In Vitro Culture of Cryptosporidium parvum Using Stem Cell-Derived Intestinal Epithelial Monolayers
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Keywords:
Cryptosporidium parvum
Monensin
Cryptosporidium parvum
Parasitology
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The Hippo pathway regulates organ size, regeneration, and cell growth by controlling the stability of the transcription factor, YAP (Yorkie in Drosophila). When there is tissue damage, YAP is activated allowing the restoration of homeostatic tissue size. The exact signals by which YAP is activated are still not fully understood, but its activation is known to affect both cell size and cell number. Here we used cultured cells to examine the coordinated regulation of cell size and number under the control of YAP. Our experiments in isogenic HEK293 cells reveal that YAP can affect cell size and number by independent circuits. Some of these effects are cell autonomous, such as proliferation, while others are mediated by secreted signals. In particular CYR61, a known secreted YAP target, is a non-cell autonomous mediator of cell survival, while another unidentified secreted factor controls cell size.
CYR61
Hippo signaling pathway
HEK 293 cells
Cell fate determination
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Objective:To study the relationship between the cell density and the cell proliferation phenotype. Methods: Plate clonality assays was used to measure the impact of cell density to cell clonality and cell cycle in BT325、786-0、293、C6 and NIH3T3 cell lines. Results: The clonality decreased when the cells grown to confluence in NIH3T3 and 7860 cell lines respectively.It seem need more cells to decrease the clonality in 293 cell line but there is no relationship between cell density and cell clonality in BT325 and C6 cell lines.Cell cycle analysis show that cell density have no effect on BT325 and C6 but on 786-0、293 and NIH3T3 cell lines. Conclusion: There might exist preventer or preventers,which is proportional to the number of cells,of immortal stem cell to expand.In addition,the rate of stem cell expansion is proportional to that of cell mitosis in immortal cell lines.
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Microcarrier
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We report a novel mechanism of cellular growth control. Increasing the density of endothelial or smooth muscle cells in culture increased cell‐cell contact and decreased cell spreading, leading to growth arrest. Using a new method to independently control cell‐cell contact and cell spreading, we found that introducing cell‐cell contact positively regulates proliferation, but that contact‐mediated proliferation can be masked by changes in cell spreading: Round cells with many contacts proliferated less than spread cells with none. Physically blocking cell‐cell contact or inhibiting PI3K signaling abrogated cell‐cell induced proliferation, but inhibiting diffusible paracrine signaling did not. Thus, direct cell‐cell contact induces proliferation in these cells.
Contact inhibition
Cell Signaling
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Over the past decades, the increase in maximal cell numbers for the production of mammalian derived biologics has been in a large part due to the development of optimal feeding strategies. Engineering of the cell line is one of probable approaches for increasing cell numbers in bioreactor. We have demonstrated that the over-expression of the c-myc gene in immortalised CHO cells can increase proliferation rate and maximal cell density in batch culture compared to the control. The changes were attributed to a rapid transition into S-phase from a shortened duration of G1 phase and to the uncoupling of cell size from cell proliferation. To achieve the >70% increase in maximal cell density without additional supply of nutrients the cells underwent an overall reduction of 14% in size as well as a significant decrease in glucose and amino acid consumption rate. Consequently, the total biomass accumulation did not show a significant change from the control. The amount of hSEAP-hFc activity of the over expressing c-myc cell line was found to be within 0.7% of the control. It is shown that the manipulation of cell cycle kinetics and indirectly cell metabolism gives higher cell densities in CHO batch cultures. The unaltered apoptotic rate supported the proposition that the increase in cell number was a result of enhance cell cycle kinetics and cellular metabolism rather than increasing viability. Production of hSEAP-hFc from a constitutive c-myc over-expressing cell line did not increase with the increase in cell number.
Cell size
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Abstract Previous combo treatment experiments on BLM human melanoma cell line identified two combos vit-A + RU-486 and Cur+P+RU as the best combinations to decrease melanoma cell growth as well as IL-8 secretion in -vitro. In order to confirm this finding, experiments were repeated on two other human melanoma cell lines 1205Lu and WM238. Initial single or solo compound treatment on 1205Lu cells resulted in cell growth in the range of 37–59% compared to untreated control 1205Lu cell growth at 100%. A double combo treatment with combinations such as D+P, D+RU, A+P, A+RU, A+D resulted in cell growth in the range of 17 to 37% on the 1205Lu cell line. Whereas on WM238 cell line resulted in cell growth in between 18–22%. A triple combo such as combinations of three compounds (Cur+P+RU, Cur+A+P, Cur+D+RU) resulted in cell growth in the range of 19-30% on 1205Lu cells, whereas on WM238 cells resulted in between 19–20% cell growth. Elisarray of the supernatants from the combos which showed a significant decrease in cell growth revealed the regulation only in IL-8 secretion. IL-8 secretion in the supernatants of combos treated with A+RU and Cur+P+RU was17.5 and 8. 0 pg/ml respectively on the 1205Lu cell line. Whereas, IL-8 secretion was 0.515 and 0.926 pg/ml respectively in the supernatants of A+RU and Cur+P+RU combo treatments on the WM238 cell line. Conclusion Based on cell growth, Elisarray and IL-8 quantitation, two combos A+RU and Cur+P+RU also fared well on the two other cell lines 1205Lu and WM238. Presentation: No date and time listed
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Cell-cell interactions are of crucial importance for the formation of tissues, homeostasis and regeneration processes as well as reactions on foreign bodies including implants. So far, however, the importance of heterotypic cell-cell interactions in the in vitro evaluation of implant surfaces has been largely neglected. This work aims to develop an in vitro methodology that enables the in-depth investigation of heterotypic cell-cell interactions in a mixed co-culture system, and to validate it with a primary adult human bone-derived osteoblast cells (HBCs) - abdominal fibroblasts (HAFs) system. The methodology proposed combines a simple live labelling step, semiautomated fluorescence image acquisition and analysis to characterize the interactions between different cell types (cell population dynamics) in co-culture in terms of cell proliferation and cell spatial distribution of each cell type. In this co-culture system, direct cell-cell contacts between the two cell types were permitted while the determination of cell-type specific responses could still be elucidated. We could show that HAF proliferation was reduced in a way negatively correlated with the seeding HBC/HAF ratio, i.e., a high proportion of HBC in the co-culture had an inhibitory effect on HAF proliferation. In all cultures segregation was found after 4 and 7 days of co-culture. HBCs were segregated at low ratios while HAFs were segregated at high ratios. Cell-cell distances depended on the total cell number in the co-culture but the dependence was different for each cell type.
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AIM To study the effects of crude extracts of Aspongopus chinensis Dallas on cell proliferation and cell cycle of SGC-7901 and HepG2 cell lines.METHODS The crude extracts were extracted by chloroform infusion.The antiproliferative effects of crude extracts were assessed by MTT assays.The cell cycle was measured by flow cytometry.RESULTS The crude extracts could inhibit the proliferation of SGC-7901 and HepG2 cell lines in a dose-dependent manner.SGC-7901 and HepG2 cell lines were sensitive to crude extracts with an IC50 of 1 193.52 and 964.34 μg/mL,respectively.From the result of flow cytometry analysis,crude extracts,as compared with the untreated cells,caused significant reduction in cell ratio at the S and G2/M phases,and an increase in cell ratio at the G0/G1 phases.CONCLUSION The crude extracts could inhibit proliferation of both cancer cell in vitro,and especially arrest human liver cancer cells at the G0/G1 phase of the cell cycle for HepG2.
MTT assay
G1 phase
IC50
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Using standardized media, incubation, and parasite inoculating procedures, we compared development of Crytosporidium parvum between Madin-Darby bovine kidney (MDBK) cells and 10 additional host cell lines available through the American Type Culture Collection. Parasite development was assessed by counting parasite numbers atop monlayers in 25 random oil fields 68 h post-infection using Nomarski interference-contrast optics. Results revealed that the human ileocecal adenocarcinoma (HCT-8) cell line supported nearly twice the number of parasite developmental stages than MDBK cells or any of the other host cell types.
Cryptosporidium parvum
Incubation period
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