Characterization of platelet-derived extracellular vesicles induced by differential platelet activation pathways / 中国输血杂志

【Objective】 To investigate the effects of platelet activation pathways on the characterization of platelet-derived extracellular vesicles(PEVs). 【Methods】 Whole blood from healthy donors was prepared into platelet suspension by centrifugation. Platelets were randomly divided into six groups, the ctrl group added no extra stimulus, and the other five groups were treated with collagen, adenosine diphosphate(ADP), thrombin, Ca2+ ionophore and freeze-thaw cycles (F-T) to activate platelets. Platelet-derived exosomes(PEXOs) and microvesicles(PMVs) were isolated by differential centrifugations, and then were determined by nano-flow cytometry and electron microscopy. The protein markers of PEXOs also were identified by western-blot. The protein concentration and content of PEXOs were also detected. 【Results】 CD9, CD81, TSG101 proteins were detected in all of the PEXOs, which had no calnexin. Both PEXOs and PMVs had CD41; PEXOs were cup-holder-like bilayer membrane vesicles under a transmission electron microscope, while PMVs were irregular membranous structure; 85%-95% of PEXOs were<100nm, 87%-94% of PMVs were 100-300nm. The concentration of exosomes and microvesicles in the F-T group was the highest(205.67±65.27 and 102.73±15.48), followed by the Ca2+ ionophore group(44.42±17.07 and 11.4±4.81). Although in the same size range, the numbers of PEVs induced by different activation conditions varied. The protein concentration of PEXOs in the F-T group(1.11±0.51) was higher than that in the control group(0.32±0.39), ADP group(0.41±0.31) and thrombin group(0.38±0.37), while the total protein(125.40±58.32) was higher than that in other three groups(the ctrl group 25.53±25.96 vs ADP group 37.21±15.73 vs thrombin group 36.28±24.18 vs Ca2+ ionophore group 47.09±23.29). 【Conclusion】 The biological characteristics of PEVs are affected by platelets activation pathways, whose ability to induce protein-packing into exosomes may also be relevant to the particle size. The freeze-thaw cycles can induce high concentrations of extracellular vesicles, which may be an ideal method for the preparation of PEVs.

Adaptive evolution and selection of stress-resistant Saccharomyces cerevisiae for very high-gravity bioethanol fermentation

Background: In industrial yeasts, selection and breeding for resistance to multiple stresses is a focus of current research. The objective of this study was to investigate the tolerance to multiple stresses of Saccharomyces cerevisiae obtained through an adaptive laboratory evolution strategy involving a repeated liquid nitrogen freeze­thaw process coupled with multi-stress shock selection. We also assessed the related resistance mechanisms and very high-gravity (VHG) bioethanol production of this strain. Results: Elite S. cerevisiae strain YF10-5, exhibiting improved VHG fermentation capacity and stress resistance to osmotic pressure and ethanol, was isolated following ten consecutive rounds of liquid nitrogen freeze­thaw treatment followed by plate screening under osmotic and ethanol stress. The ethanol yield of YF10-5 was 16% higher than that of the parent strain during 35% (w/v) glucose fermentation. Furthermore, there was upregulation of three genes (HSP26, HSP30, and HSP104) encoding heat-shock proteins involved in the stress response, one gene (TPS1) involved in the synthesis of trehalose, and three genes (ADH1, HXK1, and PFK1) involved in ethanol metabolism and intracellular trehalose accumulation in YF10-5 yeast cells, indicating increased stress tolerance and fermentative capacity. YF10-5 also showed excellent fermentation performance during the simultaneous saccharification and fermentation of VHG sweet potato mash, producing 13.40% (w/ v) ethanol, which corresponded to 93.95% of the theoretical ethanol yield. Conclusions: A multiple-stress-tolerant yeast clone was obtained using adaptive evolution by a freeze­thaw method coupled with stress shock selection. The selected robust yeast strain exhibits potential for bioethanol production through VHG fermentation.

Optimization for extraction of urine exosomes and effects of freezing on exosomal RNA content / 临床检验杂志

Objective@#To optimize the existing methods of isolation and purification for exosomes from urine and explore the effects of different storage conditions on the content of exosomal RNA in urine. @*Methods@#The exosomes in human urine samples were extracted by different precipitation method, i.e., precipitation following first concentrating and direct precipitation, respectively, and the separation efficiency and cost of the two methods were compared. ExoQuick-TCTM precipitation kit was used to extract exosomes. Nanoparticle tracking analysis technique (NTA) was used to detect the concentration and particle size distribution of exosome. Dynamic light scattering (DLS) was used to detect the potential of exosome. Transmission electron microscopy (TEM) was used to observe morphology of exosomes. western blot was used to analyze the exosomal marker molecules CD63 and Alix. The extraction method of the precipitation following first concentrating was used to verify the reliability of the optimized method in 10 clinical urine samples . Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of exosomal RNA marker let-7c and PSA mRNA in the urinary exosomes from 20 patients with prostate cancer after repeated freeze-thaw (0 [i.e., fresh], 1 , 3 and 5 times) and 9 patients with prostate cancer frozen at -80 ℃ for different time (0 [i.e., fresh], 1, 2 and 4 weeks), and were statistically analyzed by Wilcoxon rank sum test for differences between the 2 groups. @*Results@#The size distribution of exosomes extracted by the two methods was 30 to 150 nm by NTA, both of which were displayed as single peaks. The results of DLS showed that the potentials of exosome extracted by the two methods were negative values. The size of the exosomes extracted by the two methods was consistent observed under TEM namely the diameter distribution was 30 to 150 nm. western blot analysis confirmed that CD63 and Alix, the exosome labeling molecules, existed in the optimized method. The concentration of exosomes extracted from the 10 urine samples all reached 10 9 to 10 11 particles/mL. The contents of let-7c and PSA mRNA in exosomes decreased significantly after 5 freeze-thaw cycles, and the Z values were -1.79 and -1.73, respectively (P＜0.05). The RNA content of the exosomes remained stable after freezing at -80 ℃ for 1 month. @*Conclusion@#The optimized exosome extraction method could reduce greatly the cost under the premises of ensuring the concentration and quality of exosomes. The isolated exosomes may keep stable RNA content after freezing at -80 ℃ for a short time, but could not be frozen and thawed repeatedly for more than 5 times.

Solution pH jump during antibody and Fc-fusion protein thaw leads to increased aggregation / 药物分析学报

Freeze-thaw cycles impact the amount of aggregation observed in antibodies and Fc-fusion proteins. Various formulation strategies are used to mitigate the amount of aggregation that occurs upon putting a protein solution through a freeze-thaw cycle. Additionally, low pH solutions cause native antibodies to unfold, which are prone to aggregate upon pH neutralization. There is great interest in the mechanism that causes therapeutic proteins to aggregate since aggregate species can cause unwanted im-munogenicity in patients. Herein, an increase in aggregation is reported when the pH is adjusted from pH 3 up to a pH ranging from pH 4 to pH 7 during the thaw process of a frozen antibody solution. Raising the pH during the thaw process caused a significant increase in the percent aggregation observed. Two antibodies and one Fc-fusion protein were evaluated during the pH jump thaw process and similar ef-fects were observed. The results provide a new tool to study the kinetics of therapeutic protein ag-gregation upon pH increase.

Effect of Freeze Thaw Cycles Abuse and Refrigerated Thawing on Microbial Quality of Chevon.

Present study was conducted to evaluate the effect of three freeze thaw cycles and refrigeration temperature thawing on microbial quality of chevon. The muscle comprising of Semitendinosus, Semimembranosus and Bicep femoris from chevon carcass were collected from local retailer of Nagpur city in insulated ice box. For freeze thaw cycle study, samples were distributed, weighed, packaged, labeled and transferred to deep freeze (-18 ±2˚C). The frozen chevon samples were thawed at every 5th day by refrigeration method of thawing at (4±1˚C) temperature.The exposed freeze thaw cycles chevon evaluated for microbial quality like Total Plate Count, Psychrophilic Count and E coli count. Freeze thawed chevon was prone to microbial growth as evident from significant increase in total plate count (TPC). However, coliform count decreased after 3rd freeze thaw cycle while psychrophilic count increased significantly. Thus, it could be concluded that chevon exposed to freeze thaw cycle could be acceptable up to 3rd freeze thaw cycle.

Effects of Platelet Lysate Preparations on the Proliferation of HaCaT Cells

BACKGROUND: Standard protocols are lacking for the preparation of platelet lysates (PL) as an alternative to using fetal bovine serum as a cell culture supplement. This study aimed to establish optimum conditions for preparing PL for use in cell cultures. METHODS: Cell density in three pooled platelet concentrates (PC) were adjusted to 1x10(12)/L and 2x10(12)/L. PL was prepared from PC by 1 to 3 freeze-thaw (FT) cycles. HaCaT cells were cultured in media supplemented with 5% or 10% PL. Cell numbers were estimated using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan). Growth factors were quantified by using the Luminex 200 system (Luminex Corporation, USA). RESULTS: Cell proliferation rates in the presence of PLs were similar when prepared from PCs of both cell densities. The rates were higher in media containing 5% PL than 10% PL when prepared by two FT cycles. Concentrations of vascular endothelial growth factor (VEGF), platelet-derived growth factor-AB/BB (PDGF-AB/BB), PDGF-AA, and epidermal growth factor (EGF) were significantly higher in PL prepared from PC with a cell density of 2x10(12)/L than 1x10(12)/L PC. However, only VEGF and PDGF-AA concentrations in PLs were correlated with HaCaT cell counts. CONCLUSIONS: The 5% PL from PC with a cell density of 1x10(12)/L prepared by two FT cycles treatment was the most effective condition that supported steady HaCaT cell proliferation. Our finding may be useful for preparing PL-supplemented cell culture media.

Effect of the activation of lymphocyte between freeze-thaw tumor cell and the supernatant of tumorcell culture / 肿瘤研究与临床

Objective To compare the effect of freeze-thaw tumor cells and the supernatant from tumor cell culture on the activation of lymphocytes. Methods Malignant melanoma B16 cells were prepared as tumor cell vaccine and the supernatant from tumor cell culture was collected at different time point.Transwell method was used to determine the chemotaxis of lymphocyte attracted by freeze-thaw tumor cells and the supernatant from tumnor cell culture. The cytotoxic activity of lymphocyte was detected by CCK-8 method. Results Freeze-thaw tumor cells and the supernatant from more than 2 h of tumor cell culture were found to show chemotaxis of lymphocyte. The chemotaxis of tumor cell culture more than 4 h was stronger than freeze-thaw tumor cells. Each group of chemotactic lymphocytes demonstrated to have the activity of killing tumor cells. The ability of killing tumor cells induced by the tumor cell culture more than 4 h was stronger than that induced by freeze-thaw tumor cells.In a certain range,the ability of lymphocyte chemotaxis and activation were enhanced over time. Conclusion The chemotaxis and cytotoxic activation of lymphocyte induced by the supernatant from tumor cell culture for a certain time are stronger than those by freeze-thaw tumor cells. The supernatant from tumor cell culture can be used as tumor antigen to get better immune activation instead of the freeze-thaw tumor cells.

Effects of Mesenchymal Stem Cells Feeder Layer, Culture Sera and Freeze-thaw Lysates on Expansion and Differentiation of Cord Blood CD34~+ Cells in Vitro / 中山大学学报(医学科学版)

[Objective]To investigate the effects of mesenchymal stem cells (MSC) feeder layer, culture sere and freeze-thaw lysates on expansion and differentiation of cord blood CD34~+ cells in vitro. [Methods] MSC were isolated from human bone marrow and cultured until the third passage. Sera were obtained from the cultured MSC, and freeze-thaw lysates were obtained by repeated freeze-thaw procedures. Cord blood CD34~+ cells were isolated by magnetic cell separation system, and were co-cultured with the MSC feeder layer, culture sera, freeze-thaw lysates and hematopeietic growth factors (HGFs), respectively. The nucleated cells, CD34~+ cells, CD34~+CD38~- cells, CD41~+ cells and CD3~+ cells in the above culture system were detected by flow cytometry on day 6 and day 12. [Results] ①MSC feeder layer had a strong effect on nucleated cells, CD34~+,CD34~+CD38~- cells expansion. The MSC sera and freeze-thaw lysates had similar effect on cell expansion, but the effect was weaker than that of feeder layer (P<0.05). ② Both MSC sera and feeder layer inhibited cord blood CD34~+ cells differentiation toward CD3~+ cells or CD19~+ cells, and no significant differences were found between these two groups (P>0.05). ③ Both MSC sera and feeder layer promoted cord blood CD34~+ cells differentiation toward CD41~+ cells, and the effect was stronger in the feeder layer than that of the sera (P<0.05). ④ Freeze-thaw lysates had no effect on cell expansion and differentiation, and were similar with that of HGFs (P>0.05). [Conclusions] The MSC sera have positive effects on expansion of cord blood CD34~+ and CD34~+CD38~- cells, moreover they have the ability of promoting cord blood CD34~+ cells differentiation toward CD41~+ cells.