Hepatocytes are the principal functional cells of the liver, playing a critical role in various metabolic processes, drug metabolism, and detoxification. Generating functional hepatocytes from stem cells is a significant milestone in regenerative medicine and drug development. Creative Biolabs presents a comprehensive protocol for stem cell differentiation to generate hepatocytes. This protocol provides detailed guidelines on the materials required, the step-by-step procedure, and important notes for successful hepatocyte differentiation.
Maintain iPSCs in pluripotent stem cell maintenance medium. Change medium daily and monitor cell morphology. Passage cells as required to maintain sub-confluent culture. Assess pluripotency using immunofluorescence or flow cytometry for pluripotent markers.
Passage pluripotent stem cells to a new Matrigel-coated dish or plate. After reaching 70-80% confluency, replace pluripotent stem cell medium with definitive endoderm induction medium. Culture cells for 3-5 days, changing medium daily. Observe the formation of definitive endoderm cells with a typical cobblestone-like morphology.
Induce hepatoblast formation by changing the medium to hepatocyte differentiation medium. Continue daily medium changes and culture for 7-10 days. Observe cell morphology for the presence of hepatoblast-like cells.
Culture hepatoblasts in hepatocyte differentiation medium for an additional 2-4 weeks. Monitor cell morphology, and assess hepatocyte marker expression via immunofluorescence, real-time PCR, and Western blotting. Assess functional hepatocyte properties, including Albumin and CYP activity. Cryopreserve differentiated hepatocytes if necessary.
In conclusion, this protocol provides a guideline for the differentiation of stem cells into functional hepatocytes. By following these steps and considering the provided notes, researchers can successfully generate hepatocytes for various applications, including disease modeling, drug screening, and regenerative medicine.
For more detailed and tailored protocols or access to specialized reagents, consult with the experts at Creative Biolabs. Our team is dedicated to advancing your research goals and ensuring the success of your stem cell differentiation projects. Contact us today to explore our comprehensive services and expertise in stem cell-related applications.
Reference
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