E14 Stem Cell Project
January 30, 2020
Cell culture is the process of removing cells from their natural conditions, then growing and manipulating them in a controlled environment. Cell culture offers a model of cellular and molecular systems where the normal physiology and biochemistry of cells can be studied, along with the effects of drugs, toxic compounds, and other conditions. The biomanufacturing industry also uses cell culture to produce biological compounds such as vaccines, therapeutic proteins, and transplantation using stem cells. Cell clones make the results of cell culture consistent and reproducible.
This internship project was part of the preparation for the Cell Culture Techniques course (BITC-2431). In Cell Culture Techniques, the laboratory emphasizes the principles and practices of initiation, cultivation, maintenance, and preservation of mammalian cell lines. Students maintain aseptic conditions to prevent contamination of their cells throughout the semester, and design and complete an independent research project. One of the research topics offered is working with embryonic stem cells; undefined cells that can multiply and differentiate into different cell types.
The mouse embryonic stem cells (E14) used in this project were given to the Biotechnology Department by Golnar Afshar, PhD. Frozen cells were thawed and plated with media that contained LIF (a cell signaling factor that inhibits stem cell differentiation). The LIF kept the stem cells in a pluripotent state and the treated flask surface encouraged cells to grow attached to the plastic. Enough cells were grown to preserve 19 vials in liquid nitrogen, which will be available for student use in the course.
These stem cells may be allowed to differentiate into cardiomyocytes (heart muscle cells) or neurons (nerve cells). To begin the differentiation process, the cells were plated in a flask treated with gelatin (Figure 1) to encourage semi-attachment. After they had grown to confluence, the cells were moved to non-treated plate where cells could grow in suspension in the media. This media did not contain the LIF (which was preventing differentiation in the previous cultures). Retinoic acid, which encourages the development of neurons, was added to the neuron plate. After the embryoid bodies formed by about day 5 (Figure 2), the cells were plated back onto gelatin in a 24 well plate. On day 18 of the experiment, the cardiomyocytes began beating (Figure 3, also see video). On day 19, neuro cells were observed (Figure 4).
Timelapse of the Experiment