James - it will greatly depend on which tissue your stem cell is derived - e.g., for hematopoietic stem cells, this is definitely possible.

Which cells are you trying to grow?

 If you mean human embryonic stem cells, well, it will be generally a low efficiency process but can happen. Try adding ROCK inhibitor. Some cell lines have already been adapted for single cell passaging, so they are also better at that.

Definitely grow. But it  depends upon the media and growth factor. You may use knock out DMEM+ 5% FBS+ ROCK inhibitor for culture purpose.

You can use conditioned media (collect half and add half fresh) but it is not necessary. I agree that it really depends on the type of stem cell you are working with as well as the direction of your experiment. If you intend to differentiate stem cells then you probably will add serum to the media and plate at high density in 96-well plates  coated with PureCoat or Poly-D-Lysine. If you want to keep them in undifferentiated state than you need to grow them in untreated plates. If you are doing some tests, then I suggest that  following the plating you allow cells to settle and leave them 24 hrs undisturbed. When working with neural stem cells, for example, you will have to dissociate the whole sphere to get new colonies (neurospheres).

I agree with previous suggestions, mTESR with conditioned medium and rock could improve the survival, I heard laminin-521 has very good success rate, I have never tried personally though!

Thank you to all for your insight. In response to your inquiry: I have a Human Marrow Stromal Cell line (hMSCs).  I do not wish that they differentiate but rather plan to introduce a site-specific mutation (bi-allelic single gene KO via CRISPR) but in the absence of a reliable cell surface marker wanted to clone and screen via sequencing to identify sequence-supported genetic KO before phenotyping.  Again I appreciate all your response, I am really only used to working with somatic cell lines. If this community feels that I will have high attrition starting from single cells, I may consider co-transfecting with GFP marker and co-culturing single CRISPR-treated cell with WT stem cells then sorting later. 

Best, 

James 

I agree with most of previous suggestions: 1) It is possible for hMSCs, 2) iROCK will improve your survival, 3) A specific medium for hMSCs also will help (several at LifeTechnologies) 4) Try to do not stress them too much before the sorting/limiting dilution process. You can use Tryple (Invitrogen/LifeTechnologies) to detach them in a gentle way.

I feel that growing single human MSCs cell in 96 well plate is difficult as cell density matter for their growth. But you can try human fibroblast feeder layer for growing single human stem cell at serial dilution . You can supplement your growth media with EGF, Transferine, BSA, Hydrocortisone & Insulin as a growth factors. I agree with other  scientists statement that its depend on type of cell you are using for cloning . If it is haematopoetic cells you can able to clone single cell too.

Definitively you can. The easiest is to sort them at 1 cell/well and you can get up to 50 wells growing on a 96 well plate. The efficiency can also be lower. In general you do not need conditioned media, simply put media (we use DMEM:F12) with 10% FCS.

One should use a fresh culture medium to grow single cells which is more or less the same as the one that is used to grow a confluent culture, but not the spent medium of an old culture; it should ideally be the medium of the lab that is well standardized / validated for effective growth.

Hi James, I will be doing some CRISPR work in hESCs/hIPSCs and then attempt clonal expansion of the resulting cells using Laminin 521 and Tsr E8 media. will update if i get some decent results. Cheers

Hi James, cell density is VERY IMPORTANT for your culture. You should really use a co-culture approach, but it also depend on the cell type you are using.

if those are bone marrow derives mesenchymal stem cells by definition they are adherent to tissue treated plastic and are colony forming. And if as you say you are using an immortalized cell line they should be pretty robust. Your only issue will be in the starting efficiency of the seeding and first cell adherence. To support adhesion and expansion alphaMEM + 10% FBS + 5% PSF + 5ng/ml of FGF2 is general a pretty good starting recipe.

Hi James. It depends on the stem cell type. If your cells are pluripotent stem cells, e.g. hES cells or iPS cells grown in a medium with bFGF, the efficiency of cloning in separate wells will be low. You can use:

1) ROCK inhibitor treatment.

2) Culturing on Laminin521 /E-cadherin substrata (9:1 w/w).

The latter method has been developed in our lab (check up our Nat Prot paper). No co-culturing or a conditioned medium is needed.

Dear Sergey, I will be attempting clonal expansion of gene edited single hiPSCs using your protocol. I was wondering if you think Laminin 521 in combination with E8 media and ROCK inhibitor would be enough to derive cell lines?

Hi Sergey, is there any easier way to create the dialyzed albumin/medium solution ? 

Why is albumin so important? Which albumin should we order (cat no?)

when i plate a 96 well plate with matrigel and rock, how many single clones can i expect, how many can i expect with laminin521?

thanks