ENVR 421 Laboratory #7: Introduction to techniques for cell culture

Transcription

1 ENVR 421 Laboratory #7: Introduction to techniques for cell culture Introduction Using human and animal cells grown in culture is an extremely useful technique for studying the biology of live human and animal viruses. Cell culture is used for studying viral biology, disease diagnostics, vaccine production, and many other applications in virology and parasitology. Cells are grown in special dishes, with media specifically formulated to meet their metabolic needs. Working with cells in culture can be challenging, as they are extremely sensitive to contamination from bacteria and fungi, which will kill them. In this lab, you will practice some basic techniques with cell culture media to ensure that you have proper aseptic technique before working with real cells. You will also look at cell cultures under the microscope to familiarize yourself with the appearance of cells in culture. Aseptic technique with cell culture media Purpose The purpose of this laboratory exercise is to familiarize you with the aseptic techniques required for working with human and animal cells in culture, as well as correct use of a biological safety cabinet (BSC). Principle Cell culture dishes To culture cells, we use specially treated dishes. You cannot use just any petri dish or 24-well tray for cell culture; cells will not adhere to the dish and form a monolayer. Dishes sold as cell culture treated are treated with chemicals so that cells will stick to the bottom of the dish and form a monolayer. We are using 24 well trays. Multiwell trays like this allow cell culture assays to be done in Most Probable Number format, similar to the way we detected fecal coliforms and E. coli in the water analysis lab, using wells instead of tubes. Cell culture media Cells in culture are grown under a layer of liquid media containing ingredients that meet their metabolic needs. Most media is made starting with a basal medium, containing glucose and salts. This is usually called Minimum Essential Medium (MEM). Other ingredients are added to the basal medium depending on the needs of particular cells. Our medium contains the following, suitable for a wide range of types of cells:

2 MEM bicarbonate (to buffer the medium and maintain a stable ph) Fetal Calf Serum (provides proteins and other constituents to support cell growth) Hepes (also a buffer) glutamine and other amino acids (to support cell protein synthesis) gentamicin/kanamycin (antibiotics to suppress bacteria) nystatin (an antifungal) The color of the media is an important indicator of cell health. MEM also contains a ph indicator. At the optimal ph for cell growth (around neutral), it is a deep red color. As cells grow, they make the media more acidic, causing a shift to an orange color. Note this color when we examine growing cells- if you are trying to grow cells in culture and this orange shift does not take place, they have likely failed to grow. If there is contamination, the media will undergo an acidic shift (bright yellow), or an alkaline shift (bright pink). Neither of these is a good thing. Although we do add antibiotics and antifungals, they will not compensate for poor aseptic technique; you must be very careful about contamination when handling cultures. Bacteria and viruses feed on the ingredients in cell culture medium, but their growth rates are much faster than human or animal cells; they will rapidly take over the culture and kill the cells. Methods Later in the semester, we will do infectivity assays for cells. To infect cells, you draw the media off of them with a vacuum source, then add fresh media. This must be done very carefully so that you do not scratch the cell layer with the tip. You will practice this to get the technique down before we use actual cells. Use of the BSC To minimize chances of contamination, all work with cells will be done inside the biological safety cabinet. Cells are vulnerable to fungal contamination, which is commonly present in air in the laboratory. This is also done in order to work safely with viruses. The BSC establishes an airflow pattern that both protects you from the materials you have inside, and protects the materials inside from outside contamination. Don t move the sash in front- it is preset at the correct level for maximum protection. The grate in the front of the BSC creates protective airflow. Don t work over this surface or place anything on it. Work as far back in the BSC as your arms can comfortably reach. Try to have all your materials ready inside to minimize reaching in and out of the BSC while working.

3 Materials: Vacuum flask connected to vacuum source Blue pipette tips 24 well tray 1 50 ml tube of cell culture medium Protocol First day: inoculation 1. Set out your materials inside the BSC 2. Spray the cell culture media tube with ethanol and let dry 3. Spray your gloves with ethanol and rub your hands together 4. Add media to the 24 well tray, 1 ml in each well 5. Using the blue pipette tip, draw the media out of each well 6. Add another 1 ml to each well 7. Place the tray in the incubator Second day: Examination of the 24 well tray for signs of contamination Examine the appearance of your media. It should be deep red in color and totally clear. If it is orange, yellow, or cloudy, you have contamination. Examine the tube of medium that has E. coli added to it for an example of what bacterial contamination looks like. Microscopic Examination of Cells in Culture Purpose Although PCR, immunofluorescent antibodies, and other techniques are used to measure growth of viruses in culture, interpretation of cell culture results is still commonly done by examination of the appearance of the cells and changes in their appearance. This takes experience and time spent becoming familiar with the way various cells look in culture normally and when infected with viruses. For this segment of the lab, you will examine cells growing in culture to familiarize yourself with their appearance. Principle When placed in a tissue-culture-treated dish or flask, cells will attach to the bottom and form a monolayer. This is a layer of cells one cell thick. Establishment of the monolayer takes a few to several days, depending on the cell type. You will examine large flasks with cells in different

4 stages of growth. A healthy monolayer will cover the bottom of the flask with no gaps. The cells may be round or elliptical. There may be some cells floating in the media, but there should not be many. Note the orange color of the medium, a sign of active cell metabolism. The cells we are observing are A549 cells, a human lung cancer cell line used for the study of respiratory adenoviruses. Materials Cell flasks inverted (dissecting) microscope Figure 1. The A549 cell line in culture

5 Figure 2. Vero cells (a type of cell line used to grow SARS and poliovirus) Figure 3. HeLa cells, a human cancer cell line used for research