The art of generating single cell clones

Making mutations in mammalian cell lines is becoming much easier, especially with advanced molecular engineering techniques such as CRISPR/Cas9, among others. However, after making a mutation, do you know if all of the cells contain the same mutation with the same expression profiles, and are therefore homogenous? If you have 100% transfection efficiency using a GFP reporter, for example, then at least you know that all of your cells were targeted for the mutation. And if all you care about is that the message is knocked down, then a western blot looking at protein expression might also suffice. However, the cells I generally work with are difficult to transfect (much less than 100% transfection efficiency), so I need to isolate single cell clones from WT clones.

How Do You Generate Single Cell Clones?

A single cell clone is essentially generated from an original ‘multiclonal’ population, but has been separated from the rest in order to create a pure, clonal population that is genetically identical. This sounds difficult, but is actually quite possible! Just a little time consuming. Below are two methods that I have personally used to successfully generate single cell clones.

Serial dilution method:  This method is exactly as it sounds – you create a serial dilution in order to get down to approximately 1 cell. However, it is pretty tedious, and you must work relatively quickly at first. After the cells have recovered from a technique to induce a mutation, use the serial dilution method as follows:

1. Count the cells in order to put ~16,000 cells into the first well (A1) of a 96-well plate containing 200 µL of medium. The other 95 wells should contain 100 µL of the respective medium. (Perform this set-up with at least 2 96-well plates per mutation in order to generate at least 10-20 clones).
2. Remove 100 µL from A1 containing cells and mix with B1, pipetting up and down 3x to mix well before removing 100 µL and mixing with C1, etc. until reaching H1, which now has 200 µL of medium.
3. Quickly add 100 µL of medium to A1-G1 (so that all wells in column 1 will contain a total of 200 µL). Using a multichannel pipette, dilute the cells 1:1 across the rows starting with column 1, mix up and down 3x, and then move to column 2 and so forth.
4. After the dilutions, wells are filled with another 100 µL of medium before being incubated. All wells now have 200 µL of medium.
5. This approximates that at least 15 wells should contain 1 cell. However, this is not always perfect and therefore monitoring the wells are very important to ensuring 1 cell per well.

Tips for Monitoring Serial Dilutions:

I have found that some of the single cells actually do not survive, and sometimes cells that I didn’t know were there actually are. Therefore, I tend to monitor wells that contain anywhere from 0-3 cells, which are usually in the bottom right hand corner of the 96 well plate. Two days after plating, I start to identify which wells contain 0 cells, 1-3 cells, and > 5 cells. I continue to follow the wells containing 0-3 cells, eventually determining if any of these wells really contained a single cell. This can be a relatively slow process waiting for 1 cell to proliferate enough to fill 1/3 of well in order to trypsinize. In the meantime, I usually feed these wells with spent medium (medium used previously by healthy, growing cells and is combined 1:1 with fresh medium to ensure all the factors have not been used) once a week and have observed that this helps keep the single cell clones from dying. After the single cell clones have been identified, it usually takes some time before the cells can proliferate into large enough of a clonal population (about 1/3 of the 96 well) to be transferred to a larger surface area to really grow and expand.

Cloning rings/cylinders:  I have only used the cloning rings a few times now, but prefer this method over the serial dilution method. Cloning rings are quite small and can be made from polystyrene or Pyrex, and look like the end of a Pasteur pipette was cut off. Below is the protocol I use for the cloning rings:

1. Count the cells so that there are no more than 20 cells per 10cm dish. This allows the cells to be spread out in the dish.
2. The next day, mark the single cells that have attached to the plate and seem far enough away from the other cells. Remember: the cells will need to be monitored for clonal expansion.
3. Once there are a few single cell colonies in the dish, use the cloning ring to isolate them.
4. To do this, carefully aspirate the medium from the dish. Using forceps, take the sterilized cloning ring that has grease on one side and place it very carefully around the tiny growing colony of single cell clones.
5. Add approximately 40 µL of trypsin into the ring and incubate the cells for about five minutes in order to carefully detach the cells. Wash and resuspend the cells into a new dish with spent medium to allow them to grow and expand.

Concerns of Single Cell Cloning

How do you know when you have a true single cell clone? No matter which method you use, there are still some downsides to single cell cloning.

1. The cloning ring method does not have a barrier and therefore cannot prevent cells in one colony from migrating and growing with another colony; this may result in not having a true single cell clone. However, that is why I think monitoring the cells to generate a single cell clone is so important.
2. A single cell will grow much slower than if there is more than 1 cell around.
3. The cells I have worked with generally grow fairly tightly together if they are from a single cell, so I tend to look for tightly compacted colonies.
4. Sequencing results from TOPO cloning experiments has also helped determine whether I have a single cell or not. If I end up with multiple sequences after TOPO cloning, this indicates that I actually may have more than 1 clone. Although, to figure this out, I have to run a lot of DNA sequencing reactions.
5. In some cases, I have taken a cloned colony that I suspect may actually have more than one clone, and performed another round of serial dilutions/ cloning ring to ensure that it is single cell cloned.

Benefits to Single Cell Cloning

It may not seem efficient to single cell clone a mutant cell line, as it is time consuming and can take about two months to complete, depending on the growth of the cell lines. Sometimes I have even ended up with a single cell that had the GFP reporter, but turns out to have a WT sequence. However, that is exactly why I single cell sort after a transfection: the potential presence of a WT sequence would severely confound my data. Not only that, but I would be afraid that the WT could possibly even outcompete the growth of a mutant clone over time, which would also not be good. Thus, I think it is important to isolate single cell clones after engineering mutations, especially with something like CRISPR/Cas9 that can generate heterogeneous mutants.

Please let me know how these techniques worked out for you, or if you have additional methods for sorting viable single cells.