Week 2 of Research at Leeds: All About Cell Culture

I started my second week with planning out the research design to properly start my project soon (where I plan on looking at how adipocytes are affected by postmenopausal hormone levels). I drew out the cell plates and noted what tests we would carry out on each group of cells. I also had to read through a bunch of research papers to find out what the hormone concentration values I should choose, and planned how I would make solutions of those concentrations. But now it was time to get off my desk and look at the actual cells in the lab! 

The process of acquiring mature adipocytes starts with undifferentiated preadipocytes. While viewing them under the microscope, they seem to have long spindle-like arms which are filaments they use to stretch and reach each other. Being treated with a growth medium in a T-flask, the cells love to proliferate and also stick to each other, which is why more and more of the vessel surface gets covered by them. This is known as confluency, and although it is approximated, it's a very important metric! For example, when my supervisor saw that the preadipocytes are reaching higher than 70% confluency, she chose to split the cells (known as passaging) in order to decrease the confluency so they can keep growing. This is because when cells get too populated and are in contact with each other, they stop dividing. 

The process of passaging cells is quite simple. Firstly, it must be noted that the cells are stuck to the bottom surface of the flask due to adhesion. Thus, after removing the medium inside, some trypsin can be added to loosen the cells. The trypsin breaks down the bonds that stick the cells to the surface. Once the cells are loose, however, trypsin is no longer desired as it may start breaking down the very cells. Trypsin can be deactivated by blood serum, which is thankfully present in the growth medium! So, new medium is added into the flask. 

I have left out a super important part so far- we have been talking a lot about preadipocytes but what is actually needed for the project are differentiated adipocytes. Thus, differentiation medium needs to be added when the confluency is high enough. Ideally confluency should be 100%, where the preadipocytes have grown to their maximum. However, the cells do not always reach that level in reality, so we decided to start differentiation at 85% confluency. The differentiation takes 2 weeks after which mature adipocytes with strikingly different appearances are made! For example, here is a side-by-side comparison of the differentiating cells on the day the differentiation medium was added and just a week later. There are so many visible lipid droplets (which look like the black dots) in the second picture! 

It has been so exciting to watch the cells grow and change right in front of my eyes! I cannot wait to run my experiment on the cells that we have worked so hard to harvest over two weeks.