Mutations associated with clonal hematopoiesis of indeterminate potential (CHIP) have been linked to cardiovascular disease (CVD), with DNA methyltransferase 3A (DNMT3A) being the most commonly mutated gene. (Jaiswal ...
Background & AimCRISPR/Cas9 technology is a leading method for efficient gene editing, enabling the creation of isogenic pairs of mutant and control human induced pluripotent stem cells (hiPSCs). While hiPSCs prov...
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Background & AimCRISPR/Cas9 technology is a leading method for efficient gene editing, enabling the creation of isogenic pairs of mutant and control human induced pluripotent stem cells (hiPSCs). While hiPSCs provide valuable opportunities for disease modeling and personalized medicine, challenges remain in distinguishing specific mutation effects from genetic backgrounds. Manual genome editing processes often face scalability and standardization limitations. The StemcellFactory, a modular robotic system, has been developed to automate the reprogramming, expansion, and differentiation of hiPSCs, enhancing the application of CRISPR/Cas9 in research. MethodologyHiPSCs were cultured in StemMACS iPS-Brew XF medium, with daily medium changes and mycoplasma testing. For CRISPR/Cas9 editing, target gene-specific CRISPR-RNA and tracrRNA formed ribonucleoprotein complexes, utilizing an optimized nucleofection protocol. HiPSCs were dissociated, counted, and nucleofected through manual and automated processes. The automated StemcellFactory workflow included cell preparation, nucleofection, and cell seeding, managed by the COPE software for data visualization. Genotyping of polyclonal hiPSCs was performed via Sanger sequencing to assess editing efficiency, with automated isolation of monoclonal colonies conducted using an integrated cellCelector. ResultsThe StemcellFactory successfully integrated a 4D-Nucleofector with a 96-well shuttle device for automated CRISPR/Cas9 editing. Adjustments replaced manual consumables with scalable formats, enhancing efficiency. The CM150 electroporation program yielded high cell viability (83.5%) and indel rates (81.7%). Optimal culture conditions combined Laminin 521 with CloneR-supplemented iPS-Brew medium, supporting hiPSC survival and colony formation. The automated system allowed parallel processing of up to 24 conditions, demonstrating high editing efficiencies, with indel rates exceeding 80% across all tested cell lines, comparable to ma
The number of individuals suffering from neuropsychiatric disorders (NPDs) has increased worldwide, with 3 million disability-adjusted life-years calculated in 2019. Though research using various approaches including ...
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