MAMMALIAN CELL CULTURING

Background

Tissue or cell culturing is a very valuable research tool.  Many complex cellular processes can be studied in detail in cell culture, because the conditions can be manipulated easily and mutants can be obtained that would not be possible in intact animals.  Also, cell culture can be used for testing potential drugs or manufacturing reagents such as antibodies for use in the lab or in medicine.  In this lab, we will be growing cells over a period of days and determining their different rates of growth in various media.   

Culturing mammalian cells is a tricky process because the lab conditions must be very sterile so that bacteria and fungus do not start to grow in the culture.  Some hints for maintaining sterility:

      Work slowly and plan ahead!  Proper sterile technique requires precision and care.

      Wear gloves when handling cells or tissue culture equipment, and spray your gloves frequently with 70% EtOH.  Do not do bacteria work before cell culture on the same day.

      Start by turning on the hood and cleaning all of your equipment and surfaces with 70% EtOH.  Do not lean your head into the hood at any time after it is turned on.

      Gather all the supplies and equipment you will need: media, pipets, cell scraper, tubes, flasks etc.  Spray around the cap of all bottles with 70% EtOH. 

      Spray your gloves again if needed and retrieve your cells from the incubator by first tightening the cap completely, then moving the flask gently without getting media near the mouth.  Check the incubator to ensure that it has water in the bottom basin, and that nothing unauthorized is growing in it.  Also notice if the shelves appear dirty or have liquid spilled on them, and clean it up if needed.

      When transferring media or solutions, first loosen the caps of any containers you will be opening, then obtain a pipet and open it from the back end (the end that goes into the pipettor).  Place it into the pipettor with the plastic sheath still on, then carefully remove the sheath without touching the inside of the sheath.  NEVER touch the pipet with your gloves or the outside of any equipment, even if the equipment has been treated with EtOH.  Insert the pipet into containers without touching the outside or the lip of the container.  The same protocol should be followed for opening cell scrapers or any other instrument.

      Containers should be closed unless they are in immediate use, and be careful not to pass your arm or hand over the top of a container or flask, even if there is currently nothing in the flask.  If you must set a cap down, place it face up in the back of the hood so you will not pass your hand over it. 

      When placing cells back into the incubator, loosen the cap so that gas exchange may occur. 

      Clean up after yourself in the hood: wipe up any spills, spray with 70%EtOH, and put away equipment the next person will not need.  Trash should be placed in the waste container, and unused bags of flasks or pipets taped closed for the next person. 

Cells are maintained in media that is designed to replicate the conditions the cells would be in if they were in the body.  Media differs depending on the organism the cell is derived from, the developmental stage of the cell, and the specific requirements of that cell type.  In general, media contains salts so as to be isotonic, buffers to help maintain correct pH, amino acids as building blocks for cell growth, and carbohydrates such as glucose to provide energy.  The media that we will use contains antibiotics (penicillin and streptomycin) to help combat possible contaminants.  Most cells also require fetal bovine serum (FBS) to be added to the media, as a source of growth hormones and essential nutrients.  You will notice that the media is a reddish-pink color.  The color is due to an indicator (phenol red) which will begin to turn orange and then yellow if pH becomes too acidic.  This happens when too much carbonic acid from CO2 builds up, and is generally an indicator that the cells have gotten too dense.  The media will turn more pink if the cells are subjected to high oxygen content, raising the pHfor instance, if your cells are left on the bench or if there is a problem with the CO2 incubator.

            We will be working with HEK293 cells, which are a cell line (meaning they grow continuously in culture) derived from human embryonic kidney.  They are adherent (stick to the plate) and have an irregular shape with several protrusions. 

Procedure

DAY 1

1.  Practice transferring liquid using sterile technique.  At the stations provided, transfer 2 ml of the colored liquid from the tube to the flask, using the procedure described above.  Then close the flask and place it properly on its side and check that it is on the correct side, with the neck tilting up.  Your lab partner should watch you carefully to determine any possible source of contamination.

2.  Use the hemocytometers and microscope to determine the concentration of the sample culture, using the worksheet provided.

DAY 2

1.  Label your 3 sterile 25ml tissue culture flasks on the top and in the corner with your group initials, the cell type, the date and the type of media.

2.  Using sterile technique, transfer 5mls of culture media to each of your flasks. 

3.  Now transfer 0.25 ml of the starting stock cell culture to three of your flasks.  What dilution is this?  Label the flasks with this dilution.

4.  Put your flasks in the incubator, loosening the caps to allow air flow.  Be sure to close both doors of the incubator!

5.  Now you will count the cells from the stock culture to know how many cells you are starting with.

DAY 3-5

1.  Observe all of your flasks with the inverted microscope.

2.  Take one of your flasks and scrape the bottom of the flask with a cell scraper to loosen the cells.  You will do this with a different flask each day.

3.  Mix the media many times by pipetting up and down.  Try to avoid introducing bubbles into the media.  Remove 1ml of media, in a sterile fashion, and put the media into a non-sterile tube so that you can count the cells. 

4.  Make a count of your cells.  Keep a careful record of how many cells per ml you count each day.  Share the count with the group.

5.  Make a chart of the cell counts that each group in the class got for each day.

6.  Make a graph using semi-log paper to show the average growth rates obtained by the class.  Also show your own data as a separate line.