Stem Cell Culture

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Stem Cell Culture

Human embryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo. To culture these stem cells under controlled environments, one first must have a growth medium. This growth medium is made with DMEM/F12, which is cell culture medium, a 20 percent Knockout Serum Replacement, 100 μM β-Mercaptoethanol, 1 percent non-essential Amino Acid, and a Basic Fibroblast Growth Factor. This medium is then changed out everyday. This is done threw aspiration of the growth medium. One then can add 2mL of fresh medium per one well of a six well culture plate. Then you can passage the cells every 5 days. Cell passaging or splitting is a technique that enables an individual to keep cells alive and growing under cultured conditions for extended periods of time. In order to do this, you must remove the culture medium and add ½ mL of collagenase IV to each well of the 6 well plates. Then, the cells can incubate for 2-4 minutes. You then gently aspirate the collagenase and gently wash the cells with PBS. You then remove the PBS from the well and add 1mL of fresh culture medium to each well. Then pipette the medium up and down in well to detach colonies completely from the surface. You also manually scrape the tissue culture plate with the pipette tip. Finally, you take the prepared culture plate with feeder cells and split the human embryonic stem cells at a 1:6 ratio. There is also a Feeder-Free human embryonic stem cell culture. The cell plates are coated with Geltrex. These cells are passaged the same way as mentioned above and then placed into the Geltrex-Coated plates. The growth medium is MEF-conditioned medium, and it is supplemented with 1μL/mL medium of bFGF. The medium is then changed daily. He next types of stems cells one can culture are Human Mesenchymal Stem Cells. Human Mesenchymal Stem Cells are multipotent stromal cells that can differentiate into a variety of cell types.

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