Cell Counting for Bioprocessing Applications

1. You have been tasked to split a T25 containing CHO cells into one T75 flask. To do this you have washed your cells with PBS and added 1 mL of trypsin to dislodge the cells. Then 5 mLs of F12 media was added to the flask to neutralize the trypsin. The cells were then pelted by spinning in a centrifuge. All the media was removed and the cells were re-suspended in 5 mls of fresh media. 100 ul of cells were removed and added to 100 ul of trypan blue and used for cell counting using a standard hemocytometer. The results of the live cell count are listed below. Using the results below determine the volume of cells that would be needed to make 15 mLs of a new cell suspension at 1 x 105 cells/mL for the T75 flask.
The 4 corner squares were counted on sides A and B:

Side A:� � � � � � � � � � � � � � � � � � � � � � � � � � � �Side B:
1. 238 � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1) 246
2. 261 � � � � � � � � � � � � � � � � � � � � � � � � � � � � 2) 233
3. 247 � � � � � � � � � � � � � � � � � � � � � � � � � � � � 3) 272
4. 254 � � � � � � � � � � � � � � � � � � � � � � � � � � � � 4) 249

2. You need to split a cell line being cultivated in a T-25 flask containing 5 mL of media into a T-75 flask with 15 mL of media at a final concentration of �6*10^5 cells/mL. The cells were scraped from the T-25 flask and centrifuged. The supernant was removed and replaced with 5 mL of fresh media. The cells were re-suspended and 100 ul of cells were diluted in 400 uL of PBS. The resulting 500 ul of cells was mixed with 250 ul of trypan blue and used for cell counting. 313 cells and 327 cells (Total of the 4 corners, not the average) were counted on sides A and B of the hemocytometer, respectively. What volume of cells from the T-25 flask would you need?

�

3. You would like to split CHO cells into a T-25 flask with 5 mL of media at a final concentration of 10^5 cells/ml and freeze 10^7 your CHO cells in 1 ml of cryopreservation media. To do this CHO cells were grown in a T-75 flask in 15 ml of F-12 media. Once the cells were approximately 95% confluent all the F-12 media was removed and the cells were washed with 5 ml of PBS. The PBS was removed and 10 ml of fresh media was added. The cells were then scraped and re- suspended in the fresh media. 50 ul of cell suspension was removed and added to 150 ul of PBS and mixed. 10 ul of this cell suspension was then added to 10 ul of trypan blue. Cells were loaded onto a hemocytometer and counted. Side A had 137 cells and Side B had 113 cells (Total of the 4 corners, not the average) .
What volume of cells from the T-75 flask would you need for the new T-25 flask?

4. You would like to split CHO cells into a T-25 flask with 5 mL of media at a final concentration of 10^5 cells/ml and freeze 10^7 your CHO cells in 1 ml of cryopreservation media. To do this CHO cells were grown in a T-75 flask in 15 ml of F-12 media. Once the cells were approximately 95% confluent all the F-12 media was removed and the cells were washed with 5 ml of PBS. The PBS was removed and 10 ml of fresh media was added. The cells were then scraped and re- suspended in the fresh media. 50 ul of cell suspension was removed and added to 150 ul of PBS and mixed. 10 ul of this cell suspension was then added to 10 ul of trypan blue. Cells were loaded onto a hemocytometer and counted. Side A had 137 cells and Side B had 113 cells (Total of the 4 corners, not the average).

What volume of cells from the T-75 flask would you need to cryopreserve 10^7 CHO cells?