If you have an experience about the biological experiment, you may familiar with cell isolation and cell culture. These two step is essential for biological analysis. So, I will explain the detail at a cell biology level.
cell isolation
At first glance, it seems that you can get individual cells just by getting tissues, but in reality, it's not that simple.
The reason is that most of the cells are connected to each other.
The skin tissue in the picture above is a representative example.
Especially if you look at the epithelial tissue at the top of the skin tissue, you can see that between the cells are tightly connected by something, as indicated by the red square.
These connections are called adherence junctions.
If epithelial cells that make up epithelial tissue are not connected by adhesion, Our skin will run through all kinds of foreign substances into our bodies.
Therefore, adhesion in epithelial tissue helps the skin act as a kind of barrier.
These connections are important for the various tissues that make up living organisms to function in their own way, but they can be a big problem for researchers who want to study cells.
The reason is that it is impossible to isolate each cell as long as the cells are connected by junctions.
So scientists went through a lot of trials and errors to figure out how to separate connected cells into isolated cells.
In this post, we will learn how to separate epithelial cells.
The three elements required to isolate epithelial cells are:
1. trypsin
2. collagenase
3. EDTA(ethylenediaminetetraacetic acid)
Let's start with trypsin.
Trypsin is a type of protease secreted by an organ called pancreas.
Because this protein is protease, It can break down proteins.
Trypsin is especially the one that hydrolyzes the back of arginine and lysine.
When trypsin is added, the binding proteins that connect cells can be degraded.
As a result, it can weaken the binding between cells.
Next, let's look at collagenase.
This enzyme is the one that breaks collagen.
Collagen is also a type of protein, so it is made by connecting numerous amino acids, and collagenase acts as a break between these amino acids.
Then, Why do you put this in when you separate the cells?
There's space between cells. This space is called an extracellular matrix (ECM).
One of the most common substances in extracellular matrix is this collagen.
Collagens look like bundles of fibers and also play a role in connecting cells.
For this reason, collagenase is used for cell separation.
Finally, let's look at EDTA (ethylenediaminetetraacetic acid).
EDTA is a compound that binds to metal ions and sequesters them.
The figure above shows EDTA surrounding the metal ion (M).
In this way, we can say that EDTA is chelating M ion.
So why do you put EDTA when you separate cells?
To understand this, you need to know about cadherin, a protein involved in adherens junction.
The bar-shaped one shown in the picture above is cardherin.
If you look at it, you can see that it is a protein attached to the cell membrane.
In the figure above, only the cardherin monomer is shown, but in reality, two cardherins extending from the membranes of the other two cells are combined to form a cardherin dimer. As a result, an attachment connection can be formed.
If you look closely at the outer part of the cell membrane of the cardherin, that is, the round part, you can see small gray grains embedded in between. This is calcium ion (Ca2+).
With this calcium ion, the cardherin can become stiff outside the cell.
If the calcium ion concentration is low and the cardherin is droopy, of course it will not bind well to the cardherin of other cells.
This is why EDTA is included.
In other words, when EDTA chelates calcium ions, the adhesion between the intercellular cardherins interaction will weaken.
In the next post,
I will explain cell culture and flow cytometry, which are representative ways to find the type of cells we want!