Producing standardized human pluripotent stem cell (hPSC)-derived cardiomyocyte aggregates in stirred spinner flasks is a crucial step in the field of regenerative medicine and drug discovery. These aggregates, also known as cardiac spheroids, mimic the three-dimensional structure and function of the heart, making them an invaluable tool for studying cardiac development, disease modeling, and drug screening. In a recent article published in Nature Protocols, researchers have outlined a detailed protocol for generating high-quality hPSC-derived cardiomyocyte aggregates in stirred spinner flasks. Here, we will provide an overview of the key steps involved in this protocol.
First and foremost, it is essential to start with high-quality hPSCs that have been properly maintained and characterized. These cells should be cultured in a pluripotent state using appropriate culture media and conditions. Once the hPSCs are ready, they can be differentiated into cardiomyocytes using a well-established cardiac differentiation protocol. This typically involves the sequential addition of growth factors and small molecules that mimic the signaling pathways involved in cardiac development.
After the cardiomyocytes have been generated, they can be seeded into stirred spinner flasks for aggregate formation. The use of stirred spinner flasks allows for efficient mixing and oxygenation of the cells, which is crucial for promoting uniform aggregate formation. The researchers recommend using a specific type of spinner flask with a magnetic stir bar for optimal results.
To promote aggregation, the cardiomyocytes are typically cultured in a suspension culture medium that contains essential nutrients and growth factors. The researchers suggest using a defined medium that supports cardiomyocyte survival and maturation. It is also important to monitor the pH and oxygen levels in the culture medium to ensure optimal conditions for aggregate formation.
During the culture period, the researchers recommend gently shaking the spinner flasks at a low speed to promote aggregate formation without disrupting the cells. It is also important to regularly check the size and morphology of the aggregates under a microscope to ensure uniformity and quality. The researchers provide guidelines for determining the optimal seeding density and culture duration to achieve standardized cardiomyocyte aggregates.
In conclusion, producing standardized hPSC-derived cardiomyocyte aggregates in stirred spinner flasks is a complex process that requires careful attention to detail and adherence to specific protocols. By following the guidelines outlined in the Nature Protocols article, researchers can generate high-quality cardiac spheroids that can be used for a wide range of applications in cardiovascular research. This protocol represents a significant advancement in the field of stem cell biology and holds great promise for advancing our understanding of cardiac development and disease.
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