BASIC CELL CULTURE - III

I. INTRODUCTION
The purpose of this laboratory exercise, as was the previous one, is to acquaint you with some of the fundamentals of culturing cells in vitro . In this exercise each "lab" will be given a different cell culture. You will replicate the previous laboratory exercise only this time, using your own cell culture. 

 In this exercise you will begin to familiarize yourself with your particular cell culture. You will:

 
1. Observe the cell culture provided;
2. transfer your culture and set up your own stock cultures for subsequent exercises;
3. count cells to determine how many cells were in the original flask, in order to properly dilute the cell suspension and seed new stock cultures; and
4. freeze your cells in liquid nitrogen for storage/safe keeping.

II. MATERIALS

 
1. Cultures of HeLa, Early passage RL-PR-C, Late passage RL-PR-C and SCOR 1a cells. (Each "lab" will have, and must maintain on a regular basis, one of these cultures.)
2. Culture medium:DME₉₀ ABS₁₀, (for HeLa and SCOR1a) or F-12₉₀FBS₁₀ (for Early and Late RL-PR-C).
3. Pipettes - 1 ml, 5 ml, and 10 ml.
4. T-25 culture flasks - three per student.
5. Hemocytometer.
6. Hand tally counter.
7. Medium disposal flask.
8. Trypsin (0.05%) - Versene (EDTA) (0.02%) solution.
9. Non-sterile test tubes for cell counting.
10. Pasteur pipettes.
11. Centrifuge tubes (15.0 ml).
12. Calcium and magnesium free phosphate buffered saline (CMF-PBS).
13. Versene (EDTA) solution.
14. Microscope.
15. Test tube racks.
16. Trypan blue.

 

 

 III. PROCEDURE
Trypsinize your cells:

1. Each "lab" will have one of the cell cultures. Examine its macroscopic appearance and observe the culture microscopically to determine cellular morphology, appearance of the culture (whether or not cells look "healthy" or are granular, sloughing, etc.) and whether or not confluency has been reached. Record your observations.
2. Thaw and warm the trypsin-Versene, and medium solutions to 37C.
3. Aseptically remove the spent culture medium as completely as possible into a sterile beaker.
4. Add 5.0 ml of warm CMF-PBS to the side of the flask opposite the cells and gently tilt flask to wash the monolayer and adjacent sides of the flask, (WHY?) taking care not to splash the solution into the neck of the flask.
5. Remove the CMF-PBS into the aspirator bottle..
6. Add 3.0ml (for a T-25 flask, 5.0ml for a T-75 flask) trypsin/versene solution to the side of the flask opposite the cells then rotate to bring the entire monolayer in contact with the solution.
7. Remove most of the trypsin-versene (that is, when using aspirator, you do not have to be quantitative and remove every drop.), bring the residual solution in contact with the monolay and then place the cultures in the incubator until the monolayer detaches. (It is helpful to observe the cells frequently during this stage because you will then have the opportunity to observe how the cells come off the plastic.) You can tell when this stage is reached by holding the flask up to the light and observing that the monolayer has become opaque. By bringing the residual solution up and over the disintegrating monolayer and shaking or gently hitting the flask against your hand, you can see the monolayer break up and flow down the plastic.
8. When all of the cells have detached, add 5.0 ml of growth medium (for a T-25 flask; 10.0 ml to a T-75 flask) to the flask (this time to the very side on which the cells were attached) so as to wash the cells down to the bottom of the flask. With the same pipette, draw up the cell suspension and expel the solution back against the side or bottom of the flask. Try not to discharge the suspension into the liquid in the flask or foaming will occur. The pulling up of the solution and then blowing it back into the flask is known as trituration. Continue this trituration process 5-7 times in order to obtain a well dispersed cell suspension. Be sure the cells are well dispersed at this point by examining under the microscope and looking to see if the cells are separate or in clumps. If a significant number of cell clumps or aggregates are present, resume trituration and reexamine.
9. Place 0.5 ml trypan blue into a small nonsterile tube. Then place 0.1 ml of cell suspension into it. (What level of dilution of the cells does this effect?) Triturate with a Pasteur pipette and charge one side (one chamber) of a hemocytometer. Triturate once again and charge the other chamber of the hemocytometer. Count the cells in both chambers. The total number of cells in both chambers x 6 (to account for the six-fold dilution in trypan blue) x 1000 (hemocytometer factor used when counting both chambers [x 2000 if counting only one chamber]) = no. of cells/ml of the cell suspension in the flask. Multiplying the number of cells/ml of the cell suspension by the number of mls of the cell suspension = the number of cells in the monolayer of the bottle trypsinized for this count If the number of cells exceeds 2-5 x 105 cells/ml (20-50 cells per square of the hemocytometer) a further dilution of the cell suspension in CMF-PBS should be made because counting larger numbers of cells than this tends to be inaccurate. Thus, if you routinely dilute six-fold in the trypan blue and count one chamber of the hemocytometer, you would multiply the number of cells in the five squares x 12,000 to get the number of cells/ml of the cell suspension.
10. Seed three T-25 flasks as follows:

11. Incubate the cultures in the CO₂ incubator. 

12. Observe and count the cells in one bottle after 24 hrs., 48 hrs., and 72 hrs. Record the following at each interval:

                 (a.) Appearance of the cells (presence of floating cells, morphology of cells, intracellular granulation).
                 (b.) Estimate the percentage of the growing surface occupied by the cells.
                 (c.) The number of cells/ml AND per bottle for each day.
                 (d.) For the cell line you cultured, what level would you seed if you needed the cells at half monolayer and wanted to use the cells in two                                days? In one week?

13. Maintain a culture of your cells by passaging cells from one of the three flasks you have been observing.

FROM HERE ON, IT IS YOUR LABS RESPONSIBILITY TO MAINTAIN YOUR CULTURE STOCK; PASSAGING, WHEN NECESSARY, AND PLANTING SUFFICIENT CELLS TO HAVE SUFFICIENT NUMBERS READY FOR SUBSEQUENT EXERCISES. YOU WILL NOT SIMPLY PLANT ONE LARGE BOTTLE AT EACH PASSAGE FOR MAINTENANCE AS THIS IS WASTEFUL. HOWEVER, IF YOU DO USE ONE YOU MUST BE READY TO JUSTIFY WHY YOU DID SO.

GO TO THE SECTION ON FREEZING AND UNFREEZING CELLS.