I am having a bit of difficulty understanding the cell seeding process and concepts, so as a result, I am having difficulty understanding how many flasks of cells I would need to passage each week for our assays requiring cell seeding/well plating.
I normally passage one 75-cm^2 culture flask with a total volume of 25 mL of media and 0.5 mL of ST cells. When I passage the cells, I seed wells at the same time. So here is my cell passaging process:
- Make the required amount of media
- 5 mL of PBS into the culture flask to be passaged; then dump
- 5 mL of 1x Trypsin wash into the culture flask to be passaged; then dump
- Another 5 mL of 1x Trypsin, then put the culture flask in an incubator for 4 minutes to detach
- Pipette the detached cells into 20 mL of media to then centrifuge
- The supernatant is tossed out and then 10 mL of new media is added to resuspend the cells.
I then count the cells from that 10 mL of resuspended cells. Using a formula to estimate how much volume of total concentrated cells I would need to seed in my wells, I get a total of ~41 mL of concentrated cells needed for cell seeding, does this mean I will need 4 flasks of cells each week since I get 10 mL of resuspended cells from each flask (gets me a total of around 40 mL of concentrated cells)?
I would really like to know if I am understanding this correctly or if I am gravely mistaken before I use up more media than we are already using for cell passaging. Thank you very much!
Hello
The growth rate differ for every cell line. You need to find how long does it take for ST cells to become 80-90% confluent in T-75 flask. You also need to know how many cells you can obtain from one T-75 flask on attaining confluency by taking the cell count using hemocytometer.
Once you know the number of days taken to reach 80-90% confluency in T-75 flask, and the number of cells obtained from a T-75 flask on reaching confluency, you can seed that many T-75 flasks to obtain the required number of cells for your assay.
For instance, ST cells become confluent after 4days and the number of cells obtained from one T-75 flask is 5 million. If I need 30 million cells for my assay, I will seed 7 flasks (one flask extra) to obtain 35 million cells. On the 4th day I am already aware that my flasks will become confluent. So I will prepare for the assay on the 4th day. I will trypsinize the cells from the flasks and plate them in wells for the assay.
Information on trypsinization.
When you seed cells in T-75 flask, you need to add 10-12ml of complete culture medium and not 25ml. The next day you need to replace the old medium with fresh medium. Do not keep old medium in the flask. Replace the entire old medium. Feed the cells with fresh medium every two days.
When cells are 80-90% confluent, remove the culture medium and give a wash with either PBS or plain medium without FBS. Then rinse with 3-4ml trypsin and discard. Add the same amount of trypsin and incubate for 5mins at 37 degree celsius. After 5mins add 10ml complete medium and tap the flask at the sides if cells are still attached to the substratum. Pipette the cell suspension in a centrifuge tube and centrifuge at
2000 rpm for 5 minutes to obtain a cell pellet.
If you have many T-75 flasks to trypsinize you can use two or more 50ml centrifuge tubes. After centrifugation discard supernatant and tap cell pellet. Add 1ml complete medium in each 50ml centrifuge, and pool the cell suspensions in one 50ml centrifuge tube. Again centrifuge the pooled 50ml centrifuge tube. Discard supernatant and tap cell pellet. To the pooled 50ml tube add just 1ml complete medium. Take the cell count using haemocytometer. You should take 10ul cell suspension and 10ul trypan blue for cell count with dilution factor 2. Do not take 100ul of each.
The cell count you obtain say X will be X/ml. Let us say you have trypsinized 7 T-75 flasks and obtained X/ml cells which is the total cell count, and this count should be higher than what you actually need for plating in the well format for your assay. Then you can calculate how much volume of cell suspension you will require from the 1ml cell suspension which you have kept ready above. Use the formula
N1 x V1 = N2 x V2
Where
N1 is the total cell number which is X
V1 is the volume needed to be taken from N1 (say a).
N2 is the cell number you require for your assay in each well which is known.
V2 is the volume in each well which is 1ml for 24-well plate.
Therefore
a= N2 X V2/N1
Now 'a' is the volume of cell suspension for one well. So depending upon the number of wells required you can multiple the value 'a'. Say you need 200 wells then the volume of cell suspension needed will be
'a' x 200.
Always consider two wells extra because the last well will not have sufficient amount of cell suspension because of pipetting error.
I hope this information is helpful.
Good Luck.
DON'T USE NUMBERS they are mostly useless!
- You don't need to centrifuge for every passage, culture medium also eliminates the harmful effects of the trypsin.
- Do not rely on the exact number which the companies gave, numbers are not reliable for the culture conditions,
- Rely on your observations by microscope (observe the confluency) and colour changes of the culture medium.
- Once it begins the return to yellow, change the medium, you can also decide how many cells you should plant to your flask by using the only volume without counting the cells. For instance, I used 1 mm of cells (no need to count) inside the 4-5 mm of medium (T25 flask) is enough, with breast cancer cell lines.
- I used this method with a minimum loss of material and good culture results. Counting cells are just a waste of time for the routine passaging.
- You can use the same flask you don't have to change the flask for each passage.
- Each cell type has a unique doubling time, this will affect the passage frequency in a week. For instance, MCF-7 doubling time 30-35 hours. Two times passaging (in T25 flask) is enough in a week in this case.
- Just use the doubling time information to decide the passage number in a week.
Why they are useless because the cells are changing their growth profile in each passage. Most of the researchers obtain the cells from another lab, instead of buying and you can't guess how many times passaged. Numbers and calculations are just deceiving yourself.
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