TISSUE CULTURE PURPOSE

The main aim of this experiment is to generate Hela cell culture and investigate cells that is reproduced in culture.

INTRODUCTION
Cell culture is a process that cells grow and mature in specific conditions in vitro. This technique includes removing cells from living organisms.1 After, definite conditions should be prepared. Tissue cultures are classified by the origin of the cells: Primary culture, extended culture - cell strain, established (transformed) cell lines.2 Primary culture is the lift of cells directly from an animal or plant tissue. It can be composed of some different cell types. Cell lines are permanently established cell culture that will proliferate indefinitely given appropriate media and space. The difference between the primary cells and the cell lines is passaging. Primary cells are with no passage but cell lines have at least one passage. The other cell type is immortalized cell. Immortalized cell lines are readily available and can be expanded without limitation.3 They continue to grow and divide for a very long time. Hela cells are example for this group. However, cell lines may differ from the in vivo situation in important aspects. Some conditions should be prepared to live and grow cells: Media: Media is a liquid that is support to cell for living and growing. It includes aminoacids, glucose, pH indicator, salts, vitamins etc. and some chemicals like FBS. FBS provide cells growth signal. Laminar Flow Hood :

Laminar flow hood is a piece of equipment which makes sterile working procedures. That is
full carefully enclosed bench. CO2 Incubator: Specially designed for growing cell and tissue cultures in the most stable and safe conditions possible. Plastic plates: Sterile and specially treated plates that are used to provide suitable conditions for cells. Chemicals: Trypsin: Trypsin is a chemical, serine protease, found in the digestive system of many vertebrates. That is used to cleave protein bonds between cultured cells and dish. PBS(Phosphate buffered saline): PBS is a buffer solution that is used to wash. It doesnt damage cells. DMEM(10% FBS) -1-

MATERIALS AND METHODS

1. 2. 3. 4. 5. 6. 7. PBS Penicilin-Strep Trypsin Pipettes Falcon tubes Petri dish SH-SYSY cells Plates

Media is prepared including % FBS + 1%Pen/Strep + Basal media. Old media is removed from petri dish. The plate is washed with 2ml PBS. 0.5 ml Trypsin is added to plate and waited 5 minutes for breaking bonding. 9 ml DMEM is added. 1:5 is splitted to new plate. The left suspension is collected to centrifuge.

RESULT
We prepared cell culture shown in Figure 2.

Figure 1: SH-SYSY cells in old media

DISCUSSION
First of all, we learned how make cell culture and what are the suitable conditions for cell culture. In the beginning of the experiment, we take Hela cells in old media. Three days ago, media was old because of carbondioxide. So, we should had to remove the old media and prepare a new media. We were careful to remove media about not take cells with pipette, therefore we dont touch bottom of petri dish with pipette. New media is prepared according to determined situations. Media doesnt include growth factor; so we added FBS to give growth signal. We sterilized with Penisilin/Strep to escape contamination. Some bacteria has resistance to this chemical matter, thereby, we shoud be careful so much. Confluence is crucial in this part. If we dont dilute and take another place, cells may grow in new media but every cell cannot live, so we had selected some of them like nature-selection. In this condition, live cells have more resistant. -2-

Secondly, we washed cells with PBS. PBS is a buffer solution and doesnt damage cells. Cells adhere surface of petri dish with proteins. We used a protease Trypsin to break this bonds. We waited approximately 5 minutes in room temperature. If the temperature was 37C we would wait less than 5 minutes. But Trypsin begins to damage and break cells essence bonds more than 5 minutes in room temperature. We added media again to neutralize effect of Trypsin. Because media includes FBS. While cells remove from bottom of petri dish, sticks each other. To prevent adhering, we shake and did pipetting. Otherwise, they reproduce over and over on each other. Lastly, we split ratio of 1:5 of all. 1 ml of all is added to plate and 4 ml media is added to new plate. We drew figure 8 to distribute to every region equally. Suspension that left is collected to centrifuge. As a result, we learned how to make media and cell culture. It is very sensitive work, hence it must be realized in hood and be careful.

REFERENCES
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http://caat.jhsph.edu/pubsthe The University of Texas at Arlington http://www.uta.edu/biology/wilk/classnotes/tissue_culture/Primary%20culture.pdf

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Published, MCP Papers in Press, October 23, 2008, DOI 10.1074/mcp.M800258-MCP200

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