Abstract
Human induced pluripotent stem cells (hiPSCs) are widely used for disease modeling, tissue engineering, and clinical applications. Although the development of new disease-relevant or customized hiPSC lines is of high importance, current automated hiPSC isolation technologies rely largely on the fluorescent labeling of cells, thus limiting the cell line development from many applications. The objective of this research was to develop a platform for high-throughput hiPSC cytometry and splitting that utilized a label-free cell sensing approach. An image analysis pipeline utilizing background subtraction and standard deviation projections was implemented to detect hiPSC colonies from bright-field microscopy data. The pipeline was incorporated into an automated microscopy system coupling quad microraft cell-isolation arrays, computer-based vision, and algorithms for smart decision making and cell sorting. The pipeline exhibited a hiPSC detection specificity of 98% and a sensitivity of 88%, allowing for the successful tracking of growth for hundreds of microcolonies over 7 days. The automated platform split 170 mother colonies from a microarray within 80 min, and the harvested daughter biopsies were expanded into viable hiPSC colonies suitable for downstream assays, such as polymerase chain reaction (PCR) or continued culture. Transmitted light microscopy offers an alternative, label-free modality for isolating hiPSCs, yet its low contrast and specificity for adherent cells remain a challenge for automation. This novel approach to label-free sensing and microcolony subsampling with the preservation of the mother colony holds the potential for hiPSC colony screening based on a wide range of properties including those measurable only by a cell destructive assay.
Highlights
Induced pluripotent stem cells hold the potential to revolutionize research in disease modeling, drug screening, tissue engineering, and personalized medicine by virtue of their ability to be readily differentiated into somatic cell types replicating the functions of primary cells
This novel approach to label-free sensing and microcolony subsampling with the preservation of the mother colony holds the potential for Human induced pluripotent stem cells (hiPSCs) colony screening based on a wide range of properties including those measurable only by a cell destructive assay
Several reports describe the use of slightly defocused bright-field imaging to enhance the contrast of adherent cells for straightforward cell detection.[22,23,24]
Summary
Induced pluripotent stem cells (iPSCs) hold the potential to revolutionize research in disease modeling, drug screening, tissue engineering, and personalized medicine by virtue of their ability to be readily differentiated into somatic cell types replicating the functions of primary cells. The production and maintenance of iPSCs from precursor cells such as erythroblasts or fibroblasts are complex, multistep processes that continue to require numerous manual steps. Even in optimized culture conditions, iPSC cultures have a propensity for spontaneous differentiation, and differentiated cells must be identified for removal at the earliest stage to maintain high-quality cultures.[1] accuracy in iPSC sensing is critical, to ensure safety in clinical applications and to prevent failed cultures and reduce the costs of culture optimization.[2,3] Microscopic observation provides a rich sensing modality for detecting iPSC-relevant phenotypes such as nuclear-tocytoplasmic ratios, colony border definition, cellular compaction, apoptotic cells, and other morphologies.[4,5] the expert manual microscopic observation of iPSCs remains the gold standard for iPSC sensing despite its limited throughput and precision
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