RESUMO
Phenotypic heterogeneity is commonly observed in diseased tissue, specifically in tumors. Multimodal imaging technologies can reveal tissue heterogeneity noninvasively in vivo, enabling imaging-based profiling of receptors, metabolism, morphology, or function on a macroscopic scale. In contrast, in vitro multiomics, immunohistochemistry, or histology techniques accurately characterize these heterogeneities in the cellular and subcellular scales in a more comprehensive but ex vivo manner. The complementary in vivo and ex vivo information would provide an enormous potential to better characterize a disease. However, this requires spatially accurate coregistration of these data by image-driven sampling as well as fast sample-preparation methods. Here, a unique image-guided milling machine and workflow for precise extraction of tissue samples from small laboratory animals or excised organs has been developed and evaluated. The samples can be delineated on tomographic images as volumes of interest and can be extracted with a spatial accuracy better than 0.25 mm. The samples remain cooled throughout the procedure to ensure metabolic stability, a precondition for accurate in vitro analysis.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Túbulos Renais/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Miocárdio/química , Tomografia por Emissão de Pósitrons/métodos , Extratos de Tecidos/isolamento & purificação , Tomografia Computadorizada por Raios X/métodos , Animais , Feminino , Heterogeneidade Genética , Genômica , Túbulos Renais/química , Túbulos Renais/metabolismo , Metabolômica , Miocárdio/metabolismo , Proteômica , RNA/genética , RNA/isolamento & purificação , RNA/metabolismo , Extratos de Tecidos/químicaRESUMO
Handling and dosing of cells comprise the most critical step in the microfabrication of cell-based assay systems for screening and toxicity testing. Therefore, the immediate drop-on-demand technology (I-DOT) was developed to provide a flexible noncontact liquid handling system enabling dispensing of cells and liquid without the risk of cross-contamination down to a precise volume in the nanoliter range. Liquid is dispensed from a source plate within nozzles at the bottom by a short compressed air pulse that is given through a quick release valve into the well, thus exceeding the capillary pressure in the nozzle. Droplets of a defined volume can be spotted directly onto microplates or other cell culture devices. We present a study on the performance and biological impact of this technology by applying the cell line MCF-7, human fibroblasts, and human mesenchymal stem cells (hMSCs). For all cell types tested, viability after dispensing is comparable to the control and exhibits similar proliferation rates in the absence of apoptotic cells, and the differentiation potential of hMSCs is not impaired. The immediate drop-on-demand technology enables accurate cell dosage and offers promising potential for single-cell applications.
