11 replies to this topic

[JWChung]

Hi. I'm new to stem cell culture, as is my lab, hence i have no one to refer to. i have problem in passaging the MEF. the strain i use is ICR mice. initial culture is fine, but they don't attach to the culture dishes in passage 1. a senior was able to obtain passage 1 once, but by using 20% FBS instead of 10% to passage. even then, she could not proceed to passage 2. any comments? every journal i read uses 10% FBS. this problem is rather unusual. what could be wrong? below is my protocol:

MEF Derivation

1. Remove 13.5 day pups and transfer into PBS- solution.
2. Remove limbs, internal organs etc.
3. Wash in another PBS- solution.
4. Mince using fine forcep and blade for 5 minutes.
5. Transfer into trypsin-EDTA solution. Further mince for a few minutes.
6. Transfer the solution into a beaker and stir with magnetic stirrer for 20 minutes.
7. Filter into conical tube using nylon (as substitute for cell strainer)
8. Add DMEM with 3% penicilin-streptomycin and 10% FBS at ratio of 1:1.15 DMEM
9. Centrifuge for 5 minutes.
10. Prepare culture dish by adding DMEM solution
11. Aspirate the supernatant out from the conical tube. Add DMEM into the pellet and suck in and out to produce single cells.
12. Transfer the cells to the culture dishes. Incubate.
13. The medium is replaced the next day.

MEF Passaging

1. Remove media from MEF culture dishes.
2. Wash 2-3 times with PBS-.
3. Deatch the cells using trypsin for 3-5 minutes.
4. Add 1ml of DMEM with 1% penicilin-streptomycin and 10% FBS into conical tube.
5. Fill the conical tubes with detached cells. Add with DMEM.
6. Centrifuge for 5 minutes.
7. Prepare culture dishes by adding DMEM.
8. Aspirate and discard supernatang from conical tubes.
9. Add 2 ml DMEM into tube and suck in and out to dissociate the cells.
10. Place the cells into culture dishes.
11 Incubate and replace the medium 2 days later.

i'm trully grateful of any advices. thank you.

Edited by JWChung, 02 September 2010 - 07:30 AM.

[leelee]

Do you also remove the heads in step 2 of your MEF prep?

And your trypsin step seems a little long to me- our MEF lift really easy so I typically incubate no more than a minute or so.

[leelee]

Also, how hard do you split the cells when passaging? Our MEF can't cope with more than a 1 to 6 split, but are happiest with 1 to 4.

[laurequillo]

Yeah I agree, they dont like trypsin, and they like to be crowed...to use 20% FCS helps

[JWChung]

Thank you for the replies.

I did remove the head when preparing the MEF. for the trypsinization, i basically spray the trypsin onto the cells on the dish, then suck n spray again until they detach. i don't incubate. is my method bad for the cells? do u think i should just place the trypsin into the culture dish, incubate for 3 mins, then let them detach themselves? also, when passaging, i split them to 1 to 3 or 1 to 4.

perhaps you can suggest a quick, efficient way of trypsinization? i have also thought of reducing the concentration of trypsin. any comments? thanks

[leelee]

Hmm not sure about the constant "suck n spray" method, I think you are better off leaving the tryp on for say 1 min, tapping the edge of the dish to loosen the cells and then checking under the microscope for detachment. If you monitor them as they are lifting under the microscope, you should be able to see what time is optimal for them.

That said, I'm not sure if this is the cause of your problem.

Any chance of posting a pic of your cells to see if there is anything unusual that we could pick up?

Also, I routinely culture without antibiotics. For several reasons: 1. that antibiotics can affect your cells, 2. that antibiotics can maintain low level contamination undetectable (so called cryptic contamination), 3. it keeps me on my toes with my aseptic technique.

Sorry I can't be more help to you.

[JWChung]

Thanks for your advice. I haven't been able to obtain the pics yet, but once i did, i will upload it. Also, i'll keep on trying and will post the progress.

[JWChung]

Attached are pictures taken during primary culture, and first passage. the first picture clearly shows attachment, while the same cannot be said for the 2nd picture. they were taken 3 days after passaging. pls advice. thx

Attached Thumbnails

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[leelee]

It is kind of hard to see from the pics, but in the second one- the cells look attached to me, but at a very low density.
You said you are splitting them 1 to 3 or 1 to 4, so the density shouldn't be this low. Are you sure all of your cells have lifted before you passage them to fresh plates/dishes? Do you check under the microscope?
With MEF at this low a density, I'm not surprised they aren't growing.

[Rnotk]

its hard to see but the morphology of the first pic is bit odd to me,
maybe because of confluency. I usually will not make my MEF 100 % confluent like that.
Maybe you should culture less cell for P0 to give cell a room to grow.

[JWChung]

thx for ur advice. I agree with you regarding it being too confluent. But for the occasions when i passage them at 80% confluency, they don't grow. I don't understand. That's the reason i wait it until they're fully confluent before passaging. Is my problem due to the quality of my cells? Btw, i only manage to passage up to P7. And most of the cells that i freeze-thaw barely survive. Any advice? thx

[leelee]

I'm not surprised that you can't passage further than P7, I never passage my MEF further than P4. With primary cells, you should limit the number of passages.
After that, they start to change morphology, grow slower, quite often not reaching 100% confluent, and they also don't support our virus growth as well as for earlier passages.

Also, our MEF are fine being at 100% confluent (and even left that way for a day) before splitting. I doubt that is your problem (would be, of course, if you were working with an immortal cell line with no contact inhibition).