Welcome to the first ‘Cellfie’ of 2017!
This post might be slightly different to previous ‘Cellfie’ of the month posts because as many of the regular readers of my blog will know – I work on embryonic stem cells and they need to be fed and looked after every day. As I am still working at a university, the building I work in shuts down over the Christmas break. That combined with the fact that all of us in the lab don’t want to come into work over Christmas – we stop growing our cells during the holidays and freeze them down before we all go away!
But what that does mean is that when we come back in January – there are no cells to do experiments on! We need to get out cells up and running from scratch!
So, two weeks into the lab life of 2017 – our cell culture incubators currently look like this:
They are basically empty! We have three incubators that we use and we need them to look more like this before we can start doing some experiments:
As a side note – I am loving the new feature on Snapchat where you can make your own stickers. It made making this image of what we want our incubators to look like inside so much easier – even if it is a little messy!
Full of plates of cells! Enough for everyone in the lab to be doing all their own different experiments on!
But as with science – it is not as simple as getting the cells out of the freezer again and then we are away again! Oh no! For us working on embryonic stem cells, it is a much longer process for various reasons – as I will try and explain more now.
Firstly – when you thaw some cells out they grow slower than normal, so you need to give them some time to get growing at a normal pace.
Next – for our stem cells to grow, we need to prepare the feeder layers that they grow on. But what are these feeder layers? Well, we use cells called mouse embryonic fibroblasts or MEFs for short. Basically we need to plate these cells into the wells of our cell culture plates BEFORE we add our stem cells! If we put our stem cells onto plates without the feeder layer, they just wouldn’t stick down and wouldn’t grow. So, the feeder layers are basically a sticky surface that the stem cells can attach to so they can keep growing and growing. Another use of our feeder layers is that they produce different proteins called growth factors and cytokines that help our stem cells to grow too.
Here is a look down the microscope at our MEF feeder layer – my ‘cellfie’ of the month photo:
It probably doesn’t look like much, but you can probably notice there is a layer of cells at the bottom of our plate. But when we look under the microscope at a higher magnification you can see what these cells actually look like:
The third reason why it takes a bit of time for our stem cells to get up and running is that, we actually need to take the cells back OFF of the feeder layers so we can use them for experiments. Yes really!
In this case, instead of growing our stem cells on MEFs we grow them on a different layer that doesn’t involve cells. This layer does the same job as the MEFs and is a sticky surface for our stem cells to stick to, but as it is not made of cells doesn’t give our stem cells the proteins they need for growth. But we can ONLY use the cells grown on these layers in our experiments because the MEF feeder layers contain cells and therefore DNA and proteins which would affect the results we see. The cells also need to be taken off the MEFs and grown on this new cell-free layer for about 2 weeks before we can use them.
Another reason that it takes time before we have cells to use it that we often want to use cells that grow under 5% or low oxygen conditions. When we take our cells out of the freezer and start them growing again we do this under 20% or high oxygen concentrations. So, when we put cells at the lower oxygen conditions to use, we also need to give them time to adapt – and allow the cells to start making different proteins so they can continue to grow under these lower oxygen conditions. Much like with the cell-free feeder layer, the stem cells need to be at 5% oxygen for about 2 weeks before we can use them in experiments.
The final reason it is going to take some time for me to get some cells to start doing experiments on is bulking up. To save on resources, all the above is done with as few cells as possible in as few plates as possible. Once they are on the cell-free feeder layer and have been at the right oxygen concentration for about 2 weeks, we need to start increasing the amount of cells we have so I can get some for experiments, so all my lab mates can have cells to do experiments on AND there are still some stock plates going in case we have any problems.
So – we are trying to reduce this time as much as possible. At the moment we have cells at both 5% and 20% oxygen concentrations and they have been there for nearly long enough now – but I only put them onto our cell-free layer this weekend, so it will be at least another week before there is cells ready for experiments, but fingers crossed it is all going to plan so far and I will have cells by the end of the month!
Hopefully that is a quick insight into starting up embryonic stem cell culture from scratch, and hasn’t confused anyone too much! If it has got a bit complicated, I am more than willing for you to ask me some questions so I can help you understand. Don’t be afraid to get in contact with me. You can comment below or contact me on Facebook, Twitter or Instagram.