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BACKGROUND:Many types of mammalian cells aggregate and display three-dimensional multicellular spheroids when they are in normal physiological conditions. In order to observe and explore cellular natural states, many researchers try to use spherical cellculture in vitro, a common three-dimensional culture pattern. OBJECTIVE:To use three different methods for spherical culture in vitro of adipose-derived stem cells and to observe their biological features. METHODS:Adipose-derived stem cells were confirmed by the analysis of the markers for cellphenotypes as wel as adipogenic and osteogenic differentiation potential assays. Three different methods of sphere cultures were used as fol ows:(1) ultra low attachment culture;(2) hanging-drop culture and (3) Eppendorf tube culture. The sphere formation was compared among above three methods. We used Imagej to calculate mean areas of these spheres. And we used Viability/Cytotoxicity Assay Kit for Animal Live&Dead cells to detect their vitality. RESULTS AND CONCLUSION:(1) Adipose-derived stem cells were confirmed by the analysis of the markers for cellphenotypes, CD29, CD44, CD59 were positive, as wel as adipogenic and osteogenic differentiation potential assays were positive. The conventional monolayer cultures of adipose-derived stem cells showed spindle and cloning growth within three passages. (2) Ultra low attachment culture, hanging-drop culture, Eppendorf tube culture al could elicit adipose-derived stem cells spherical growth. However, spherical size, shape and uniformity differed depending on cellnumbers, culture time and spherical culture methods. The ultra low attachment culture was comparatively difficult to control spherical shape and uniformity of adipose-derived stem cells. But hanging-drop culture and Eppendorf tube culture were able to form even cellspheres. (3) Spherical formation of adipose-derived stem cells using our three methods displayed good cellvitality.
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Objective: To investigate the anti-apoptosis competence and the expression of glutathione- S-transferase-π (GST-π) of tumor spheres from lung cancer cell line A549. Methods: The tumor spheres were achieved from lung cancer cell line A549 cultured in serum-free medium. The apoptosis rates of both tumor spheres and primary A549 cells after treatment with cisplatin were detected by immunofluorescence assay with Annexin V-FITC+PI and JC-1 stainings, and the expressions of GST-π protein were also examined in the tumor spheres and the primary A549 cells as well as their grafted tumors in nude mice by Western blotting and fluorescence immunohistochemistry, respectively. Results: Immunofluorescence assay showed that the apoptosis rate was significantly higher in tumor spheres than that in primary A549 cells. Compared with the primary A549 cells and their corresponding grafted tumors, the expression levels of GST-π protein were significantly higher in the tumor spheres and their corresponding grafted tumors. Conclusion: Tumor spheres from lung cancer cell line A549 have a strong capability of drug-resistance, which is probably associated with the up-regulated expressions of drug-resistance proteins in these tumor spheres.
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Objective: To generate tumor spheres from colorectal cancer cell line HCT116 in serum-free medium (SFM) and to observe the location of the cancer stem cell surface marker CD133 as well as the ratio of CD133+ cells so as to investigate the possible relationship between the epithelial-mesenchymal transition and metastatic ability of tumor spheres. Methods: Human HCT116 colorectal cancer cells were cultured in SFM supplemented with cell growth factors. The location of cancer stem cell marker CD133 in tumor spheres was observed by confocal laser scanning microscopy, and the content of CD133 + cells in tumor spheres was detected by flow cytometry (FCM). Transwell chamber assay, immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR) were applied to examine the metastatic ability of tumor spheres in vitro and the expressions of epithelial-mesenchymal-related key markers. Results: The surface marker CD133 was located on cell membrane, and the content of CD133+ cells was significantly increased in HCT116 tumor spheres. The expressions of mesenchymal markers N-cadherin and Vimentin were highly elevated, while the expression of epithelial marker E-cadherin was reduced. The metastatic ability of cell sphere was higher than that of monolayer cells. Conclusion: HCT116 tumor spheres are enriched with CD133+ cells. The surface marker CD133 is located on the cell membrane. The cell spheres may get higher metastatic ability through epithelial-mesenchymal transition. Copyright© 2011 by TUMOR.
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Objective:To isolate breast cancer stem/progenitor cells from human breast cancer and study their proliferation and differentiation biological characteristics over long-term passages in vitro. Methods:Human breast cancer stem/progenitor cells were enriched in suspension cultures as nonadherent mammospheres(MS). Serial sphere formation assay was performed to determine self-renewal ability of mammosphere-derived cells (MSDC). Differentiation was induced by culturing MSDC in DMEM-F12 supplemented with serum but without growth factors. The ratio of CD44~+/CD24~(-/low) cell population was evaluated by flow cytometry(FCM). Results:The mammospheres were formed after inoculation of primary breast cancer cells into culcutre medium with growth factors but without serum. The mammospheres contained undifferentiated cells similar to stem cells, which had self-renewal and extensive proliferation capabilities. With increasing passages, the cells tended to adhere and differentiate. The number of adhering and differentiating cells increased, and the amount and size of mammospheres decreased. The CD44~+/CD24~(-/low) cell population was enriched in the basal-like molecular subtype of human breast tumors. The biological behaviors of mammospheres varied between different specimens.Conclusion:Cancer cells with stem cell properties of self-renewal, indefinite proliferation and multi-lineage differentiation widely existed in human breast cancer tissues. The biological behaviors varied because of different origin of specimens and changed under the effects of environmental factors.
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Objective To investigate the effect of the ubiquitin-proteasome pathway inhibitor MG132 on the natural resistance to cisplatin in the human cervical cancer line (HCE1) muhicellular spheroid (HCE1/MCS) model and to probe it if MG132 could reverse the HCE1/MCS resistance to cisplatin, as well as the possible mechanism of drug resistance.Methods (1) HCE1/MCS was obtained using liquid overlay and rotating technique.(2)Four groups were established (MG132 group, cisplatin group, MG132 + cisplatin group, the control group).Cell viability were measured by trypan blue exclusion assay.Cell cycle and apoptosis were detected by flow cytometry.(3) The expression of nuclear factor (NF) kB of both HCE1 monolayer cells (HCEI/MC) and HCE1/MCS was detected by western blot, and the expression of B cell lymphoma/leukemia-2 (bcl-2) was detected by immunohistochemistry.Results (1) HCE1/MCS was established successfully.(2) Cell inhibited rate of HCE1/MC and HCE1/MCS was: in MG132 group, (11.67 ± 2.34) % vs (10.78 ± 1.17) % (P > 0.05) ; in MG132 + cisplatin group, (92.67 ± 2.52)% vs (91.33 ±2.18)% (P>0.05); in cisplatin group, (45.01±7.44)% vs (9.45±5.98)% (P<0.05).(3)The rate of apoptosis of HCE1/MC and HCE1/MCS were: in MG132 group, 8.14% and 5.97% ; in MG132 + cisplatin group, 99.01% and 95.22% ; in cisplatin group, 33.61% and 0.88%.(4)The expression level of NF-kB and the high expression rate of bcl-2 were: in HCE1/MCS of control group, 0.67 and 60% ; in HCE1/MCS of cisplatin group, 0.85 and 83% ; in HCE1/MCS of MG132 group, 0.39 and 20% ; in HCE1/MCS of MG132 + cisplatin group, 0.47 and 33%.Conclusions (1) HCE1/ MCS present natural resistance to cisplatin and may become a good model for the study of cervical cancer drug resistance in vitro.(2) MG132 could induce the inhibition and apoptosis of HCE1/MCS cells and partially reverse the natural resistance of HCE1/MCS to cisplatin, of which partially reverse the natural resistance may be in relation to the down-regulation of NF-kB and bcl-2 expression.
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Objective To investigate the feasibility of pancreatic cancer muhieellular spheroids in chemosensitivity study. Methods Pancreatic adenocarcinoma cell lines SW1990, PCT-3 and ASPC-1 were cultured in collagen gel. When the muhicellular spheroids were formed, the drug chemosensitivities of Fluorouracil ( 5-FU ), Gemzar ( GEM ) and Oxaliplatin ( OXA ) were detected by CD-DST and CCK-8 methods. The results were compared with those of scattered cells model. Results The chemosensitivities of three pancreatic lines in form of multicellular spheroids were significantly lower than those of scattered cells model (P < 0. 05). The inhibitory rates of 50 μg/ml 5-FU on SW1990, PCT-3, ASPC-1 were (53.96 ±4.32)% ,(58.49±5.98)%, (49.57±4.36)% ;the inhibitory rates of 25 μg/ml GEM on SWI990,PCT-3, ASPC-1 were (53.02 ± 4.06) %, (61.90 ± 4.80) %, (38.09 ± 4.88 ) % ;the inhibitory rates of 10 μg/ml OXA on SW1990, PCT-3, ASPC-1 were (57.33 ± 6.27 ) %, (50.90 ± 4.80) %, (47.26 ± 4.29) % ;which were significantly lower than those of scattered cells model ( P < 0.05 ). Conclusions In collagen gel, formation of the multicellular spheroids of pancreatic cells caused less chemosensitivity and promoted anticancer drug resistance, which was consistent with in vivo state.