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.

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.

MALDI coupled with laser-postionization and trapped ion mobility spectrometry contribute to the enhanced detection of lipids in cancer cell spheroids

Cancer cell spheroids(CCS) are a valuable three-dimensional cell model in cancer studies because they could replicate numerous characteristics of solid tumors. Increasing researches have used matrix-assisted laser desorption/ionization mass spectrometry imaging(MALDI-MSI) to investigate the spatial distribution of endogenous compounds(e.g., lipids) in CCS. However, only limited lipid species can be detected owing to a low ion yield by using MALDI. Besides, it is still challenging to fully characterize the structural diversity of lipids due to the existence of isomeric/isobaric species. Here, we carried out the initial application of MALDI coupled with laser-postionization(MALDI-2) and trapped ion mobility spectrometry(TIMS) imaging in HCT116 colon CCS to address these challenges. We demonstrated that MALDI-2 is capable of detecting more number and classes of lipids in HCT116 colon CCS with higher signal intensities than MALDI. TIMS could successfully separate numerous isobaric/isomeric species of lipids in CCS. Interestingly, we found that some isomeric/isobaric species have totally different spatial distributions in colon CCS. Further MS/MS imaging analysis was employed to determine the compositions of fatty acid chains for isomeric species by examining disparities in signal intensities and spatial distributions of product ions. This work stresses the robust ability of TIMS and MALDI-2 imaging in analyzing endogenous lipids in CCS, which could potentially become powerful tools for future cancer studies.

In situ biosynthesis of bacterial cellulose hydrogel spheroids with tunable dimensions

Bacterial cellulose(BC)hydrogel spheroid plays a significant role in diverse fields due to its spatial 3D structure and properties.In the present work,a series of BC spheroids with controllable size and shape was obtained via an in situ biosynthesis.Crucial factors for fabricating BC spheroid in-cluding inoculum concentration of 1.35×10^(3)CFU/mL,shaking speeds at 100 r/min,and 48-96 h incubation time during the biosynthetic process,were comprehensively established.An operable mechanism model for tuning the size of BC spheroids from 0.4 to 5.0 mm was proposed with a fresh feeding medium strategy of dynamic culture.The resulting BC spheroids exhibit an inter-active 3D network of nanofibers,a crystallinity index of 72.3%,a specific surface area of 91.2 m^(2)/g,and good cytocompatibility.This study reinforces the understanding of BC spheroid forma-tion and explores new horizons for the design of BC spheroids-derived functional matrix materials for medical care.

Multicellular tumor spheroids bridge the gap between two-dimensional cancer cells and solid tumors: The role of lipid metabolism and distribution

Previous studies demonstrated that three-dimensional(3D) multicellular tumor spheroids(MCTS) could more closely mimic solid tumors than two-dimensional(2D) cancer cells in terms of the spatial structure, extracellular matrix-cell interaction, and gene expression pattern. However, no study has been reported on the differences in lipid metabolism and distribution among 2D cancer cells, MCTS, and solid tumors. Here, we used Hep G2 liver cancer cell lines to establish these three cancer models. The variations of lipid profiles and spatial distribution among them were explored by using mass spectrometry-based lipidomics and matrix-assisted laser desorption/ionization mass spectrometry imaging(MSI). The results revealed that MCTS, relative to 2D cells, had more shared lipid species with solid tumors. Furthermore,MCTS contained more comparable characteristics than 2D cells to solid tumors with respect to the relative abundance of most lipid classes and mass spectra patterns. MSI data showed that 46 of 71 lipids had similar spatial distribution between solid tumors and MCTS, while lipids in 2D cells had no specific spatial distribution. Interestingly, most of detected lipid species in sphingolipids and glycerolipids preferred locating in the necrotic region to the proliferative region of solid tumors and MCTS. Taken together, our study provides the evidence of lipid metabolism and distribution demonstrating that MCTS are a more suitable in vitro model to mimic solid tumors, which may offer insights into tumor metabolism and microenvironment.

Using pipette tips to readily generate spheroids comprising single or multiple cell types

Three-dimensional(3D)cell culture methods have been validated that can replicate the tumor environment in vivo to a large extent,providing an effective tool for studying tumors.In this study,we demonstrated the use of standard laboratory pipette tips as micro vessels for generating 3D cell spheroids.No microfabrication or wet-chemistry surface modifications were involved in the procedure.Spheroids consisting of single or multiple cell types were generated within 24 h just by pipetting and incubating a cell suspension in pipette tips.Scanning electron microscope and optical microscope proved that the cells grew together tightly,and suggested that while gravity force might have initiated the sedimentation of cells at the bottom of the tip,the active aggregation of cells to form tight cell-cell interactions drove the formation of spheroids.Using common laboratory micropipettes and pipette tips,the rate of spheroid generation and the generation reproducibility was characterized from five boxes each with 80 tips.The ease of transferring reagents allowed modeling of the growth of microvascular endothelial cells in tumor spheroids.Moreover,the pairing and fusion of tumor spheroids could be manipulated in the pipette tips,suggesting the potential for building and assembling heterogeneous micro-tumor tissues in vitro to mimic solid tumors in vivo.This study demonstrated that spheroids can be readily and cost-effectively generated in standard biological laboratories in a timely manner using pipette tips.

Extracellular magnetic labeling of biomimetic hydrogel-induced human mesenchymal stem cell spheroids with ferumoxytol for MRI tracking

Labeling of mesenchymal stem cells(MSCs)with superparamagnetic iron oxide nanoparticles(SPIONs)has emerged as a potential method for magnetic resonance imaging(MRI)tracking of transplanted cells in tissue repair studies and clinical trials.Labeling of MSCs using clinically approved SPIONs(ferumoxytol)requires the use of transfection reagents or magnetic field,which largely limits their clinical application.To overcome this obstacle,we established a novel and highly effective method for magnetic labeling of MSC spheroids using ferumoxytol.Unlike conventional methods,ferumoxytol labeling was done in the formation of a mechanically tunable biomimetic hydrogel-induced MSC spheroids.Moreover,the labeled MSC spheroids exhibited strong MRI T2 signals and good biosafety.Strikingly,the encapsulated ferumoxytol was localized in the extracellular matrix(ECM)of the spheroids instead of the cytoplasm,minimizing the cytotoxicity of ferumoxytol and maintaining the viability and stemness properties of biomimetic hydrogel-induced MSC spheroids.This demonstrates the potential of this method for post-transplantation MRI tracking in the clinic.

Therapeutic strategies of three-dimensional stem cell spheroids and organoids for tissue repair and regeneration

Three-dimensional(3D)stem cell culture systems have attracted considerable attention as a way to better mimic the complex interactions between individual cells and the extracellular matrix(ECM)that occur in vivo.Moreover,3D cell culture systems have unique properties that help guide specific functions,growth,and processes of stem cells(e.g.,embryogenesis,morphogenesis,and organogenesis).Thus,3D stem cell culture systems that mimic in vivo environments enable basic research about various tissues and organs.In this review,we focus on the advanced therapeutic applications of stem cell-based 3D culture systems generated using different engineering techniques.Specifically,we summarize the historical advancements of 3D cell culture systems and discuss the therapeutic applications of stem cell-based spheroids and organoids,including engineering techniques for tissue repair and regeneration.

Cellulose nanofibril matrix drives the dynamic formation of spheroids

Multicellular spheroids,which mimic the natural organ counterparts,allow the prospect of drug screening and regenerative medicine.However,their application is hampered by low processing efficiency or limited scale.This study introduces an efficient method to drive rapid multicellular spheroid formation by a cellulose nanofibril matrix.This matrix enables the facilitated growth of spheroids(within 48 h)through multiple cell assembly into size-controllable aggregates with well-organized physiological microstructure.The efficiency,dimension,and conformation of the as-formed spheroids depend on the concentration of extracellular nanofibrils,the number of assembled cells,and the heterogeneity of cell types.The above strategy allows the robust formation mechanism of compacted tumoroids and hepatocyte spheroids.

Function of hepatocyte spheroids in bioactive microcapsules is enhanced by endogenous and exogenous hepatocyte growth factor

The ability to maintain functional hepatocytes has important implications for bioartificial liver development,cell-based therapies,drug screening,and tissue engineering.Several approaches can be used to restore hepatocyte function in vitro,including coating a culture substrate with extracellular matrix(ECM),encapsulating cells within biomimetic gels(Collagen-or Matrigel-based),or co-cultivation with other cells.This paper describes the use of bioactive heparin-based core-shell microcapsules to form and cultivate hepatocyte spheroids.These microcapsules are comprised of an aqueous core that facilitates hepatocyte aggregation into spheroids and a heparin hydrogel shell that binds and releases growth factors.We demonstrate that bioactive microcapsules retain and release endogenous signals thus enhancing the function of encapsulated hepatocytes.We also demonstrate that hepatic function may be further enhanced by loading exogenous hepatocyte growth factor(HGF)into microcapsules and inhibiting transforming growth factor(TGF)-β1 signaling.Overall,bioactive microcapsules described here represent a promising new strategy for the encapsulation and maintenance of primary hepatocytes and will be beneficial for liver tissue engineering,regenerative medicine,and drug testing applications.

Co-culture of tumor spheroids and monocytes in a collagen matrix-embedded microfluidic device to study the migration of breast cancer cells

Recapitulating the tumor microenvironment is a major challenge in the development of in vitro tumor model for the study of cancer biology and therapeutic treatments. 3D multicellular tumor spheroids (MCTS) have been used as reliable models of mimicking in vivo solid tumors. Macrophages and extracellular matrix (ECM), regarded as two key factors of the tumor microenvironment, play significant roles in tumor progression and drug resistance. In order to investigate their effects on tumor cell migration, a microfluidic chip-based 3D breast cancer model was developed by co-culturing monodisperse MCTS with monocytes in 3 D collagen matrix. A reversible bonding technique was employed for the fabrication of the microfluidic chip, which made it easier for MCTS formation and tailoring the MCTS co-culture conditions. When co-culturing monocytes with low invasive T47D spheroids or high invasive MD-MBA-231 spheroids, we found that T47 D cells with the stimulation of macrophage colony-stimulating factor (M-CSF) and MD-MBA-231 cells could polarize monocytes into tumor-associated macrophages (TAMs). The increased stiffness via increasing collagen concentration decreased tumor cell migration, whereas the presence of TAMs enhanced the migration ability of cells.Moreover, M-CSF-activated TAMs promoted the migration of T47 D tumor cells via the regulation of TGFβ1. Overall, this 3D co-culture microfluidic model may be useful for studying tumor progress and may offer a reliable and low-cost method for evaluation of drug efficiency.

Engineering subcellular-patterned biointerfaces to regulate the surface wetting of multicellular spheroids

Studying the wetting behaviors of multicellular spheroids is crucial in the fields of embryo implantation, cancer propagation, and tissue repair. Existing strategies for controlling the wetting of multicellular spheroids mainly focus on surface chemistry and substrate rigidity. Although topography is another important feature in the biological micro-environment, its effect on multicellular spheroid wetting has seldom been explored. In this study, the influence of topography on the surface wetting of multicellular spheroids was investigated using subcellular- patterned opal films with controllable colloidal particle diameters （from 200 to 1,500 nm）. The wetting of hepatoma carcinoma cellular （Hep G2） spheroids was impaired on opal films compared with that on flat substrates, and the wetting rate decreased as colloidal particle diameter increased. The decrement reached 48.5% when the colloidal particle diameter was 1,500 nm. The subcellular-patterned topography in opal films drastically reduced the cellular mobility in precursor films, especially the frontier cells in the leading edge. The frontier cells failed to form mature focal adhesions and stress fibers on micro-patterned opal films. This was due to gaps between colloidal particles leaving adhesion vacancies, causing weak cell-substrate adhesion and consequent retarded migration of Hep G2 spheroids. Our study manifests the inhibiting effects of subcellular-patterned topography on the wetting behaviors of multicellular spheroids, providing new insight into tissue wetting-associated treatments and biomaterial design.

MoS_(2)nanosheets and bulk materials altered lipid profiles in 3D Caco-2 spheroids

MoS_(2)nanosheets(NSs)are novel 2D nanomaterials(NMs)with potential uses in many areas,and therefore oral exposure route to MoS_(2)NSs is plausible.Currently,MoS_(2)NSs are considered as biocompatible NMs,but there is lacking of systemic investigations to study the interactions of MoS_(2)NSs with intestinal cells.In this study,we exposed the 3D Caco-2 spheroids to MoS_(2)NSs or MoS_(2)powders(denoted as MoS_(2)-bulk),and investigated the potential adverse effects of MoS_(2)-materials based on transcriptomics and lipidomics analysis.As expected,both MoS_(2)NSs and MoS_(2)-bulk were dose-dependently internalized into 3D Caco-2 spheroids but did not induce cytotoxicity,membrane disruption or decrease of thiols.However,the Gene Ontology(GO)and Kyoto Encyclopedia of Gene and Genomes(KEGG)analysis indicated that nutrient absorption and metabolism was decreased.One of the most significantly decreased KEGG pathways is fat digestion and absorption(map04975),and Western blotting analysis further showed that fatty acid binding protein 1 and apolipoprotein A1,key proteins involved in fat digestion and absorption,were down-regulated by MoS_(2)NSs or MoS_(2)-bulk.In addition,BODIPY 493/503 staining suggested that exposure to MoS_(2)NSs and MoS_(2)-bulk decreased lipid levels in the spheroids.However,lipidomics data indicated that MoS_(2)materials only decreased 8 lipid classes,including lysophosphatidylcholine,lysodimethylphosphatidylethanolamine,N-acylethanolamine,ceramide phosphoethanolamines,gangliosides,lysosphingomyelin and sulfatide,whereas most of the lipid classes were indeed increased.In addition,MoS_(2)NSs was more potent to decrease the lipid classes compared with MoS_(2)-bulk.Combined,the results from this study showed that MoS_(2)NSs and bulk materials were non-cytotoxic but altered lipid profiles in 3D Caco-2 spheroids.

A novel DPSS filter optimization scheme to reduce the intrinsic interference of FBMC-QAM systems

In order to reduce the intrinsic interference of the filter bank multicarrier-quadrature amplitude modulation(FBMC-QAM)system,a novel filter optimization scheme based on discrete prolate spheroidal sequences(DPSS)is proposed.Firstly,a prototype filter function based on DPSS is designed,since the eigenvalue can be used as an indicator of the energy concentration of DPSS,so a threshold is set,and the sequence with the most concentrated energy is selected under the threshold,that is,the sequence with the eigenvalue higher than the threshold,and the prototype filter function is rewritten as a weighted sum function of multiple eigenvectors.Under the energy constraints of the filter,the relationship between the eigenvectors and the intrinsic interference function is established,and the function problem is transformed into an optimization problem for the weighted coefficients.Through the interior point method,the most suitable weight is found to obtain the minimum intrinsic interference result.Theoretical analysis and simulation results show that compared with the prototype filters such as Type1 and CaseC,the DPSS filter applying the proposed optimization algorithm can effectively suppress the intrinsic interference of the system and obtain a better bit error rate(BER)performance.

Agar coating in multicellular spheroids culture of rat hepatocytes

Aggregation of freshly isolated adult rat hepatocytes in vitro is important for the construction of artificial liver support system. In the experiment, agar has been used as an extracellular matrix substrate and results demonstrate that hepatocytes in serum-free culture