A non-enzymatic method for isolating human adipose tissue-derived stromal stem cells

Background aimsThe isolation of human adipose stromal/stem cells (ASCs) currently relies on the use of the enzyme collagenase, which digests the triple helix region of peptide bonds in the collagen of adipose tissue. Collagenase is an expensive reagent derived from a bacterial source, and its use in isolating ASCs is a time-consuming procedure. This experiment evaluated the extraction of ASCs without an enzymatic digest.MethodsWe used a simple method of washing adipose tissue to isolate and characterize the cells and compared this method with the enzymatic procedure in terms of processing time, stem cell yield, differentiation potential and immunophenotype.ResultsBased on fluorescence activated cell sorting analysis, the stromal vascular fractions isolated with the washing method displayed a distinct and potentially favorable immunophenotype relative to the collagenase digestion. This difference may reflect the absence of chemical alteration of the cells by collagenase digestion. Independent of the isolation procedure, the resulting passaged ASCs were comparable based on immunophenotype and adipogenic and osteogenic differentiation potential.ConclusionsAlthough using collagenase substantially increases cell yield, the two methods yield a similar cell product.     

Effect of cell culture media on extracellular vesicle secretion from mesenchymal stromal cells and neurons

BackgroundExtracellular vesicles (EVs) secreted by neuronal cells in vitro have promising therapeutic potential for brain diseases. Optimization of cell culture conditions and methodologies for high-yield isolation of EVs for preclinical and clinical applications, however, remains a challenge.ObjectiveTo probe the cell culture conditions required for optimal EV secretion by human-derived neuronal cells.MethodologyFirst, we optimized the EV purification protocol using human mesenchymal stromal cell (MSC) cultures. Next, we compared the effects of different variables in human pluripotent stem cell (hPSC)-derived neuronal cultures on EV secretion. EVs were isolated from cell conditioned media (CCM) and control media with no cells (NCC) using ultrafiltration combined with size-exclusion chromatography (SEC). The hPSC neurons were cultured in 2 different media from which EVs were collected at 2 maturation time-points (days 46 and 60). Stimulation with 25 mM KCl was also evaluated as an activator of EV secretion by neurons. The collected SEC fractions were analyzed by nanoparticle tracking analysis (NTA), protein concentration assay, and blinded transmission electron microscopy (TEM).ResultsA peak in cup-shaped particles was observed in SEC fractions 7–10 of MSC samples, but not corresponding media controls, indicating successful isolation of EVs. Culture medium had no significant effect on EV yield. The EV yield of the samples did not differ significantly according to the culture media used or the cell maturation time-points. Stimulation of neurons with KCl for 3 h reduced rather than increased the EV yield.ConclusionsWe demonstrated successful EV isolation from MSC and neuronal cells using an ultrafiltration-SEC method. The EV yield from MSC and neuronal cultures exhibited a large batch effect, apparently related to the culture media used, highlighting the importance of including NCC as a negative control in all cell culture experiments.     

Combined use of N-acetylcysteine and Liberase improves the viability and metabolic function of human hepatocytes isolated from human liver

Background aimsSuccessful hepatocyte isolation is critical for continued development of cellular transplantation. However, most tissue available for research is from diseased liver, and the results of hepatocyte isolation from such tissue are inferior compared with normal tissue. Liberase and N-acetylcysteine (NAC) have been shown separately to improve viability of isolated hepatocytes. This study aims to determine the effect of Liberase and NAC in combination on human hepatocyte isolation from normal and diseased liver tissues.MethodsHepatocytes were isolated from 30 liver specimens through the use of a standard collagenase digestion technique (original protocol) and another 30 with the addition of NAC and standard collagenase substituted by Liberase (new protocol). Viability and success, defined as maintenance of cell adhesion and morphology for 48 hours, were assessed. Metabolic function was assessed by means of albumin and urea synthesis.ResultsBaseline factors were similar for both groups. The delay to tissue processing was slightly shorter in the new protocol group (median, 2 versus 4 hours; P = 0.007). The success rate improved from 12 of 30 (40.0%) to 21 of 30 (70.0%) with the use of the new protocol (P = 0.037), and median viable cell yield increased from 7.3 × 10⁴ to 28.3 × 10⁴ cells/g tissue (P = 0.003). After adjusting for delay, success rate (P = 0.014) and viable cell yield/g tissue (P = 0.001) remained significantly improved. Albumin and urea synthesis were similar or superior in the new protocol group.ConclusionsNAC and Liberase improve the success of hepatocyte isolation, with a significantly higher yield of viable cells. The use of these agents may improve the availability of hepatocytes for transplantation and laboratory research.     

A chemically defined biomimetic surface for enhanced isolation efficiency of high-quality human mesenchymal stromal cells under xenogeneic/serum-free conditions

Background aimsMesenchymal stromal cells (MSCs) are one of the most frequently used cell types in regenerative medicine and cell therapy. Generating sufficient cell numbers for MSC-based therapies is constrained by (i) their low abundance in tissues of origin, which imposes the need for significant ex vivo cell expansion; (ii) donor-specific characteristics, including MSC frequency/quality, that decline with disease state and increasing age; and (iii) cellular senescence, which is promoted by extensive cell expansion and results in decreased therapeutic functionality. The final yield of a manufacturing process is therefore primarily determined by the applied isolation procedure and its efficiency in isolating therapeutically active cells from donor tissue. To date, MSCs are predominantly isolated using media supplemented with either serum or its derivatives, which poses safety and consistency issues.MethodsTo overcome these limitations while enabling robust MSC production with constant high yield and quality, the authors developed a chemically defined biomimetic surface coating called isoMATRIX (denovoMATRIX GmbH, Dresden, Germany) and tested its performance during isolation of MSCs.ResultsThe isoMATRIX facilitates the isolation of significantly higher numbers of MSCs in xenogeneic (xeno)/serum-free and chemically defined conditions. The isolated cells display a smaller cell size and higher proliferation rate than those derived from a serum-containing isolation procedure and a strong immunomodulatory capacity. The high proliferation rates can be maintained up to 5 passages after isolation and cells even benefit from a switch towards a proliferation-specific MSC matrix (myMATRIX MSC) (denovoMATRIX GmbH, Dresden, Germany).ConclusionIn sum, isoMATRIX promotes enhanced xeno/serum-free and chemically defined isolation of human MSCs and supports consistent and reliable cell performance for improved stem cell-based therapies.     

CD133 immunomagnetic separation: effectiveness of the method for CD133(+) isolation from umbilical cord blood

Background aimsUmbilical cord blood (UCB) is a rich source of stem cells, the characterization and isolation of which requires specific stem cell markers and reliable and reproducible protocols.MethodsWe assessed CD133 isolation in 39 UCB samples, using a commercial immunomagnetic cell-sorting protocol, and, because of its non-reproducibility, we applied optimized protocols in an effort to improve it. These included extra-labeling of the selected CD133⁺ subpopulation and indirect labeling using anti-phycoerythrin (PE) microbeads, goat anti-mouse IgG microbeads or a combination of both. The CD34 isolation was used as a control.ResultsThe mononuclear cell fraction expressed 0.53 ± 0.06% CD133. The corresponding value for CD34 was 1.64 ± 0.15%. Following the manufacturer's instructions, the CD34 isolation resulted in a population expressing 93 ± 1.25% CD34 while, after the corresponding process, CD133⁺ expression ranged from 10% to 85% (median 60%). The optimized isolation protocols did not result in improved CD133⁺ yield. The variation in the purity of the CD133 population cannot be attributed to the different clones of CD133 used, because they do not cross-block, while other factors such as glycosylation, which could possibly interfere, do not apply in normal hematopoietic stem cells (HSC).ConclusionsCD34 isolation by the immunomagnetic method results in highly pure CD34⁺ population, while the efficient and reproducible yield of a pure CD133⁺ population is not feasible. Therefore quantification of the positive cells should follow each isolation procedure in order to confirm the number of CD133⁺ cells.     

Age influence on stromal vascular fraction cell yield obtained from human lipoaspirates

Background aimsThe adipose stromal vascular fraction (SVF) is a heterogeneous population of mononuclear cells that includes approximately 1–10% mesenchymal stromal cells. These SVF cells can be freshly obtained from human lipo-aspirates and represent and ideal candidate for regenerative medicine applications. In the present study, we analyzed the SVF yield as a function of the patient's age.MethodsAdipose tissue samples from 52 informed subjects (all women) were processed by means of an innovative point-of-care technology for SVF isolation (GID platform). After enzymatic dissociation of adipose tissue and SVF pellet resuspension, we measured the concentration of mononucleated cells as well as other cell quality analyses on the cell suspension obtained. We then calculated the cell yield as total nucleated cells per milliliter of dry adipose processed.ResultsThe mean SVF yield obtained was 7.19 × 10⁵ ± 2.11 × 10⁵ total nucleated cells per milliliter of adipose tissue. Our results show that there is a clear statistically significant decline in SVF cell yield with increasing age.ConclusionsBecause all samples were obtained from the same donor area and the isolation technique used was the same in all cases, we conclude that the SVF cell yield in women is affected by patient age. Specific age-related factors should be analyzed in the future to explain these results.     

A novel Ca2+-signal transduction inhibitor,kujigamberol C,isolated from Kuji amber

ABSTRACT

A novel labdane type diterpenoid, 15-nor-8-labden-13-ol, named kujigamberol C, was isolated from Kuji amber using a modified isolation method to increase the yield of biologically active compounds. The structure was determined using HREIMS, 1D and 2D NMR. Kujigamberol C showed growth-restoring activity against mutant yeast via Ca²⁺-signal transduction.     

4. Preparative Immunoabsorption Electrophoresis

ABSTRACT

A method for separating a component from a mixture of antigens is described. The component, which may be a virus or subfraction of a virus, is isolated by driving the mixture by electrophoresis through a gel containing precipitating antibodies directed against the unwanted components. The method is illustrated by the isolation of hepatitis B antigen from whole serum and by the separation of wild cucumber mosaic virus from a strain of tobacco mosaic virus.     

Polymer-mediated cyclodehydration of alditols and ketohexoses

The polymer PEDOT⁺ (1 or 2) mediates a cyclodehydration reaction with alditols 3, 5, 7, 9, in hydrocarbon solvents, to give cyclic ethers 4, 6, 8, or 10, respectively, in high yield with a trivial isolation protocol. Polymers 1 or 2 also mediate the cyclodehydration of ketohexoses such as d-fructose, but not aldohexoses, to the important industrial intermediate 5-hydroxymethylfurfural (17), under milder conditions when compared to reactions mediated by mineral acids. A cascade reaction with ketohexoses is observed in toluene via cyclodehydration followed by Friedel–Crafts alkylation of the initially formed benzylic alcohol to give 16.     

High Yield Isolation of Nuclei from Plant Protoplasts

Nuclei were isolated from protoplasts obtained from Parthenocissus tricuspidata Crown Gall callus tissues. The effect of various isolation procedures, detergent or ultrasonication, on yield and quality of nuclei was studied. A standard procedure, based on the use of 5 × 10^?3% Triton × 100 — 6% PVP — 20% glycerol, may be carried out in 30 min and gives 80 to 90% yield of nuclei in which RNA polymerase activity is retained.     

Procedure for Isolation of the 20,000-Dalton Variant of Human Growth Hormone

Abstract

An isolation procedure for the 20,000-dalton variant of human growth hormone has been devised to improve the yield of the final product. The improvement involved elimination of cumbersome steps that decreased yield, and modification of chromatography on DEAE-cellu-lose to provide better separation of the variant from the major form of growth hormone.

We reported (1) an isolation procedure for the 20,000-dalton variant of human growth hormone (hGH_20K) (2) which provided quite homogeneous material but the yields were not optimum. The initial steps were cumbersome and losses resulted from them. In addition, there was the problem that in order to completely remove the major 22,000-dalton form of the hormone (hGH) during the final chromatography step (DEAE-cellulose), only the leading edge of the hGH_20k?peak could be used. The trailing part of the peak was always mixed with hGH and reworking this mixture resulted in losses. We have modified the procedure so that time-consuming steps were eliminated and the final chromatography step was improved so that now the hGH_20K can be separated from hGH by a single column.     

Picomole Syntheses of High Quality Oligonucleotide Primers in Combination with the Preparation of Oligonucleotide Arrays

Abstract

The establishment of a new synthesis procedure for the preparation of oligonucleotide arrays is described. A modified phosphoramidite chemistry allowed the in situ synthesis of oligomer arrays on specially derivatized polypropylene membranes which can be used both for hybridisation experiments and for the isolation of the individual oligonucleotides.     

Exercise in Isolation- A Countermeasure for Electrocortical,Mental and Cognitive Impairments

IntroductionMental impairments, including deterioration of mood and cognitive performance, are known to occur during isolation and space missions, but have been insufficiently investigated. Appropriate countermeasures are required, such as exercise, which is known to prevent mood disorders for prolonged space and isolation missions. Based on the interaction of brain activity, mood and cognitive performance, this study aims to investigate the effect of long-term isolation and confinement and the long-term effect of exercise on these parameters.MethodsEight male volunteers were isolated and confined for about eight month during the winter period at the Antarctic Concordia Station. Every six weeks electroencephalographic measurements were recorded under rest conditions, and cognitive tests and a mood questionnaire were executed. Based individual training logs, subjects were afterwards separated into an active (> 2500 arbitrary training units/interval) or inactive (< 2500 arbitrary training units/interval) group.ResultsA long-term effect of exercise was observed for brain activity and mood. Regularly active people showed a decreased brain activity (alpha and beta) in the course of isolation, and steady mood. Inactive people instead first increased and than remained at high brain activity accompanied with a deterioration of mood. No effect of exercise and isolation was found for cognitive performance.ConclusionThe findings point out the positive effect of regularly performed voluntary exercise, supporting subjective mental well-being of long-term isolated people. The choice to be regularly active seems to support mental health, which is not only of interest for future isolation and space missions.     

Genetic differentiation of Albizia lebbeck (L.) Benth. populations estimated by RAPD and ISSR markers

Abstract

Albizia lebbeck (L.) Benth., a popular multi‐purpose legume tree species, has a wide distribution throughout the Indian subcontinent. The species is highly valued for its quality timber, gum yield and therapeutical uses. However, the increasing social and economical pressures are devastating the natural stands of A. lebbeck forests. So, there is a need to estimate the genetic variation present in A. lebbeck populations. Both RAPD and ISSR molecular markers were used to analyse 172 individuals representing eight populations of the species in different geographical range. The within‐population genetic diversity ranged from 1.23 to 1.38. The total gene variability was 0.34 of which 0.17 (50%) accounted for within‐population gene variability. The genetic differentiation between populations (Gst = 0.49) was significantly correlated to geographical distance (r = 0.61, p < 0.001). An UPGMA clustering suggests a high level of genetic isolation due to distance. This study revealed the genetic differentiation which will provide a template for adaptation and evolution of the populations and species.     

青藏高原阿里、那曲和海西地区土壤可培养放线菌的多样性

【目的】为了研究青藏高原北部地区土壤可培养放线菌的多样性,并比较不同选择性分离培养基对高原土壤放线菌的分离效果。【方法】使用9种分离培养基,并尝试添加藤黄微球菌发酵液,对采集自阿里、那曲和海西地区的14份土壤样品中的放线菌进行选择性分离。通过16S r RNA基因序列分析对分离菌株进行初步分类鉴定,并在不同分类水平上统计所分离得到的放线菌多样性。【结果】分离得到去重复后的放线菌255株,分布于放线菌门的8个目,14个科,23个属,包含94个可能的物种。其中至少25个物种可能为新种,分布于13个属。链霉菌属的菌株108株,可能的物种28个,是最主要的优势菌属。分离培养基中添加藤黄微球菌发酵液明显增加了放线菌分离菌株的数量和多样性,稀释的葡萄糖酵母麦芽汁培养基适合分离链霉菌,淀粉甘油脯氨酸培养基、丙酸钠酪蛋白培养基等则适合分离稀有放线菌。【结论】青藏高原北部土壤放线菌多样性非常丰富,并且存在较多的新颖放线菌类群;添加藤黄微球菌发酵液是提高放线菌分离效率的有效手段。     

抗性基因报告系统辅助选育厦门霉素A高产菌株

【背景】厦门霉素A是厦门链霉菌(Streptomyces xiamenensis) 318菌株的主要次级代谢产物,具有显著的抗纤维化活性及药用潜力。但野生型菌株中厦门霉素A的产量仅有14 mg/L,其生产水平亟待提升。【目的】通过随机诱变-抗性标记筛选获得高产菌株并进行培养基优化,以提高厦门霉素A的产量。【方法】在厦门霉素A的生物合成基因簇后融合一个抗性基因,用于报告整个基因簇的表达水平。对构建的基因工程菌株进行常压室温等离子体(atmospheric and room temperature plasma,ARTP)诱变,从抗性水平高的突变菌株中筛选高产菌株,并通过培养基优化,使厦门霉素A产量显著提升。【结果】构建携带卡那霉素抗性标记的产厦门霉素A的工程菌MT-XN作为出发菌株,对该菌株进行一轮ARTP诱变,使用90 mg/L卡那霉素筛选,得到了厦门霉素A产量为101.7 mg/L的突变菌株MA-8。进一步通过响应面法优化培养基配方,在最佳培养基中MA-8菌株产生的厦门霉素A达到134.2 mg/L,较野生型菌株提高了845.1%。【结论】采用随机诱变-报告基因筛选系统,可快速筛选出厦门霉素A产量大幅提升的高产菌株,为后续的药物开发奠定良好的基础。     

小鼠肝血窦内皮细胞分离与鉴定新方法

目的:改进小鼠原代肝血窦内皮细胞的分离方法。方法:经过小鼠肝脏的原位灌洗、消化制备单细胞悬液、差速离心、密度梯度离心以及免疫磁珠分选等步骤,分离获得小鼠原代肝血窦内皮细胞,再通过流式细胞仪鉴定、细胞内吞功能染色以及对细胞超微结构的电子显微镜观察,对分离出的肝血窦内皮细胞进行鉴定。结果:肝血窦内皮细胞的平均得率为5.6×10~6个/只小鼠,细胞活性比率约为96%左右;细胞流式鉴定结果显示新鲜分离出的肝血窦内皮细胞VEGFR3阳性率达到95.8%,VEGFR2+CD31+双阳性细胞阳性率达到93.7%。分选出的LSECs能够有效吞噬FITC-FSA和Dil-Ac-LDL。培养1天后肝血窦内皮细胞的微观结构,可见其特征性的窗孔和筛板。结论:本文总结的分离方法可以稳定、高效地获得小鼠原代肝血窦内皮细胞。     

Severely damaged kidneys possess multipotent renoprotective stem cells

Background aimsTissue-specific stem cells are a promising target for kidney regeneration, because it has been shown that they play a primary role in kidney repair. Several methods have been developed for the isolation of stem/progenitor cells from healthy kidneys but the existence of these cells in chronically damaged kidneys has not been noticed so far.MethodsA mouse model of chronic kidney failure was developed by ligation of the left ureter for 5 months, and then isolation of stem cells from this tissue as well as normal kidneys was attempted.ResultsWe found that multipotent stem cells could be isolated from both types of tissue. In addition, the cells isolated from damaged kidneys showed potential for homing to the site of injury and a renoprotective effect in an animal model of cisplatin-induced nephropathy.ConclusionsThese results show that multipotent renoprotective stem cells exist in severely damaged kidneys, which could be a target for designing new therapies.     

Extremely low genetic diversity indicating the endangered status of Ranodon sibiricus (Amphibia: Caudata) and implications for phylogeography

BackgroundThe Siberian salamander (Ranodon sibiricus), distributed in geographically isolated areas of Central Asia, is an ideal alpine species for studies of conservation and phylogeography. However, there are few data regarding the genetic diversity in R. sibiricus populations.Conclusion/SignificanceOur findings suggest that the genetic diversity in the R. sibiricus populations is the lowest among all investigated amphibians. We conclude that the isolation of R. sibiricus populations occurred recently and was a result of recent human activity and/or climatic changes. The Pleistocene glaciation oscillations may have facilitated intraspecies genetic homogeneity rather than enhanced divergence. A low genomic evolutionary rate and elevated inbreeding frequency may have also contributed to the low genetic variation observed in this species. Our findings indicate the urgency of implementing a protection plan for this endangered species.