This document summarizes an experiment testing the effects of methotrexate on t-PA production in CHO cells over 10 days. Two flasks of CHO cells were cultured, one with 1-3x10-7M methotrexate and one without. Cell viability, growth, metabolism, and t-PA production were measured and compared between the two conditions. The results showed that cells without methotrexate had higher viability and t-PA production. Some complications with the experimental design are noted that could impact the conclusions.

1. t-PA Assay
Experiment
Chi Lo
May 3, 2007
Independent Study
Kompala Lab

2. Experiment Description
v Sacrifice two 25 cm2 T-flasks for 10 days
(plus Day 0 – seed culture)
v 1 - 3 x 10-7 M methotrexate
v 1 without methotrexate
v Procedure:
v Remove cells + supernatant from flask, Trypsin
for attached cells
v Centrifuge for 7 minutes at 1200 rpm
v Remove supernatant samples for assay and
measurements
v Mix cells with PBS, Trypsin to break up clumps

3. Methods
Cell counting Hemocytometer
(the slide) with
Trypan Blue dye
exclusion
Glucose and
glutamine
measurements
YSI 2700 Select
Biochemistry
Analyzer
Ammonia
measurements
Fisher Scientific
Accumet Basic
t-Pa Assay Perkin Elmer HTS
7000 Bio Assay
Reader

4. Cell Viability
Viability
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 50 100 150 200 250
Experiment Hours
Viabbility(%)
With Methotrexate Without Methotrexate
Viable Cells
0
500000
1000000
1500000
2000000
2500000
0 50 100 150 200 250
Experiment Hour
NumberofCells
With Methotrexate Without Methotrexate
Total Number of Cells
0
500000
1000000
1500000
2000000
2500000
3000000
0 50 100 150 200 250
Experiment Hour
NumberofCells
With Methotrexate Without Methotrexate
Cell Death
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
0 50 100 150 200 250
Experiment Hour
NumberofCells
With Methotrexate Without Methotrexate

5. Glucose/Glutamine/Ammonia
Glucose
0
1
2
3
4
5
6
7
8
9
10
0 50 100 150 200 250
Experiment Hour
Concentration(g/L)
With Methotrexate Without Methotrexate
Glutamine
0
1
2
3
4
5
6
7
8
9
10
0 50 100 150 200 250
Experiment Hour
Concentration(mmol/L)
With Methotrexate Without Methotrexate
Ammonia Concentration
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
0 50 100 150 200 250
Experiment Hour
AmmoniaConcentration(M)
With Methotrexate Without Methotrexate

6. t-PA Assay (With 3 x 10-7 M
Methotrexate)
Day 0
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 1
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 2
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 3
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 4
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 5
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 6
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 7
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 8
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 9
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 10
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Blank
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)

7. t-PA Assay (With 3 x 10-7 M
Methotrexate)
Day 0 y = 2E-05x + 0.0799
R2
= 0.9989
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 1 y = 7E-05x + 0.1022
R2
= 0.9907
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 2 y = 0.0002x + 0.1432
R2
= 0.9909
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 3 y = 0.0004x + 0.2414
R2
= 0.991
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 4 y = 0.0007x + 0.4106
R2
= 0.9911
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 5 y = 0.001x + 0.7119
R2
= 0.997
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 6 y = 0.0007x + 0.7646
R2
= 0.9877
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 7 y = 0.0013x + 0.8638
R2
= 0.9971
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500
Time (Sec)
Absorbancy(405nm)
Day 8 y = 0.0012x + 0.8299
R2
= 0.9925
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 9 y = 0.0015x + 1.0187
R2
= 0.9986
0
1
2
3
4
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 10
y = 0.0015x + 1.2241
R2
= 0.9963
0
1
2
3
4
5
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)

8. t-PA Assay (Without Methotrexate)
Day 0
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 1
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 2
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 3
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 4
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 5
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 6
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 7
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 8
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 9
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Day 10
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)
Blank
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500 2000
Time (Sec)
Absorbancy(405nm)

9. Results
v More t-PA production with CHO cells without
Methotrexate
v Without methotrexate - more viable cells
t-PA Chromozyme Activity
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
0.0012
0.0014
0.0016
0.0018
0.0020
0 50 100 150 200 250
Experiment Hour
Slope
With Methotrexate Without Methotrexate
Viable Cells
0
500000
1000000
1500000
2000000
2500000
0 50 100 150 200 250
Experiment Hour
NumberofCells
With Methotrexate Without Methotrexate

10. Possible Complications
v Flasks are separated from one another
v Not a true indicator of progression of a single
culture
v Cell counting - may not have taken
accurate sample
v Glucose membrane may need
replacement
v Need more replicates

11. Thank You