There are occasions where one would like to be able to grow cells under serum-free conditions. One such occasion would be where the serum component of the medium interferes, in some way, with the action of some biologically active agent whose effect on cells one wishes to study. In this situation, one would have to have previously adapted an existing, serum grown culture to grow under conditions where the serum has been removed. To see how this might be done, you will examine your own "lab’s" cell culture with respect to serum dependence:  i.e. the saturation density obtained by  growing the cells in media containing 5% and 0.5% fetal bovine serum, and serum-free media supplemented with growth factors. (Question: Why is the serum-free medium supplemented with growth factors?)

Growth and Characterization of Serum Dependence

II. MATERIALS

You will need to have two T75 flasks of cells which were planted three days earlier with 2 x 10⁶ cells in 15ml medium.

CELL LINES:      Laboratory #1 - SCOR 1A
                          Laboratory #2 - RL-PR-C (early passage)
                          Laboratory #3 - RL-PR-C (late passage)
                          Laboratory #4 - HeLa or KB

MEDIA:       Medium 1 - DME-F12 +2.5% FBS
                   Medium 2 - DME-F12 + 0.5% FBS
                   Medium 3 - DME-F12 + Growth Factors (insulin-5m g/ml; transferrin-5mg/ml;  selenous acid-4ng/ml)

Trypsin Inhibitor (Stock solution = 1.0 mg/ml)
Versene
Trypsin/Versene
CMF-PBS
P60 Petri dishes
T25 flasks
Haemocytometer
Hand tally counter
Giemsa stain
 

III. PROCEDURE

A. Your cultures will have been grown in T75 flasks.

B. Trypsinize cells, resuspend in  CMF-PBS + 1.0ml Trypsin Inhibitor (TI) and count .  Why is TI used?
      Adjust to 5 x 10 ⁶ cells in 5.0 ml of CMF-PBS + 1.0ml Trypsin Inhibitor (TI). (= 1 x 10 ⁶ cells/ml)

1. Plant 9-T25 flasks with 4.5ml of medium (three T25 with medium 1; three with medium 2 and three with medium 3) + 0.5ml of cell suspension. (What is the dilution factor of the cells? What cell number has been planted in each T-25 flask?) Be certain of an even distribution of the cells and then incubate.
2. At the same time, plant 6 P60 Petri dishes with 4.5 ml of medium (2 P60's with medium 1, 2 with medium 2 and 2 with medium 3) + 0.5ml of a 1:1000 dilution of the original cell suspension made in the CMF-PBS-TI buffer. (What cell number was planted in the P-60?) Swirl gently to distribute cells evenly and then incubate.

Day 0:   Set up experiment and record the time you completed the setup.

Day 2 (48hrs.):  At the same time as you finished on Day 0, observe, record your observation and possibly* count one T25 flask of each of the three different media.  (* If the cells on the plastic appear to be only a very few (1-3) per field, record this observation but it is not necessary to count the cells in the flask. There would be far too few to be able to count. Reincubate the flask however and observe on subsequent days along with the others.)

Day 4 (96hrs.):  Same as on Day 2 for a second T25.

Day 6 (144hrs.): Same as on Day 4 for a third T25.

Day 7:  Remove the medium from all the Petri Plates, wash twice with 3ml CMF-PBS, fix twice with 3ml methanol (add the methanol and let stand for approximately 1 minute.  After removing the methanol a second time,  air dry the plate. Add 3ml Giemsa stain and stain for 20 minutes. Wash with water, dry and count the colonies.
 

III. RESULTS AND DISCUSSION

Analysis

1. Calculate cell number per flask, per square centimeter of growing surface and per ml of culture medium. Check distribution of cells for uniform growth over surface of flask.
2. Plot cell concentration on a log scale, against time on a linear scale.
3. Determine the lag time, population doubling time, and plateau (saturation density)
4. Based upon the number of cells planted in the Petri dishes and the number of colonies counted, calculate the plating efficiency of the cells in the various media. What can you conclude?