3 replies to this topic

[Gunnar]

I´m looking for methods to make a single cell suspension from a solid human tumor.
Does anybody know of an effective method?

Thanks in advance,  Gunnar.

[gfischer]

Gunnar, on Jun 25 2010, 10:17 AM, said:

I´m looking for methods to make a single cell suspension from a solid human tumor.
Does anybody know of an effective method?

Thanks in advance,  Gunnar.

Generally, one would dissociate the tissue with enzymes including collagenase, trypsin, papain, etc.  Some mechanical dissociation may be necessary as well, for instance trituration through a pipette of a defined diameter to get a true single cell suspension.  More details about the exact tissue type and your downstream plans for the cells would be helpful for more detailed advice.  Also, check the literature for any published  methods for what you're trying to do.

[Gunnar]

gfischer, on Jun 25 2010, 07:32 PM, said:

Gunnar, on Jun 25 2010, 10:17 AM, said:

I´m looking for methods to make a single cell suspension from a solid human tumor.
Does anybody know of an effective method?

Thanks in advance,  Gunnar.

Generally, one would dissociate the tissue with enzymes including collagenase, trypsin, papain, etc.  Some mechanical dissociation may be necessary as well, for instance trituration through a pipette of a defined diameter to get a true single cell suspension.  More details about the exact tissue type and your downstream plans for the cells would be helpful for more detailed advice.  Also, check the literature for any published  methods for what you're trying to do.

Thank you for your reply Gfischer.
The tissue types im working with are from tumors and are variable. Im looking for a general protocol that I might tweak to better suit what im doing which is chemosensitivity tests. Up to this point I have been unsuccessful to find accurate lab protocols online for making cell suspensions. Maybe the internet is not the best place for finding those kinds of things? Im rather new at this and would be grateful with any kind of advice!

[Rsm]

Not tried for tumours, but good for single cells from skin:

Cut your tissue into 1mm2 small pieces ( I like rotary cutters, they really rock the boat!), put into a 15ml Falcon tube filled with 3mg/ml Collagenase P (Roche) and 3mg/ml Dispase dissolved in 10ml of medium of your choice (like DMEM +10%FCS). Incubate at 37C with gentle shaking (e.g. hybridization oven). Dissociate further by pipetting with 10ml pipett. Incubate another 30mins at 37C. Add EDTA to 5mM final, filtrate through cell strainer, spin 1500rpm x 5min.

Routinely gives me 2x 10E7 cells from 2cm2 skin sections. Should be applicable for solid tumours as well. However, I recommend to optimize this protocol for your purpose, like increase the incubation time etc.

Personally, I wouldn't use Trypsin or EDTA, because this is rather bad for the cells. You'd need to put the cells in PBS, which is not good, and Trypsin also digests everything, including cell membranes. With Collagenase/Dispase you can leave your cells overnight, no problem.

Cheers,

Minna