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A feeder layer consists of inactivated mouse embryonic fibroblast cells that provide necessary intercellular interactions and nutrients to cultured stem cells. This layer prevents spontaneous differentiation, allowing stem cells to maintain their undifferentiated state while dividing and spreading across the culture dish to form colonies.

Once stem cell colonies grow to the required size, they are removed gently and subjected to passage, where they are replated or subcultured multiple times. This process establishes a pure and continuously growing cell line capable of generating millions of stem cells in vitro for research and therapeutic applications.

Stem cells are extracted from adult tissues, embryonic tissues, umbilical cord blood, and deciduous teeth. Adult stem cells are multipotent and limited to differentiating into cell types found in their tissue of origin, while embryonic stem cells are pluripotent with broader differentiation potential.

Induced pluripotent stem cells (iPSCs) are genetically reprogrammed adult cells that function like embryonic stem cells, capable of generating cells from all three germ layers. They avoid the legal, ethical, and immunological problems associated with embryonic stem cells, making them a promising advance for cell-based therapy applications.

Culture media is supplemented with growth hormones and essential nutrients that nourish stem cells, enabling them to divide and spread across the culture dish. These components maintain optimal conditions for stem cell proliferation and colony formation in vitro for extended periods.

Once an established cell line is generated, batches of cultured stem cells can be frozen for future use. This preservation method allows researchers and clinicians to maintain viable stem cell populations without continuous culture, supporting long-term storage for regenerative medicine applications.

Embryonic stem cells may trigger immune rejection because the patient's body recognizes them as foreign, and their isolation raises ethical concerns. Adult stem cells avoid immunological issues but have limited differentiation potential, requiring researchers to balance therapeutic capability with safety and ethical considerations.