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Lab 9: Mammalian Cell Culture Jacob Feste 010617389 4/13/15 Objective The objective of this experiment is to learn and perform mammalian cell culture techniques including cell passage, cell counting, and culture maintenance. This experiment utilizes NIH- 3T3 mammalian cells with Trypsin for a dissociation agent during cell passaging. Cell counting will be performed using a hemocytometer with Trypan blue solution for staining. It is also requires strict sterilization techniques to be followed, such as the use of ethanol and chemical hoods to reduce the chances of contamination for sensitive cells such as mammalian cells. NIH- 3T3 cells are mammalian mouse embryonic fibroblast cells from a continuous cell line. Mammalian cells typically proliferate or divide until there is no longer room for them to do so, and are considered to be at confluency. Upon the removal of cells the remaining cells will divide, again until there is no longer room. Cell passaging is the process of removing cells from a culture and into a sub culture, giving way to cell lines. For this experiment, Trypsin will be used as a dissociation agent due to its ability to dissociate the anchor the cells make with the surface to allow removal. NIH-3T3 cells are not altered or damaged by Trypsin, making it ideal to use as a dissociation agent for these cells. Cell lines utilize this property to “harvest” cells from a culture of cells with this property, allowing them to be used over and over. NIH-3T3 cells have genetically been modified to induce immortality for the cells as long as they remain uncontaminated, allowing them to infinitely be harvested over time and therefore classifying the NIH-3T3 cell line as continuous. These properties make mammalian cells such as NIH-3T3 and cells from other continuous cell lines desirable for 1
