Months Of Cryopreservation Research Articles

Effect of cryopreservation on pollen viability, fertility and morphology of different Psidium species

Cryopreservation of pollen is a complementary conservation strategy and can be used for conserve the diversity in the genus Psidium. The present study aims to cryopreserve the pollen of Psidium species to overcome asynchronous flowering. The pollen of different Psidium species were germinated in vitro in an optimized medium of germination. In vitro/in vivo pollen viability assessment and SEM analysis were carried out to determine the changes after cryopreservation. The in vitro pollen viability was determined at monthly intervals starting from fresh pollen until six months of cryopreservation. The in vivo fertility tests were carried out by pollination using both fresh and cryopreserved pollen. The cryopreserved pollen showed in vitro germination ranging from 1.78% (in P. molle) to 81.67% (in “H 12–5”) compared to fresh pollen (2.16% in P. molle to 86.08% in P. guineense). In vivo fertility was tested by controlled pollination using six-month-old cryopreserved pollen and it resulted in fruit setting ranged between 3.33% (P. cattleianum var. cattleianum) and 27.66% (P. chinensis) as compared to fresh pollen between 4.0% (P. cattleianum var. cattleianum) and 30.66% (P. chinensis). Seed set and germination was also recorded in all the crosses attempted using cryopreserved pollen. These in vitro and in vivo results indicated that cryopreservation is an effective technique for breeding and conserving the haploid gene pool in cryo genebank. Scanning Electron Microscopic studies of pollen revealed no significant variation in shape and size after cryopreservation when compared to fresh pollen.

Impact of Exposure Period to Liquid Nitrogen Vapor on Criteria of Human Spermatozoa Cryopreserved in New Cryopreservation Technique

Background: The emptied sheep’s ovarian follicles recently used as a container for spermatozoa during cryopreservation, it was found a proper carrier to cryopreserving spermatozoa in vapor-dependent cryopreservation. The aim of this study was to evaluate the effect of two periods of exposure to liquid nitrogen (LN2)vapor on the parameter of spermatozoa during cryopreservation in this technique. Method: The study was conducted on 30 semen samples from patients with oligozoospermia diagnosed by semen analysis according to the standard criteria of World Health Orgnization (WHO) 2010. Sheep’s ovarian follicles obtained from local slaughterhouse and prepared by slicing the ovaries and evacuating the follicular fluid and oocyte. Each semen sample diluted 1:1 with cryosolution (glycerol 10%) and injected within eight emptied sheep’s ovarian follicles. The first four follicles represent P1; exposed to LN2 vapor for 7.5 minutes and the other four follicles represent P2; exposed to LN2 vapor for 15 minutes before emerged in liquid nitrogen. Sperm progressive motility, total motility, normal morphology and DNA fragmentation index (DFI) were analyzed for all samples pre-freezing and post-thawing. Results: After two months of cryopreservation, sperm progressive motility and total motility significantly (P<0.01) increased post-thawing in P2 as compared with P1, while both of P1 and P2 significantly (P<0.01) decreased as compared with pre-freezing. Normal morphology significantly (P<0.01) decreased post thawing in both P1 and P2 as compared with pre-freezing, while no significantly difference foundbetween P1and P2. DFI significantly (P<0.01) increased post-thawing in P1 and P2 as compared with pre-freezing, while in P2 DFI was significantly lower than in P1. Conclusions: The exposure to liquid nitrogen vapor for 15 minutes in emptied ovarian follicles technique gives a better results than exposure to the vapor for 7.5 minutes regarding sperm progressive motility, total motility and DFI.

Mechanical Stability of Cryopreserved Split-Thickness Tectonic Corneal Grafts.

To evaluate the influence of cryopreservation on the pressure-strain relationship of microkeratome dissected anterior stromal grafts (ASGs). Partial thickness ASGs were created from 7 pairs of human corneas and randomized to immediate grafting or grafting after 3 months of cryopreservation at -80°C into a whole globe ex vivo corneal perforation model. High frequency ultrasound speckle tracking was used to calculate the cross-sectional axial and lateral strains in each graft at increasing intraocular pressure (IOP) from 5 to 30 mm Hg. The mean axial and lateral strains were compared between the paired groups. The mean axial and lateral strains were not significantly different between the cryopreserved and noncryopreserved ASGs. The mean lateral strains at 30 mm Hg in the noncryopreserved and cryopreserved grafts were 2.4% ± 2.1% and 1.4% ± 0.7% (P = 0.294), respectively. The mean axial strains at 30 mm Hg in the noncryopreserved and cryopreserved grafts were -7.8% ± 3.3% and -5.5% ± 3.0% (P = 0.198), respectively. A linear pressure-strain relationship was found for all grafts at physiologic IOP. ASGs cryopreserved at -80°C maintain their IOP-strain relationship compared with noncryopreserved ASGs at physiologic pressures, supporting the potential use of cryopreserved human corneal stroma for patch grafting procedures.

Cryopreservation and functional analysis of cardiac autonomic neurons

BackgroundGenerally, primary neurons are isolated and seeded within hours of isolation, but cryopreservation, documented for a small number of central and peripheral neuronal subtypes, can contribute to improved utility and reduce the cost of developing new in vitro models. The preservation of cells of the autonomic nervous system (ANS), specifically sympathetic and parasympathetic neurons, has not been explored. New MethodIn this work, we establish a method for preserving cardiac ANS neurons as well as evaluating the phenotypical changes of dissociated superior cervical ganglia (sympathetic neurons) and intracardiac ganglia (parasympathetic neurons) for up to a month of storage in liquid nitrogen. ResultsNeuron populations maintained a viability of at least 35%, and the extent of neurite outgrowth was not different from fresh cells, regardless of the storage duration studied. Expression of tyrosine hydroxylase and choline acetyl transferase were maintained over one month of cryopreservation in sympathetic and parasympathetic populations, respectively. Electrophysiological recordings for both neuron types indicate sustained characteristic resting potentials, excitability, and action potentials after more than one month in liquid nitrogen. Comparison with Existing MethodsPrimary cultures of the autonomic nervous system have been previously established for in vitro investigations. This is the first example of preserving primary ANS neuron cultures for long-term on-demand use. ConclusionsThis report describes a readily implemented method for cryopreserving sympathetic and parasympathetic neurons that does not alter neither morphological nor electrophysiological characteristics. This methodology expands the utility of ANS cultures for use in morphological and functional assays.

Analysis of freezable water content by DSC for apple dormant bud cryopreservation

Dormant bud cryopreservation is a valuable tool for the germplasm of temperate trees preservation. Freezable water content, as an important cryopreservation characteristic, was analysed by DSC. Scions of modern apple tree varieties (Malus domestica) were collected during the winter season from orchards, freeze-dehydrated to 26–32% of water content at –5°C and cryopreserved by the two-step procedure. Samples were rehydrated in moist peat after 4 months of cryopreservation and grafted on rootstocks in a field. Regeneration by sprouting buds on rootstocks was evaluated. The varieties were divided into three groups according to their water content and percentage of crystallized water. The regeneration in the three groups decreased with decreasing bud water content. Regeneration was higher than 80% in most of the varieties and reached 100% in almost 1/3 of the tested varieties. In only one variety, was regeneration lower than 40%, which is still a high regeneration rate for considering apple genotypes as successfully cryopreserved. According to this study, the two-step cryoprotocol was successfully optimized for Central European conditions.

Improvement of in vitro potency assays by a resting step for clinical-grade chimeric antigen receptor engineered T cells

BackgroundChimeric antigen receptor engineered T (CAR-T) cell therapy is a promising approach currently revolutionizing the field of cancer immunotherapy. However, data concerning clinical-grade CAR-T cell stability and functionality after months of cryopreservation have not been released by companies so far. To investigate the effect of cryopreservation on CAR-T cells and to further optimize the potency assays, we performed this study. MethodsA third generation of CD19 CAR-T cells was manufactured according to Good Manufacturing Practice (GMP) requirements, which is applied to patients in an ongoing clinical phase 1 study. Quality control tests for sterility, endotoxin and mycoplasma were performed for each batch. Stability in terms of viability, recovery, transduction efficiency and functional capacity was determined using microscopy, multiparametric flow cytometry as well as chromium-51 release tests. ResultsUp to 90days of cryopreservation had no influence on viability, recovery and transduction efficiency of CAR-T cells. However, higher cell concentration for cryopreservation could alter the cell viability and recovery but not the transduction efficiency. Moreover, directly after thawing, both the quantity and quality of the functionality of CAR-T cells were transiently hampered by the negative effects of cryopreservation. Notably, the impaired functionality could be fully restored and even strengthened after an overnight resting process. DiscussionCryopreservation is a challenge for the functional activity of CAR-T cells. However, CAR-T cells regain their potency by overnight incubation at 37°C, which mimics the clinical application setting. Therefore, an overnight resting step should be included in in vitro potency assays.

Long Term Effect of Cryopreservation on Primary Human Skin Cells

Cryopreservation is essential for tissue engineering and regenerative medicine. This study was carried out to assess the effect of cryopreservation on skin cells and evaluate the performance of cells after 12 months of cryopreservation. Redundant skin tissue samples were obtained from surgery with consent from patients. The tissue was cleaned, processed and cultured until passage 3. Upon confluency, cells were trypsinised and total cell yield and viability were determined before and after being cryopreserved. Sterility and immunocytochemistry analysis for collagen type I (Col-1) and cytokeratin 14 (CK14) antibodies were also performed on cells cryopreserved for one, three, six and twelve months. There is no significant difference in growth rates for cryopreserved cells for 1 to 12 months, except for fibroblasts at 6 months. Cell viability for both keratinocytes and fibroblasts decreased with time (65%± 3.5% - 89%± 4.5%). Sterility testing showed no contamination after 12 months of cryopreservation. Immunocytochemistry analysis showed positive expression for CK14 (keratinocytes) and Col -1 (fibroblasts) after 12 months of cryopreservation. Morphologically, keratinocytes and fibroblasts were able to retain its phenotype. The loss in viability is consistent in all samples and possibly due to thermal-cycling effect. Immunocytochemistry and consistent cell growth analysis showed that keratinocytes and fibroblasts were able to retain their characteristics in cryopreservation condition. These preliminary findings show that primary skin cells can be stored via cryopreservation and still retain their characteristics. However, further investigations using longer periods of cryopreservation (24 months, 48 months) should be conducted.

Establishment and characterization of a cell line derived from the kidney of tilapia, Oreochromis niloticus

PDF HTML阅读 XML下载 导出引用 引用提醒 罗非鱼肾脏细胞系的建立及其生物学特性 DOI: 作者: 作者单位: 1. 中国水产科学研究院长江水产研究所, 湖北 武汉 430223;2. 中山大学海洋学院, 广东 珠海 519082 作者简介: 赵建青(1991-),硕士,研究实习员,从事水产养殖病害研究.E-mail:964117942@qq.com;贾鹏(1989-),博士研究生,从事海洋经济鱼类体细胞研究及应用.E-mail:825143529@qq.com 通讯作者: 中图分类号: S947 基金项目: 湖北省科技支撑计划项目（2015BBA234）；国家大宗淡水鱼产业技术体系专项资金（CARS-45-16）；中国水产科学研究院基本科研业务费专项（2017HY-ZD1005）. Establishment and characterization of a cell line derived from the kidney of tilapia, Oreochromis niloticus Author: Affiliation: 1. Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China;2. School of Marine Sciences, Sun Yat-sen University, Zhuhai 519082, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:采用组织块移植法，对尼罗罗非鱼（）的肾脏组织细胞进行原代培养，建立了罗非鱼肾脏细胞系，已稳定传代培养50代以上，命名为TiK。罗非鱼肾脏细胞系为纤维样细胞，其最佳培养基为DMEM，最适培养温度为28℃，最适血清浓度为15%。在最适培养条件下，罗非鱼肾脏细胞系的群体倍增时间为45.8 h。细胞经液氮冷冻保存6个月后进行复苏，经台盼蓝染色，约（89.84±3.48）%的细胞具有活性，复苏后细胞生长旺盛。染色体分析显示，第32代罗非鱼肾脏细胞系染色体数目分布在20~66之间，众数为48。使用本实验室分离鉴定的罗非鱼病毒感染罗非鱼肾脏细胞，可产生典型的细胞病变效应，表明罗非鱼肾脏细胞对该病毒敏感。该细胞系的建立为罗非鱼病毒病防控技术研究提供了重要的实验材料。 Abstract:Tilapia is an important aquaculture species world-wide, and China is the largest producer of farmed tilapia. In recent years, alongside the rapid development of tilapia aquaculture in the world, diseases in this fish have become a great concern, including viral diseases caused by beta Nodavirus, herpes simplex virus, and tilapia lake virus, which lead to huge economic losses. So far, research studies on tilapia viral pathogens are dependent on the E-11 cell line derived from grouper and tilapia primary brain cells; thus, a cell line derived from the host of tilapia needs to be urgently established for applications. Therefore, in the present study, by using tissue explant techniques, a novel cell line (designated TiK) derived from the kidney of tilapia (), was established and fully characterized. The TiK cell line are fibroblast-like cells, which have been stably subcultured for more than 50 passages. The optimal growth conditions for the TiK cell line are DMEM medium, 28℃, and 15% FBS. Under the optimal conditions, the doubling time of TiK cells is about 45.8 h. After 6 months of cryopreservation in liquid nitrogen, TiK cells exhibit a viability of about (89.84±3.48)%, as measured by trypan blue staining. Chromosome analysis of TiK cells showed that the number of chromosomes ranged from 20 to 66, and the modal number is 48 at the 32th passage. The TiK cell line was infected with tilapia virus isolated by our laboratory, and showed typical cytopathic effects, demonstrating that the TiK cell line was susceptible to infection by tilapia viruses. The establishment of the TiK cell line will contribute to research on the control of tilapia viral diseases. 参考文献 相似文献 引证文献

Performance Verification of Alternative Quality Control Materials for Urine Albumin Assessment.

Accurate detection of urine albumin is important for evaluating the progression of diabetic kidney disease. However, two levels of daily quality control may not be practically feasible in some small clinical laboratories owing to a small number of patient samples and high costs. We aimed to prepare homemade quality control material (HQM) to measure urine albumin and then verify its performance. Normal saline solution and fresh mixed urine samples from five donors with serious kidney disease were used to prepare two levels of HQM (HQM1 and HQM2). Anhydrous ethylene glycol and sodium azide were used as antifreeze and as a preservative, respectively. Before being separated into Eppendorf tubes, 20 tests for HQM1 and HQM2 were performed, resulting in mean ± SD of 19.52 ± 0.91 mg/L and 105.28 ± 3.71 mg/L, respectively. After having been divided, the vial-to-vial variations of HQM1 and HQM2 were small (4.93% and 3.70%, respectively). The stability of HQM1 and HQM2 stored at 2 - 8°C was about 2 months and 80 days, respectively, and when stored at -20°C, remained stable for more than 8 months. After 1 - 8 months of cryopreservation at -20°C, once opened, the HQM in every Eppendorf tube could be kept for at least five days (CV < 6.1%). Our HQM stored at -20°C remained stable for a long time, and so could be considered as an alternative to standard QMs in the clinical laboratory.

First attempt on somatic cell cryopreservation of critically endangered Camelus bactrianus of India

The double-humped camel (Camelus bactrianus) of India is critically endangered since the population is around two hundred only. Immediate measures are required for its conservation so as to prevent loss of this precious germplasm forever. This is the first report on development of ear marginal tissue fibroblast cell line from Bactrian camels using primary explant technique followed by cryopreservation. The cells showed typical fusiform morphology with centrally located oval nuclei with radiating, flame like or whirlpool like migrating patterns. Four different culture media MEM, DMEM-Hi glucose, DMEM/Ham's F-12 and fibroblast specific media (HiFibroXL™) were tested with respect to the potential to support growth of camel fibroblasts. Fibroblast specific media was selected for the primary culture. Maximum cell multiplication during secondary culture was supported by the DMEM/Ham's F-12 as total cell count with same seeding (80,000 cells) and under similar culture conditions was 6,75,000, 3,05,000, 13,90,000 and 10,05,000cells/mL for MEM, DMEM-Hi glucose, DMEM/Ham's F-12 and fibroblast specific media (HiFibroXL™), respectively. Cells followed a typical sigmoid growth curve with population doubling time of 26.13h. Epithelial and fibroblast cell initially grew together. However, fibroblast cells outgrew their epithelial counterparts in subsequent passages. Reverse transcriptase PCR was performed for individual samples at different passages by designing primers specific to Cytokeratin19 (CK19) and Osteopontin (OPN). Osteocytes were absent from the very beginning whereas, epithelial cells were eliminated after the first passage. Thus, the cells were cryopreserved from 4th to 6th passages. Chromosome karyotyping showed normal diploid (2n=74) number and the cells were free from microbial contaminations. Cells had more than 90% viability in the culture after one month of cryopreservation. These cells were morphologically indistinguishable from the cell stocks prior to freezing. Thus, genetic resource of Bactrian camel has been preserved at cellular level. The repository can also serves as a valuable resource for genome, post genome and somatic cell cloning research.

P241 A study of immunocomplex capture fluorescence analysis using lymphocyte and monocyte fraction by freezing isolation

Aim Immunocomplex capture fluorescence analysis (ICFA) is a novel method to predict rejection/acceptance responses in organ transplantation. The method is less susceptible to the effect of drug compared with traditional methods. The method uses fluorescent beads to sensitively detect antibodies directed against HLA I/II antigens on blood cells.In this study, the changes in mean fluorescence intensity (MFI) control values of lymphocyte and monocyte fraction from donor cells before cryopreservation and donor cells after one month of cryopreservation were monitored using ICFA for a lymphocyte crossmatch. Methods The subjects were patients who were going to undergo living-donor kidney transplantation. ICFA method for a lymphocyte crossmatch was performed. The stability of MFI control values was monitored for donor cells before cryopreservation and those after one month of cryopreservation. Results The monitoring results showed that the MFI control value of lymphocyte and monocyte fraction from donor cells after one month of cryopreservation was 9.4% higher than that of lymphocyte and monocyte fraction from donor cells before cryopreservation. On the other hand, the MIF control value of leukocytes after one month of cryopreservation using the conventional method was 100%. Conclusions More reliable donor-specific HLA antibody test results can be obtained by lymphocyte crossmatch test using lymphocyte and monocyte fraction by freezing isolation from cryopreserved donor cells.

The effect of different concentrations of Dimethyl sulfoxide (DMSO) and glycerol as cryoprotectant in preserving Vero cells

Different concentrations of dimethyl sulfoxide (DMSO) and glycerol were used for cryopreservation of Vero cells. After total cell count Vero cells were preserved in liquid nitrogen. Two frozen stocks were made simultaneously from the cell suspensions of same concentrations using DMSO or glycerol at concentrations of 2.5%, 5%, 10% and 15%. After six months of cryopreservation both frozen stocks were used providing same nutrients and environment for the viability of the Vero cells. The cell viability analysis was performed immediately after thawing by Trypan Blue Exclusion Test. Both cryoprotectants showed a protective effect on Vero cells. When Glycerol was used, a maximum cell viability rate of 89.4% and a lowest cell viability rate of 63% were achieved at concentrations of 10% and 2.5%, respectively. On the other hand, DMSO at a concentration of 10% had the highest effect on cryoprotectivity and showed highest cell viability (75%), while at 15% concentration it showed the lowest cell viability (53%). It is suggested that DMSO and glycerol are appropriate protective materials for the cryopreservation of Vero cells. The solutions at concentration of 10% of DMSO and glycerol could be the best choice of cryoprotectant for long-term (6 months) preservation of Vero cells.Bangl. vet. 2016. Vol. 33, No. 1, 1-7

The effects of cryopreservation on the expression of canine regulatory T‐cell markers

Regulatory T (Treg) cells have been described as key regulators in various immunological processes and are of growing interest in veterinary allergy. Cryopreservation of immune cells is performed routinely in human basic science research and in clinical studies. As such, it allows batch testing of collected samples at a single time point, resulting in a significant reduction in sample variability. Data which describe the effects of cryopreservation on Treg cell frequency and functionality in the canine species are important to inform future research. The purpose of this study was to establish a robust freeze/thaw procedure and flow cytometric staining protocol for canine Treg cells, and to compare the frequencies of different canine Treg cell phenotypes before and after cryopreservation. Nine privately owned dogs. Peripheral blood mononuclear cells were isolated and Treg cells stained and analysed by flow cytometry, before and after three months of cryopreservation. The recovery percentages and the corresponding correlations (fresh versus cryopreserved) for CD4+ CD25+ , CD4+ FOXP3+ and CD4+ CD25+ FOXP3+ cell populations were calculated. A high recovery rate of 97.2 (r = 0.94, P < 0.0001), 93.9 (r = 0.77, P < 0.01) and 101.7% (r = 0.99, P < 0.0001) for CD4+ CD25+ , CD4+ FOXP3+ and CD4+ CD25+ FOXP3+ cell populations, respectively, was observed. This study demonstrates an optimized protocol for freezing, thawing and quantifying canine Treg cells. These results indicate that cryopreservation does not substantially affect the expression of surface and intracellular markers of canine Treg cells; however, additional studies will be necessary to assess whether functionality of the cells is also maintained.

Conditioned medium: a new alternative for cryopreservation of equine umbilical cord mesenchymal stem cells.

Cryopreservation is a feasible alternative to maintaining several cell lines, particularly for immediate therapeutic use, transportation of samples, and implementation of new in vitro studies. This work parts from the hypothesis that the medium of cryopreservation composed by 90% of conditioned medium (CM) supports cryopreservation of equine umbilical cord intervascular matrix mesenchymal stem cells (UCIM-MSCs), allowing the maintenance of the biological properties for the establishment of cell banks intended for therapeutic use and in vitro studies. Thus, we evaluated the viability, apoptosis/necrosis rates, immunophenotypic profile (IP), chromosomal stability, clonicity, and differentiation potential of UCIM-MSCs cryopreserved with four different mediums (with FBS: M1, M3, M4 and without FBS: M2). After 3 months of cryopreservation, samples were thawed and analyzed. The potential of differentiation in the mesodermal lineages, clonicity, and the chromosomal stability were maintained after cryopreservation of UCIM-MSCs with medium containing FBS. Changes (P < 0.05) at IP for some markers were observed at cells cryopreserved with medium M1-M3. Only the UCIM-MSCs cryopreserved with the CM (M4) had similar viability post-thaw (P = 0.23) when compared with fresh cells. We proved the hypothesis that the medium of cryopreservation containing CM supports the cryopreservation of UCIM-MSCs, at the experimental conditions, being the medium that better maintains the biological characteristics observed at fresh cells. Thus, future studies of UCIM-MSCs secretome should be conducted to better understand the beneficial and protective effects of the CM during the freezing process.

Reproductive stage-dependent effects of additional cryoprotectant agents for the cryopreservation of stallion germ cells

The main objective of this study was to evaluate the efficacy of an additional cryoprotectant in 10% dimethyl sulfoxide (DMSO) on cryopreserving germ cells from stallions at different reproductive stages. Testicular samples were obtained from pre-pubertal (1–1.5 yr, n=6) and post-pubertal (3–7 yr, n=5) stallions. Germ cells were isolated using a two-enzyme digestion procedure and cryopreserved in minimal essential medium alpha containing 10% fetal bovine serum and 10% DMSO with or without addition of trehalose (50, 100, or 200mM) or polyethylene glycol (PEG, 2.5, 5, or 10%). Viability, cell population, and viable population were assessed after 1 and 3 months of cryopreservation. The viable UTF1-positive population of pre-pubertal stallion germ cells was also measured using immunocytochemistry after 1 and 3 months of cryopreservation. As expected, the viability, cell population, and viable cell population were significantly reduced after 1 and 3 months of cryopreservation. At the pre-pubertal stage, the addition of trehalose or PEG to 10% DMSO did not show any effect on the viability, cell population, viable cell population, or viable UTF1-positive germ cells at either 1 or 3 months after cryopreservation. However, at the post-pubertal stage, the viable population was significantly higher in germ cells that were cryopreserved with 5% or 10% PEG, than in the cells cryopreserved with 10% DMSO only. In conclusion, PEG at 5% or 10% added to 10% DMSO serves as an optimal cryoprotectant agent for the cryopreservation of germ cells from post-pubertal stallions.

Preparation of Internal Quality Control Material for Lymphocyte Subset Analysis.

Lymphocyte subset analysis is widely used in clinical laboratories, and more than two levels of daily QC materials are required for reliable results. Commercially available, expensive QC materials have short shelf lives and may not be suitable in resource-poor settings. We compared different methods for preparing homemade QC material, including fixation with 1%, 2%, or 4% paraformaldehyde (PFA); freezing with 10% dimethylsulfoxide (DMSO), 0.1% bovine serum albumin-phosphate buffered saline, or after ethanolic dehydration; and using cryopreservation temperatures of -20℃, -80℃, or -196℃. We found an optimal experimental condition, which is 'fixation with 4% PFA, freezing with 10% DMSO, and storage at 80℃'. To evaluate long-term stability of QC materials prepared in this optimal condition, two levels of QC materials (QM1 and QM2) were thawed after 30, 33, 35, 37, 60, 62, 64, and 67 days of cryopreservation. Lymphocyte subset was analyzed with BD Multitest IMK kit (BD Biosciences, USA). QM1 and QM2 were stable after 1-2 months of cryopreservation (CV <3% for CD3, CD4, and CD8 and 5-7% for CD16/56 and CD19). We propose this method as an alternative cost-effective protocol for preparing homemade internal QC materials for lymphocyte subset analysis in resource-poor settings.

Mesenchymal stem cells derived from human umbilical cord and adipose tissue retain their properties after 24 months of cryopreservation

Mesenchymal stem cells derived from human umbilical cord and adipose tissue retain their properties after 24 months of cryopreservation

Utilization of stored autologous PBSCs to support second autologous transplantation in multiple myeloma patients in the era of novel agent therapy.

Outcomes in multiple myeloma (MM) have improved significantly with novel agent therapy and autologous stem cell transplantation (ASCT). ASCTs are typically planned as either tandem or a single transplant with additional stored PBSCs available for a second salvage transplant. To accommodate these strategies, many centers routinely collect and store adequate PBSCs for two ASCTs. We analyzed the cost associated with this practice by determining the expenses of PBSC collection, cryopreservation and storage, and the ultimate use of additional cryopreserved PBSCs in patients who had undergone at least one ASCT. There were 889 MM patients transplanted between 1993 and 2011 at our center. Most (N=726) had residual PBSCs in storage after their first ASCT (ASCT1). Only 135 patients underwent a second ASCT within a median of 14 months after ASCT1. The percentage of patients receiving a second ASCT declined over time. The resources required to collect and store unused PBSCs added up to 336 extra patient days of apheresis and 41 587 extra patient months of cryopreservation, translating into an average extra cost per patient of US$4981.12. A reconsideration of conventional PBSC collection and storage practices would save significant cost for the majority of MM patients who never undergo a second ASCT.

Microparticle-loaded neonatal porcine Sertoli cells for cell-based therapeutic and drug delivery system.

Neonatal porcine Sertoli cells (NPSC) are immune privileged cells showing innate phagocytic and antibacterial activities. NPSC have been shown capable of immunoaltering the body's response and possess lung homing capacity. These properties encourage investigation of NPSC as functional components of cell-based therapeutic protocols to treat lung infections and related complications. In this work, for the first time, NPSC were tailored to carry an antibiotic drug loaded into poly(d,l lactic) acid microparticles (MP). A loading protocol was developed, which afforded 30% drug uptake and high stability over time, with little or no effects on NPSC viability, morphology, reactive oxygen species production and DNA integrity. FSH receptor integrity, and TGFβ (transforming growth factor β) and AMH (anti-Müllerian hormone) expressions were unchanged after 1month of cryopreservation. Protein tyrosine kinase activation due to phagocytosis may have had resulted in changes in inhibin B expression. The activity of MP-loaded or NPSC alone against Pseudomonas aeruginosa was maintained throughout 1month of storage. NPSC couple an innate antibacterial activity with the capacity to embody drug loaded MP. We showed for the first time that engineered NPSC can be cryopreserved with no loss of their basic properties, thereby possibly representing a novel approach for cell-based therapeutic and drug delivery system.

Optimized cryopreservation of mixed microbial communities for conserved functionality and diversity.

The use of mixed microbial communities (microbiomes) for biotechnological applications has steadily increased over the past decades. However, these microbiomes are not readily available from public culture collections, hampering their potential for widespread use. The main reason for this lack of availability is the lack of an effective cryopreservation protocol. Due to this critical need, we evaluated the functionality as well as the community structure of three different types of microbiomes before and after cryopreservation with two cryoprotective agents (CPA). Microbiomes were selected based upon relevance towards applications: (1) a methanotrophic co-culture (MOB), with potential for mitigation of greenhouse gas emissions, environmental pollutants removal and bioplastics production; (2) an oxygen limited autotrophic nitrification/denitrification (OLAND) biofilm, with enhanced economic and ecological benefits for wastewater treatment, and (3) fecal material from a human donor, with potential applications for fecal transplants and pre/probiotics research. After three months of cryopreservation at −80°C, we found that metabolic activity, in terms of the specific activity recovery of MOB, aerobic ammonium oxidizing bacteria (AerAOB) and anaerobic AOB (AnAOB, anammox) in the OLAND mixed culture, resumes sooner when one of our selected CPA [dimethyl sulfoxide (DMSO) and DMSO plus trehalose and tryptic soy broth (DMSO+TT)] was added. However, the activity of the fecal community was not influenced by the CPA addition, although the preservation of the community structure (as determined by 16S rRNA gene sequencing) was enhanced by addition of CPA. In summary, we have evaluated a cryopreservation protocol that succeeded in preserving both community structure and functionality of value-added microbiomes. This will allow individual laboratories and culture collections to boost the use of microbiomes in biotechnological applications.