Hello,
I always learn a lot thanks to researchgate members.
This time I wonder about the cell passage.
I often split HUVECs and add passage to splitted HUVECs whenever my splittings are over.
But I am concerned about what I thought as splitted HUVECs of same passage are actually not the same passage by variable factors.
Example : I splitted HUVECs of passage 3 so I consider the seeded HUVECs in new culture tools as passage4 but I wonder whether some of splitted cells can display characteristics of cells in Passage other than passage4 by unnoticed variables. so I attached file of my method of cell split.
I want you to evaluate my method in the file and give comments what you think about my procedure in cell split.
Thank you!
Hi Lee - unfortunately I cannot open your protocol as my computer does not recognise the file type.
However I can answer your question in a general manner and perhaps give you some tips.
"Passage" is not a very well defined term and "passage 5" cells from one person may be very different to "passage 5" cells from another person.
The reason for this is that the term "passage" does not take into account
a) the seeding density of the cells
b) the harvest number/density of the cells (at time of splitting)
c) the type of flask the cells are grown in.
For example: I put 10 000 passage 4 (p4) cells in a T25 and grow to 80% confluent and I also put 10 000 p4 cells in a T175 and grow to 80% confluence. Obviously the cells have doubled more times in the T175 (as this is bigger flask) than in the T25.
So despite both cells now being p5 when I harvest the cells from the T175 they are biologically "older"
Also from the above example the initial seeding density of the cells in the T175 is much lower than that in the T25 (57 cells/cm2 versus 400 cells/cm2) - a low seeding density can cause the emergence of "sub-populations" of more rapidly growing cells.
Rather than growing the cells using 1:3 or 1:4 "splits" it is better to use a fixed seeding density for expansion (i.e. transfer 1x10e6 cells into a T175 at each passage ~ 5700 cells/cm2). Also count the cell numbers when the cells are harvested.
This allows you to calculate the Population Doubling (PD) of the cells in each passage
PD = (log harvest number/log seeding number)/log2.
If you maintain a record of the PD of the cells during expansion at each passage you can add them together at the time of an experiment to give the cells cumulative Population Doubling (cPD) which is a much more accurate way of calculating the "age" of the cells (it also allows you to calculate the doubling time of the cells at each passage).
We have found that the later passages of some cell types (such as CaCo2) perform very differently in experiments (barrier function in the case of CaCo2 cells) than cells from earlier passages - so yes, depending upon the cPD cells from the same "passage" can be very different to each other!
When you get a new cell line in the lab I recommend you generate a "master cell bank" of 2-4 vials of early passage then expand them a bit more to generate a "working cell bank" of vials of sufficient number for your set of experiments.
If you use all of the working bank up another working bank of the same passage/cPD (+/- 1 PD) can generate for more experiments from the master bank vials. This will give a better comparability between your experiments and reduce experimental variability.
Finally I would recommend that you perform your experiments on cells at the same density in whatever format (24 well, 6 well etc.).
The density you perform experiments at (i.e. 80%) is a bit cell type dependent and requires empirical determination for your line.
As an example if you seed "fibroblast" cells at 10 000 cells/cm2 (19 000 cells per well of a 24 well plate) the next day they are generally ~50% confluent. So 2 days or so later (day of experiment) they will be ~ 80% confluent.
I hope this helps,
Gary
Dear Gary Morley,
Thank you so much for your information.
I was impressed by your knowledge.
Thank you again!
Dear Gary
I had the same problem and I am impressed with your knowledge. Appreciated.
Maryam
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