Cell Culture Labs

Procedure

Preparation

For the following set of labs you will be provided with these solutions. Keep them refrigerated and as sterile as possible.

	Culture flasks and dishes. These sterile plastic containers have been specially treated to allow cells to adhere to them.
	Dulbecco's Modified Eagles Medium with L-Glutamine, fetal bovine serum, and an Antibiotic/Antimycotic added (DMEM++). This medium contains all the essential nutrients and growth factors necessary for the growth of many mammalian cells. The Antibiotic/Antimycotic is added as a safety measure to infections by bacteria and fungi. It is NOT a replacement for good sterile technique.
	Calcium and Magnesium Free Phosphate Buffered Saline (CMF-PBS). This is an isotonic saline solution buffered to the cell's natural pH.
	Trypsin/EDTA in CMF-PBS solution. Trypsin is a proteolytic enzyme which breaks the attachment between the cells and the growing surface of the culture flasks. This frees up the cells for subculturing. Chemicals in the DMEM++ will inactivate the trypsin if present.

Day 1 (Lab Day)

Today you will be provided with a culture flask containing human fibroblast cells which have grown to confluency and created a monolayer on the bottom of the flask. Your task is to perform a 1:2 split of the culture. That is, you will be detaching the cells from the bottom of the flask, suspending them in 10 ml of fresh growth medium, and transferring the cells into two new, sterile culture flasks. These cells will reattach and divide until they reach confluency.

See the instructions below labeled Subculturing Monolayer Cultures.

USE STERILE TECHNIQUE when performing the split.

Days 2 through 6

Eyeball your cultures each day. You will need to feed your cultures on the third or fourth day.

Day 7 (plus or minus a day)

You will need to do splits on both your culture flasks. This time you will do 1:2 splits into a flask and a culture dish for each monolayer. That is, when you are done you will have two dishes and two flasks.

Day 8 through 14

Eyeball the cultures each day. You will need to feed your cultures on day 11 or 12. Feeding dishes is essentially the same as feeding flasks.

Subculturing Monolayer Cultures (1:2 Split)

Sterilize your work environment with 70% ethanol.

Collect the supplies you need and place them adjacent to you sterile work field. For EACH flask you are going to subculture you will need:

	Two sterile culture flasks (or one culture flask and one growth plate).
	DMEM++, CMF-PBS, Trypsin/EDTA solution
	A couple of 2ml, 5ml and 10ml individually wrapped pipets with pipetors to fit.
	Waste beaker.

Carefully open the culture flask containing the monolayer and poor the medium into the waste beaker.

Pipet 5 ml of CMF-PBS into the flask, swirl it around carefully then pour it into the waste beaker. Rinse the culture well since any medium left in the flask will prevent the trypsin from working in the next step.

Pipet 1 ml of Trypsin/EDTA solution into the flask, swirl it over the culture and then set the flask flat on the counter for 10 minutes to allow the cells time to detach. Eyeball the cells before the next step to be sure the cells are detaching.

Pipet 10 ml of fresh medium into the flask. This will deactivate the trypsin. Reach in with the pipet and draw up the liquid and spray it against the monolayer to break the cells free. Repeat this several times.

Draw up all the liquid one final time and put half into each of two new culture flasks (or one flask and one dish). It is assumed half the cells will go into each of the new flasks. Put the flasks in the culture chamber. Don't forget to loosen the lids.

Feeding Cultures

Follow steps 1 through 4 in the procedure above (you won't need the two new flasks).
After rinsing pipet 5 ml of fresh medium into the flask.
Return the culture flasks to the culture chamber. Don't forget to loosen the caps.

Eyeballing your cultures

First note the appearance of the culture medium. Phenol Red (a pH indicator) is a constituent of Dulbecco's media. As acid byproducts build up the indicator will turn from red to orange then yellow. If your cultures' media has begun to turn a distinct orange it is probably past due to be fed.

The medium should be clear. A cloudy medium often indicates contamination by bacteria or fungi.

Next, look at the shape of the cells under the inverted scope. The cells should be long and thin and adhered to the bottom of the flask. Except when dividing, cells should not be loose and round. If there are a significant number of large round cells it could indicate a problem. Start by feeding the culture and check them again the next day. Individual cells should look something like:

fibroblast.jpg (10211 bytes)

Aseptic Technique

Whenever possible, work in a cell culture hood. This will prevent airborne organisms from entering your cultures.
Work areas should be wiped clean with 70% ethanol before and after use. It may help to rinse your hands with ethanol as well or to wear gloves.
Never leave sterile flasks, bottles, or petri dishes, open to the environment. Do not remove the cover until the instant you are ready to use it, and replace the cap as soon as you are finished.
Never place caps removed from bottles or flasks down on the bench surface. Instead, grasp it in the pinky finger of one hand, and keep it facing down. Once a bottle is open, keep it tilted so that airborne microorganisms will not fall directly into the medium.
Sterile pipettes should not be removed from their container until just before use. They should be kept at the sterile bench or in the culture hood only.
Do not pipette by mouth. Use a pipette bulb or automatic pipettor when working with solutions. Even though pipettes are plugged with cotton, mycoplasmas from your mouth can still squeeze through the cotton and contaminate your cultures.
Use a separate sterile pipette for each manipulation. Do not draw from a bottle more than once with the same pipette.
Do not talk, sing, chew gum, or breathe directly on your culture when performing sterile technique.
Techniques should be performed as rapidly as possible to minimize exposure to microorganisms.

Last Updated: 4/16/08