Unknown,Transcriptomics,Genomics,Proteomics

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Comparing gene expression of human cardiac stem cells between automatic cell culture and manual handling culture

ABSTRACT: BACKGROUND: Cell-based regeneration therapies hold great promise and potential for new area in clinical medicine, although some obstacles still remain to be overcome for a wide range of clinical applications. One of the major impediments in this field is difficulties in large-scale production of cells of interest with reproducibility. Current protocol of cell therapy requires a long-time laborious manual process. To solve this problem, we focused on the robotics of automated and high throughput cell culture system. To date, an automated robotic cultivation of stem or progenitor cells in clinical trials has not been reported. METHODS: The system, AutoCultureÂ®, used in this study can automatically replace the medium, centrifuge cells, split the cells, and take a photograph for their morphology. We examined the feasibility to use it in the clinical field by comparing the growth rate and the characteristics of cardiac stem cells (CSCs) cultivated by AutoCulture and the manual handling culture, which protocol is the exact same as current performing clinical trial in our institute. RESULTS: We demonstrated similar characteristics in both culture methods, in terms of growth rates, gene expression profiles, cell surface profiles by FACS, carbon hydrate structures on the cell surface, and genomic DNA stability. IMPLICATIONS: The results of this study showed that AutoCulture is feasible to cultivate human cells for regenerative medicine. An automated cell-processing machine for cell therapy will play more important role, as it will be widespread from multi-center trials to off-the-shelf cell products. The cells were cultured by manual handling or by the automatic cell culture apparatus. Total RNA was extracted from these cells at day 7 by the RNeasy Plus Mini Kit (QIAGEN). Gene expression analysis was performed using the Agilent Whole Human Genome Microarray chips G4112F (Agilent). Raw data were normalized and analyzed by GeneSpring GX11 software.

ORGANISM(S): Homo sapiens

SUBMITTER: Akihiro Umezawa

PROVIDER: E-GEOD-44032 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Ex vivo activation and expansion of natural killer (NK) cells is a strategy to produce sufficient numbers of these effector cells for adoptive immunotherapy. Therefore we aimed at the development of an automated, clinical scale NK cell expansion process and compared NK cells after manual and automated expansion.We found only a small subset of 124 genes that significantly varied between both sample groups. These genes were associated with movement of leukocytes were identified including a group of genes for NK cell movement (CMKLR1, CX3CR1, S1PR5, GNLY and CXCR1) which were slightly higher expressed after automated compared to manual expansion. Nevertheless, the expansion profiles of NK cells after automated or manual expansion were rather similar in comparison to the remarkable difference between primary and expanded NK cells in general represented by the vast majority of regulated genes between the analyzed sample groups. Pathway analysis revealed that most of regulated genes upon expansion were associated with cell cycle regulation, DNA replication, DNA recombination and DNA repair, regulation of apoptosis and cell survival as well as cytokine signaling. Furthermore, the expression of many NK cell relevant markers was changed upon expansion with the strongest effects on up regulation of TRAIL and FASL, inhibitory TIGIT and chemokine receptors CCR2, CCR5 and CXCR6. In addition, granzyme M was down regulated but other important effector molecules like TNFa, perforin and granzymes A, B and K were up regulated. In summary we could show that manual and automatically expanded NK cells show a similar expansion profile while the differ significantly from primary NK cells. Up regulation of many NK cell relevant markers indicate for an enhanced NK cell functionaility after ex vivo activation and expansion. 24 samples were analyzed in total. 6 biological replicates represented by cells from different donors. 4 different samples per donor: freshly isolated NK cells (d0) and NK cells expanded for 14 days under 3 different conditions: in co-culture with EBV-LCL feeder cells and 500 U/mL IL2 either cultivated manually in T75 flasks (T) or automatically using the CliniMACS Prodigy system (P); NK cells cultured with 500 U/ml IL2 without feeder cells (I)

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Comparing gene expression of human cardiac stem cells between automatic cell culture and manual handling culture

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