Long-term Cryopreservation Research Articles

Effect of alpha-lipoic acid on oxidative stress, viability and motility in the common carp (Cyprinus carpio) spermatozoa after short-term storage and cryopreservation

As known for different metabolic functions, α-lipoic acid (ALA) has been tested for spermatozoa preservation of animals as well as of human, but not for fish spermatozoa. The present study determined the effects of ALA on short and long-term (cryopreservation) preservation of common carp (Cyprinus carpio) spermatozoa, for the first time. For that, spermatozoa were diluted in extenders containing 0 (control), 0.025, 0.05, 0.1, 0.5, 1, 2, 5, and 10 mM of ALA concentrations in both short-term preservation and cryopreservation. Spermatozoa motility parameters by computer-assisted semen analysis, viability, lipid peroxidation and catalase activity in spermatozoa were conducted in both 2nd and 120th hours of short-term storage and post-thaw samples. Higher percentages of total spermatozoa motility (80 ± 3) and viability (87 ± 3) were observed in 0.5 mM ALA group after 120 h of incubation. In post-thaw samples, higher percentages of these parameters were in 1 mM ALA group (74 ± 3 and 83 ± 2, respectively). Moreover, the results have shown that the addition of ALA until concentrations of 2 mM improved especially spermatozoa curvilinear velocity, maintained viability, and suppressed excessive lipid peroxidation during the preservations. In conclusion, the additions of 0.5 mM ALA for short-term preservation and 1 mM ALA for cryopreservation were the optimal concentrations, and shown the protective effects on common carp spermatozoa, when considering all measured parameters together.

Cryopreservation: Paper‐Based Cell Cryopreservation (Adv. Biosys. 3/2020)

In article number 1900203, Mohammad A. Qasaimeh and co-workers utilize filter paper as a unique platform for long-term cryopreservation of mammalian cells. Paper fibers (blue) offer 3D-structural support for cells during cryopreservation and maintain the growth of 3D cell culture and spheroid formations before and after cryopreservation. The confocal image shows a HeLa spheroid formed post-cryopreservation and grown within the paper platform (red: F-actin, blue: nuclei, green: tubulin).

Long-term sperm cryopreservation does not affect post-thaw survival rates.

Objective:To compare cryosurvival rates of human spermatozoa in a prolonged period of cryopreservation.Methods:This retrospective study involved 33 cryopreserved semen samples from patients with cancer, between 2002 and 2011. The semen sample was obtained by masturbation and initial semen analysis was performed. The cryoprotectant solution was added and samples were frozen in liquid nitrogen in a slow step-wise process. For thawing, the samples were incubated at 25.0ºC for 15 min, followed by incubation at 36.7ºC for 15 min. The cryosurvival rate (CS) was calculate by CS= [(% total motile sperm post-thaw) x100/(% total motile sperm/tube)]. Each study sample was divided into three aliquots (Study Group; n=23): (I) official patient sample, which was kept cryopreserved for subsequent Assisted Reproduction procedure, cryopreserved between 2002 and 2011; (II) sample destined to post-thaw tests, performed after the sample had been kept cryopreserved for 24 hours; and (III) study sample. Only in 2014, after 3-12 years of cryopreservation, the study samples were thawed and evaluated. To validate the study design, a Validation Group was created including 10 samples obtained between 2014 and 2016, using the same methodology in the study samples. The data was analyzed using the T-test, with a significant p-value of 5%.Results:The mean age was 29.93±9.57 years in the Study Group and 21.80±6.49 years in the Validation Group. No significant difference between the Validation and Study Groups was found in the initial semen analysis (p>0.05). After 24 hours of cryopreservation, the cryosurvival rate was 26.11±46.36% in the Study Group and 23.71±57.06% in the Validation Group. Aliquots of the same sample preserved from 3-12 years demonstrated 23.71±57.06% of cryosurvival rate. Thus, no significant difference was found vis-à-vis the cryosurvival rates (p=0.56).Conclusion:We concluded that the method introduced in the late 1990s, which enables the removal of debris, potentially toxic elements and generators of reactive oxygen species from the seminal sample before cryopreservation, exhibited efficiency in maintaining the same cryosurvival rate after an extended period.

Assessment of Post-thaw Quality of Dental Mesenchymal Stromal Cells After Long-Term Cryopreservation by Uncontrolled Freezing.

Cryopreservation abilities of dental tissue-derived mesenchymal stromal cells (DMSCs) including dental pulp stem cells (DPSCs) and dental follicle stem cells (DFSC) play an important role in the applications of these cells in clinical settings. In this context, we checked whether storage at - 80°C in 10% DMSO for a longer period has any adverse effect on the functionality and genetic stability. We carried our studies on DPSC and DFSC samples that were revived after a maximum of 5years of cryopreservation. We observed that even after long-term uncontrolled freezing at - 80°C, these cells survived and proliferated efficiently. The assessment was made based on their post-thaw morphology, immunophenotypes, differentiation potential, growth kinetics, and genetic features. These cells retained the expression of stemness markers, differentiation ability and maintained their normal karyotype. Our results indicated no significant morphological or immunophenotypic differences between the cryopreserved DMSCs and the fresh DMSCs. Our study implies that mesenchymal stromal cells derived from the dental tissue origin are very robust and do not require any sophisticated preservation protocols. Thus, these can be an ideal source for research, stem cell banking, as well as successful clinical applications in tissue engineering and cell-based therapeutics. Graphical Abstract Schematic diagram showing the cryopreservation of DMSCs by uncontrolled freezing at -80 c has no adverse effects on their functionality and genetic stability.

Stem Cells from Human Trabecular Meshwork Hold the Potential to Develop into Ocular and Non-Ocular Lineages After Long-Term Storage.

Stem cells from the eye hold a great potential for vision restoration and can also be used for regeneration in other tissues. In this study, we characterized the stem cell properties of Trabecular meshwork stem cells (TMSCs) after long-term cryopreservation (∼8 years). TMSCs derived from four donors were examined for their viability and proliferation, as well as stem cell marker expression. Spheroid formation, colony formation, and multipotency were investigated. We observed that TMSCs were fully viable with variable proliferation ability. They expressed the stem cell markers CD90, CD166, CD105, CD73, OCT4, SSEA4, Notch1, KLF4, ABCG2, Nestin, and HNK1 detected by flow cytometry, quantitative polymerase chain reaction, or immunofluorescent staining. They could form spheroids and colonies after thawing. All TMSCs were able to differentiate into osteocytes, neural cells, and trabecular meshwork (TM) cells, but not adipocytes. Differentiated TM cells responded to dexamethasone treatment with increased expression of myocilin and angiopoietin-like 7 (ANGPTL7). In a nutshell, our study demonstrated that TMSCs retain their stem cell properties after long-term cryopreservation and hence can be an effective cell therapy source for various clinical applications.

Effects of Me2SO and Trehalose on the Cell Viability, Proliferation, and Bcl-2 Family Gene (BCL-2, BAX, and BAD) Expression in Cryopreserved Human Breast Cancer Cells.

Long-term cryopreservation of the viability and metabolic state of cells in cancer cell/tissue specimens has significant implications for diagnostic verification of disease progression in cancer patients and selection of effective treatment options via development of the patient-derived xenograft (PDX) models for drug screening. The purpose of this study is to investigate the effects of cryoprotectant agents (CPAs) on the expression of BCL-2 family genes (BCL-2, BAX, and BAD) that are involved in the growth and development of breast cancers. MCF-7 cells were cryopreserved in Dulbecco's modified Eagle's medium (DMEM) with 20% (v/v) fetal bovine serum, using 10% (v/v) Me2SO (dimethyl sulfoxide, DMSO) or 7.5% (v/v) Me2SO with 100is-300 mM trehalose as cryoprotectant solutions. After storage at -80°C for 7 days, the cells were thawed for evaluation. The use of Me2SO and trehalose has affected cell survival, proliferation, apoptotic state, as well as BCL-2 family gene expression. The conventional 10% (v/v) Me2SO method yields ∼80% post-thaw cell survival and good cell proliferation, but it drastically alters the pattern of the BCL-2 family gene expression. The antiapoptotic gene BCL-2 is downregulated, whereas two proapoptotic genes BAX and BAD are upregulated. The partial substitution of Me2SO with 200 or 300 mM trehalose enhances cell proliferation of survived cells after cryopreservation. The presence of trehalose upregulates the expression of both the antiapoptotic gene BCL-2 and proapoptotic genes BAX and BAD. Cryopreservation could tip off the checkpoint of the apoptotic pathway regulated by the BCL-2 family members, and the effect may be protectant dependent. The findings of this study demonstrate the importance of paying attention to the potential change of gene expression and metabolic state of cancer cells after cryopreservation in an attempt to development of the PDX models from cryopreserved cancer cells or tissue specimens.

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

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

The vitality of the Serbian spruce (Picea omorika) pollen during the long-term cryopreservation

This article studies differences in the vitality (germination rate and pollen tube length) of fresh pollen and pollen stored at −20 °C for 14 years of Serbian spruce (Picea omorika [Pančić] Purkyne). Fresh pollen was sampled from 24 trees from a Serbian spruce seed stand area on the site of Bela zemlja (43° 48′ 32″ N, 19° 44′ 25″ E) in the area of Mount Zlatibor (Serbia) in two successive years. Six nutrition media with different sucrose concentrations (0%, 5%, 10%, 15%, 20% and 25% aqueous solution) were used to assess the vitality of the pollen. Variance analysis shows significant differences among years of pollen sampling, years of cryopreservation, trees, and sucrose treatments for pollen germination for both germination rate and pollen tube length. The results indicate that climate conditions preceding the pollen maturation period had an impact on the pollen quality as well as on the pollen vitality during the years of cryopreservation. Inter-individual variability (differences between trees genetic variability) was very pronounced and enabled selection of pollinator trees that retained their initial vitality during the period of cryopreservation. This periodicity in Serbian spruce full flowering might have significant practical application in the breeding of this important species.

Fidelity of long-term cryopreserved adipose-derived stem cells for differentiation into cells of ocular and other lineages

Adipose-Derived Stem Cells (ADSCs) have an important contribution in regenerative medicine ranging from testing stem cell therapy for disease treatment in pre-clinical models to clinical trials. For immediate use of stem cells for therapy, there is a requirement of the high dose of stem cells at different time points which can be met by cryopreservation. In this study, we evaluated the characteristics of long-term cryopreserved ADSCs and their regenerative potential after an average of twelve-year cryopreservation. Revived ADSCs were examined for cell viability and proliferation by trypan blue, Calcein/Hoechst and MTT assay. Expression of stem cell markers was examined by flow cytometry, immunostaining and qPCR. Colony forming efficiency and spheroid formation ability were also assessed. Multilineage differentiation potential was evaluated by induction into osteocytes, adipocytes, neural cells, corneal keratocytes and trabecular meshwork (TM) cells. Post-thaw, ADSCs maintained expression of stem cell markers CD90, CD73, CD105, CD166, NOTCH1, STRO-1, ABCG2, OCT4, KLF4. ADSCs retained colony and spheroid forming potential. These cells were able to differentiate into osteocytes, confirmed by Alizarin Red S staining and elevated expression of osteocalcin and osteopontin; into adipocytes by Oil Red O staining and elevated expression of PPARγ2. ADSCs could differentiate into neural cells, stained positive to β-III tubulin, neurofilament, GFAP as well as elevated expression of nestin and neurofilament mRNAs. ADSCs could also give rise to corneal keratocytes expressing keratocan, keratan sulfate, ALDH and collagen V, and to TM cells expressing CHI3L1 and AQP1. Differentiated TM cells responded to dexamethasone treatment with increased Myocilin expression, which could be used as in vitro glaucoma model for further studies. Conditioned medium from ADSCs was found to impart a regenerative effect on primary TM cells. In conclusion, ADSCs maintained their stemness and multipotency after long-term cryopreservation with variability between different donors. This study can have great repercussions in regenerative medicine and pave the way for future clinical trials using cryopreserved ADSCs.

Phenotypical and functional analysis of donor lymphocyte infusion products after long-term cryopreservation

Donor lymphocyte infusions, collected from peripheral blood by apheresis, are regularly used to re-establishing disease control in patients with impending or full relapse after allogeneic cell transplantation. The cryopreservation and thawing processes of the cellular products, required for clinical needs, result in a decreased cellular recovery. The aim of this study was to perform an integral analysis of phenotypic and functional characteristics in different cell populations, within cryopreserved products used for therapeutic purposes.A total of 77 cryopreserved products were analysed. Cell viability and subpopulations such as CD3, CD4, CD8, CD14 and CD56 cells were quantified by FACS. Cell proliferation, cytotoxic capacity and CD4 intracellular ATP content were evaluated. A significant loss of cell viability was observed. CD56 cells were significantly reduced when compared with mononuclear cells without cryopreservation. Cell proliferation was also significantly reduced in the cryopreserved products. Cytotoxic capacity was decreased as well although it did not reach statistical significance. However, CD4 intracellular ATP was increased in the cryopreserved products. The analysed functional cell properties showed a wide distribution range although the apheresis, cryopreservation and thawing procedures were similar in all the analysed samples. Our findings may be useful for an improved characterisation of cryopreserved products to be used as donor lymphocyte infusion for therapeutic purposes.

Current perceptions and decisions of patients about cryopreserved embryos in Argentina.

Worldwide there is an increase in the number of assisted reproduction treatments. The developments in culture media and drugs, associated with the occurrence of rapid freezing or vitrification techniques has improved the procedures encouraging transfers of a single embryo per cycle. This generates a greater number of viable surplus embryos. An embryo disposition decision making is a complex phenomenon. Method: This descriptive exploratory study investigates the perceptions and opinions of a sample of 60 participants who have started an assisted reproduction treatment in a specialized Fertility Center of the City of Buenos Aires. The absence of children in the majority of the sample (70.6% still have no children) makes the prevailing option to “continue cryopreservation” (36.67%). The majority, 38.4% (23 people), considers that the option that best represents the embryo is "a child". Although there are relatively few people who have decided to donate their embryos (5 participants), several study participants report having reflected on it (35%). The vast majority (86.6%) of respondents would not be willing to face the costs of maintaining a long-term cryopreservation. After more than three decades of assisted reproduction, Argentinean patients still face a high degree of uncertainty regarding what to do with the surplus embryos. Gaining more in-depth knowledge regarding the decision-making process can help to improve the communication strategies. The normative absences generate that the ARTs users are "disengaged" from their embryos; this hinders the decision making, increasing the risk of accumulating more "abandoned" embryos

Chamber probe position effects on controlled rate freezing

Background & Aim Background: Controlled Rate Freezers (CRF) enable long-term cryopreservation of cellular therapy products. Freezing curves record that cryopreservation temperatures are following optimized protocols. Two abnormal freezing curves were investigated. Methods, Results & Conclusion Method Retrospective case reviews. Results In the first case, the standard freezing protocol was followed with minor cyclic oscillations. Product integrity was not compromised. The CRF has a wire chamber temperature probe that makes a 90 degree bend and descends through a six inch metal straw which prevents liquid nitrogen vapor from directly contacting the probe. The insulated probe is uncoated for 3 mm on the distal protruding end allowing for accurate measurements. The straw and probe end are not visible in the CRF due to a metal plate. Upon inspection by the vendor after first abnormal curve, the chamber probe was not protruding far enough out of the straw. Repositioning counteracted the observed oscillations. Several months later, a second abnormal freezing curve was observed. Ten minutes into the run, it was noted that the chamber temperature was having drastic, wide-ranging swings. The sample temperature did not show the irregular swings, but indicated warming. Upon inspection, the uncoated tip of the probe was making contact with the metal straw. Both types of oscillation were reproducible by varying the position of the chamber probe. The exposed temperature probe allows the CRF to measure the temperature of the chamber and inject liquid nitrogen vapor into the system to allow for cooling. When the probe is not in the proper position it is either insulated by the straw (probe within the straw) or the probe is grounded and produces erroneous temperature swings (when the probe touches the metal straw). In the latter case, erroneously cold chamber temperatures occurred, producing large-scale temperature oscillations on the chamber probe which caused the CRF to respond with rapid warming. Product integrity was compromised by the second, more severe event. The vendor was called to discuss the findings and they reported other clients had similar experiences with the grounding of the probe. Currently, we ensure the chamber probe is pulled at least 1 cm through the straw and bent at a 90 degree angle. Conclusion: Probe position is essential for proper function of the CRF which may affect cryopreservation of products. Any oscillations in the freezing curve necessitate evaluation of the chamber probe position.

The effect of cryopreservation on mitochondrial function in freshwater mussel tissue samples (Bivalvia: Unionida)

In non-thermoregulating and sessile organisms, such as the imperiled freshwater mussels (Bivalvia: Unionida), thermal sensitivity of mitochondria is a key factor for survival to global warming. Given the protected status of many unionids, non-destructive biopsies and subsequent cryopreservation are advisable procedures for further investigation of their mitochondrial function. To address whether long-term cryopreservation affects mitochondria in freshwater mussels, the mitochondrial respiration in permeabilized somatic cells of Elliptio complanata has been fully characterized through high-resolution respirometry. Our results indicate that cryopreservation does affect the absolute rate of respiration, which significantly decrease compared to fresh tissues, independently of substrates combination, respiratory state and normalizing factor. However, the negative impact is not reflected at the level of flux control ratios, suggesting that, even in front of a sharp decline in the aerobic capacity, cryopreserved tissues preserve the mitochondrial organization and could be thus employed for the qualitative analysis of mitochondrial function.

Cryobiotechnology: A Double-Edged Sword for Obligate Plant Pathogens.

Pathogen-free stock plants are required as propagation materials in nurseries and healthy materials are needed in germplasm exchange between countries or regions through quarantine programs. In addition, plant gene banks also prefer to maintain pathogen-free germplasm collections. Shoot tip cryotherapy is a novel biotechnology method whereby cryopreservation methods are used to eradicate obligate pathogens from vegetatively propagated plants. Long-term preservation of pathogens is necessary in all types of virus-related basic research and applications such as antigen preparation for virus detection by immunology-based methods, production of plant-based vaccines, genetic transformation to produce virus-derived resistant transgenic plants, and bionanotechnology to produce nano drugs. Obligate plant pathogens such as viruses and viroids are intracellular parasites that colonize only living cells of the hosts. Therefore, their long-term preservation is difficult. Cryotreatments cannot completely eradicate the obligate pathogens that do not infect meristematic cells and certain proportions of plants recovered from cryotreatments are still pathogen-infected. Furthermore, cryotreatments often fail to eradicate the obligate pathogens that infect meristematic cells. Cryopreservation can be used for the long-term cryopreservation of the obligate plant pathogens. Thus, cryobiotechnology functions as a double-edged sword for plant pathogen eradication and cryopreservation. This review provides updated a synthesis of advances in cryopreservation techniques for eradication and cryopreservation of obligate plant pathogens.

Quality assessment on the long-term cryopreservation and nucleic acids extraction processes implemented in the andalusian public biobank.

Human samples are commonly collected and long-term stored in biobanks for current and future analyses. Even though techniques for freezing human blood are well established, the storage time can compromise the cell viability as well as the yield and quality of nucleic acids (RNA and DNA) extracted from them. In this study, a protocol to obtain peripheral blood mononuclear cells (PBMCs) from 70 subjects, which were stored at - 196°C from EDTA tubes for a long-term, was assessed. In parallel; a protocol to obtain DNA from the same subjects, which were stored at - 80°C from citrate tubes, was also studied. Samples stored from 2008 to 2012 were studied and the results obtained showed that there were no statistically significant differences in the RNA or DNA extracted in terms of purity, integrity and functionality The freezing protocol used by the Málaga Biobank shows that viable PBMCs and DNA could be kept for a period of, at least, 10years, with a high quality and performance. Furthermore, RNA extracted from these PBMCs presents also a good quality and performance. Therefore, the samples frozen according to the conditions of the protocols assessed in this study could be optimal for biomedical research.

Thawing methods do not affect cell viability of CD45+ and CD34+ cells, but long-term cryopreservation of umbilical cord blood units generally decreases cell viability

IntroductionUmbilical cord blood units (UCBUs) are collected and cryopreserved in biobanks for a future transplant. Hematopoietic stem cells and hematopoietic progenitor cells (HSC/HPC) present in UCB can be damaged due to factors such as the cryopreservation process, the thawing process, and prolonged storage time. MethodsUCBUs (n = 27) were obtained from the Biobank of the National Center of Blood Transfusion (NCBT) from Mexico. They contained three attached segments of UCBU, including 1.0–2.3 × 106 CD34+ cells prior to cryopreservation and were stored during the period from 2003 to 2015. Each UCB segment was thawed with three different methods and CD34 cells, CD45 cells, and 7-AAD were identified by flow cytometry. Furthermore, we carried out CFU assays, and trypan blue staining. ResultsViable CD45+ (vCD45+) cells, vCD34+ cells, CFU, and percentage of E-Clone were not statistically significant among three different thawing methods. The number of vCD45+ and vCD34+ cells diminished in the three thawing methods compared with the same cells prior to their cryopreservation (p < 0.0001). vCD45+ and vCD34+ cells diminished in the ≥10-year cryopreservation group (p < 0.001). In addition, percentage of recovery of vCD45+ and vCD34+ cells diminished in this same group (p = 0.013 and p < 0.0001, respectively). ConclusionThe thawing methods did not affect either cell viability (vCD45+ and vCD34+ cells) or pluripotency (CFU, percentage of E-Clone) in attached segments of UCBUs. The ≥10-year cryopreservation time in attached segments of UCBUs alters the number of vCD45+ and vCD34+ cells; however, it does not affect their pluripotency.

Current perspectives on pollen cryopreservation in horticultural species

Cryopreserved pollen collections provide long-term security for wildflora, ornamentals, fruit and vegetable crops, medicinal herbs and endangered species. Technology has been developed for long-term cryopreservation of pollen in the form of nuclear genetic diversity (NGD) of mango, amla, pomegranate, jackfruit, guava, passion fruit, jamun, different species and cultivars of citrus, papaya (different species), grape, tomato, eggplant (Solanum gene pool), onion, capsicum, rose, gladiolus, gerbera, carnation and RET species of medicinal plants. Pollen collecting is usually done on a bright sunny day between 10-11 am. Pollen collections are subjected to viability indexing by germination in vitro by the hanging drop or cellophane techniques. Pollen samples are packed either in gelatin capsules or butter paper packets, sealed air tight in polyethylene aluminium laminated pouches and lowered into a canister of a cryoflask. The fertility of stored pollen is tested under field conditions through controlled pollination. The pollen cryobank is managed by periodic replenishment of the cryogen. With the recent advances in allied bio-scientific areas in which cryopreserved pollen could be used in general crosses, shuttle breeding maintenance of B line in male sterile based hybrid seed production. Security of male parent is also ensured in the cryobank. Pollen cryopreservation protocols developed for different horticultural species will be discussed in details.

Long-Term Cryopreservation and Revival of Tissue-Engineered Skeletal Muscle.

The ability to freeze, revive, and prolong the lifetime of tissue-engineered skeletal muscle without incurring any loss of function represents a significant advancement in the field of tissue engineering. Cryopreservation enables the efficient fabrication, storage, and shipment of these tissues. This in turn facilitates multidisciplinary collaboration between research groups, enabling advances in skeletal muscle regenerative medicine, organ-on-a-chip models of disease, drug testing, and soft robotics. Furthermore, the observation that freezing undifferentiated skeletal muscle enhances functional performance may motivate future studies developing stronger and more clinically relevant engineered muscle.

What was the fate of human embryos following long-term cryopreservation (≥12 years) and frozen embryo transfer?

Do human embryos survive long-term cryopreservation (CP) (≥12 years) and implant after frozen embryo transfer (ET)? Human embryos remain usable after long-term CP. Several cohort studies have reported the live birth rate or neonatal outcomes of human embryos after CP for up to 5 years. Only a few case reports have described successful live births from human embryos after long-term CP up to 12 years. This retrospective observational study in China included 20 patients (128 embryos) from March 2016 to April 2017. Twenty patients who had at least one live birth during their previous IVF/ICSI treatments and had surplus embryos cryopreserved were observed. Data concerning frozen embryo recovery, pregnancy and obstetric outcomes following frozen ET were recorded. A total of 128 embryos of 20 patients were observed. The embryo storage duration was 12.0-17.1 years, with a mean of 13.9 ± 1.73 years. In all, 115 embryos were thawed to transfer, with a survival rate of 74%. Sixty embryos were further cultured, which resulted in 20 blastocysts with a blastocyst formation rate of 33%. There were 21 cleavage-stage embryos and 13 blastocysts transferred in a total of 12 and 11 cycles, respectively, which resulted in one biochemical pregnancy, one first trimester miscarriage, two ectopic pregnancies, three singletons and one case of twins, with a clinical pregnancy rate of 25% (D3 ET) and 36% (blastocyst transfer) and a live birth rate of 17% (D3 ET) and 27% (blastocyst transfer). Two of the four patients who had live birth developed gestational diabetes mellitus. One of the five live births was a preterm delivery. The sample size was small due to the unique study population, and all the embryos underwent slow freezing. The fate of long-term cryopreserved embryos after vitrification is still unclear. The results provide evidence to support the use of embryos after extended CP to preserve patients' fertility. This study was supported by grants from the National Key Research and Development Programme of China (2016YC1000205) and the Guangzhou Scientific Programme (201508020006). None of the authors has any conflicts of interest to declare.

Subsets of CD4+, CD8+, and CD25hi Lymphocytes Are in General Not Influenced by Isolation and Long-Term Cryopreservation.

Several key factors can affect the outcome of immunological studies; isolation/cryopreservation can possibly alter T, B, NK, and T-regulatory (Treg) cell marker expression patterns. Blood samples from 50 blood donors supplemented with Na-heparin or K2EDTA were handled within 4 and 24 h after blood sampling. PBMC were isolated with different density gradients. Flow cytometric analysis of intracellular and extracellular CD markers was performed on blood samples freshly isolated PBMC, and PBMC was thawed 6 and 12 mo post-cryopreservation for the purpose of identifying B, NK, Th, T-cytotoxic, and Treg cells. No differences were observed in the percentages for CD3+, CD3+CD4+, CD3+CD8+, CD19+, or CD56+CD16+ cells within 24 h of sampling regardless of which supplement or isolation techniques were used. Differentiated (diff) CD4+ cells were in general less affected by isolation and cryopreservation than diff CD8+ cells. Terminally diff effector CD4+ and CD8+ cells were not affected by either isolation of lymphocytes or cryopreservation. In contrast, naive and early-diff effector memory CD4+ and CD8+ cells were affected by isolation and cryopreservation. The percentages of Treg cells defined as CD4+CD25hi expressing CD101 or CD129, CD4+CD25hiCD127-, and CD4+CD25hiCD127-FOXP3+, respectively, remained stable after isolation and cryopreservation. Subsets expressing CD127, with or without FOXP3, were not affected by isolation/cryopreservation. Subsets expressing CD39, contrary to CD45RA, on CD4+CD25+CD127- cells with or without FOXP3 were not affected by either isolation or cryopreservation. In conclusion, subsets of CD4+, CD8+, and CD25hi lymphocytes are in general not influenced by isolation and long-term cryopreservation.