Population doubling time of keratinocyte,etc. - (Mar/01/2005 )

my preliminary test will be calculating the differences of PDT between the treated and untreated keratinocyte culture.. because my hypothesis will be the treated keratinocyte culture with some kinds of wounf dressing materials will enhance the PDT and thus will serve a better wound healing material.

i'm wondering to know what is the precise confluency level for me to start putting the test substances and counting the doubling populatiom time (PDT) of human keratinocyte culture?? i gotta do it in triplicates. my time interval will be 24 hours to 96 hours...and what passage would be suitable to put test substances and start doing the PDT??


anyone here would suggest any better idea,websites or journals that there were people had done it before, at least regarded.

ur suggestions will be great appreciated!!

thanks!!

hi

for my experiments, i started with a "cell solution" which i calculated the concentration (cells/ml for example). I put about 500 000 cells on 15 cm plates and calculate the number of cells after 24h 48h and 72h.

Due to the fact i should replicate assays, my colleague help me with a cell sorter. it's a faster method but available in few labs.

But my first experiments gave me good results.

Fred

thanks fred!! but i'm wondering also if u put 500 000 cells into a culture plate, those cells which adhered might be less than 500 000 cells, let say 50 000 cells only get adhered, and others might be adhered 2 or 3 days later...how do u deal with it?? once u trypsinize ur cells for number calculation, do u reculture ur calculated cells into the plates??

if u do not reculture, that means u make some plates for other time intervals,right?? let say u need to calculate the cells at time 72 hours..96hours..and so on. if u had 3 interval time to do, that means u have 3 culture plates and each of them has 500 000 cells, right??


thanks for ur answers!!!

hi
first i want to apologize for my long answer, that is probably confused. I'll try to be clear.

you told : ---"if u do not reculture, that means u make some plates for other time intervals,right?? let say u need to calculate the cells at time 72 hours..96hours..and so on. if u had 3 interval time to do, that means u have 3 culture plates and each of them has 500 000 cells, right??"

->you're right.
I needed to compare three conditions of culture, and wanted to check at 24 48 72h. That's why i seeded three plate for 24 h (1/condition of culture) 3 for the 48h-measure and 1 for 72h-measure.
As oi wanted more repruductibility, i made my experiments three times.

You said that i put 500 000 cells on plate but less than 500 000 cells adhered. I'm ok with that. But it's not troubling me for two reasons.
The first one is that all my polls of cells have been treated in the same conditions. Hence, i assume that if 5-10% of cells in condition 1.24h did not adered, the same percentage of conditions 2.24h 3.24H not adhered. so for a comparison of these conditions it's ok.
The second reason is that i measure my doubling time on three days. if we admit that the first measure is a little wrong (restart of cell division, and stuff like this) we should consider that these problems are minimized in the assays at day 2 and day 3.

Finally, when i analyzed my measurements, there was not a big difference of calculations on these three separate days. That's why i took the average doubling time.

But if you think fir your experiments the 24h calculation is too aproximate, do not take this one and make your experiment either at 48h 60h 72h or 48 72 96H...

I hope my answer was helpful for you.
Do not hesitate to mail me for more.

Fred

thanks a lot ,Fred!! i really appreciated ur prompt reply.

i have done the same procedures as what u clarified to me indeed. so, here..again can i know which passage u started to use or initialize ur population doubling time (PDT) calculation?? in my thought was the second passage, because i might be using some chemical substances to treat on the cultures and compare the PDT between the treated and untreated cultures.

so, if i started the treatment on cultures at the second passage, and only then i do the PDT...is that any consequences?? pls list ur opinions??

futher more, if i do the treatment on the culture monolayer using the direct contact method, what do u think of the best or more suitable cultures confluency level before i initialize the treatment, and start the PDT??

thanks again to u!!

hi
i use 293 cells for my calculation. That's why i can't really speak about "passage".
But for my experiments, i take 2 cares (not sure if it's the right word...)

First, i seed a first plate like a preculture (two or three, depending on the number of further assays you want to do). When cells are 70-80% confluency, i trypsinise cells, centrifuge (1000 rpm 5') and resuspend them in 20ml media. I calculate the cell concentration and then seed the plates for my experiments.

Second, i do not use cells that have been just defrozed from nitrogen or cels that have stayed at 100% confluency. That's why i do this kind of "preculture".

That's why i think that second passage is ok for your experiments.

For the treatment :
you said the treatment will increase the PDT.You must take in mind the aproximate doubling time of your cells. For my experiments, know that it is feasible for a 293cell to make a full population. For example, IMR90cells are not able to. I don't know for keratinoytes.

But for a 15cm plate, i think that 500 000 cells are ok, assuming the fact that this plate ma have more than 10millions cells

The best advice i can tell you for you, is to make a "dummy experiment".
Try to seed with 250 000, 500 000, 1million and 2 millions of cells the plate you will use for further experiments, and analyze if after 72 or 96h (depending on the longer time for our experiment) if cells are less than 80% confluency.
Then you will get the max seed quantity of cells for the "untreated" cells.

Let say your treatment will divide by 2 the PDT.
Then, you seed all plates by half of the quantity which you get by your "dummy" assay

Hope i helped you.

nice, fred!! thanks!!

so, there i can see ur way to deal with ur PDT, all of them were done under one flask or culture dish...cos u never subcultured ur cells. and ur PDT of ur 293 cell line will be calculated in only one culture dish, but u get it in mean of 3 culture dishes..namely u get the average PDT.

here again, let say i do it at the 2nd passage. both the flasks were seeded with the similar density. after 2 days, the attached cells were detached by trypsin,and then get counted. so, from here i got the number of previous living cells.

then, i proceeded the culture. of course the trypsinized cells were thrown. i assumed the similar results happened in another treated cultures. so, here, do i need to wait until the 40% of confluency level and start the treatment or, i start the treatment right away?? in my thought was do the treatment immediately as the cells might have entered log phase. then, i followed the time intervals accordingly to get the PDT between the treated and untreated cells. my hypothesis would be the treated cells might have faster PDT than the untreated cells. what's ur opinion??

thanks in advanece!!

hi
i think that if you want to get best resutls :
day 1 : seed plates at 30% confluency
day 2 : count cell number on (minimum) 2 plates : you'll get the number of cells that did survive after the seeding of the plate and the number of cells that could be used for your experiment.
Start the treatment at this day.

Day 3 : count cells
Day 4 : count cells
Day 5 : count cells (if it is possible as you say your treatment will decrease the PDT (more divisions in the same interval of time)
Do the averages and estimate your PDT in the different conditions.

At the day 5, if you can't count cells of your "treated cells" condition, (take care of confluency at the day 5), that means that you should do more experiments (more plates or more tries, the best is more tries)...

Fred

hi! thanks for ur valuable comments & opinions!!

i have just done the almost similar thing as u mentioned. i seeded the cells at the 2 X 10**5 cells/ml at a culture flask. and counted the living cells (recognized by trypan blue and adherent) after 2 to 3 days as the cells might be started to proliferate and survive. i cant do it after 1day of the seeding cos most of the cells might be taking time to get adhered.

so, others were similar to urs. i counted the cells everyday until 5th day. but the treated cells might have to undergo the subculture. then i only stopped the PDT. then, everything was re-done as above. do u think any problems if i do it as mentioned above??

thanks for ur opinions again!!take care!!

hi
your experiment should e ak as mentionned above.
I would add a commentary of my colleague who said that cells need 3-5 hours to become adherent but 18-24 hours are needed if you want to transfect or add treatment. (if i'm wrong, anyone, please correct me)
Good luck to you and tell me the results !

Fred