Arrhythmogenic right ventricular cardiomyopathy(ARVC)is a progressive disease characterized by adipose and fibrous replacement of the myocardium.While elevated testosterone levels have been implicated in the pathologi...Arrhythmogenic right ventricular cardiomyopathy(ARVC)is a progressive disease characterized by adipose and fibrous replacement of the myocardium.While elevated testosterone levels have been implicated in the pathological process of ARVC,its exact contribution to cardiac fibrosis in ARVC remains unclear.In this study,we analyzed the potential contribution of gender-based differences on the distribution of the low-voltage area in an ARVC cohort undergoing an electrophysiological study,which was indicated by feature selection.Additionally,we established engineered cardiac spheroid models in vitro using patient-specific induced pluripotent stem cell(iPSC)-derived cardiomyocytes(iPSC-CMs)and iPSC-derived cardiac fibroblasts(icFBs).We elucidated the pathogenicity of abnormal splicing in the plakophilin-2(PKP2)gene caused by an intronic mutation.Additionally,pathogenic validation of the desmoglein-2(DSG2)point mutation further confirms the reliability of the models.Moreover,testosterone exacerbated the DNA damage in the mutated cardiomyocytes and further activated myofibroblasts in a chain reaction.In conclusion,we designed and constructed an in vitro three-dimensionally-engineered cardiac spheroid model of ARVC based on clinical findings and provided direct evidence of the fibrotic role of testosterone in ARVC.展开更多
Xerostomia(dry mouth)is frequently experienced by patients treated with radiotherapy for head and neck cancers or with Sjögren’s syndrome,with no permanent cure existing for this debilitating condition.To this e...Xerostomia(dry mouth)is frequently experienced by patients treated with radiotherapy for head and neck cancers or with Sjögren’s syndrome,with no permanent cure existing for this debilitating condition.To this end,in vitro platforms are needed to test therapies directed at salivary(fluid-secreting)cells.However,since these are highly differentiated secretory cells,the maintenance of their differentiated state while expanding in numbers is challenging.In this study,the efficiency of three reversible thermo-ionically crosslinked gels:(1)alginate–gelatin(AG),(2)collagen-containing AG(AGC),and(3)hyaluronic acid-containing AG(AGHA),to recapitulate a native-like environment for human salivary gland(SG)cell expansion and 3D spheroid formation was compared.Although all gels were of mechanical properties comparable to human SG tissue(~11 kPa)and promoted the formation of 3D spheroids,AGHA gels produced larger(>100 cells/spheroid),viable(>93%),proliferative,and well-organized 3D SG spheroids while spatially and temporally maintaining the high expression of key SG proteins(aquaporin-5,NKCC1,ZO-1,α-amylase)for 14 days in culture.Moreover,the spheroids responded to agonist-induced stimulation by increasingα-amylase secretory granules.Here,we propose alternative lowcost,reproducible,and reversible AG-based 3D hydrogels that allow the facile and rapid retrieval of intact,highly viable 3D-SG spheroids.展开更多
AIM:To investigate the efficacy of Eleutherine bulbosa(Mill.)Urb.bulb extract(EBE)on the 3D human retinoblastoma cancer cells(WERI-Rb-1)spheroids and explore its apoptotic mechanism.METHODS:The 3D WERI-Rb-1 and human ...AIM:To investigate the efficacy of Eleutherine bulbosa(Mill.)Urb.bulb extract(EBE)on the 3D human retinoblastoma cancer cells(WERI-Rb-1)spheroids and explore its apoptotic mechanism.METHODS:The 3D WERI-Rb-1 and human retinal pigmented epithelium cells(ARPE-19)spheroids were developed using type 1 murine collagen that was excised from the rat tail tendon and cultured via hanging drop and embedded techniques.The cytotoxic activity was examined by Alamar blue assay meanwhile,the morphological characteristics were assessed by 4’,6-diamidino-2-phenylindole(DAPI)and scanning electron microscopy(SEM).The mRNA and protein expressions of apoptotic and antioxidant signal transduction pathways were explored to ascertain its molecular mechanisms.The statistical analysis was carried out using GraphPad Prism.RESULTS:The Alamar blue assay portrayed higher half maximal inhibitory concentration(IC50)values of EBE and cisplatin on 3D WERI-Rb-1 model as compared to the previous study on 2D model.The results of DAPI and SEM illustrated apoptotic features upon treatment with EBE and cisplatin in a dose-dependent manner on 3D WERI-Rb-1 model.The mRNA and protein levels of apoptotic and antioxidant-related pathways were significantly affected by EBE and cisplatin,respectively(P<0.05).The regulation of gene and protein expressions of 3D WERI-Rb-1 spheroids differed from the 2D study,suggesting that the tumor microenvironment of extracellular matrix(ECM)collagen matrix hindered the EBE treatment efficacy,leading to apoptotic evasion.CONCLUSION:A significant inhibition effect of EBE is observed on the 3D WERI-Rb-1 spheroids.The presence of ECM causes an increase in cytotoxic resistance upon treatment with EBE and cisplatin.展开更多
Surface modification may have important influences on the penetration behavior of nanoscale drug delivery system. In the present study, we mainly focused on whether cell targeting or cell penetration could affect pene...Surface modification may have important influences on the penetration behavior of nanoscale drug delivery system. In the present study, we mainly focused on whether cell targeting or cell penetration could affect penetration abilities of nanostructured lipid carriers（NLC）. Real--time penetration of folate--or cell penetrating peptide（CPP）-modified NLC was evaluated using a multicellular tumor spheroid（MTS） established by stacking culture method as an in vitro testing platform. The results suggested that CPP modification had a better penetration behavior both on penetration depth and intensity compared with folate-modified NLC at the early stage of penetration process.展开更多
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.Her...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.展开更多
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 u...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.展开更多
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 ...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.展开更多
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...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.展开更多
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 call...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.展开更多
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...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.展开更多
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.Thi...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.展开更多
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...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.展开更多
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...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.展开更多
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 r...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.展开更多
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 dif...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.展开更多
基金supported by the National Natural Science Foundation of China(Nos.82370322 to CC,82200352 to FZ,82300352 to YZ,22275034 to HX,and 82070343 to MLC)the Natural Science Foundation of Jiangsu Province of China(Nos.BK20220710 to FZ and BK20230733 to YZ)Postgraduate Research&Practice Innovation Program of Jiangsu Province(No.JX13414086 to HYC).
文摘Arrhythmogenic right ventricular cardiomyopathy(ARVC)is a progressive disease characterized by adipose and fibrous replacement of the myocardium.While elevated testosterone levels have been implicated in the pathological process of ARVC,its exact contribution to cardiac fibrosis in ARVC remains unclear.In this study,we analyzed the potential contribution of gender-based differences on the distribution of the low-voltage area in an ARVC cohort undergoing an electrophysiological study,which was indicated by feature selection.Additionally,we established engineered cardiac spheroid models in vitro using patient-specific induced pluripotent stem cell(iPSC)-derived cardiomyocytes(iPSC-CMs)and iPSC-derived cardiac fibroblasts(icFBs).We elucidated the pathogenicity of abnormal splicing in the plakophilin-2(PKP2)gene caused by an intronic mutation.Additionally,pathogenic validation of the desmoglein-2(DSG2)point mutation further confirms the reliability of the models.Moreover,testosterone exacerbated the DNA damage in the mutated cardiomyocytes and further activated myofibroblasts in a chain reaction.In conclusion,we designed and constructed an in vitro three-dimensionally-engineered cardiac spheroid model of ARVC based on clinical findings and provided direct evidence of the fibrotic role of testosterone in ARVC.
基金support from Fonds de Recherche du Québec Santé(FRQS,grant no.281271)support from FRQS doctoral award #304367funding from CFI,Rheolution Inc.,and Investissement Québec.
文摘Xerostomia(dry mouth)is frequently experienced by patients treated with radiotherapy for head and neck cancers or with Sjögren’s syndrome,with no permanent cure existing for this debilitating condition.To this end,in vitro platforms are needed to test therapies directed at salivary(fluid-secreting)cells.However,since these are highly differentiated secretory cells,the maintenance of their differentiated state while expanding in numbers is challenging.In this study,the efficiency of three reversible thermo-ionically crosslinked gels:(1)alginate–gelatin(AG),(2)collagen-containing AG(AGC),and(3)hyaluronic acid-containing AG(AGHA),to recapitulate a native-like environment for human salivary gland(SG)cell expansion and 3D spheroid formation was compared.Although all gels were of mechanical properties comparable to human SG tissue(~11 kPa)and promoted the formation of 3D spheroids,AGHA gels produced larger(>100 cells/spheroid),viable(>93%),proliferative,and well-organized 3D SG spheroids while spatially and temporally maintaining the high expression of key SG proteins(aquaporin-5,NKCC1,ZO-1,α-amylase)for 14 days in culture.Moreover,the spheroids responded to agonist-induced stimulation by increasingα-amylase secretory granules.Here,we propose alternative lowcost,reproducible,and reversible AG-based 3D hydrogels that allow the facile and rapid retrieval of intact,highly viable 3D-SG spheroids.
基金Supported by Universiti Putra Malaysia,Serdang,Selangor,Malaysia(UPM/700-2/1/GPB/2017/9549900).
文摘AIM:To investigate the efficacy of Eleutherine bulbosa(Mill.)Urb.bulb extract(EBE)on the 3D human retinoblastoma cancer cells(WERI-Rb-1)spheroids and explore its apoptotic mechanism.METHODS:The 3D WERI-Rb-1 and human retinal pigmented epithelium cells(ARPE-19)spheroids were developed using type 1 murine collagen that was excised from the rat tail tendon and cultured via hanging drop and embedded techniques.The cytotoxic activity was examined by Alamar blue assay meanwhile,the morphological characteristics were assessed by 4’,6-diamidino-2-phenylindole(DAPI)and scanning electron microscopy(SEM).The mRNA and protein expressions of apoptotic and antioxidant signal transduction pathways were explored to ascertain its molecular mechanisms.The statistical analysis was carried out using GraphPad Prism.RESULTS:The Alamar blue assay portrayed higher half maximal inhibitory concentration(IC50)values of EBE and cisplatin on 3D WERI-Rb-1 model as compared to the previous study on 2D model.The results of DAPI and SEM illustrated apoptotic features upon treatment with EBE and cisplatin in a dose-dependent manner on 3D WERI-Rb-1 model.The mRNA and protein levels of apoptotic and antioxidant-related pathways were significantly affected by EBE and cisplatin,respectively(P<0.05).The regulation of gene and protein expressions of 3D WERI-Rb-1 spheroids differed from the 2D study,suggesting that the tumor microenvironment of extracellular matrix(ECM)collagen matrix hindered the EBE treatment efficacy,leading to apoptotic evasion.CONCLUSION:A significant inhibition effect of EBE is observed on the 3D WERI-Rb-1 spheroids.The presence of ECM causes an increase in cytotoxic resistance upon treatment with EBE and cisplatin.
基金National key Basic Research Program(Grant No.2013CB932501)National Natural Science Foundation of China(Grant No.81273454 and 81473156)+1 种基金Beijing National Science Foundation(Grant No.7132113)Doctoral Foundation of the Ministry of Education(Grant No.20130001110055)
文摘Surface modification may have important influences on the penetration behavior of nanoscale drug delivery system. In the present study, we mainly focused on whether cell targeting or cell penetration could affect penetration abilities of nanostructured lipid carriers（NLC）. Real--time penetration of folate--or cell penetrating peptide（CPP）-modified NLC was evaluated using a multicellular tumor spheroid（MTS） established by stacking culture method as an in vitro testing platform. The results suggested that CPP modification had a better penetration behavior both on penetration depth and intensity compared with folate-modified NLC at the early stage of penetration process.
基金funded by the National Natural Science Foundation of China(Grant No.:21874156)the Chinese Academy of Medical Science(CAMS)Innovation Fund for Medical Sciences(Grant No.:2021-1-I2M-028).
文摘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.
基金supported by the National Natural Science Foundation of China (Nos. 21675096 and 21475073)Youth Scientific Research Funds from Graduate School at Shenzhen, Tsinghua University (No. QN20160002)
文摘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.
基金National Taiwan University Core Consortium,No.NTU-CC-110L892501Ministry of Science and Technology,No.MOST 110-2218-E-002-037.
文摘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.
基金supported by the National Natural Science Foundation of China(No.32171401)the Natural Science Foundation of Chongqing(No.CSTB2022NSCQ-MSX0808)the Specific Research Fund of the Innovation Platform for Academicians of Hainan Province(No.YSPTZX202126),China.
文摘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.
基金the Coordination for the Improvement of Higher Education Personnel(CAPES),No.88882.366181/2019-01the Carlos Chagas Filho Foundation for Research Support of the State of Rio de Janeiro(FAPERJ),No.E-26/202.682/2018National Council for Scientific and Technological Development(CNPq),No.467513/2014-7
文摘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.
基金supported by the National Natural Science Foundation of China (11372243, 11532009, and 11522219)the China Postdoctoral Science Foundation (2013M540742)+2 种基金the Doctoral Program of Higher Education of China (20130201120071)the Natural Science Basic Research Plan in Shaanxi Province of China (2014JQ1004)the Fun- damental Research Funds for the Central Universities
文摘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.
基金supported by the National Natural Science Foundation of China(No.32071347)the ZJU-Hangzhou Global Scientific and Technological Innovation Center,Zhejiang University(No.02020200-K02013008)the Joint Laboratory Grant from Jiangsu Wuzhong Aesthetics Biotech Co.,Ltd.,and the Starting Grant of ShanghaiTech University.
文摘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.
基金supported by National Natural Science Foundation of China (Nos. 22036001, 22106130 and 91843301)Research Grant Council (Nos. 463612 and 14104314) of Hong Kong。
文摘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.
基金financially supported by National Natural Science Foundation of China(No.51803055)Natural Science Foundation of Hunan Province(No.2019JJ50372)Hunan Provincial Key Research and Development Program(No.2018GK2062)。
文摘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.
文摘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.
文摘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.
