Short-term Cryopreservation Research Articles

IPPRAS Cryobank for the Conservation of Orthodox Seeds of Rare, Endangered, Medicinal, and Ornamental Plant Species-Current Research.

Plant cryobanks play a significant role in modern science and breeding. They contribute to the recovery of lost species, the emergence of new plant varieties, and help preserve and explore the diversity of the plant world. The IPPRAS Cryobank collection is constantly supplemented with new samples, while, at the same time, the stored samples are being monitored. In order to test seed germination, seeds of Allium and Veratrum species were thawed. Rare Allium species seeds, such as A. nutans, A. schoenoprasum, and A. victorialis were stored in liquid nitrogen for 17, 19, and 30 years, respectively. Long-term cryopreservation decreased germination rates for A. nutans from 96.55 to 50.00%, for A. schoenoprasum from 72.00 to 62.75%, and for A. victorialis from 90.00 to 83.05%. Seeds of a rare medicinal species, Veratrum lobelianum, were stored in liquid nitrogen for 18 years; the seed germination rate during this storage period has been significantly decreased from 75.00 to 14.81%. V. nigrum seeds were also collected and frozen in liquid nitrogen for 3 days. Short-term cryopreservation did not result in a statistically significant change in germination rates (from 79.71 to 82.69%). The seeds of an endangered ornamental species, Cypripedium calceolus, were collected and kept frozen for 3 days. After cryopreservation, the seeds were planted on three different media, as follows: ½ MS, MS with 10% coconut milk, and BM1. On ½ MS medium, 24.98% seeds formed protocorms, while on MS medium with 10% coconut milk, this number was 10.02%, and on BM1 medium, it was 15.02%, respectively; however, after 2.5 months, all of the protocorms died. Thus, it appears that the existing protocol for seed cryopreservation of C. calceolus needs further improvement. The size, weight, and free water content (WC) of six previously cryopreserved Stipa species and three Allium species were measured. For all the Allium and Stipa species studied, we found no correlation between seed size, WC, and cryotolerance. We also found no correlation between the life form, which reflects the water requirement of the species, and cryotolerance.

Impact of long- and short-term cryopreservation on intactness of precision cut lung slices

Impact of long- and short-term cryopreservation on intactness of precision cut lung slices

Long-Term Cryopreservation May Cause Genomic Instability and the Premature Senescence of Cells.

Cryopreservation is an essential step for utilizing various cell types for biological research and medical purposes. At the same time, there is a lack of data on the effect of cryopreservation, especially when prolonged, on the karyotype of cells. In the present work, we analyzed the genetic stability of cells subjected to a cryopreservation procedure. The objects were immortalized Chinese hamster lung fibroblasts (CHL V-79 RJK line) and human endometrial mesenchymal stem/stromal cells (eMSCs). We showed that short-term cryopreservation in liquid nitrogen for up to 6 months did not affect the karyotype stability of CHL V-79 RJK and eMSCs. On the contrary, karyotyping of G-banded metaphase chromosomes in cells underwent 10-year cryopreservation, which revealed genomic instability in both cell lines associated with the variability of chromosome number in cells, random chromosomal rearrangements, and condensation disorder in homologs. In addition, we found out that long-term cryopreservation of eMSCs does not affect the expression of their typical surface markers and morphology, but results in a significant reduction in proliferative potential and early manifestation of cellular senescence features upon eMSCs culturing. Thus, we concluded that the long-term cryopreservation of cells of different types and biological origin can lead to irreversible changes of their karyotype and acceleration of cellular senescence.

Evaluation of Seed Germination of Six Rare Stipa Species following Low Temperature Stress (Cryopreservation).

Cryopreservation is one way to preserve rare, endangered species. However, during the cryopreservation process, plant cells undergo considerable stress, which may lead to cell death. In our work, orthodox Stipa seeds of six rare species were cryopreserved: S. sareptana, S. ucrainica, S. tirsa, S. dasyphylla, S. adoxa, and S. pulcherríma. Short-term cryopreservation (14 days) stimulated germination of all Stipa species studied. Prolonged cryopreservation (70 days and more) decreased the germination of all Stipa seeds except S. sareptana. The decrease in germination progressed over time as a result of the cumulative stress of cryopreservation rather than the initial stress. To stimulate germination, seeds were stratified and treated with GA3, KNO3, NaOH, and H2O2. After four years of seed cryopreservation, it was possible to obtain seedlings of all the Stipa species studied with 30 days of stratification and 180 days of germination. After five years of cryopreservation and seed treatment with 30% NaOH for one hour, the best germination was obtained in S. adoxa and S. pulcherrima. After treatment with 5% H2O2 for 20 min, the best germination was obtained in S. sareptana, S. ucrainica, and S. dasyphylla. S. sareptana seeds germinated in all the aforementioned experiments. S. sareptana has a non-deep physiological dormancy and is the most widespread and drought-tolerant Stipa species studied. The best habitat adaptation and stress tolerance correlated with this species'cryotolerance. S. sareptana was recommended for further cryopreservation, while storage protocols for the other Stipa species studied need further improvements.

Exploring the feasibility of cryopreserving larvae of the common cockle (Cerastoderma edule) for hatchery production

The decline of natural populations of the common cockle (Cerastoderma edule) through the European coast is posing a threat to local small-scale fisheries. These declines are primarily attributed to the prevalence of several pathogens and the disseminated neoplasia in cockle populations. The institution of a biobank of cryopreserved larvae could enhance hatchery production and help the restocking. The present work aimed at the development of a cryopreservation protocol for larvae of the common cockle using the mollusk cryopreservation protocols designed in our laboratory. Toxicity bioassays and short-term cryopreservation experiments were performed for protocol optimization according with cellular tolerance. Once settled, the viability of cryopreserved larvae was studied long term. Toxicity tests evidenced high tolerance of larvae against detrimental effects of Cryoprotecting Agents (CPAs). Cryopreservation of 48 h-old D-larva showed a 100% survival when increasing the equilibrium time from 15 to 60 min and using Propylene-Glycol (PG) + 0.4 M Trehalose (TRE) in Filtered Sea Water (FSW) and 60 min of exposure to CPA solution before slow-cooling. However, when cryopreserving the older larvae, the variation in equilibrium times hardly showed any effect but 10% Ethylene-Glycol (EG) + 0.4 M TRE and 60 min of exposure yielded the best relative survivorship (100%). Cryopreservation caused a significant delay on the growth rate of the latest larval stage. However, cryopreserved larvae survived to day 4–6, while 30 ± 12.17% of control larvae developed into pediveliger stage, of which 50% settled and transformed into juvenile cockles. These results demonstrated the role of the cell-type specificity in cryopreservation and highlight the importance of studying potential long-term effects of this tool to ensure the viability of the protocols.

The effect of short-term cryopreservation on the properties and functionality of platelet-rich plasma

Platelet Rich Plasma (PRP) is a biological treatment which, thanks to its enhanced growth factors content, is widely used in the field of regenerative medicine for its reparative effects. Although it is usually used fresh immediately after preparation, its freezing for preservation for future usage could be key in increasing its versatility and new applications. To assess the suitability of freezing, after collecting PRP and platelet lysates (PL) from 6 patients, they were preserved for 1 or 3 months at temperatures of −20ºC and −80°C. Measurements were then made on platelet number and integrity, growth factor levels, biomechanical properties of the clot and its bioactivity on cultured cells. Fresh PRP and PL were used as controls. The results showed an increase in platelet size (p < .01) and clot elasticity (p < .01), as well as decrease in levels of PDGF (P < .05) and VEGF (p < .05), though the overall bioactivity was not affected as culture cells showed the same responsiveness to both frozen and fresh PRP and PL in terms of cell viability. Based on these results, it could be assumed that preservation of PRP by freezing is a feasible and suitable option for its further use.

Effect of Cryoprotectant and Pre-Freezing on the Sperm Motility, Viability and Fertility of Goldfish Carassius Auratus (Pisces: Cyprinidae) Post Short-Term Cryopreservation

BACKGRUND: Goldfish Carassius auratus is a popular ornamental fish extensively cultured worldwide. Sperm cryopreservation is a common fish breeding method that ensures sperm availability around the year. Studies on cryopreservation of goldfish sperm, especially on the suitability of cryoprotectant types and pre-freezing time, are scarcely available. OBJECTIVE: To determine the most suitable type of cryoprotectant and pre-freezing for the successful cryopreservation of goldfish sperm. MATERIALS AND METHODS: A completely randomized design with two factors was utilized in this study. The first factor is the type of cryoprotectants, which included methanol, ethanol, ethylene glycol, glycerol, and DMSO. The second is pre-freezing times of 10, 20, 30, and 40 min at each of the pre- freezing temperatures of 4°C, −10°C, and −79°C, meaning that the total times for the ramping down of temperature were 30, 60, 90 and 120 min, respectively. The Ringer solution and 10% egg yolk were used as extender and extracellular cryoprotectant. The sperm was stored at −179°C for 7 days. RESULTS: The ANOVA test showed that cryoprotectants and pre-freezing significantly affected the motility, viability, and fertility of goldfish sperm after freezing in liquid nitrogen for 7 days (ρ&lt;0.05). Furthermore, 10% DMSO combined with 15% egg yolk with an pre-freezing time of 20 min can maintain sperm motility, viability, and fertility higher than other treatments, by 79%, 80%, and 33%, respectively. The agarose gel electrophoresis showed no DNA fragmentation in all samples, including fresh sperm. CONCLUSION: We conclude that 10% DMSO combined with 15% egg yolk and 20 min pre-freezing is the best treatment for goldfish sperm cryopreservation.

Banking of perinatal mesenchymal stem/stromal cells for stem cell-based personalized medicine over lifetime: Matters arising.

Mesenchymal stromal/stem cells (MSCs) are currently applied in regenerative medicine and tissue engineering. Numerous clinical studies have indicated that MSCs from different tissue sources can provide therapeutic benefits for patients. MSCs derived from either human adult or perinatal tissues have their own unique advantages in their medical practices. Usually, clinical studies are conducted by using of cultured MSCs after thawing or short-term cryopreserved-then-thawed MSCs prior to administration for the treatment of a wide range of diseases and medical disorders. Currently, cryogenically banking perinatal MSCs for potential personalized medicine for later use in lifetime has raised growing interest in China as well as in many other countries. Meanwhile, this has led to questions regarding the availability, stability, consistency, multipotency, and therapeutic efficiency of the potential perinatal MSC-derived therapeutic products after long-term cryostorage. This opinion review does not minimize any therapeutic benefit of perinatal MSCs in many diseases after short-term cryopreservation. This article mainly describes what is known about banking perinatal MSCs in China and, importantly, it is to recognize the limitation and uncertainty of the perinatal MSCs stored in cryobanks for stem cell medical treatments in whole life. This article also provides several recommendations for banking of perinatal MSCs for potentially future personalized medicine, albeit it is impossible to anticipate whether the donor will benefit from banked MSCs during her/his lifetime.

Short-term cryopreservation and thawing have minimal effects on Plasmodium falciparum ex vivo invasion profile.

Ex vivo phenotyping of P. falciparum erythrocyte invasion diversity is important in the identification and down selection of potential malaria vaccine targets. However, due to the lack of appropriate laboratory facilities in remote areas of endemic countries, direct processing of P. falciparum clinical isolates is usually not feasible. Here, we investigated the combined effect of short-term cryopreservation and thawing processes on the ex vivo invasion phenotypes of P. falciparum isolates. Ex-vivo or in vitro invasion phenotyping assays were performed with P. falciparum clinical isolates prior to or following culture adaptation, respectively. All isolates were genotyped at Day 0 for parasite clonality. Subsequently, isolates that were successfully culture-adapted were genotyped again at Days 7, 15, 21, and 28-post adaptation. Invasion phenotyping assays were performed in isogenic isolates revived at different time points (3, 6, and 12 months) post-cryopreservation and the resulting data were compared to that from ex-vivo invasion data of matched isogenic parental isolates. We also show that short-term culture adaptation selects for parasite clonality and could be a driving force for variation in invasion phenotypes as compared to ex vivo data where almost all parasite clones of a given isolate are present. Interestingly, our data show little variation in the parasites’ invasion phenotype following short-term cryopreservation. Altogether, our data suggest that short-term cryopreservation of uncultured P. falciparum clinical isolates is a reliable mechanism for storing parasites for future use.

SVF-GEL Cryopreserved for Different Times Exhibits Varied Preservation and Regeneration Potential After Transplantation in a Mouse Model

BackgroundMatrix vascular component (SVF) gels derived from fat preserve tissue integrity and cell viability under cryopreserved conditions, making them easy to inject again for later use. Here, we compared the preservation power and regeneration potential of SVF-gel under different cryopreservation times.MethodsThe SVF-gel stored under − 20 °C, without cryoprotectant cryopreservation for 5, 15, and 45 days, with fresh SVF-gel as control. We evaluated the rate of volume retention after thawing the SVF-gel and the apoptosis rate of adipose-derived stem cells. Next, we analyzed retention rated, adipogenesis, angiogenesis, and connective tissue hyperplasia of the grafts, one month after subcutaneously transplanting the specimen into immunodeficient mice.ResultsSVF-gel cryopreserved for 5 and 15 days exhibited no significant different in apoptosis rates relative to the control group. Extending the cryopreservation time to 45 days resulted in significantly increased and decreased apoptosis and volume retention rates of SVF-gel, respectively. SVF-gel grafts cryopreserved for 5 and 15 days exhibited no significant differences from those in the control group, although their weights and volumes still fluctuated. Extending the cryopreservation time to 45 days resulted in significantly decreased retention rates of the grafts. Histologically, extending freezing time resulted in a gradual decline in the graft’s health adipose tissue, as well as decreased angiogenesis, and connective tissue hyperplasia.ConclusionSimple freezing of SVF-gel at − 20 °C conferred them with sufficient cell viability. Notably, short-term cryopreservation did not significantly increase the apoptosis rate, and it still had a certain regeneration after transplantation. However, prolonging freezing time to 45 days resulted in increased apoptosis rate and worsened transplantation effect.No Level AssignedThis journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Effects of vitrified cryopreservation duration on IVF and neonatal outcomes

BackgroundIn this study, we aimed to evaluate the impact of the duration of cryopreservation storage on embryo viability, implantation competence, pregnancy outcome and neonatal outcomes.MethodsWe retrospectively evaluated the outcomes of patients who underwent IVF with vitrified cryopreserved embryos between January 2004 and August 2019 by following the first frozen embryo transfer cycles within the study period. A total of 31,143 patients met the inclusion criteria and were grouped according to the embryo storage time as follows: Group 1 (n = 20,926),1–90 days; Group 2 (n = 6,472), 91–180 days; Group 3 (n = 2,237), 181–365 days; Group 4 (n = 746), 366–730 days; and Group 5 (n = 762), > 731 days.ResultsThe embryo survival rate decreased significantly with longer durations of cryopreservation. The highest and lowest survival rate was recorded in Group 1 and Group 5, respectively (34853/35338; 98.63% vs. 1281/1801; 71.13%; P < 0.01). The human chorionic gonadotropin (HCG) detection and clinical pregnancy rate was highest in Group 1 (57.85% and 55. 26%, respectively; P < 0.01). Short-term cryopreservation (≤ 3 months) is associated with higher rates of clinical pregnancy. There were no significant differences in neonatal birth weight, neonatal height and congenital anomalies among the groups (P > 0. 05).ConclusionThe prolonged storage time of vitrified embryos negatively affected survival rate and clinical pregnancy rate. It did not have a significant influence on neonatal health. This study provides new findings about the relationship between prolonged storage time of vitrified embryos and clinical outcomes and offers evidence for the safety of using long-stored embryos after vitrification.

Epidemiological investigation and reinfection evaluation of Toxoplasma gondii in chickens in Shandong Province, China

Toxoplasma gondii infects almost all warm-blooded animals, and toxoplasmosis is a common zoonotic parasitic disease worldwide. A nested PCR with high specificity and sensitivity was developed in this study based on the data collected on the infection rate of toxoplasmosis in chickens in Shandong province, and the effect of low temperature on the infectivity of tachyzoites was investigated. The sampling data showed that the total prevalence of T. gondii in Shandong province was 12.3%, and the positive rate varied in different regions, ranging from 6.7% to 21%. Chickens were infected with T. gondii under laboratory conditions, and positive chicken hearts were stored under various cold conditions to infect mice for reinfection evaluation. The results demonstrated that the parasites maintained high infectivity in mice even after 6 h of storage at −20 °C ambient temperature, indicating that short-term cryopreservation is not effective in reducing the risk of T. gondii transmission. These results form the basis for assessing the risk of toxoplasmosis contamination in consumed chicken products and provide information on the prevention of parasite transmission from animals to humans.

Effects of Different Cryoprotectants, Cryoprotectant Concentrations and Extenders for Short Term Cryopreservation of African Catfish Clarias gariepinus (Burchell, 1822) Milt

Abstract Aquaculture of Clarias gariepinus is on the increase in Africa. Among the challenges to its production is the scarcity and high cost of broodstocks and sacrifice of the male during induced breeding. Cryopreservation of the milt of the species is a solution to this challenge. However, cryopreservation success depends on right extender, cryoprotectant and concentration. Phosphate buffered solution (PBS) and freshwater ringer solution (extenders), Dimethyl sulphoxide (DMSO), glycerol and glucose (cryoprotectants) at 5% and 10% concentrations were used to investigate the best cryoprotectants, cryoprotectant concentrations and extenders for short term cryopreservation of Clarias gariepinus milt. Control using fresh milt was also set up. Fifteen each of matured male and female broodstocks of C. gariepinus, weighing between 1.1 – 2.0kg were used for the experiment. analysis of variance (ANOVA), followed by comparison of means using Duncan’s Multiple Range Test. Percentage data were calculated prior to analysis. All statistical analyses were performed using SPSS software (Version 16.0, 2005). Dimethyl sulphoxide + phosphate buffered solution (DP) had the highest fertilization rate of 65% and 78% for 5 and 10% cryoprotectant concentrations respectively. Lowest fertilization rate was in glucose + freshwater ringer solution (GF) with 5 and 10% concentration giving 5% and 8% fertilization. Fertilization in control was 88%. Hatching and survival followed fertilization trend with DP 10% &gt; DP 5% &gt; DF 10% &gt; GLP 10% &gt; DF 5% &gt; GLP 5 % &gt; GLF 10% &gt; GLF 5% &gt; GP 10% &gt; GP 5% &gt; GF 10% &gt; GF 5%. Good quality sperm used, non-toxicity and high permeability of DMSO, osmolality effect of PBS on milt were some of the factors responsible for the success of the cryopreservation. 10% DMSO and PBS are the best cryoprotectant and extender for short term cryopreservation of African catfish milt for at least 5 days.

Effect of Fetal Bovine Serum on the Sperm Quality of Depik (Rasbora tawarensis) after Short-term Cryopreservation

Background: Sperm cells are susceptible to oxidative stress during cryopreservation. Therefore, an antioxidant is necessary to protect them from damages. Fetal bovine serum (FBS) is one of potent antioxidants for fish sperm cryopreservation. Hence, the aims of this study are to examine the effect of FBS on sperm quality after a short period and to determine its optimum concentration on depik (Rasbora tawarensis). Methods: Depik fish were obtained from the Fish hatchery of Lukup Badak, Aceh Tengah District, Indonesia. Sperms collected from the fish were diluted in Ringer extenders containing FBS concentration of 10% (P1), 20% (P2), 30% (P3), 40% (P4), 50% (P5) and 60% (P6), filled into 2 ml cryotubes and equilibrated prior immersed into liquid nitrogen for 15 days. The parameters observed were sperm motility, consistency, pH, fertilization and hatching rates and DNA fragmentation post-thawing. Result: The ANOVA test indicates that the application of FBS in Ringer had a significant effect on sperm motility, fertilization and hatching rates (P less than 0.05). The highest motility (58.33%) was recorded at FBS 60% and significantly different from those at other concentrations. The laddering analysis showed that applying FBS protected the integrity of depik sperms DNA. It is concluded that the optimum concentration of FBS on depik sperm led to a short-term cryopreservation of 60%.

Long-term preservation of modern Russian potato cultivars in the VIR cryobank

Cryopreservation of shoot-tips (apexes) excised from in vitro plants is used for long-term preservation of potato cultivars. The most widely used method for creating potato cryo-collections is droplet-vitrification of shoot-tips which, together with its numerous modifications, is widely used in genebanks. A modified protocol of droplet vitrification method is used at VIR for cryopreservation of shoot tips from in vitro potato plants. This paper presents the results of cryopreservation of modern cultivars which were released by seven Russian breeding centers. In vitro clones used in the cryopreservation experiments were genetically identical to the cultivars’ nomenclatural standards and herbarium vouchers.The frequency of post-thaw regeneration in control experiments after short-term cryopreservation varied from 23.3 to 53.3%, depending on the genotype. Five out of 16 accessions (‘Varâg’, ‘Gusar’, ‘Evpatij’, ‘Solnečnyj’, ‘Tango’)* had low post-cryogenic regenerative capacity from 20 to 30%; the regeneration rate exceeded 30% in 11 accessions, and 8 cultivars (‘Grand’, ‘Zlatka’, ‘Lina’, ‘Safo’, ‘Siverskij’, ‘Signal’, ‘Utro’, ‘Ûna’) and ‘Аlyj Parus’ breeding clone had regeneration rate above 40%. The regeneration rate in the studied subset was genotype independent according to the ANOVA results (p=0.711). Viability and regeneration rate were significantly correlated (r=0.86). As a result of the experiments, explants of 14 modern cultivars and two breeding clones with the known post-thaw regeneration rate were successfully cryopreserved in the VIR cryobank. Four cultivars (‘Grand’, ‘Gusar’, ‘Signal’, ‘Utro’) were monitored for their regeneration capacity after the long-term (seven months) preservation in the VIR cryobank. On an average, these four cultivars demonstrated a post-thaw regeneration capacity of 41.8%. It can be concluded that the use of the modified method of droplet vitrification is relevant for increasing the VIR potato cryo-collection.

Effects of Cryopreservation on Cell Metabolic Activity and Function of Biofabricated Structures Laden with Osteoblasts.

Biofabrication and maturation of bone constructs is a long-term task that requires a high degree of specialization. This specialization falls onto the hierarchy complexity of the bone tissue that limits the transfer of this technology to the clinic. This work studied the effects of the short-term cryopreservation on biofabricated osteoblast-containing structures, with the final aim to make them steadily available in biobanks. The biological responses studied include the osteoblast post-thawing metabolic activity and the recovery of the osteoblastic function of 3D-bioprinted osteoblastic structures and beta tricalcium phosphate (β-TCP) scaffolds infiltrated with osteoblasts encapsulated in a hydrogel. The obtained structures were cryopreserved at −80 °C for 7 days using dimethyl sulfoxide (DMSO) as cryoprotectant additive. After thawing the structures were cultured up to 14 days. The results revealed fundamental biological aspects for the successful cryopreservation of osteoblast constructs. In summary, immature osteoblasts take longer to recover than mature osteoblasts. The pre-cryopreservation culture period had an important effect on the metabolic activity and function maintain, faster recovering normal values when cryopreserved after longer-term culture (7 days). The use of β-TCP scaffolds further improved the osteoblast survival after cryopreservation, resulting in similar levels of alkaline phosphatase activity in comparison with the non-preserved structures. These results contribute to the understanding of the biology of cryopreserved osteoblast constructs, approaching biofabrication to the clinical practice.

SUL-109 Protects Hematopoietic Stem Cells from Apoptosis Induced by Short-Term Hypothermic Preservation and Maintains Their Engraftment Potential.

The newly developed 6-hydroxychromanol derivate SUL-109 was shown to provide protection during hypothermic storage of several cell lines, but has not been evaluated in hematopoietic stem cells (HSCs). Hypothermic preservation of HSCs would be preferred over short-term cryopreservation to prevent cell loss during freezing/thawing and would be particularly useful for short-term storage, such as during conditioning of patients or transport of HSC transplants. Here we cultured human CD34+ umbilical cord blood (UCB) cells and lineage-depleted (Lin-) Balb/c bone marrow (BM) cells for up to 7 days in serum-free HSC expansion medium with hematopoietic growth factors. SUL-109-containing cultures were stored at 4°C for 3 to 14 days. The UCB cells were tested for viability, cell cycle, and reactive oxygen species (ROS). DMSO-cryopreserved Lin- BM cells or Lin- BM cells maintained for 14 days at 4°C were transplanted into RAG2-/- Balb/c mice and engraftment was followed for 6 months. The addition of SUL-109 during the hypothermic storage of expanded CD34+ UCB cells provided a significant improvement in cell survival of the immature CD34+/CD38- fraction after 7 days of hypothermic storage through scavenging of hypothermia-induced ROS and was able to preserve the multilineage capacity of human CD34+ UCB cells for up to 14 days of cold storage. In addition, SUL-109 protected murine BM Lin- cells from 14 days of hypothermic preservation and maintained their engraftment potential after transplantation in immune-deficient RAG2-/- mice. Our data indicate that SUL-109 is a promising novel chemical for use as a protective agent during cold storage of human and murine HSCs to prevent hypothermia-induced apoptosis and promote cell viability.

Efficacy of mesenchymal stem cell therapy in systolic heart failure: a systematic review and meta-analysis

BackgroundHeart failure (HF) is the end stage of most heart disease. Mesenchymal stem cells (MSCs), with their specific biological effects, have been applied in several clinical trials to evaluate the efficacy in HF therapy. We performed this meta-analysis to review the clinical evidence of their therapeutic effect on HF.MethodsThree databases were searched. The outcomes of interest were death, readmission, the 6-min walk test (6MWT), New York Heart Association (NYHA) class and left ventricular ejection fraction (LVEF). The relative risk (RR) and weighted mean difference (WMD) were calculated to evaluate the effects of MSCs on HF compared to placebo.ResultsA total of nine studies were included, involving 612 patients who underwent MSCs or placebo treatment. The overall rate of death showed a trend of reduction of 36% (RR [CI] = 0.64 [0.35, 1.16], p = 0.143) in the MSC treatment group. The incidence of readmission was reduced by 34% (RR [CI] = 0.66 [0.51, 0.85], p = 0.001). The patients in the MSC treatment group realised an average of 40.44 m (WMD [95% CI] = 40.44 m [19.07, 61.82], p < 0.0001) improvement in 6MWT. The NYHA class was reduced obviously in the MSC group (WMD [95% CI] = − 0.42 [− 0.64, − 0.20], p < 0.0001). The changes of LVEF from baseline were significantly more than 5.25% (WMD [95% CI] = 5.25 [3.58, 6.92], p < 0.0001) in the MSCs group, unlike in the placebo group.ConclusionsOur results suggested that MSC treatment is an effective therapy for HF by improving the prognosis and exercise capacity. SCs derived from allosomes have superior therapeutic effects, and intracoronary injection is the optimum MSC delivery approach. Short-term cryopreservation is feasible in MSCs storage or transport.

Active bacterial communities of pig fecal microbiota transplantation suspension prepared and preserved under different conditions

Although fecal microbiota transplantation (FMT) has become a research hotspot, studies on comparison of the active fecal bacteria suspension under different preparation conditions are limited. This study investigated the abundances of active bacterial community in pig FMT suspension that produced under different oxygen concentrations or cryopreservation conditions. Fecal samples from a Landrace × Yorkshire sow were used to prepare fecal bacteria suspension under the anaerobic (AN group) and aerobic conditions (AE group), respectively. And then half of the anaerobic fecal bacteria suspension was cryopreservation in − 80 °C (AN-CR group) for 1 week. The microbial RNA in the fecal bacteria suspension was extracted before and after cryopreservation, and reverse transcribed into cDNA. MiSeq sequencing 16S rRNA gene of bacterial cDNA showed that the bacterial diversity in the AN group was significantly higher than that in the AE group. Comparing with the sows’ fecal sample, the relative abundances of Lactobacillus johnsonii, Lactobacillus coleohominis and Parabacteroides merdae in AN, AE and AN-CR groups were reduced. The short-term cryopreservation had low impact on the structure of the active bacterial community in the fecal bacterial suspension. These results suggest that fecal bacteria suspension can be better prepared under strict anaerobic condition, and that fecal bacteria suspension can be cryopreserved in − 80 °C for a short time.

Freezing of dendritic cells with trehalose as an additive in the conventional freezing medium results in improved recovery after cryopreservation.

Dendritic cell (DC) vaccination involves administration of multiple doses. Cryopreservation of tumor antigen-pulsed DCs can provide a ready to use vaccine source and eliminate the need of frequent withdrawal of the patient's blood for vaccine preparation. The aim of this study was to assess the effect of addition of trehalose in the freezing medium on the recovery of DCs after cryopreservation. DCs were generated from mononuclear cells from apheresis samples of healthy donors. For long-term storage of 6 months, cells were frozen with a rate-controlled programmable freezer and stored in liquid nitrogen. For short-term storage of 1 month, cells were frozen and stored at -80°C. DCs frozen with Iscove's Modified Dulbecco's Medium + 10% dimethyl sulfoxide + 20% fetal bovine serum served as the control group, while the test group was additionally supplemented with 50 μg/mL of trehalose. After revival of control and test DCs, they were assessed for viability, morphology, phenotype, and functions. The addition of trehalose to the conventional freezing medium helped to preserve the viability and functionality of DCs better than dimethyl sulfoxide alone in both long- and short-term cryopreservation. Trehalose also protected the mitochondrial membrane potential and cytoskeleton integrity of DCs, which are necessary for their functionality. Mediators of the intrinsic apoptotic pathway like Caspase-9 and Bim-1 were found to be low in the test. Supplementation of conventional freezing medium with trehalose results in better quality of DCs revived after cryopreservation. This finding could help improve DC vaccine preparation for cancer immunotherapy.