Spatiotemporal pharmacometabolomics based on ambient mass spectrometry imaging to evaluate the metabolism and hepatotoxicity of amiodarone in HepG2 spheroids

Three-dimensional(3D)cell spheroid models combined with mass spectrometry imaging(MSI)enables innovative investigation of in vivo-like biological processes under different physiological and pathological conditions.Herein,airflow-assisted desorption electrospray ionization-MSI(AFADESI-MSI)was coupled with 3D HepG2 spheroids to assess the metabolism and hepatotoxicity of amiodarone(AMI).High-coverage imaging of>1100 endogenous metabolites in hepatocyte spheroids was achieved using AFADESI-MSI.Following AMI treatment at different times,15 metabolites of AMI involved in Ndesethylation,hydroxylation,deiodination,and desaturation metabolic reactions were identified,and according to their spatiotemporal dynamics features,the metabolic pathways of AMI were proposed.Subsequently,the temporal and spatial changes in metabolic disturbance within spheroids caused by drug exposure were obtained via metabolomic analysis.The main dysregulated metabolic pathways included arachidonic acid and glycerophospholipid metabolism,providing considerable evidence for the mechanism of AMI hepatotoxicity.In addition,a biomarker group of eight fatty acids was selected that provided improved indication of cell viability and could characterize the hepatotoxicity of AMI.The combination of AFADESI-MSI and HepG2 spheroids can simultaneously obtain spatiotemporal information for drugs,drug metabolites,and endogenous metabolites after AMI treatment,providing an effective tool for in vitro drug hepatotoxicity evaluation.

Bioprinting-Based High-Throughput Fabrication of Three-Dimensional MCF-7 Human Breast Cancer Cellular Spheroids

Cellular spheroids serving as three-dimensional(3D) in vitro tissue models have attracted increasing interest for pathological study and drug-screening applications. Various methods, including microwells in particular, have been developed for engineering cellular spheroids. However, these methods usually suffer from either destructive molding operations or cell loss and non-uniform cell distribution among the wells due to two-step molding and cell seeding. We have developed a facile method that utilizes cellembedded hydrogel arrays as templates for concave well fabrication and in situ MCF-7 cellular spheroid formation on a chip. A custom-built bioprinting system was applied for the fabrication of sacrificial gelatin arrays and sequentially concave wells in a high-throughput, flexible, and controlled manner. The ability to achieve in situ cell seeding for cellular spheroid construction was demonstrated with the advantage of uniform cell seeding and the potential for programmed fabrication of tissue models on chips. The developed method holds great potential for applications in tissue engineering, regenerative medicine, and drug screening.

Changes of cell membrane fluidity for mesenchymal stem cell spheroids on biomaterial surfaces

BACKGROUND The therapeutic potential of mesenchymal stem cells(MSCs)in the form of threedimensional spheroids has been extensively demonstrated.The underlying mechanisms for the altered cellular behavior of spheroids have also been investigated.Cell membrane fluidity is a critically important physical property for the regulation of cell behavior,but it has not been studied for the spheroid-forming cells to date.AIM To explore the association between cell membrane fluidity and the morphological changes of MSC spheroids on the surface of biomaterials to elucidate the role of membrane fluidity during the spheroid-forming process of MSCs.METHODS We generated three-dimensional(3D)MSC spheroids on the surface of various culture substrates including chitosan(CS),CS-hyaluronan(CS-HA),and polyvinyl alcohol(PVA)substrates.The cell membrane fluidity and cell morphological change were examined by a time-lapse recording system as well as a highresolution 3D cellular image explorer.MSCs and normal/cancer cells were prestained with fluorescent dyes and co-cultured on the biomaterials to investigate the exchange of cell membrane during the formation of heterogeneous cellular spheroids.RESULTS We discovered that vesicle-like bubbles randomly appeared on the outer layer of MSC spheroids cultured on different biomaterial surfaces.The average diameter of the vesicle-like bubbles of MSC spheroids on CS-HA at 37℃ was approximately 10μm,smaller than that on PVA substrates(approximately 27μm).Based on time-lapse images,these unique bubbles originated from the dynamic movement of the cell membrane during spheroid formation,which indicated an increment of membrane fluidity for MSCs cultured on these substrates.Moreover,the membrane interaction in two different types of cells with similar membrane fluidity may further induce a higher level of membrane translocation during the formation of heterogeneous spheroids.CONCLUSION Changes in cell membrane fluidity may be a novel path to elucidate the complicated physiological alterations in 3D spheroid-forming cells.

Cartilage and bone tissue engineering using adipose stromal/stem cells spheroids as building blocks

Scaffold-free techniques in the developmental tissue engineering area are designed to mimic in vivo embryonic processes with the aim of biofabricating,in vitro,tissues with more authentic properties.Cell clusters called spheroids are the basis for scaffold-free tissue engineering.In this review,we explore the use of spheroids from adult mesenchymal stem/stromal cells as a model in the developmental engineering area in order to mimic the developmental stages of cartilage and bone tissues.Spheroids from adult mesenchymal stromal/stem cells lineages recapitulate crucial events in bone and cartilage formation during embryogenesis,and are capable of spontaneously fusing to other spheroids,making them ideal building blocks for bone and cartilage tissue engineering.Here,we discuss data from ours and other labs on the use of adipose stromal/stem cell spheroids in chondrogenesis and osteogenesis in vitro.Overall,recent studies support the notion that spheroids are ideal"building blocks"for tissue engineering by“bottom-up”approaches,which are based on tissue assembly by advanced techniques such as three-dimensional bioprinting.Further studies on the cellular and molecular mechanisms that orchestrate spheroid fusion are now crucial to support continued development of bottom-up tissue engineering approaches such as three-dimensional bioprinting.

Unexpected Occurrence of Fetal Hemophagocytic Syndrome in a Patient with Hereditary Diffuse Leukoencephalopathy with Spheroids

Colony stimulating factor-1 receptor (CSF1R) plays important roles in the differentiation and proliferation of macrophage and microglia in systemic organs and the brain. A genetic defect in CSF1R causes hereditary diffuse leukoencephalopathy with spheroids (HDLS). HDLS mainly affects the cerebral white matter and shows pre-senile cognitive decline, motor disturbance, and epilepsy. However, systemic manifestations outside the brain have not yet been described in patients with HDLS. Here, we report the case of a 41-year-old man with HDLS carrying the p. K793T mutation in CSF1R, who unexpectedly died of sepsis and hemophagocytic syndrome shortly after the onset of HDLS. The fetal sequence of sepsis and hemophagocytic syndrome was triggered by enterocolitis. An autopsy revealed that focal inflammation in the intestine had almost resolved. Most strikingly, massive infiltration of cluster of differentiation (CD) 68- and CD163-immunopositive macrophages with hemophagocytosis was observed in the bone marrow, spleen, and liver. Less abundant infiltration of CD68- and CD204-immunopositive macrophages without hemophagocytosis was also seen in the lung and intestine. At present, the pathogenetic link between CSF1R mutation and hemophagocytic syndrome in this patient is unclear. Our case, however, clearly shows that even in patients with HDLS, aberrant activation of functional macrophages can be induced under certain conditions in visceral organs.

Characterization of three-dimensional multipotent adipose-derived stem cell spheroids

Human adipose stem cells(hADSCs)are reliable sources for cell therapy.However,the clinical applications are limited by the decrease in activity during in vitro culture.We used a knockout serum replacement(KSR)medium,Eppendorf(EP)tube culture,and a simulated microgravity(SMG)culture system to establish hADSC spheroids.We found that hADSCs aggregated and formed spheroids in the KSR culture medium.The EP tube culture method revealed many biological cell characteristics,such as good cell viabilities,rough surfaces,polar growth,fusion phenomenon,and injectability.The findings show its advantages for hADSCs spherical cultures.When cultured in SMG,hADSC spheroids produced large-scale spheroids.Additionally,confocal examination and viability assays revealed that SMG-cultured hADSC spheroids had higher cell viabilities and looser spherical structures,relative to those cultured in EP tubes.hADSC spheroids in static EP tube culture had tighter structures and more dead cells with rough and irregular surfaces,while hADSC spheroids in dynamic SMG condition exhibited looser structures and better cell viabilities with flat and regular surfaces.Therefore,the KSR media promotes spherical formation by hADSCs,which showed polar growth,fusion,and injectability in vitro.The dynamic SMG culture enhances the formation of a looser structure and better cell viabilities for hADSC spheroids.

Formation Mechanism of Reduction Spheroids with Dark Cores in Cretaceous Red Beds in Jiaolai Basin, China

Red beds are not entirely red sometimes, in which grey-green spheroids or irregular spots can be found. However, the formation mechanism of grey-green spheroids or irregular spots in red beds is not clear so far. Samples taken from well JK1 in Jiaozhou area of Jiaolai Basin displayed that the reduction spheroids have more Vanadium (V) element, less TFe3O4 and Lead (Pb) element, almost the same content of other elements such as FeO and so on, comparing the red parts of the samples. The existence of organisms can explain the existence of green reductive spheres in the red beds formed under the oxidation environment.

Artificial Equilibrium Points in the Low-Thrust Restricted Three-Body Problem When Both the Primaries Are Oblate Spheroids

This paper studies the existence and stability of the artificial equilibrium points (AEPs) in the low-thrust restricted three-body problem when both the primaries are oblate spheroids. The artificial equilibrium points (AEPs) are generated by canceling the gravitational and centrifugal forces with continuous low-thrust at a non-equilibrium point. Some graphical investigations are shown for the effects of the relative parameters which characterized the locations of the AEPs. Also, the numerical values of AEPs have been calculated. The positions of these AEPs will depend not only also on magnitude and directions of low-thrust acceleration. The linear stability of the AEPs has been investigated. We have determined the stability regions in the xy, xz and yz-planes and studied the effect of oblateness parameters A1(0A1?and ?A2(0A2<1) on the motion of the spacecraft. We have found that the stability regions reduce around both the primaries for the increasing values of oblateness of the primaries. Finally, we have plotted the zero velocity curves to determine the possible regions of motion of the spacecraft.

3D （Three-Dimensional） Caco-2 Spheroids： Optimized in vitro Protocols to Favor Their Differentiation Process and to Analyze Their Cell Growth Behavior

3D （Three-dimensional） Caco-2 spheroids closely recapitulating in vivo physiological organization of intestinal epithelial cells, provide an excellent in vitro model system to study their pathophysiology and their response to stressful stimuli. The objective of this technical note is to provide optimized in vitro experimental protocols for culturing 3D Caco-2 spheroids and for analyzing their cell growth features. An optimized 3D Caco-2 spheroid culturing technique based on a new configuration of the culture medium is provided A methodological approach to determine the distribution of the cell cycle phases in disaggregated Caco-2 spheroids by using cytofluorimetric analysis is also described. The optimized culturing protocol favors 3D Caco-2 spheroid differentiation process, as evaluated by the number of well-differentiated spheroids with a single hollow lumen. The cytofluorimetric analysis allows rapid collection of cell cycle phase data from high numbers of spheroid samples, thus, permitting to estimate their growth dynamics in a relatively short time. The optimized technical approaches described here can be applied in systematic manner to a variety of research activities utilizing 3D Caco-2 spheroids. Ease of use, time and economic saving advantages deriving from these protocols further highlight their potential.

桥梁缆索用87MnSi钢线材微观组织演变对力学性能的影响

针对桥梁缆索用87MnSi钢线材的异常断裂现象,对其盘条和生产工艺过程中钢丝的微观组织和力学性能演变规律进行系统分析。结果表明,盘条组织由珠光体和少量的先共析铁素体组成,且渗碳体片层随机分布。冷拉拔后,渗碳体片层发生扭转弯曲或减薄纤维化,且部分渗碳体发生破碎溶解,溶解量为1.40%(占比9.69%)。热镀锌后,溶解的渗碳体发生球化。盘条(329.8HV)在冷拉拔-热镀锌-稳定化处理过程中的显微硬度呈现先增大(450.2HV)后减小(447.9HV)再增大(469.6HV)的变化趋势;冷拉拔过程中溶解的渗碳体使得钢丝硬度值上升至468.3HV,而热镀锌过程渗碳体的球化,导致硬度值下降至439.8HV,并恶化拉伸性能。当边部的渗碳体球化程度明显时,使得钢丝内外组织和力学性能存在较大差异,并在稳定化处理过程中因受力不均匀而导致断裂。

TiVNbTa高熵合金球形粉末制备及选区激光熔化成形

利用氢化脱氢、机械球磨、等离子体球化技术成功制备了TiVNbTa难熔高熵合金球形粉末,并采用选区激光熔化(SLM)技术将此球形粉末制备成TiVNbTa难熔高熵合金块体试样,研究不同SLM工艺参数对合金显微组织和力学性能的影响。结果表明:所制备的粉末球形度高、分散良好,平均粒度为48μm;SLM成形的TiVNbTa难熔高熵合金具有超细晶组织,其熔池心部为胞状晶区,熔池边缘为柱状晶区。随着激光功率的增加,合金的压缩屈服强度整体变化不大,但塑性应变呈下降趋势;然而,随着扫描速率的增加,合金的压缩屈服强度和塑性应变持续下降。当激光功率为175 W、扫描速率为400 mm/s时,沉积态合金具有最低的孔隙率,XOY面和YOZ面的孔隙率分别降低至0.26%和1.42%,压缩屈服强度σ_(0.2)为1001 MPa,最大抗压强度σ_(bc)为1741 MPa,塑性应变ε_(p)为10.0%。

椭球体绕流阻力数值模拟研究

颗粒在无界均匀流场中的阻力是颗粒多相流领域最基本的问题之一。在相关的工程应用与科学研究中,颗粒基本都是非球形。早期基本以相同体积的圆球阻力来近似非球颗粒的实际阻力,近来则提出了多种基于实验数值拟合的非球颗粒阻力经验公式。本文通过高精度的计算方法,系统模拟了从扁平到细长的回转椭球颗粒,在常见的颗粒雷诺数范围内(0.1~1000),对于各种来流攻角条件下的阻力,数值模拟结果得到了已知理论解的充分验证。在不存在理论解的大多数条件下,将数值结果与多种流行的经验公式结果进行了系统比较。一般而言,当颗粒以最大迎风面积置于来流中时,其所受阻力最大。但是数值模拟发现了特例,在流动进入牛顿阻力区时(雷诺数1000),扁平的椭球在回转轴与流向一致时(此时具有最大迎风面积),其阻力反而小于倾斜置于流场中的结果。对流动结构的分析表明,这是因为在非轴对称条件(颗粒倾斜)下,流动更容易失稳,造成紊乱的尾流,使得压差阻力上升。综合分析表明,对于给定的颗粒形状,数值模拟相对于通过各种实验数据拟合得到的适用于一般非球颗粒的经验公式具有更高的精度。累积更大量的高精度数值模拟结果,有望给出具有更高精度、更具适应性的阻力公式。

细胞球可控制备的微流控芯片

基于热发泡打印原理,采用互补金属氧化物半导体-微电子机械系统(CMOS-MEMS)技术设计了一种可以产生细胞球的微流控芯片,通过调整打印次数控制细胞球的直径。该芯片通过控制底层的CMOS电路驱动电阻发热,从而带动MEMS微结构中的细胞悬液被喷射到指定位置并形成细胞球。实验结果表明:打印过程中,该芯片对CHO细胞活性基本无损伤,且可以制备出平均直径250.4~449.1μm范围内的细胞球,细胞球直径均一性良好,变异系数(CV)为6.17%~10.22%,实现了直径均一的细胞球的可控制备。

ZnO/ZnSe复合材料的制备、 表征及催化特性

对传统的两步水合热法进行改进,采用更加经济、安全、简便的方法在室温下合成ZnO/ZnSe核壳结构。调节原料比例,实验温度,优化了制备ZnO/ZnSe核壳结构的最佳生长与修饰条件,使其具有良好的分散性,时间稳定性,良好的发光特性。研究采用醋酸锌作为锌源,硒粉作为硒源,通过调整原料比例以及实验温度制备出球形核壳结构的ZnO/ZnSe复合材料,用透射电子显微镜(TEM),X射线粉末衍射仪(XRD),紫外可见分光光度计(UV-Vis),光致发光光谱(PL),X射线光电子能谱(XPS)对晶体结构进行了表征,同时对光催化性能进行了研究。结果表明,改变实验条件,原料比例为Zn∶Se=1.6∶1,温度为80℃时生成的ZnO/ZnSe核壳结构对甲基橙溶液有较好的吸光效果。ZnSe过厚的壳层生长会阻碍复合材料对甲基橙的降解效率。实验数据结合理论分析得到了实验最佳条件。

基于热泡喷墨技术制备均匀细胞球的创新方法

细胞球在药物发现、生理病理学研究和组织工程等多个领域发挥重要作用。然而,传统的制造细胞球的方法限制了其应用。本文提出了一种基于热泡喷墨打印技术的系统性方法,称为聚集打印,它可以快速、高通量且低成本地产出均匀尺寸的球体。聚集打印可以在16 min内产出864个直径均匀的细胞球。本文以CHO细胞作为研究对象,仔细分析了打印后的细胞活性。同时,为了提高细胞球体尺寸的均匀性,提出3种优化方法,分别可以将细胞球体直径的变异系数(CV值)降低17.04%~48.38%不等,最后研究了打印后的细胞球培养0、1、4、7 d的生长情况。实验结果充分证明了聚集打印方法在细胞球体制造方面的优势性,本方法也为类器官的制备提供参考。

球形粉体制备技术研究进展

【目的】球形粉体由于具有更好的流动性和均匀性被广泛应用于增强材料、涂料、陶瓷、3D打印等领域。为了满足不同行业对粉体材料的表面特性和物理性能要求,提升产品性能,降低工艺损失,促进绿色制造,对球形粉体制备技术的研究现状进行总结和思考。【研究现状】综述球形粉体常见制备技术,包括传统物理法(机械整形法和喷雾干燥法)、化学法(气相化学反应法、沉淀法、水热合成法、溶胶凝胶法及微乳液法)和高温熔融法(等离子体球化法、雾化法和气体燃烧火焰成球法),总结上述方法的优势及目前的局限性,重点阐述不同球形粉体材料制备技术的基本原理与应用领域的研究进展。【展望】对球形粉体制备技术的发展趋势进行分析与展望,认为高温熔融法是最具实现大规模工业化生产潜力的球形粉体制备技术,提出高纯超细、窄分布、粒径可控、高球化率、高效率工业智能化绿色生产是我国未来球形粉体制备技术的发展趋势。

ADI 1050-6前轴铸件工艺开发与应用

介绍了ADI 1050-6前轴为大型农机重要受力件,壁厚相差大,铸件力学性能要求高。通过采用冷铁与冒口联合补缩工艺避免了铸件缩松缺陷;同时选择的合适的化学成分,并通过有效的铁液预处理等四级孕育、球化处理的工艺方案,保证铸件满足高性能材质的基本要求;铸件经过盐浴等温淬火处理提升铸件的力学性能,达成客户要求并实现量产。

两步法制备纳米TiC_(p)/GH3536复合粉末组织性能研究

采用机械合金化(mechanical alloying, MA)结合热等离子体球化处理(thermal plasma spheroidization,TPS)两步法,制备纳米TiC_(p)/GH3536复合粉末。采用霍尔流速计、斯科特容量计、XRD、粒度粒形仪、扫描电子显微镜及透射电子显微镜等分析测试手段,研究球化处理前后复合粉末的流动性、松装密度、粒度分布、球形度及微观组织形貌。结果表明:第一步MA-5 h处理得到的预制粉末为基体GH3536表面非均匀附着纳米TiC颗粒的不规则形状粉末;相比原始GH3536粉末,预制粉末内部等轴晶数量增多,晶粒得到细化,但流动性和松装密度均降低。第二步TPS处理得到的纳米TiC_p/GH3536复合粉末为球形,原始不规则纳米TiC颗粒被球化,同时均匀分布于GH3536基体内部;去除平均粒度183.27 nm的亚微米颗粒后,复合粉末的流动性、松装密度和球形度得到进一步改善。亚微米颗粒的TEM结果显示,纳米级GH3536微球上均匀包覆着一层TiC,类似核壳结构,且粉末中出现球状TiC颗粒。两步法制备的纳米TiC_(p)/GH3536复合粉末,平均球形度达0.965,平均粒径为33.65μm,流动性为17.1 s/50 g,松装密度为4.184 g/cm^(3)。

天然鳞片石墨特征对球形加工的影响

以萝北(LB)、双鸭山(SYS)、鸡西(JX)、吉林(JL)4个地区浮选石墨精矿及其球化产品为研究对象。通过偏光显微镜(PLM)、扫描电镜(SEM)、X射线衍射(XRD)分析了脉石状态、片径大小、鳞片厚度、结晶程度,探究天然鳞片石墨特征对球形化石墨产品的影响。结果表明,4个地区石墨浮选精矿固定碳含量为JX>JL>SYS>LB,片径为JX>LB>JL>SYS,球形化时脉石矿物被包裹,恶化产品品质。一定粒径范围,大鳞片石墨成球效果更好;石墨鳞片厚成球颗粒更“实心”,鳞片薄成球颗粒内部缺陷更多。球形化过程中,石墨层间结构稳定程度高低为LB>SYS>JL>JX。LB石墨成球效果更好,更适合制备球形石墨。

Porous nanofibrous dressing enables mesenchymal stem cell spheroid formation and delivery to promote diabetic wound healing

Delayed and nonhealing of diabetic wounds imposes substantial economic burdens and physical pain on patients.Mesenchymal stem cells(MSCs)promote diabetic wound healing.Particularly when MSCs aggregate into multicellular spheroids,their therapeutic effect is enhanced.However,traditional culture platforms are inadequate for the efficient preparation and delivery of MSC spheroids,resulting in inefficiencies and inconveniences in MSC spheroid therapy.In this study,a three-dimensional porous nanofibrous dressing(NFD)is prepared using a combination of electrospinning and homogeneous freeze-drying.Using thermal crosslinking,the NFD not only achieves satisfactory elasticity but also maintains notable cytocompatibility.Through the design of its structure and chemical composition,the NFD allows MSCs to spontaneously form MSC spheroids with controllable sizes,serving as MSC spheroid delivery systems for diabetic wound sites.Most importantly,MSC spheroids cultured on the NFD exhibit improved secretion of vascular endothelial growth factor,basic fibroblast growth factor,and hepatocyte growth factor,thereby accelerating diabetic wound healing.The NFD provides a competitive strategy for MSC spheroid formation and delivery to promote diabetic wound healing.