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 sphero...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.展开更多
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 resul...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
目的构建并表征胃癌三维多细胞球体模型,用于评价化疗药物的瘤内穿透。方法将12000个小鼠胃癌MFC细胞系接种于96 U 3D细胞培养板,以构建MFC多细胞肿瘤球体(MFC MCTS);将8000个MFC细胞和4000个小鼠成纤维NIH/3T3细胞系共同接种于96 U 3D...目的构建并表征胃癌三维多细胞球体模型,用于评价化疗药物的瘤内穿透。方法将12000个小鼠胃癌MFC细胞系接种于96 U 3D细胞培养板,以构建MFC多细胞肿瘤球体(MFC MCTS);将8000个MFC细胞和4000个小鼠成纤维NIH/3T3细胞系共同接种于96 U 3D细胞培养板,以构建MFC-NIH/3T3共培养多细胞肿瘤球体(CO MCTS);通过活细胞成像表征球体的直径、圆度、面积和透光率;通过激光共聚焦成像表征球体微环境和化疗药物(LipoDOX,5.0μg/ml)的瘤内穿透。结果培养5 d时,两种球体的透光率最低,圆度>0.90,直径约为530μm,面积约为0.23 mm^(2)。两种球体模型内均检测到3种微环境荧光探针分布。LipoDOX能相对均匀地穿透MFC MCTS,却在CO MCTS内表现出外多内少的不均匀穿透,药物荧光强度降低约58.3%(P<0.01)。结论本研究构建了两种具有良好圆度、高紧实度、适中直径和面积的胃癌三维多细胞球体模型,两种胃癌球体模型均表现出酸性、缺氧和氧化还原微环境,可精准、高效地评价化疗药物的瘤内穿透。展开更多
AIM: To examine the factor(s) involved in differentiation of intestinal macrophages (IMACs) using a recently established in vitro model. METHODS: To test whether soluble or membrane bound factors induce IMAC-different...AIM: To examine the factor(s) involved in differentiation of intestinal macrophages (IMACs) using a recently established in vitro model. METHODS: To test whether soluble or membrane bound factors induce IMAC-differentiation, freshly elutriated monocytes (MO) were incubated with conditioned media or cell membranes of intestinal epithelial cells (IEC) or cultured with IEC in transwell systems. To determine the importance of an active migration of MO, three- dimensional aggregates from a 1:1-mixture of MO and IEC were examined by immunohistochemistry and flow cytometry. Apoptosis was examined by caspase-3 Western blots. Extracellular matrix production in differentiation models was compared by immunohistochemistry. RESULTS: IMAC differentiation was observed in a complex three-dimensional co-culture model (multicellular spheroid, MCS) with IEC after migration of MO into the spheroids. By co-culture of MO with conditioned media or membrane preparations of IEC no IMAC differentiation was induced. Co-culture of MO with IEC in transwell- cultures, with the two cell populations separated by a membrane also did not result in intestinal-like differentiation of MO. In contrast to IEC-spheroids with immigrating MO in mixed MCS of IEC and MO only a small subpopulation of MO was able to survive the seven day culture period. CONCLUSION: Intestinal-like differentiation of MO in vitro is only induced in the complex three-dimensional MCS model after immigration of MO indicating a roleof cell-matrix and/or cell-cell interactions during the differentiation of IMACs.展开更多
AIM： To evaluate the interplay between gliadin and LoVo cells and the direct effect of gliadin on cytoskeletal patterns.METHODS： We treated LoVo multicellular spheroids with digested bread wheat gliadin in order to ...AIM： To evaluate the interplay between gliadin and LoVo cells and the direct effect of gliadin on cytoskeletal patterns.METHODS： We treated LoVo multicellular spheroids with digested bread wheat gliadin in order to investigate their morphology and ultrastructure （by means of light microscopy and scanning electron microscopy）, and the effect of gliadin on actin （phalloidin fluorescence） and the tight-junction protein occludin and zonula occluden-1.RESULTS： The treated spheroids had deep holes and surface blebs, whereas the controls were smoothly surfaced ovoids. The incubation of LoVo spheroids with gUadin decreased the number of intracellular actin filaments, impaired and disassembled the integrity of the tight-junction system.CONCLUSION： Our data obtained from an ＂in vivolike＂ polarized culture system confirm the direct noxious effect of gliadin on the cytoskeleton and tight junctions of epithelial cells. Unlike two-dimensional cell culture systems, the use of multicellular spheroids seems to provide a suitable model for studying cell-cell interactions.展开更多
基金This research is supported by National Natural Science Foundation of China （Nos. 21425314, 21434009, and 21421061）, National Program for Special Support of Eminent Professionals, Beijing Municipal Science ＆ Technology Commission （No. Z161100000116037）, and MOST （No. 2013YQ190467）.
文摘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.
文摘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
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.
基金Supported by the Deutsche Forschungsgemeinschaft (SFB585, Ro 1236/3-2)the BMBF Kompetenznetz-CED
文摘AIM: To examine the factor(s) involved in differentiation of intestinal macrophages (IMACs) using a recently established in vitro model. METHODS: To test whether soluble or membrane bound factors induce IMAC-differentiation, freshly elutriated monocytes (MO) were incubated with conditioned media or cell membranes of intestinal epithelial cells (IEC) or cultured with IEC in transwell systems. To determine the importance of an active migration of MO, three- dimensional aggregates from a 1:1-mixture of MO and IEC were examined by immunohistochemistry and flow cytometry. Apoptosis was examined by caspase-3 Western blots. Extracellular matrix production in differentiation models was compared by immunohistochemistry. RESULTS: IMAC differentiation was observed in a complex three-dimensional co-culture model (multicellular spheroid, MCS) with IEC after migration of MO into the spheroids. By co-culture of MO with conditioned media or membrane preparations of IEC no IMAC differentiation was induced. Co-culture of MO with IEC in transwell- cultures, with the two cell populations separated by a membrane also did not result in intestinal-like differentiation of MO. In contrast to IEC-spheroids with immigrating MO in mixed MCS of IEC and MO only a small subpopulation of MO was able to survive the seven day culture period. CONCLUSION: Intestinal-like differentiation of MO in vitro is only induced in the complex three-dimensional MCS model after immigration of MO indicating a roleof cell-matrix and/or cell-cell interactions during the differentiation of IMACs.
基金Supported by the "Fondazione San Paolo" grant to "Centro perlo Studio della Celiachia"
文摘AIM： To evaluate the interplay between gliadin and LoVo cells and the direct effect of gliadin on cytoskeletal patterns.METHODS： We treated LoVo multicellular spheroids with digested bread wheat gliadin in order to investigate their morphology and ultrastructure （by means of light microscopy and scanning electron microscopy）, and the effect of gliadin on actin （phalloidin fluorescence） and the tight-junction protein occludin and zonula occluden-1.RESULTS： The treated spheroids had deep holes and surface blebs, whereas the controls were smoothly surfaced ovoids. The incubation of LoVo spheroids with gUadin decreased the number of intracellular actin filaments, impaired and disassembled the integrity of the tight-junction system.CONCLUSION： Our data obtained from an ＂in vivolike＂ polarized culture system confirm the direct noxious effect of gliadin on the cytoskeleton and tight junctions of epithelial cells. Unlike two-dimensional cell culture systems, the use of multicellular spheroids seems to provide a suitable model for studying cell-cell interactions.
