The cell line that i use proliferates at 33 degrees, and differentiates at 37 degrees. they are hardly any detailed protocols avaIlable online so i was wondering if this protocol i adapted below would work in general. Also can all cells generally be freezed down using FBS?
Plan for podocytes cell line:
Thawing from cryovial (2.25 x 10^6 cells total) upon arrival from shipping
1) Incubate RPMI media in waterbath. Have tissue with ethanol sprayed ready.
2) Transfer 8ml media into 50ml falcon tube.
3) Thaw cryovial in waterbath till small clump of ice remains.
4) Wipe cryovial 70% ethanol.
5) Transfer contents of vial into falcon tube, resuspend up and down gently.
6) Centrifuge 1500 rpm for 2 minutes.
7) Discard supernatant.
8) Resupend cell in 10ml RPMI media gently up and down.
9) Transfer everything into 10 cm dish at 33 degrees celcius incubation.
Once 80 % confluency is reached,
1) Incubate trypsin RT, media in waterbath
2) Remove media using suction.
3) Wash with 10ml 1X PBS.
4) Remove PBS with suction.
5) Add 2ml trypsin into 10cm dish.
6) Tap gently on sides to detach.
7) Observe under microscope for detachment once 50% detached, bring back into hood.
8) Neutralise with 10ml media.
9) Centrifuge at 1500rpm for 2 minutes.
10) Remove supernatant.
11) Resupend pellet with 5 ml media.
12) Add 1ml cell suspension into 10 cm proliferating plate then add 9ml media. Add 0.5ml cell suspension into 3cm plate then add 2.0ml media (this plate will be grown at 33 degrees for proliferation until 80% confluency then it is transferred to 37 degrees for differentiation.
13) Centrifuge remaining cells in tube at 1500rpm for 2 minutes.
14) Remove supernatant.
15) Resuspend pellet in 20 ml FBS.
16) Transfer 1ml of this suspension into each cryovial incubate at -80 degrees
17) Next day thaw one of the vials and grow in 10cm plate to see if it can grow.
Hi Iwan
I have few question about your protocol.
1) you are not coating the culture flasks with Collagen or Fibronection that is essential to attach the podocytes to the flask?
2) you are not adding IFN-gamma required to maintain and proliferate the podocytes in undifferentiated state at 33 degree.
I am enclosing the podocyte culture protocol that I used to culture podocytes. I hope it will help you maintaining the podocyte culture in right manner.
Good luck
Pardeep Aggarwal
hi pardeep. I am using trypsin to detach the cells. In your experience, how would you rate the difficulty in trying to detach the cells?
can you state in detail how you detach the cells? i was thinking of sufficiently covering the monolayer with trypsin followed by incubation at room temperature for 4 mins while gently tapping the cells. This is followed by neutralization with media and mechanical flushing of the still-attached cells.
hi Iwan, podocytes come off very easily with trypsin in 4-5 min, until n unless you grow them to 100% confluence. Use trypsin at 33 degree for undifferentiated cells and detach cell at 33 degree not at room temperature. Add 3ml trypsin per T-75 flask or 1ml/t-25 or 5ml/T-150 flask.
And do keep in mind if podocyte gets confluent they lose their properties.
Do not tap any type of cells, it leads to clumping.
Trypsinization and neutralization step is given in the protocol i attached.
Good luck
hi may i know what will happen if the cells are allowed to grow to 100% confluency?
It wasnt overgrown per se but the supplier providing me the cell line had given more than the number of cells in the vial thus when i had initially thawed it into the recommended number of vials, it was overcrowded for a period of 6 hours before i passaged the cells again.
Hi Iwan
Confluent Podocytes can loose the expression of key proteins including Nephrin, podocicn and WT1.
Check for their expression to confirm that podocyte retain their properties.
Hi i have 2 more questions and thanks a lot for the help
1) will confluency induce differention
2). Also may i ask while maintaining the podocytes at proliferation temperature whats the percentage of podocytes that somehow gets differentiated and morphologically is there a sure way to tell differentiation is taking/has taken place?
For both questions i will say i have "no idea". Never checked that. To check that you may use flow cytometry using podocyte differentiation specific markers.
hi pardeep, im in the midst of growing the cells in T75 flaks for subsequent freezing down. However i notice that the proliferation rate of these cells aren't high. from an initial confluence of 20% to about 60% requires about 3 days.
Is this the case for you as well? what is the approximate doubling time from your experience handling these cell lines?
it depends on the initial seeding density. if you add little no. of cells than they sometime take much longer. seed at least 4000cells/cm2.
Hi pardeep.
I tried detecting WT-1 (wilms tumour) in my undifferentiated podocyte but got no detection. I read online many conflicting reports with some suggesting that it is a marker for differentiated podocytes. I was intending to use this marker to ensure that my cell line is really of podocyte origin.
Have you tried detecting this marker in undifferentiated podocytes?
I think I never tried looking for WT-1 in undifferentiated podocytes.
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