Growing cell lines from frozen tissue samples - Is this even possible??? (Apr/28/2006 )

My lab accidently ordered frozen tumor tissue samples instead of frozen cells and are wanting me to thaw them and grow them up as cell lines. The tissue samples came with hardly any information at all (essentially just tumor designation, histologic type, animal host strain, and when it was frozen). I am wondering if it is possible to thaw a frozen tissue sample and grow up a cell line from it and if any one has a protocol or suggestions on how I could do it? Or even if you don't have a protocol maybe just some guesses as to how I could try to go about doing this ... my lab would really like for me to try and see if I can grow them up (they want me to do something with them). I have little experience working with tissue samples so any advice anything at all will be useful to me.

It is certainly possible to grow cells from frozen solid tissue so long as the tissue was frozen in a way that enables the recovery of viable cells. We used to successfully freeze small pieces of solid human tissue for later cell culture (saved us the hastle of doing the cell culture if the tissue wasn't eventually required).

If your tissue was snap frozen in liquid nitrogen there will be no viable cells and you will be wasting your time attemping to culture. If the tissue, usually small pieces (maybe 2mm cubed) was appropriately cryopreserved using DMSO then it should be possible to recover viable cells.

I'd first check will the supplier to see whether the establishment of a cell line is possible.

If the tissue was properly frozen ... Do you thaw it the same way you would frozen cells? How do you establish a cell line from the tissue sample? Do I treat it like a fresh sample? Do you have a good protocol you could recommend for me to review? Thanks

Assuming the tissue was frozen down in 10% DMSO, thaw the vial quickly as you would for frozen cells. As soon as the liquid has thawed tip/transfer the contents into a sterile 55mm petri dish and remove all liquid. Add some fresh tissue culture media and wash the tissue to remove all traces of DMSO (just swish the media around in the dish). Remove the media and repeat the wash.

I'll need to double check our method for establishing tumour cell lines but I think its pretty much the same as our non-malignant solid tissue method. I'm assuming you have enough tissue to establish at least 1 x 25cm2 flask. I'll write a few notes below but wait till I check the method when i'm back at work tomorrow - i'll add it as an attachment. We use long barrel sterile plastic transfer pipettes throughout.

Remove all media from the petri dish and wash with PBS w/o Ca & Mg. Remove the PBS and scrap the tissue into the middle of the dish with a sterile size 22 scalpel blade. Add a few drops of 0.25% trypsin solution on top of the tissue and chop it with the scalpel blade. Scrap the tissue to the side of the dish and transfer it to a 10ml sterile, conical based tube. Add another ml of trypsin to the petri dish and wash out any remaining tissue from the surface of the dish and transfer it to the tube. Top up the tube so that it contains about 2 ml of trypsin. Give it a few vigorous flicks to mix the tissue around and incubate for about 45-60 mins in a 37C incubator.
Add 5ml complete medium (supplemented with 20% FBS) to neutralize the trypsin and centrifuge the tube to pellet the tissue. Remove the supernatant and transfer the tissue using a small amount of tissue culture medium into a 25cm2 flask. With solid tissue we often scratch any larger pieces of tissue into the surface of the flask (snap the end off a sterile glass pipette and scratch/scour/anchor larger bits of tissue into the surface of the flask using the jagged end of the pipette). Gently add more complete tissue culture medium to a final volume of 5ml and incubate as usual. Maintain as for any solid tissue culture. If the tissue is viable you should see evidence of growth within several days.
As i said, let me check the solid tumour method - it might be slightly different with regards to culturing vessels and enzymatic digestion but i'll get back soon.

OK, sounds like the soft tissue tumours we deal with are handled more simply. Apparently most labs do not use enzymatic digestion although we know of one that routinely uses collagenase.

The tissue is just placed in a small sterile petri dish (eg. 55mm dish suitable for cell culture) and is chopped finely in the dish using a size 22 scalpel blade. We usually only receive about 3mm3 of malignant tissue. Some of the finely chopped tissue is usually transferred to another petri dish and both dishes are flooded with a small volume of tissue culture medium. Incubate as usual in a humidified 5%CO2 in air incubator at 37C.
Leave for about 4 days before media changing and then maintain the cultures as usual. Growth can be quite prolific early on so keep an eye on the cultures (at least for fresh samples this is often the case). Others take a while to get established.

As I said in the previous post, if the tissue has been snap frozen you will be wasting your time so i'd check this first.

Thank you. I found that the tissue was frozen using 10% DMSO 10% FBS and the appropriate growth medium (i.e. definately not snap frozen). So your info will be very helpful to me.

i suggest you inject the thawed tissues or tumor cells into nude or nod/scid mice to passage it before you make any effort to establish it into cell line. in my experience, it's very hard to establish tumor cell line from either fresh primary tumor sample or fresh xenografts.

yeah that would be the ideal situation ... it's been discussed for at least one of the cell lines ... but i don't think it will be done for all of them unfortunately.

Hello there, I also want to find out more on how to retrieve viable cells from frozen organs. I am interested in a genetically modified mouse strain from the Jackson Lab, but what I need is only the splenocytes and hepatocytes for in vitro tissue culture experiments. So, I really don't want to go through the hassles of importing live animals from the USA to the UK just to sacrifice them for their spleens/livers.

The Jackson Lab said that they can send me the organs (spleen and liver), what transport medium and condition would you recommend using? Considering that it may well take 48 hours (possibly longer, actualy I don't know) for the organs to arrive in our lab.

And would I be using the same splenocytes isolation protocol for fresh organs?

Any suggestion most appreciated.

Fai