Day THREE!

BOOM SHAKALAKA!!! It's day 3 of our research programme now and we've decided to combine our perspectives and make it into a more picture-filled post.

We met our mentor who will be taking us for the next 3 days, Ajay, who is a second year Ph.D student. briefly for the three days we'll be doing surface functionalisation, micropatterning and cell culture. All 3 would be linked closely and the steps have to be executed precisely or we will not have any cell culture.

So for today we did surface functionalisation. What we did was to prepare the glass cover slips that would be used for the next 2 days to cultivate cells on. Ajay taught us how to modify the glass cover slips to have the optimal conditions to grow our stem cells.

We added chemicals, washed away the excess, added another chemical and the steps were pretty complicated. However, Ajay did a really good job in explaining how and why each step was done, using his glove as a whiteboard too. HAHAH!! It gave us an understanding of the importance of each step in the journey to get our cover slips in the required conditions.

Basically what we did today was:

1. making the glass cover slips hydrophilic by adding Piranha solution
2. transferring it to a chemical called APTES to add a new functional group
3. adding Gluteraldehyde (GA) to provide a C=O double bond. The presence of C=O bond will allow proteins to form peptide bonds, allowing cells to bind to our cover slips.

And there were precautions to take for each step...

• Piranha solution was a mix of concentrated sulfuric acid and hydrogen peroxide, which makes it really dangerous. We had to dispose it properly or it could cause an explosion. BOOOMMMMZ!!!
• GA is light sensitive and we had to work in the semi-darkness, so that we could prevent unwanted compounds from being formed
• A lot of washing had to be done as well and there was a machine called a shaker that spins the petri dish in which the cover slips were held. It helps to swirl the distilled water so that there could be even and thorough washing. It could even take up to 1-2 hrs!!! :o
• a nitrogen blow was used to dry the cover slips, primarily to prevent the cover slips from oxidising.

In order to complete some steps, the time taken to add a new solution to the cover slips could be done in a very short time, but the waiting time after that was indeed a boring and long one (thus the blog, just kidding). The whole process of making our cell cultures would take up to 3 days and every single step is imperative to the success. For certain steps, we had to do it fast, for e.g APTES gets oxidised very easily especially with atmospheric oxygen, thus transferring the solution had to be done as fast as possible to minimise exposure.

There was not much hands-on today and we definitely look forward to some form of it tmr. :) Till then~

Transferring cover slips to a petri dish after washing it in acetone and ethanol, to get a clean surface.

That is actually a STACK of glass cover slips. One piece alone is really thin, so we had to be careful when transferring it.

Here, we are adding Piranha solution to make the cover slips hydrophilic by adding -OH groups. We had to ensure that the cover slips are fully covered in the solution. Ajay made us stand away because of how dangerous the solution is. 

All other solutions are also added in the same manner.

Heating the cover slips with Piranha solution.

This is called a well tray. We put a cover slip into each well. It is fully sterile to prevent any contamination of cells when we use the cover slips to culture them.

This is the shaker, which swirls the liquid inside whatever container you put on top such as petri dish or well trays.

Ajay is adding GA. It is done in semi-darkness, although it may not be as obvious in the picture.

Lastly, we dry the cover slips with a nitrogen blow. It is like a hairdryer, except it blows out nitrogen and it has a thinner mouth. Nitrogen is used instead to prevent the functional groups on the cover slips to get in contact with oxygen, hence preventing oxidation.