293T cells unable to revive - (Feb/05/2018 )
Hi, I am new to culturing 293T cells and was told that this cell line is particularly vulnerable.
I've received healthy stock from senior and have no problem growing them until when I keep stock and try to revive them, two attempts failed to revive cells (very little cells attached and looked very unhealthy).
My protocol for keeping cell stock:
1. Trypsinize 70% confluent 293T cells in T75 flask with 2ml trypsin, after all cells detached add 8ml fresh media and resuspend, count cell number and viability ( often >90%), spin down cells (1500 rpm, 5 min, room temp), discard media, add 700ul stock solution (10% DMSO in FCS) and resuspend with tips, transfer to cell vial, immediately stored in -80C freezer.
*One T75 flask cells for 1 vial of cell stock (about 3 - 5 millions cells)
My protocol for reviving cells:
1. After 2 days of storing in -80C, I try to revive to see if it works.
2. Melt one vial of 293T stock (about 3 - 5 millions cells) in 37C water bath, transfer cells to 10ml fresh media, spin down (1500 rpm, 5 min, room temp), discard media, add 12ml fresh media and resuspend, transfer to T-75 flask and incubate at 37C, 5%CO2.
These protocol are established one in my lab, I have also used the same protocol to keep stock and revive TZM-BL cells, it grows very well in contrast to 293T cells. So it makes me confused.
Please if anyone has the experience let me know which step possibly went wrong, or which step needs to be extra careful for a beginner like me.
Thanks.
There are a few steps where you need to be careful:
1. Re-suspending the cells in the DMSO mixture; you should do this very gently or you might lyse the cells. The DMSO releases heat during mixing with medium at this step, which can cause damage to the cells. Personally I gently re-suspend the cells in medium, cool on ice, then add ice cold 2x freezing medium (20% DMSO, 20% FCS, 60% complete medium) drop by drop ensuring the drops are mixed in.
2. Immediate and rapid freezing of the cells is bad for them - it causes large ice crystal formation, which disrupts membranes. Ideally you should freeze the cells slowly at about -1 C/minute. This rate is not absolutely critical, but it should be slow so that the DMSO has time to form what is known as a glass phase (basically solid but not crystalline). This is most commonly achieved by putting the cells into a special container called a "Mr Frosty" containing isopropanol, which slows the freezing rate.
3. Thawing the cells - at this step you want to have the cells almost thawed when you remove from the waterbath. They should still be cold to the touch; too much heating at this phase can cause lysis of the cells.
HEK293 are also notorious for being very weakly attached and taking a long time to attach after seeding. It might be that your cells are alive, just floating. You can also try seeding in a smaller area vessel (t-25) and see if this helps.
bob1 on Mon Feb 5 22:06:45 2018 said:
There are a few steps where you need to be careful:
1. Re-suspending the cells in the DMSO mixture; you should do this very gently or you might lyse the cells. The DMSO releases heat during mixing with medium at this step, which can cause damage to the cells. Personally I gently re-suspend the cells in medium, cool on ice, then add ice cold 2x freezing medium (20% DMSO, 20% FCS, 60% complete medium) drop by drop ensuring the drops are mixed in.
2. Immediate and rapid freezing of the cells is bad for them - it causes large ice crystal formation, which disrupts membranes. Ideally you should freeze the cells slowly at about -1 C/minute. This rate is not absolutely critical, but it should be slow so that the DMSO has time to form what is known as a glass phase (basically solid but not crystalline). This is most commonly achieved by putting the cells into a special container called a "Mr Frosty" containing isopropanol, which slows the freezing rate.
3. Thawing the cells - at this step you want to have the cells almost thawed when you remove from the waterbath. They should still be cold to the touch; too much heating at this phase can cause lysis of the cells.
HEK293 are also notorious for being very weakly attached and taking a long time to attach after seeding. It might be that your cells are alive, just floating. You can also try seeding in a smaller area vessel (t-25) and see if this helps.
Thanks bob1 for the advise.
1. I did not use media for stock solution. Instead, I use 700ul FCS+10% DMSO. Does it sound right?
2. Yes I used Bicell container with isopropanol to store the cells in -80. I did not realized the theory behind it so thank you for your enlightment.
3. I tried to put the vial of cell stock (3 million cells) all into a T25 flask. They do not attach well.
Btw, usually how much HEK293 cells stock to revive in T75 or T25 flask is considered appropriate?
1) should be OK, it still has the problem of localized heating when mixing though.
3) OK - check to see if they are still alive - try a simple trypan blue stain with your hemoctyometer.
for a T-75 10^6 is a good seeding point for cells that are being regularly passaged, and about 3x10^5 for a T-25. From frozen I would go a little higher for each as there will be cells that don't survive the freezing process.
bob1 on Wed Feb 7 16:32:17 2018 said:
1) should be OK, it still has the problem of localized heating when mixing though.
3) OK - check to see if they are still alive - try a simple trypan blue stain with your hemoctyometer.
for a T-75 10^6 is a good seeding point for cells that are being regularly passaged, and about 3x10^5 for a T-25. From frozen I would go a little higher for each as there will be cells that don't survive the freezing process.
Thanks. I have one more question. I also realize i directly put the cell stock into the freezing container with isopropanol that was already stored in -80C. So does it mean my cells would freeze speedly in -80C? I wonder if I should be taking the container out to RT then only put the cell stock before storing it at -80C.
If the isopropanol was at -80 already then your cells froze just as fast as if they were put straight into the -80 (possibly faster actually, due to faster heat transfer in the liquid vs air).
The isopropanol should be at room temp or fridge temp before freezing.