The embryos from which human embryonic stem cells are derived are typically four or five days old and are a hollow microscopic ball of cells called the blastocyst.

The blastocyst is formed by

• Trophoblast, which is the layer of cells that surrounds the blastocyst

• Blastocoel, which is the hollow cavity inside the blastocyst

• Inner cell mass, which is a group of approximately 30 cells.
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Culture

Growing cells in the laboratory is known as cell culture.

• Embryonic stem cells are isolated by transferring the inner cell mass into a plastic laboratory culture dish that contains a nutrient broth known as culture medium.

• The cells divide and spread over the surface of the dish. The inner surface of the culture dish is typically coated with mouse embryonic skin cells that have been treated so they will not divide. This coating layer of cells is called a feeder layer. The reason for having the mouse cells in the bottom of the culture dish is to give the inner cell mass cells a sticky surface to which they can attach. Also, the feeder cells release nutrients into the culture medium.

• Over the course of several days, the cells of the inner cell mass proliferate. When this occurs, they are removed gently and plated into several fresh culture dishes. The process of replating the cells is repeated many times and for many months, and is called subculturing.
• After six months or more, the original 30 cells of the inner cell mass yield millions of embryonic stem cells. Embryonic stem cells that have proliferated in cell culture for six or more months without differentiating, are pluripotent, and appear genetically normal are referred to as an embryonic stem cell line.

• Once cell lines are established, or even before that stage, batches of them can be frozen and shipped to other laboratories for further culture and experimentation.