Induced pluripotent stem cells (iPSCs) can differentiate into any cell type and have the capacity for unlimited self-renewal, making them valuable tools for drug discovery, regenerative medicine, and disease modeling. However, the long-term culture of iPSCs can result in genotypic and phenotypic heterogeneity, meaning that constant monitoring is required to safeguard critical quality attributes (CQAs) such as pluripotency, viability, and stability. Technologies designed to streamline existing workflows for cellular characterization promise to increase the utility of iPSCs for a broad range of research and clinical applications.
A typical iPSC research and development workflow involves a series of steps, beginning with sourcing representative somatic cells and inducing pluripotency with various methods. Next, a combination of techniques is used to identify clones meeting defined quality criteria and demonstrating the ability to differentiate into the desired cell type. Candidates of interest are then banked, before being expanded to production culture volumes using appropriate growth factors and cytokines, which are often optimized for iPSCs. Lastly, the resultant iPSC products are harvested for comprehensive characterization and quality control (QC) testing, which includes evaluating biosafety, genetic stability, pluripotency phenotype, and viability, prior to cryopreservation.
The quality of iPSC cultures can vary significantly due to a multitude of factors. These include the somatic cell source from which the iPSC culture was derived, which can impact its ability to differentiate into certain lineages, and the method used for inducing pluripotency. For example, lentiviral miRNA infection is known to produce higher yields of pluripotent cells compared to using adenoviral methods. Additionally, it is essential to use optimized methods for culturing iPSCs to limit the risk of spontaneous differentiation or reduced viability. This includes the matrices used to adhere iPSCs to culture plastic, as well as the media formulations, which should include the correct growth factors and cytokines to maintain high levels of pluripotency and cell health. To ensure that only the best-performing iPSC lines are progressed, it is common to perform initial tests for key attributes such as viability and pluripotency, as well as to check for consistent marker expression across each subsequent stage of the iPSC workflow.
Conventional methods for assessing the viability and pluripotency of iPSC cultures, such as flow cytometry, have several inherent limitations. Not only do they have lengthy workflows, involving fixation, immunostaining, and multiple wash steps, but they also necessitate using large sample volumes that reduce the number of cells available for downstream applications. In addition, the low throughput of many existing instruments reduces the capacity for intra- and inter-experimental replication, while the need to perform complicated compensation optimization and manual data analysis risks introducing errors or user bias that could compromise the accuracy of results.
The development of simpler, more robust, and standardized techniques for evaluating individual iPSC lines and monitoring iPSC differentiation has been a major driving factor behind many recent advances in stem cell research. Notably, by switching from traditional methods for characterization to using more sophisticated tools and technologies, scientists are better able to ensure that the right iPSC lines are selected for lengthy (and often expensive) downstream studies. Newer methods have also enhanced the scalability and throughput of applications utilizing iPSCs, including liver toxicity screening and efficacy ranking of novel drugs. Furthermore, the production of growth factor and cytokine-supplemented media formulations optimized for specific iPSC lines has been fundamental to improving the quality of iPSC cultures.
Combining advanced flow cytometry with live-cell analysis is a proven strategy to streamline iPSC research and development workflows. Specifically, by integrating rapid sample acquisition with low volume requirements and plate-based data analytics, advanced flow cytometry conserves time, reagents, and sample during iPSC phenotyping and viability evaluation. And, by enabling continuous morphological assessment without the need to remove cultures from the stable conditions within the CO2 incubator, live-cell analysis allows for closer monitoring of iPSC colonies from initial isolation all the way through differentiation. Overall, these benefits translate to more resources for multiple replicates, deeper insights into morphology and pluripotency changes over time, and lower attrition rates due to low-quality cell products.
Sartorius offers a comprehensive array of solutions to support iPSC research and development, including the iQue® Advanced Flow Cytometry Platform, the Incucyte® Live-Cell Analysis Platform, and an extensive selection of high-quality, animal-free, research use only (RUO) growth factors and cytokines optimized for iPSCs. To learn more, visit sartorius.com/ipscs.