB bank in Korea, we are pursuing greater efficiency in CB bank management. Thus, we analyzed whether the cryopreservation period has a negative impact on the selection of CB units for CB transplantation (CBT). Until December 2019, 468 CB units were used for transplantation. The cryopreservation period, total nucleated cell (TNC), and CD34+ cell counts were analyzed among the CB units according to the CBT-year and the donation year. The results showed that the cryopreservation period was increased in recent CBT-year groups. The transplanted CB units showed similar TNC counts irrespective of the donation year, and the mean TNC count was 13.9 × 108/unit. CB units cryopreserved for a relatively long period were transplanted consistently. The mean TNC count of CB units cryopreserved for over 10 years was 16.4 × 108/unit. The mean CD34+ cell counts were not significantly different among the CB units transplanted after CBT-2013 and among the CB units donated after CBT-2011. Through an analysis of the CB units selected by clinicians for CBT, this study revealed that clinicians placed more weight on the TNC counts than on the cryopreservation period of cryopreserved CB units. Therefore, the minimum TNC count of CB units suitable for cryopreservation should be increased up to 13.0 × 108/unit to balance the satisfaction of clinicians’ needs with the efficiency of the CB bank.

Human sperm cryopreservation prior cancer treatment: Follow up of 480 males for 23 years.

e24061 Background: Cancer treatment represents a potential risk for spermatogenesis and may compromise male fertility. Semen freezing has become a routine process for male patients prior to cancer treatment to preserve their fertility. We sought to provide information regarding what happens to the cryopreserved material and the fertility in cancer treated patients. Using a cohort from a 23-year cryopreservation program. Methods: Patients whom cryopreserved sperm to preserve fertility due to diagnosis of cancer between 1995 and 2018 in a single private center of reproductive medicine in south of Brazil were selected. A retrospective analysis of medical records was performed and clinico-pathological characteristics were collected using standard templates. Results: A total of 480 patients were evaluated for sperm cryopreservation before cancer treatment. Testis cancer was the most prevalent (59.3%), followed by hematologic malignancies (21.4%) and prostate cancer (5.4%). The age at cryopreservation varied from 14 to 72 y/o. Among them, 61.3% were up to 30 years old, and 10.8% more than 40 y/o. 8 patients decided to not cryopreserve The mean time between the freezing and the beginning of cancer treatment was 5.5 days. Eleven patients presented azoospermia; 3 of them froze testicular tissue. A shorter time between cancer diagnosis and sperm banking was observed for hematologic malignancies ( p=0.044). Out of the 472 patients, 46.8% still keep the sperm cryopreserved, 34.3% decided to discard it, 1.9% transferred it to another clinic, 8,5% abandon the specimen and 8,5% died without using it. The main reason for discard the sample was pregnancy and/or normal sperm count after treatment. The mean age of discarding was 35.7±10.3 years old. The period of maintenance cryopreservation was 5 years (min 1 month, max 23 years). The mean time of freezing before abandoning was 7.5±4.4 years Eighty-two patients achieved pregnancy. Spontaneous pregnancies occurred in 53 cases, and 47 underwent assisted reproduction with the cryopreserved sample, with 61,7% of pregnancy (n=29).15.3% of those that have children still keep the sperm cryopreserved; 11.4% gave up on having children. Out of the 133, 66.9% authorized the use of their specimen post-mortem. Conclusions: Only one third of the patients have maintained fertility after cancer treatment and 35% of pregnancies after cancer treatment were possible due to sperm preservation. Considering the high pregnancy rate with assisted reproduction techniques in patients that lost testicular functionsemen cryopreservation is warranted.