Cell Culture

Cell Culture

Summary

Cell culture refers to the process of continuously growing cells outside for of their normal environment. For our lab, we are currently growing two lines of melanoma cells (LM45 and LM45-CD47) in vitro. Cell culture requires the use of various specific conditions techniques, materials, and reagents to do properly. All cell lines are stored and handled in sterile conditions, specifically in a cell incubator devoted to storing only cell cultures. To maintain cell health, each dish of cells must be passaged regularly (~once a week), and more information about passaging can be found here. In addition to passaging cells regularly, our cultured cells are used for transfections, which refers to the introduction of nucleic acids into eukaryotic cells. More information about transfections can be read here.

Outside of passaging and transfections, additional protocols to cell culture involve deep freezing cells for use later, thawing the deep frozen cells, and observing cells under fluorescence. Supplemental information related to those techniques can be seen via the embedded links, with links to the protocols of each within their informational pages. Although most of what we do in our lab with cells pertains to passaging or transfections, it is important to understand both the purpose and how to do all cell culture protocols.

Other protocols pertaining to Cell Culture can be read here.

In our Lab

(talk about of cell culture, current goals, future goals)

Current Goals

Currently, our lab is practicing passaging and transfecting Melanoma cells provided by Dr. Baer's lab down the hall. We are growing Melanaoma cells in tissue culture petri dishes, and when the petri dish cultures are close to being 100% confluent (i.e. the cells have spread over 100% of the petri dish's surface) we essentially remove a small number of the cells and transfer them to a new dish in order to start the culture process over. This is called passaging. Transfecting is the process of introducing foreign DNA/nucleic acids into eukaryotic cells. We undergo transfection in our chosen eukaryotic cells through a reagent known as Lipofectamine. Lipofectamine allows our DNA of interest to be introduced into the target cell by forming a liposome, which will contain the DNA (to form a lipoplex), which can traverse across the cell membrane and be endocytosed (more information here). It is through this process that we introduce the CAS-9 DNA into the cells of interest.

Our current goals include practicing and perfecting our passaging, transfecting, and sterile technique methods in order for us to move from working with Dr. Baer's Melanoma cells to Calu-3 Cells (a line of lung epithelial caner cells) that Dr. Norimatsu will acquire from a colleague in Columbia. See the CRISPR Project To-Do List or the Project Progress Page for the current state of the project's timeline and what needs to be done.

Future Goals

Ideally, we will be using Calu-3 cells along with our improved sterile technique and passaging/transfection methods to utilize prime editing techniques to modify the CFTR gene.

Specific steps for how to undergo cell culture can be seen on the cell culture project protocols page.