Cryopreservation of red blood cells(RBCs)provides great potential benefits for providing transfusion timely in emergencies.High concentrations of glycerol(20%or 40%)are used for RBC cryopreservation in current clinica...Cryopreservation of red blood cells(RBCs)provides great potential benefits for providing transfusion timely in emergencies.High concentrations of glycerol(20%or 40%)are used for RBC cryopreservation in current clinical practice,which results in cytotoxicity and osmotic injuries that must be carefully controlled.However,existing studies on the low-glycerol cryopreservation of RBCs still suffer from the bottleneck of low hematocrit levels,which require relatively large storage space and an extra concentration process before transfusion,making it inconvenient(time-consuming,and also may cause injury and sample lose)for clinical applications.To this end,we develop a novel method for the glycerol-free cryopreservation of human RBCs with a high final hematocrit by using trehalose as the sole cryoprotectant to dehydrate RBCs and using core–shell alginate hydrogel microfibers to enhance heat transfer during cryopreservation.Different from previous studies,we achieve the cryopreservation of human RBCs at high hematocrit(>40%)with high recovery(up to 95%).Additionally,the washed RBCs post-cryopreserved are proved to maintain their morphology,mechanics,and functional properties.This may provide a nontoxic,high-efficiency,and glycerol-free approach for RBC cryopreservation,along with potential clinical transfusion benefits.展开更多
Teratozoospermia is an infertility issue that affects a significant number of couples of reproductive age. One of the potential causes contributing to the global decline in seminal quality includes factors such as die...Teratozoospermia is an infertility issue that affects a significant number of couples of reproductive age. One of the potential causes contributing to the global decline in seminal quality includes factors such as diet, alcohol and tobacco consumption, high levels of stress, and environmental influences. This underscores the need to preserve the fertility of these patients through cryopreservation techniques. In this review, we explore the latest methods for freezing seminal samples, highlighting their advancements and advantages in addressing the challenge of perpetuating animal species, particularly in the context of human infertility.展开更多
Poultry genetics resources,including commercial selected lines,indigenous breeds,and experimental lines,are now being irreversibly lost at an alarming rate due to multiple reasons,which further threats the future live...Poultry genetics resources,including commercial selected lines,indigenous breeds,and experimental lines,are now being irreversibly lost at an alarming rate due to multiple reasons,which further threats the future livelihood and academic purpose.Collections of germplasm may reduce the risk of catastrophic loss of genetic diversity by guaranteeing that a pool of genetic variability is available to ensure the reintroduction and replenishment of the genetic stocks.The setting up of biobanks for poultry is challenging because the high sensitiveness of spermatozoa to freezing–thawing process,inability to cryopreserve the egg or embryo,coupled with the females being heterogametic sex.The progress in cryobiology and biotechnologies have made possible the extension of the range of germplasm for poultry species available in cryobanks,including semen,primordial germ cells,somatic cells and gonads.In this review,we introduce the state-of-the-art technologies for avian genetic resource conservation and breed reconstruction,and discuss the potential challenges for future study and further extending of these technologies to ongoing and future conservation efforts.展开更多
For men with severe oligozoospermia, sperm cryopreservation can preserve surgically obtained sperm. How to cryopreserve single sperm in men is still a hot topic in assisted reproduction technology. Aim to analyze the ...For men with severe oligozoospermia, sperm cryopreservation can preserve surgically obtained sperm. How to cryopreserve single sperm in men is still a hot topic in assisted reproduction technology. Aim to analyze the laboratory and pregnancy outcomes of single sperm cryopreservation group, we retrospectively selected 38 cycles underwent single sperm cryopreservation and thawing as the study group and 618 cycles underwent conventional sperm cryopreservation and thawing as the control group, which were performed in the reproductive medicine center of the Sixth Affiliated Hospital, Sun Yatsen University, from April 2014 to October 2023. All the sperm came from microdissection testicular sperm extraction (micro-TESE), and performed intracytoplasmic sperm injection (ICSI) for fertilization. Zygotes were cultured to Day 3 embryo, which were freshly transferred to female uterus. Surplus embryos were cultured to blastosphere and cryopreserved. There was no statistical difference in female/male age, female BMI, infertility duration and female basal sex hormone (FSH, LH E2, AMH), No. of oocytes retrieved per cycle, No. of ICSI oocytes per cycle and No. of embryos transferred per cycle between the two groups (P > 0.05). No significant difference was found in two-pronuclear oocyte fertilization rate (59.23% VS 58.84%), Day 3 available embryo rate (61.81% VS 63.55%), Day 3 good-quality embryo rate (45.73% VS 50.27%), blastocyst formation rate (47.83% VS 49.46%), the implantation rate (47.37% VS 52.16%), clinical pregnancy rate (36.84% VS 47.18%), miscarriage rate (14.29% VS 12.68%) and live birth rate (85.71% VS 81.70%) between two groups (P > 0.05). In conclusion, single-sperm cryopreservation was the optimal method to preserve sperm after micro-TESE. It can increase the utilization of each sperm and lead to clinical pregnancy.展开更多
Cryopreservation is currently the only effective tool for long-term storage of semen in most species. However, it is well-recognized that, even in species that freeze well, some individuals resist cryopreservation. Wo...Cryopreservation is currently the only effective tool for long-term storage of semen in most species. However, it is well-recognized that, even in species that freeze well, some individuals resist cryopreservation. Work from this laboratory has demonstrated a relationship between maternal lipid content and the chemical constitution of the embryos they produce. The objective of the present study was to determine if a similar relationship might exist in paternal body chemistry and the animal’s semen sample and if such a difference could be determined with a simple weight test. Semen samples were obtained from cattle with known differences in body composition. The samples first underwent semen analysis and were then prepared as either cell-free (CF) or neat specimens (NS). Known volumes of each sample were weighed, and the remainder of the samples was analyzed for lipids, total proteins, and total carbohydrates using a series of spectrophotometric assays and blood chemistry techniques. As expected, weight differences were seen in the CF vs NS preparations of individual semen samples (p < 0.001). Differences were also found in triglycerides (p < 0.001), glucose (p < 0.001), total protein (p < 0.001), and fructose (p < 0.009) of individuals with differing body composition. Statistical analysis suggested a non-linear correlation between the observed weights and total protein (p < 0.047) as well as triglyceride levels (p < 0.003). Together, these data suggest it might be possible to develop an algorithm to allow adjustment in cryoprotectants based on a simple weight procedure, allowing modification of cryoprotectants on an individual basis and potentially improving outcomes for valuable animals currently classified as “poor freezers”.展开更多
Liver cell transplantation presents clinical benefit in patients with inborn errors of metabolism as an alternative,or at least as a bridge,to orthotopic liver transplantation.The success of such a therapeutic approac...Liver cell transplantation presents clinical benefit in patients with inborn errors of metabolism as an alternative,or at least as a bridge,to orthotopic liver transplantation.The success of such a therapeutic approach remains limited by the quality of the transplanted cells.Cryopreservation remains the best option for long-term storage of hepatocytes,providing a permanent and sufficient cell supply.However, isolated adult hepatocytes are poorly resistant to such a process,with a significant alteration both at the morphological and functional levels.Hence,the aim of the current review is to discuss the state of the art regarding widely-used hepatocyte cryopreservation protocols,as well as the assays performed to analyse the post-thawing cell quality both in vitro and in vivo. The majority of studies agree upon the poor quality and efficiency of cryopreserved/thawed hepatocytes as compared to freshly isolated hepatocytes.Intracellular ice formation or exposure to hyperosmotic solutionsremains the main phenomenon of cryopreservation process,and its effects on cell quality and cell death induction will be discussed.The increased knowledge and understanding of the cryopreservation process will lead to research strategies to improve the viability and the quality of the cell suspensions after thawing.Such strategies,such as vitrification,will be discussed with respect to their potential to significantly improve the quality of cell suspensions dedicated to liver cell-based therapies.展开更多
AIM To demonstrate the feasibility of cryopreservation of peripheral blood mononuclear cells(PBMCs) for prognostic circulating tumor cell(CTC) detection in gastroesophageal cancer.METHODS Using 7.5 m L blood samples c...AIM To demonstrate the feasibility of cryopreservation of peripheral blood mononuclear cells(PBMCs) for prognostic circulating tumor cell(CTC) detection in gastroesophageal cancer.METHODS Using 7.5 m L blood samples collected in EDTA tubes from patients with gastroesopheagal adenocarcinoma, CTCs were isolated by epithelial cell adhesion molecule based immunomagnetic capture using the Iso Flux platform. Paired specimens taken during the same blood draw(n = 15) were used to compare number of CTCs isolated from fresh and cryopreserved PBMCs. Blood samples were processed within 24 h to recover the PBMC fraction, with PBMCs used for fresh analysis immediately processed for CTC isolation. Cryopreservation of PBMCs lasted from 2 wk to 25.2 mo(median 14.6 mo). CTCs isolated from pre-treatment cryopreserved PBMCs(n = 43) were examined for associations with clinicopathological variables and survival outcomes.RESULTS While there was a significant trend to a decrease in CTC numbers associated with cryopreserved specimens(mean number of CTCs 34.4 vs 51.5, P = 0.04), this was predominately in samples with a total CTC count of > 50, with low CTC count samples less affected(P = 0.06). There was no significant association between the duration of cryopreservation and number of CTCs. In cryopreserved PBMCs from patient samples prior to treatment, a high CTC count(> 17) was associated with poorer overall survival(OS)(n = 43, HR = 4.4, 95%CI: 1.7-11.7, P = 0.0013). In multivariate analysis, after controlling for sex, age, stage, ECOG performance status, and primary tumor location, a high CTC count remained significantly associated with a poorer OS(HR = 3.7, 95%CI: 1.2-12.4, P = 0.03). CONCLUSION PBMC cryopreservation for delayed CTC isolation is a valid strategy to assist with sample collection, transporting and processing.展开更多
In vertebrates, most somatosensory pathways begin with the activation of dorsal root ganglion(DRG) neurons. The development of an appropriate DRG culture method is a prerequisite for establishing in vitro peripheral n...In vertebrates, most somatosensory pathways begin with the activation of dorsal root ganglion(DRG) neurons. The development of an appropriate DRG culture method is a prerequisite for establishing in vitro peripheral nerve disease models and for screening therapeutic drugs. In this study, we compared the changes in morphology, molecular biology, and transcriptomics of chicken embryo DRG cultured on tissue culture plates(T-DRG) versus three-dimensional collagen hydrogels(C-DRG). Our results showed that after 7 days of culture, the transcriptomics of T-DRG and C-DRG were quite different. The upregulated genes in C-DRG were mainly related to neurogenesis, axon guidance, and synaptic plasticity, whereas the downregulated genes in C-DRG were mainly related to cell proliferation and cell division. In addition, the genes related to cycles/pathways such as the synaptic vesicle cycle, cyclic adenosine monophosphate signaling pathway, and calcium signaling pathway were activated, while those related to cell-cycle pathways were downregulated. Furthermore, neurogenesis-and myelination-related genes were highly expressed in C-DRG, while epithelial–mesenchymal transition-, apoptosis-, and cell division-related genes were suppressed. Morphological results indicated that the numbers of branches, junctions, and end-point voxels per C-DRG were significantly greater than those per T-DRG. Furthermore, cells were scattered in T-DRG and more concentrated in C-DRG, with a higher ratio of 5-ethynyl-2′-deoxyuridine(EdU)-positive cells in T-DRG compared with C-DRG. C-DRG also had higher S100 calcium-binding protein B(S100 B) and lower α-smooth muscle actin(α-SMA) expression than T-DRG, and contained fewer terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)-positive cells after 48 hours of serum starvation. After cryopreservation, C-DRG maintained more intact morphological characteristics, and had higher viability and less TUNEL-positive cells than T-DRG. Furthermore, newly formed nerve bundles were able to grow along the existing Schwann cells in C-DRG. These results suggest that C-DRG may be a promising in vitro culture model, with better nerve growth and anti-apoptotic ability, quiescent Schwann cells, and higher viability. Results from this study provide a reference for the construction, storage, and transportation of tissue-engineered nerves. The study was approved by the Ethics Committee of Aier School of Ophthalmology, Central South University, China(approval No. 2020-IRB16), on March 15, 2020.展开更多
Sucrose is known to play an important role in the cryopreservation of sperm and female gonads; however, its effect on the cryopreservation of pig spermatogonial stem cells(p SSCs) has not been tested. The aim of this ...Sucrose is known to play an important role in the cryopreservation of sperm and female gonads; however, its effect on the cryopreservation of pig spermatogonial stem cells(p SSCs) has not been tested. The aim of this work was to study the effect of sucrose during p SSC cryopreservation and to find the most effective concentration in freezing medium. p SSCs were cryopreserved with freezing media containing different concentrations of sucrose(70, 140, 210, and 280 mmol L^(–1)) and a control group without sucrose. The survival rates, plasma membrane integrity, and mitochondrial membrane potential of thawed cells were detected by trypan blue(TB) staining, SYBR-14/propidium iodide(PI) dual staining, and JC-1 staining, respectively. All the staining results showed an obvious increase in cell survival in the sucrose-treated groups as compared to that in the control group, with the exception of 280 mmol L^(–1) sucrose. Moreover, the 210 mmol L^(–1) sucrose group yielded the highest survival rate among all the groups(P<0.05). The results of SYBR-14/PI dual staining and JC-1 staining were consistent with those of TB staining as above described. Quantitative real-time PCR(q RT-PCR) indicated that the m RNA levels of three apoptosis-promoting genes(BAX, APAF1 and CASPASE9) were significantly higher in thawed cells than in cells before freezing(P<0.05). Moreover, the mR NA level of one anti-apoptotic gene(XIAP) was significantly lower in thawed cells than in cells before freezing(P<0.05). When comparing the m RNA expression of apoptosis-related genes in thawed cells, the m RNA level of the anti-apoptotic genes in the control group was significantly lower than that in the sucrose-treated groups(P<0.05). Western blot analyses showed that the expression levels of cleaved CASPASE9, CASPASE3 and PARP-1 in the sucrose-treated groups were lower than those in the control group and were the lowest in the 210 mmol L^(–1) sucrose group. Both q RT-PCR and Western blot analyses suggested that sucrose inhibited cell apoptosis during freezing and thawing. Briefly, sucrose promoted p SSCs survival after freezing and thawing, especially at a concentration of 210 mmol L^(–1), which possibly assisted p SSC dehydration and inhibited cell apoptosis. These findings hold great promise for further studies of the regulatory mechanism of proliferation and differentiation of p SSCs.展开更多
AIM:To optimize a xeno-free cryopreservation protocol for primary human hepatocytes.METHODS:The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes.Despite several hepatocyte cry...AIM:To optimize a xeno-free cryopreservation protocol for primary human hepatocytes.METHODS:The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes.Despite several hepatocyte cryopreservation protocols being available,improvements are urgently needed.We first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the groups.Using the polystyrene box freezing,we compared two xeno-free freezing solutions for freezing of primary human hepatocytes:a new medium(STEM-CELLBANKER,CB),which contains dimethylsulphoxide(DMSO) and anhydrous dextrose,both permeating and non-permeating cryoprotectants,and the frequently used DMSO-University of Wisconsin(DMSOUW) medium.The viability of the hepatocytes was assessed by the trypan blue exclusion method as well as a calcein-esterase based live-dead assay before and after cryopreservation.The function of the hepatocytes was evaluated before and after cryopreservation by assessing enzymatic activity of 6 major cytochrome P450 isoforms(CYPs):CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP3A4 and CYP3A7.RESULTS:The new cryoprotectant combination preserved hepatocyte viability significantly better than the standard DMSO-UW protocol(P < 0.01).There was no significant difference in viability estimation between both the trypan blue(TB) and the Live-Dead Assay methods.There was a correlation between viability of fresh hepatocytes and the difference in cell viability between CB and DMSO protocols(r 2 = 0.69) using the TB method.However,due to high within-group variability in the activities of the major CYPs,any statistical between-group differences were precluded.Cryopreservation of human hepatocytes using the cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte viability.Thus,it may be a better alternative to the standard DMSO-UW protocol.Estimating CYP activities did not seem to be a relevant way to compare hepatocyte function between different groups due to high normal variability between different liver samples.CONCLUSION:The cryoprotectant combination may be a better alternative to the standard DMSO-UW protocol in primary human hepatocyte cryopreservation.展开更多
Cell cryopreservation has evolved as an important technology required for supporting various cell-based applications,such as stem cell therapy,tissue engineering,and assisted reproduction.Recent times have witnessed a...Cell cryopreservation has evolved as an important technology required for supporting various cell-based applications,such as stem cell therapy,tissue engineering,and assisted reproduction.Recent times have witnessed an increase in the clinical demand of these applications,requiring urgent improvements in cell cryopreservation.However,cryopreservation technology suff ers from the issues of low cryopreservation effi ciency and cryoprotectant(CPA)toxicity.Application of advanced biotechnology tools can signifi cantly improve post-thaw cell survival and reduce or even eliminate the use of organic solvent CPAs,thus promoting the development of cryopreservation.Herein,based on the diff erent cryopreservation mechanisms available,we provide an overview of the applications and achievements of various biotechnology tools used in cell cryopreservation,including trehalose delivery,hydrogel-based cell encapsulation technique,droplet-based cell printing,and nanowarming,and also discuss the associated challenges and perspectives for future development.展开更多
The development and application of spermatogonial stem cell technology have an important significance in animal cloning, preservation of endangered species and spermatogenesis research. In this study, the seminiferous...The development and application of spermatogonial stem cell technology have an important significance in animal cloning, preservation of endangered species and spermatogenesis research. In this study, the seminiferous epithlium cells were isolated and purified, the cells were cryopreserved after identification, and the effects of different purification and cyopreservation methods on bovine testicular cells were studied. The results showed that there were spermatogonial stem cells and sertoli cells in the neonatal bovine seminiferous tubules, differential adherent selection methods could effectively separate these two cell types. Spermatogonial stem cells were positive after AKP, C-kit, and OCT-4 identification; sertoli cells were positive after oil red O and vimentin identification. Frozen stock solution supplemented with 10% DMSO had the best effect in spermatogonial stem cell cryopreservation, while frozen stock solution supplemented with 10% of ethylene glycol and 0.1 mmol·L-1 trehalose had the best effect in sertoli cells cryopreservation.展开更多
Objective:To compare the outcomes of vitrification using cryoloop with slow-freezing meth-od for human blastocyst cryopreservation.Methods:In IVF-ET cycles,supernumerary embryos were cultured to Day 5 or Day 6,blas-to...Objective:To compare the outcomes of vitrification using cryoloop with slow-freezing meth-od for human blastocyst cryopreservation.Methods:In IVF-ET cycles,supernumerary embryos were cultured to Day 5 or Day 6,blas-tocysts were cryopreserved by vitrification using cryoloops or slow-freezing method,then blasto-cyst survival rate and pregnant rate were compared.Results:115 vitrified blastocysts from 39 cycles were warmed,104(90.4%)blastocysts sur-vived.After the transfer of 74 blastocysts in 38 cycles,28(73.7%)women got clinically preg-nant,2(7.1%)of them suffered from miscarriage,2 healthy babies were born in 2 deliveries,and the other 24 pregnancies are ongoing.As to slow-freezing method,87 blastocysts from 21 cy-cles were thawed,37(42.5%)of them survived,28 blastoeysts were transferred in 15 cycles,6(40%)women got clinically pregnant,1 of them miscarried,3 healthy babies were born in 2 de-liveries,and the other 3 pregnancies are ongoing.Conclusion:The survival rate and pregnant rate of vitrification using cryoloop are superior totraditional slow-freezing method,and the transfer cancel rate is lower than that of slow-freezingmethod.The miscarriage rate is similar in two methods.展开更多
In this study, a programmable freezing technique has been developed for strip spawned sperm in the blue mussel, M ytilus galloprovincialis. The optimized key parameters include cooling rate, endpoint temperature, thaw...In this study, a programmable freezing technique has been developed for strip spawned sperm in the blue mussel, M ytilus galloprovincialis. The optimized key parameters include cooling rate, endpoint temperature, thawing temperature, sugar addition and sperm to oocyte ratio. The sperm quality was assessed by the fertilization rate or the integrity of sperm component and organelle. The highest post-thaw sperm fertilization rate was 91%, which was produced with sperm cryopreserved in 8% dimethyl sulfoxide at the cooling rate of-4°C/min from 2°C to-30°C before being plunged into liquid nitrogen for at least 12 h, thawed in a 20°C seawater bath and fertilized at sperm to egg ratio of 50 000:1. The addition of glucose, sucrose or trehalose to 8% dimethyl sulfoxide could not further improve fertilization rates. The fluorescent assessments showed that the post-thaw sperm plasma membrane integrity and acrosome integrity were significantly damaged in comparison with fresh sperm.展开更多
Background:The application of cryopreservation and artificial insemination technology have contributed to the advancement of animal reproduction.However,a substantial proportion of spermatozoa undergoes alterations an...Background:The application of cryopreservation and artificial insemination technology have contributed to the advancement of animal reproduction.However,a substantial proportion of spermatozoa undergoes alterations and loses their fertility during cryopreservation,rendering the frozen-thawed semen impractical for routine use.Cryopreservation is known to reduce sperm lifespan and fertility.Variation in cryosurvival of spermatozoa from different sires and even with the individual sire is common in artificial insemination(AI)centers.Our goal is to improve post-thawed semen quality by optimization of cryopreservation technique through sperm selection prior to cryopreservation process.Results:Our strategy of sperm selection based on rheotaxis and thermotaxis(SSRT)on macrosale in a rotating fluid flow demonstrated the ability to maintain the original pre-freezing structural integrity,viability and biological function related to fertilization competence.This strategy has a positive effect on the cryosurvival and fertilizing abilities of spermatozoa as supported by the improvement on pregnancy rate of Japanese Black heifers and Holstein repeat breeders.This technique protected further sublethal damage to bovine spermatozoa(higher%cryosurvival than the control)and resulted in the improvement of DNA integrity.Prefreeze selected spermatozoa demonstrated slower and controlled capacitation than unprocessed control which is thought to be related to sperm longevity and consequently to appropriate timing during in vivo fertilization.Conclusions:These results provide solid evidence that improvement of post-thawed semen quality by SSRT method is beneficial in terms of cryosurvival,longevity of post-thawed sperm,and optimization of in vivo fertilization,embryo development and calving as supported by the favorable results of field fertility study.展开更多
AIM To investigate a suitable long-term culture system and optimal cryopreservation of intestinal organoid to improve organoid-based therapy by acquiring large numbers of cells.METHODS Crypts were isolated from jejunu...AIM To investigate a suitable long-term culture system and optimal cryopreservation of intestinal organoid to improve organoid-based therapy by acquiring large numbers of cells.METHODS Crypts were isolated from jejunum of C57BL/6 mouse. Two hundred crypts were cultured in organoid medium with either epidermal growth factor/Noggin/R-spondin1(ENR) or ENR/CHIR 99021/VPA(ENR-CV). F o rsubculture, organoids cultured on day 7 were passaged using enzyme-free cell dissociation buffer (STEMCELL Technologies). The passage was performed once per week until indicated passage. For cryopreservation, undissociated and dissociated organoids were resuspended in freezing medium with or without Rho kinase inhibitor subjected to different treatment times. The characteristics of intestinal organoids upon extended passage and freeze-thaw were analyzed using Ed U staining, methyl thiazolyl tetrazolium assay, q PCR and time-lapse live cell imaging.RESULTS We established a three-dimensional culture system for murine small intestinal organoids using ENR and ENR-CV media. Both conditions yielded organoids with a crypt-villus architecture exhibiting Lgr5^+ cells and differentiated intestinal epithelial cells as shown by morphological and biochemical analysis. However, during extended passage (more than 3 mo), a comparative analysis revealed that continuous passaging under ENR-CV conditions, but not ENR conditions induced phenotypic changes as observed by morphological transition, reduced numbers of Lgr5^+ cells and inconsistent expression of markers for differentiated intestinal epithelial cell types. We also found that recovery of long-term cryopreserved organoids was significantly affected by the organoid state, i.e., whether dissociation was applied, and the timing of treatment with the Rho-kinase inhibitor Y-27632. Furthermore, the retention of typical morphological characteristics of intestinal organoids such as the crypt-villus structure from freeze-thawed cells was observed by live cell imaging.CONCLUSION The maintenance of the characteristics of intestinal organoids upon extended passage is mediated by ENR condition, but not ENR-CV condition. Identified long-term cryopreservation may contribute to the establishment of standardized cryopreservation protocols for intestinal organoids for use in clinical applications.展开更多
In recent years,with the rapid development of medical research,cancer diagnosis and treatment technology have significantly improved young cancer patient’s survival rate.Anticancer therapy such as chemotherapy,radiot...In recent years,with the rapid development of medical research,cancer diagnosis and treatment technology have significantly improved young cancer patient’s survival rate.Anticancer therapy such as chemotherapy,radiotherapy,or hematopoietic stem cell transplantation can lead to premature ovarian insufficiency.The endocrine and reproductive function of the ovary is critical to women’s physical and mental health.Ovarian tissue cryopreservation and transplantation can protect not only female fertility but also preserve ovarian endocrine function.This paper interprets the guidelines for ovarian tissue cryopreservation and transplantation issued by the Chinese Society of Gynecological Endocrinology affiliated to the International Society of Gynecological Endocrinology.The purpose of this guideline’s interpretation is to promote more medical workers to understand the technology of ovarian tissue cryopreservation and transplantation,which can provide patients with more choices of fertility protection methods and improve their quality of life.展开更多
The traditional corneal cryopreservation technique was improved. We carried out an experimental study that rabbit corneas were cryop-reserved by using polyvinylpyrolidone (PVP) as cryoprotective agent and dimethlsulfo...The traditional corneal cryopreservation technique was improved. We carried out an experimental study that rabbit corneas were cryop-reserved by using polyvinylpyrolidone (PVP) as cryoprotective agent and dimethlsulfoxide (DMSO) as the control. The endothelia of cryopreserved corneas were evaluated by scanning and transmission electron microscopy and vital staining. The study shows that PVP is an excellent extracellular cryoprotective agent and has the characteristic of low toxicity or no toxicity to co...展开更多
基金the National Natural Science Foundation of China(No.82172114)the Anhui Provincial Natural Science Foundation for Distinguished Young Scholars(No.2108085J37).
文摘Cryopreservation of red blood cells(RBCs)provides great potential benefits for providing transfusion timely in emergencies.High concentrations of glycerol(20%or 40%)are used for RBC cryopreservation in current clinical practice,which results in cytotoxicity and osmotic injuries that must be carefully controlled.However,existing studies on the low-glycerol cryopreservation of RBCs still suffer from the bottleneck of low hematocrit levels,which require relatively large storage space and an extra concentration process before transfusion,making it inconvenient(time-consuming,and also may cause injury and sample lose)for clinical applications.To this end,we develop a novel method for the glycerol-free cryopreservation of human RBCs with a high final hematocrit by using trehalose as the sole cryoprotectant to dehydrate RBCs and using core–shell alginate hydrogel microfibers to enhance heat transfer during cryopreservation.Different from previous studies,we achieve the cryopreservation of human RBCs at high hematocrit(>40%)with high recovery(up to 95%).Additionally,the washed RBCs post-cryopreserved are proved to maintain their morphology,mechanics,and functional properties.This may provide a nontoxic,high-efficiency,and glycerol-free approach for RBC cryopreservation,along with potential clinical transfusion benefits.
文摘Teratozoospermia is an infertility issue that affects a significant number of couples of reproductive age. One of the potential causes contributing to the global decline in seminal quality includes factors such as diet, alcohol and tobacco consumption, high levels of stress, and environmental influences. This underscores the need to preserve the fertility of these patients through cryopreservation techniques. In this review, we explore the latest methods for freezing seminal samples, highlighting their advancements and advantages in addressing the challenge of perpetuating animal species, particularly in the context of human infertility.
基金Financial support of this study was provided by National Key R&D Program of China(No.2021YFD1200301 and 2021YFD1200305)joint research project raised by National Natural Science Foundation of China and The Egyptian Academy of Scientific Research and Technology(No.31961143028)+1 种基金China Agriculture Research Systems(No.CARS-40)National Germplasm Bank of Domestic Animals(No.2021–2022)。
文摘Poultry genetics resources,including commercial selected lines,indigenous breeds,and experimental lines,are now being irreversibly lost at an alarming rate due to multiple reasons,which further threats the future livelihood and academic purpose.Collections of germplasm may reduce the risk of catastrophic loss of genetic diversity by guaranteeing that a pool of genetic variability is available to ensure the reintroduction and replenishment of the genetic stocks.The setting up of biobanks for poultry is challenging because the high sensitiveness of spermatozoa to freezing–thawing process,inability to cryopreserve the egg or embryo,coupled with the females being heterogametic sex.The progress in cryobiology and biotechnologies have made possible the extension of the range of germplasm for poultry species available in cryobanks,including semen,primordial germ cells,somatic cells and gonads.In this review,we introduce the state-of-the-art technologies for avian genetic resource conservation and breed reconstruction,and discuss the potential challenges for future study and further extending of these technologies to ongoing and future conservation efforts.
文摘For men with severe oligozoospermia, sperm cryopreservation can preserve surgically obtained sperm. How to cryopreserve single sperm in men is still a hot topic in assisted reproduction technology. Aim to analyze the laboratory and pregnancy outcomes of single sperm cryopreservation group, we retrospectively selected 38 cycles underwent single sperm cryopreservation and thawing as the study group and 618 cycles underwent conventional sperm cryopreservation and thawing as the control group, which were performed in the reproductive medicine center of the Sixth Affiliated Hospital, Sun Yatsen University, from April 2014 to October 2023. All the sperm came from microdissection testicular sperm extraction (micro-TESE), and performed intracytoplasmic sperm injection (ICSI) for fertilization. Zygotes were cultured to Day 3 embryo, which were freshly transferred to female uterus. Surplus embryos were cultured to blastosphere and cryopreserved. There was no statistical difference in female/male age, female BMI, infertility duration and female basal sex hormone (FSH, LH E2, AMH), No. of oocytes retrieved per cycle, No. of ICSI oocytes per cycle and No. of embryos transferred per cycle between the two groups (P > 0.05). No significant difference was found in two-pronuclear oocyte fertilization rate (59.23% VS 58.84%), Day 3 available embryo rate (61.81% VS 63.55%), Day 3 good-quality embryo rate (45.73% VS 50.27%), blastocyst formation rate (47.83% VS 49.46%), the implantation rate (47.37% VS 52.16%), clinical pregnancy rate (36.84% VS 47.18%), miscarriage rate (14.29% VS 12.68%) and live birth rate (85.71% VS 81.70%) between two groups (P > 0.05). In conclusion, single-sperm cryopreservation was the optimal method to preserve sperm after micro-TESE. It can increase the utilization of each sperm and lead to clinical pregnancy.
文摘Cryopreservation is currently the only effective tool for long-term storage of semen in most species. However, it is well-recognized that, even in species that freeze well, some individuals resist cryopreservation. Work from this laboratory has demonstrated a relationship between maternal lipid content and the chemical constitution of the embryos they produce. The objective of the present study was to determine if a similar relationship might exist in paternal body chemistry and the animal’s semen sample and if such a difference could be determined with a simple weight test. Semen samples were obtained from cattle with known differences in body composition. The samples first underwent semen analysis and were then prepared as either cell-free (CF) or neat specimens (NS). Known volumes of each sample were weighed, and the remainder of the samples was analyzed for lipids, total proteins, and total carbohydrates using a series of spectrophotometric assays and blood chemistry techniques. As expected, weight differences were seen in the CF vs NS preparations of individual semen samples (p < 0.001). Differences were also found in triglycerides (p < 0.001), glucose (p < 0.001), total protein (p < 0.001), and fructose (p < 0.009) of individuals with differing body composition. Statistical analysis suggested a non-linear correlation between the observed weights and total protein (p < 0.047) as well as triglyceride levels (p < 0.003). Together, these data suggest it might be possible to develop an algorithm to allow adjustment in cryoprotectants based on a simple weight procedure, allowing modification of cryoprotectants on an individual basis and potentially improving outcomes for valuable animals currently classified as “poor freezers”.
基金Supported by The Belgian National Fund for Medical Research, the Région Wallonne-DGTRE(Grant WALEO/HEPATERA) and "La Fondation St Luc-ARC Thérapie Cellulaire"Stéphenne Xis recipient of a Grant-FNRS for cell cryopreservation
文摘Liver cell transplantation presents clinical benefit in patients with inborn errors of metabolism as an alternative,or at least as a bridge,to orthotopic liver transplantation.The success of such a therapeutic approach remains limited by the quality of the transplanted cells.Cryopreservation remains the best option for long-term storage of hepatocytes,providing a permanent and sufficient cell supply.However, isolated adult hepatocytes are poorly resistant to such a process,with a significant alteration both at the morphological and functional levels.Hence,the aim of the current review is to discuss the state of the art regarding widely-used hepatocyte cryopreservation protocols,as well as the assays performed to analyse the post-thawing cell quality both in vitro and in vivo. The majority of studies agree upon the poor quality and efficiency of cryopreserved/thawed hepatocytes as compared to freshly isolated hepatocytes.Intracellular ice formation or exposure to hyperosmotic solutionsremains the main phenomenon of cryopreservation process,and its effects on cell quality and cell death induction will be discussed.The increased knowledge and understanding of the cryopreservation process will lead to research strategies to improve the viability and the quality of the cell suspensions after thawing.Such strategies,such as vitrification,will be discussed with respect to their potential to significantly improve the quality of cell suspensions dedicated to liver cell-based therapies.
文摘AIM To demonstrate the feasibility of cryopreservation of peripheral blood mononuclear cells(PBMCs) for prognostic circulating tumor cell(CTC) detection in gastroesophageal cancer.METHODS Using 7.5 m L blood samples collected in EDTA tubes from patients with gastroesopheagal adenocarcinoma, CTCs were isolated by epithelial cell adhesion molecule based immunomagnetic capture using the Iso Flux platform. Paired specimens taken during the same blood draw(n = 15) were used to compare number of CTCs isolated from fresh and cryopreserved PBMCs. Blood samples were processed within 24 h to recover the PBMC fraction, with PBMCs used for fresh analysis immediately processed for CTC isolation. Cryopreservation of PBMCs lasted from 2 wk to 25.2 mo(median 14.6 mo). CTCs isolated from pre-treatment cryopreserved PBMCs(n = 43) were examined for associations with clinicopathological variables and survival outcomes.RESULTS While there was a significant trend to a decrease in CTC numbers associated with cryopreserved specimens(mean number of CTCs 34.4 vs 51.5, P = 0.04), this was predominately in samples with a total CTC count of > 50, with low CTC count samples less affected(P = 0.06). There was no significant association between the duration of cryopreservation and number of CTCs. In cryopreserved PBMCs from patient samples prior to treatment, a high CTC count(> 17) was associated with poorer overall survival(OS)(n = 43, HR = 4.4, 95%CI: 1.7-11.7, P = 0.0013). In multivariate analysis, after controlling for sex, age, stage, ECOG performance status, and primary tumor location, a high CTC count remained significantly associated with a poorer OS(HR = 3.7, 95%CI: 1.2-12.4, P = 0.03). CONCLUSION PBMC cryopreservation for delayed CTC isolation is a valid strategy to assist with sample collection, transporting and processing.
基金supported by the National Natural Science Foundation of China,Nos.82000871(to ZKC),81871495(to JSC)the Natural Science Foundation of Hunan Province,China,No.2020JJ5001(to ZKC)+1 种基金the Science Research Grant of Aier Eye Hospital Group,China,No.AF1913D2(to ZKC)Central South University Postdoctoral Funds,China。
文摘In vertebrates, most somatosensory pathways begin with the activation of dorsal root ganglion(DRG) neurons. The development of an appropriate DRG culture method is a prerequisite for establishing in vitro peripheral nerve disease models and for screening therapeutic drugs. In this study, we compared the changes in morphology, molecular biology, and transcriptomics of chicken embryo DRG cultured on tissue culture plates(T-DRG) versus three-dimensional collagen hydrogels(C-DRG). Our results showed that after 7 days of culture, the transcriptomics of T-DRG and C-DRG were quite different. The upregulated genes in C-DRG were mainly related to neurogenesis, axon guidance, and synaptic plasticity, whereas the downregulated genes in C-DRG were mainly related to cell proliferation and cell division. In addition, the genes related to cycles/pathways such as the synaptic vesicle cycle, cyclic adenosine monophosphate signaling pathway, and calcium signaling pathway were activated, while those related to cell-cycle pathways were downregulated. Furthermore, neurogenesis-and myelination-related genes were highly expressed in C-DRG, while epithelial–mesenchymal transition-, apoptosis-, and cell division-related genes were suppressed. Morphological results indicated that the numbers of branches, junctions, and end-point voxels per C-DRG were significantly greater than those per T-DRG. Furthermore, cells were scattered in T-DRG and more concentrated in C-DRG, with a higher ratio of 5-ethynyl-2′-deoxyuridine(EdU)-positive cells in T-DRG compared with C-DRG. C-DRG also had higher S100 calcium-binding protein B(S100 B) and lower α-smooth muscle actin(α-SMA) expression than T-DRG, and contained fewer terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)-positive cells after 48 hours of serum starvation. After cryopreservation, C-DRG maintained more intact morphological characteristics, and had higher viability and less TUNEL-positive cells than T-DRG. Furthermore, newly formed nerve bundles were able to grow along the existing Schwann cells in C-DRG. These results suggest that C-DRG may be a promising in vitro culture model, with better nerve growth and anti-apoptotic ability, quiescent Schwann cells, and higher viability. Results from this study provide a reference for the construction, storage, and transportation of tissue-engineered nerves. The study was approved by the Ethics Committee of Aier School of Ophthalmology, Central South University, China(approval No. 2020-IRB16), on March 15, 2020.
基金supported by the China Postdoctoral Science Foundation(2014M560809)the Shaanxi Province Postdoctoral Science Foundation,China+1 种基金the Fundamental Research Funds for the Central Universities,China(NWSUAF,2452015145)the National Basic Research Program of China(2014CB943100)
文摘Sucrose is known to play an important role in the cryopreservation of sperm and female gonads; however, its effect on the cryopreservation of pig spermatogonial stem cells(p SSCs) has not been tested. The aim of this work was to study the effect of sucrose during p SSC cryopreservation and to find the most effective concentration in freezing medium. p SSCs were cryopreserved with freezing media containing different concentrations of sucrose(70, 140, 210, and 280 mmol L^(–1)) and a control group without sucrose. The survival rates, plasma membrane integrity, and mitochondrial membrane potential of thawed cells were detected by trypan blue(TB) staining, SYBR-14/propidium iodide(PI) dual staining, and JC-1 staining, respectively. All the staining results showed an obvious increase in cell survival in the sucrose-treated groups as compared to that in the control group, with the exception of 280 mmol L^(–1) sucrose. Moreover, the 210 mmol L^(–1) sucrose group yielded the highest survival rate among all the groups(P<0.05). The results of SYBR-14/PI dual staining and JC-1 staining were consistent with those of TB staining as above described. Quantitative real-time PCR(q RT-PCR) indicated that the m RNA levels of three apoptosis-promoting genes(BAX, APAF1 and CASPASE9) were significantly higher in thawed cells than in cells before freezing(P<0.05). Moreover, the mR NA level of one anti-apoptotic gene(XIAP) was significantly lower in thawed cells than in cells before freezing(P<0.05). When comparing the m RNA expression of apoptosis-related genes in thawed cells, the m RNA level of the anti-apoptotic genes in the control group was significantly lower than that in the sucrose-treated groups(P<0.05). Western blot analyses showed that the expression levels of cleaved CASPASE9, CASPASE3 and PARP-1 in the sucrose-treated groups were lower than those in the control group and were the lowest in the 210 mmol L^(–1) sucrose group. Both q RT-PCR and Western blot analyses suggested that sucrose inhibited cell apoptosis during freezing and thawing. Briefly, sucrose promoted p SSCs survival after freezing and thawing, especially at a concentration of 210 mmol L^(–1), which possibly assisted p SSC dehydration and inhibited cell apoptosis. These findings hold great promise for further studies of the regulatory mechanism of proliferation and differentiation of p SSCs.
基金Supported by Grants from VINNMER,Lundin foundation, R&D Funds from Stockholm County and Karolinska Institutet (ALF),and the Swedish Research Council
文摘AIM:To optimize a xeno-free cryopreservation protocol for primary human hepatocytes.METHODS:The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes.Despite several hepatocyte cryopreservation protocols being available,improvements are urgently needed.We first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the groups.Using the polystyrene box freezing,we compared two xeno-free freezing solutions for freezing of primary human hepatocytes:a new medium(STEM-CELLBANKER,CB),which contains dimethylsulphoxide(DMSO) and anhydrous dextrose,both permeating and non-permeating cryoprotectants,and the frequently used DMSO-University of Wisconsin(DMSOUW) medium.The viability of the hepatocytes was assessed by the trypan blue exclusion method as well as a calcein-esterase based live-dead assay before and after cryopreservation.The function of the hepatocytes was evaluated before and after cryopreservation by assessing enzymatic activity of 6 major cytochrome P450 isoforms(CYPs):CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP3A4 and CYP3A7.RESULTS:The new cryoprotectant combination preserved hepatocyte viability significantly better than the standard DMSO-UW protocol(P < 0.01).There was no significant difference in viability estimation between both the trypan blue(TB) and the Live-Dead Assay methods.There was a correlation between viability of fresh hepatocytes and the difference in cell viability between CB and DMSO protocols(r 2 = 0.69) using the TB method.However,due to high within-group variability in the activities of the major CYPs,any statistical between-group differences were precluded.Cryopreservation of human hepatocytes using the cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte viability.Thus,it may be a better alternative to the standard DMSO-UW protocol.Estimating CYP activities did not seem to be a relevant way to compare hepatocyte function between different groups due to high normal variability between different liver samples.CONCLUSION:The cryoprotectant combination may be a better alternative to the standard DMSO-UW protocol in primary human hepatocyte cryopreservation.
文摘Cell cryopreservation has evolved as an important technology required for supporting various cell-based applications,such as stem cell therapy,tissue engineering,and assisted reproduction.Recent times have witnessed an increase in the clinical demand of these applications,requiring urgent improvements in cell cryopreservation.However,cryopreservation technology suff ers from the issues of low cryopreservation effi ciency and cryoprotectant(CPA)toxicity.Application of advanced biotechnology tools can signifi cantly improve post-thaw cell survival and reduce or even eliminate the use of organic solvent CPAs,thus promoting the development of cryopreservation.Herein,based on the diff erent cryopreservation mechanisms available,we provide an overview of the applications and achievements of various biotechnology tools used in cell cryopreservation,including trehalose delivery,hydrogel-based cell encapsulation technique,droplet-based cell printing,and nanowarming,and also discuss the associated challenges and perspectives for future development.
基金Supported by Scientific Research Foundation for Doctors of Northeast Agricultural University (2012RCB27)Postdoctoral Fund of Heilongjiang Provincial Academy of Agricultural Sciences (LRB04-185)
文摘The development and application of spermatogonial stem cell technology have an important significance in animal cloning, preservation of endangered species and spermatogenesis research. In this study, the seminiferous epithlium cells were isolated and purified, the cells were cryopreserved after identification, and the effects of different purification and cyopreservation methods on bovine testicular cells were studied. The results showed that there were spermatogonial stem cells and sertoli cells in the neonatal bovine seminiferous tubules, differential adherent selection methods could effectively separate these two cell types. Spermatogonial stem cells were positive after AKP, C-kit, and OCT-4 identification; sertoli cells were positive after oil red O and vimentin identification. Frozen stock solution supplemented with 10% DMSO had the best effect in spermatogonial stem cell cryopreservation, while frozen stock solution supplemented with 10% of ethylene glycol and 0.1 mmol·L-1 trehalose had the best effect in sertoli cells cryopreservation.
文摘Objective:To compare the outcomes of vitrification using cryoloop with slow-freezing meth-od for human blastocyst cryopreservation.Methods:In IVF-ET cycles,supernumerary embryos were cultured to Day 5 or Day 6,blas-tocysts were cryopreserved by vitrification using cryoloops or slow-freezing method,then blasto-cyst survival rate and pregnant rate were compared.Results:115 vitrified blastocysts from 39 cycles were warmed,104(90.4%)blastocysts sur-vived.After the transfer of 74 blastocysts in 38 cycles,28(73.7%)women got clinically preg-nant,2(7.1%)of them suffered from miscarriage,2 healthy babies were born in 2 deliveries,and the other 24 pregnancies are ongoing.As to slow-freezing method,87 blastocysts from 21 cy-cles were thawed,37(42.5%)of them survived,28 blastoeysts were transferred in 15 cycles,6(40%)women got clinically pregnant,1 of them miscarried,3 healthy babies were born in 2 de-liveries,and the other 3 pregnancies are ongoing.Conclusion:The survival rate and pregnant rate of vitrification using cryoloop are superior totraditional slow-freezing method,and the transfer cancel rate is lower than that of slow-freezingmethod.The miscarriage rate is similar in two methods.
基金Supported by the China Scholarship Council and South Australian Research and Development Institute(SARDI)
文摘In this study, a programmable freezing technique has been developed for strip spawned sperm in the blue mussel, M ytilus galloprovincialis. The optimized key parameters include cooling rate, endpoint temperature, thawing temperature, sugar addition and sperm to oocyte ratio. The sperm quality was assessed by the fertilization rate or the integrity of sperm component and organelle. The highest post-thaw sperm fertilization rate was 91%, which was produced with sperm cryopreserved in 8% dimethyl sulfoxide at the cooling rate of-4°C/min from 2°C to-30°C before being plunged into liquid nitrogen for at least 12 h, thawed in a 20°C seawater bath and fertilized at sperm to egg ratio of 50 000:1. The addition of glucose, sucrose or trehalose to 8% dimethyl sulfoxide could not further improve fertilization rates. The fluorescent assessments showed that the post-thaw sperm plasma membrane integrity and acrosome integrity were significantly damaged in comparison with fresh sperm.
基金supported by the Ministry of Agriculture,Forestry and Fisheries,Japan(MAFF)under the project name“Development of technology for enhancement of livestock lifetime productivity by improving fertility through assisted reproduction”the JSPS KAKENHI(15H04585).
文摘Background:The application of cryopreservation and artificial insemination technology have contributed to the advancement of animal reproduction.However,a substantial proportion of spermatozoa undergoes alterations and loses their fertility during cryopreservation,rendering the frozen-thawed semen impractical for routine use.Cryopreservation is known to reduce sperm lifespan and fertility.Variation in cryosurvival of spermatozoa from different sires and even with the individual sire is common in artificial insemination(AI)centers.Our goal is to improve post-thawed semen quality by optimization of cryopreservation technique through sperm selection prior to cryopreservation process.Results:Our strategy of sperm selection based on rheotaxis and thermotaxis(SSRT)on macrosale in a rotating fluid flow demonstrated the ability to maintain the original pre-freezing structural integrity,viability and biological function related to fertilization competence.This strategy has a positive effect on the cryosurvival and fertilizing abilities of spermatozoa as supported by the improvement on pregnancy rate of Japanese Black heifers and Holstein repeat breeders.This technique protected further sublethal damage to bovine spermatozoa(higher%cryosurvival than the control)and resulted in the improvement of DNA integrity.Prefreeze selected spermatozoa demonstrated slower and controlled capacitation than unprocessed control which is thought to be related to sperm longevity and consequently to appropriate timing during in vivo fertilization.Conclusions:These results provide solid evidence that improvement of post-thawed semen quality by SSRT method is beneficial in terms of cryosurvival,longevity of post-thawed sperm,and optimization of in vivo fertilization,embryo development and calving as supported by the favorable results of field fertility study.
基金a grant of the Korea Institute of Radiological and Medical Sciences,funded by Ministry of Science,ICT and Future Planning,South Korea,No.1711031810/50586-2016 and No.1711031808/50581-2016
文摘AIM To investigate a suitable long-term culture system and optimal cryopreservation of intestinal organoid to improve organoid-based therapy by acquiring large numbers of cells.METHODS Crypts were isolated from jejunum of C57BL/6 mouse. Two hundred crypts were cultured in organoid medium with either epidermal growth factor/Noggin/R-spondin1(ENR) or ENR/CHIR 99021/VPA(ENR-CV). F o rsubculture, organoids cultured on day 7 were passaged using enzyme-free cell dissociation buffer (STEMCELL Technologies). The passage was performed once per week until indicated passage. For cryopreservation, undissociated and dissociated organoids were resuspended in freezing medium with or without Rho kinase inhibitor subjected to different treatment times. The characteristics of intestinal organoids upon extended passage and freeze-thaw were analyzed using Ed U staining, methyl thiazolyl tetrazolium assay, q PCR and time-lapse live cell imaging.RESULTS We established a three-dimensional culture system for murine small intestinal organoids using ENR and ENR-CV media. Both conditions yielded organoids with a crypt-villus architecture exhibiting Lgr5^+ cells and differentiated intestinal epithelial cells as shown by morphological and biochemical analysis. However, during extended passage (more than 3 mo), a comparative analysis revealed that continuous passaging under ENR-CV conditions, but not ENR conditions induced phenotypic changes as observed by morphological transition, reduced numbers of Lgr5^+ cells and inconsistent expression of markers for differentiated intestinal epithelial cell types. We also found that recovery of long-term cryopreserved organoids was significantly affected by the organoid state, i.e., whether dissociation was applied, and the timing of treatment with the Rho-kinase inhibitor Y-27632. Furthermore, the retention of typical morphological characteristics of intestinal organoids such as the crypt-villus structure from freeze-thawed cells was observed by live cell imaging.CONCLUSION The maintenance of the characteristics of intestinal organoids upon extended passage is mediated by ENR condition, but not ENR-CV condition. Identified long-term cryopreservation may contribute to the establishment of standardized cryopreservation protocols for intestinal organoids for use in clinical applications.
基金supported by Capital's Funds for Health Improvement and Research of China(Grant No.2020-2-2112)Beijing Municipal Administration of Hospitals’Ascent Plan of China(Grant No.DFL20181401)the Beijing Natural Science Foundation of China(Grant No.7202047),References。
文摘In recent years,with the rapid development of medical research,cancer diagnosis and treatment technology have significantly improved young cancer patient’s survival rate.Anticancer therapy such as chemotherapy,radiotherapy,or hematopoietic stem cell transplantation can lead to premature ovarian insufficiency.The endocrine and reproductive function of the ovary is critical to women’s physical and mental health.Ovarian tissue cryopreservation and transplantation can protect not only female fertility but also preserve ovarian endocrine function.This paper interprets the guidelines for ovarian tissue cryopreservation and transplantation issued by the Chinese Society of Gynecological Endocrinology affiliated to the International Society of Gynecological Endocrinology.The purpose of this guideline’s interpretation is to promote more medical workers to understand the technology of ovarian tissue cryopreservation and transplantation,which can provide patients with more choices of fertility protection methods and improve their quality of life.
文摘The traditional corneal cryopreservation technique was improved. We carried out an experimental study that rabbit corneas were cryop-reserved by using polyvinylpyrolidone (PVP) as cryoprotective agent and dimethlsulfoxide (DMSO) as the control. The endothelia of cryopreserved corneas were evaluated by scanning and transmission electron microscopy and vital staining. The study shows that PVP is an excellent extracellular cryoprotective agent and has the characteristic of low toxicity or no toxicity to co...
