PMC:4996362 / 13816-18030 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4996362","sourcedb":"PMC","sourceid":"4996362","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4996362","text":"4. Discussion\nIn 2005, Ferrer et al. and Waterworth et al. [7,18] developed and immunophenotyped a panel of paraffin-embedded cell lines using five human adenocarcinoma lines. In 2006, Andersson et al. described cell TMAs (cells and tissues) as a tool for antibody-based proteomics using 46 frequently used cell lines in addition to 12 patient cell samples [12]. Therefore, microarrays generated from formalin fixed and paraffin embedded cultured cell lines can serve as a platform for in vitro analysis of protein expression profiles. To our knowledge, no “cell microarrays” (CMAs) have yet been reported to analyze antigen expression, by immunohistochemistry, of human pluripotent stem cells to demonstrate the utility of CMAs as a source for the rapid screening of molecules of interest. Yamazoe and Iwata have used CMAs to screen feeder cells in differentiation and candidate cells for specific ES cell differentiation using the PA6-mouse ES cell co-culture system [19]. The rapid and efficient screening of iPS derived cell clones, using CMA technology, is very advantageous considering that each reprogramming event generates a high number of abortive clones [20] all of which need to be molecularly characterized. In this respect, the CMA platform offers several advantages, including minimal consumption of valuable biological samples and reagents, the costs associated with pluripotent stem cells expansion and differentiation are very high. The in vitro assembly of cultured iPS cells in a CMA opens up possibilities to analyze not only the expression of stem cell biomarkers but also to analyze the manipulation of the cells prior to fixation, such as check for the presence/absence of the reprogramming vehicle, cell phenotype and heterogeneity, morphological features, shape, nucleus/cytoplasm ratio and number of intracellular components. Fixing harvested cells in agarose molds decreases the number of cells necessary for marker expression analysis, when compared to coverslip growth (Figure 1). This approach is very advantageous considering that the proliferation rate of fully differentiated stem cells is very low [21]. With only one cell pellet a large panel of antigens can be tested since from a single recipient block over a hundred of slices can be sectioned and tested [22]. Moreover on a single recipient block different samples, representing several iPS clones and/or multiple differentiation statues, can be inserted. Thus, TMA technology advantages, such as time-saving in analysis, labor and reagent costs [3], can be effectively translated to cellular samples. Therefore, CMAs can be classified as a highthroughput technique when dealing with iPS derived clones that need to be characterized [20]. \nAn important aspect of CMA technology is to preserve cell morphology. Using the AF22 neuralized iPS cell line as a cellular model to validate the method, we investigated two experimental conditions: cell trypsinization and mechanical scraping using a rubber policeman. Here, we show that mechanical scraping is able to maintain cell morphology in a comparable way to those cells grown on coverslip. Indeed trypsin treatment cleaves the binding of anchorage protein to the dish, generating round cells with poor morphology that does not reflect the original one, increasing the possibility to misinterpret immunofluorescence staining (i.e., false negative). Otherwise, mechanical scraping after fixation with PFA enables better preservation of cell morphology and thus nucleus/cytoplasm ratio is maintained, leading to better analysis of the images. The advantages in the application of CMA platform are connected to the precision in the spot definitions and to the traceability of a large number of samples and digital reporting. This automation is highly important when dealing with iPS clones that can be simultaneously analyzed as a whole set of cells, minimizing any possibility of error [23].\nAltogether, these results support the use of CMA platform for the screening of large number of cell lines. In particular morphology preservation techniques enhance the power of this technology and introduce CMA as a new tool for stem cell research and specifically iPS clone screening. 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