My Salzburg Secondment Journey

12 July 2019

My Salzburg Secondment Journey
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Friday 12th July 2019

Guten morgen! I’m writing to you from the beautiful city of Salzburg in Austria.  I just arrived last week for my 2-month secondment as a PhD student and I’m loving it already! The city is within a beautiful area of mountains and the river Salzach. It’s full of good vibes, bikes and beer gardens.

The work has started slowly as it normally does but I’m very excited to start characterising the metabolic behaviour of my colon organoids and tumoroids! Everyone that I have met so far at Dr. Barbara Kofler’s lab at the University Clinic of Child and Adolescent Medicine, Paracelsus Medical University Salzburg, have been nice and welcoming. There has even already been a rooftop party!

 I will start doing some oxygen consumption and glycolysis assays soon, measuring mitochondrial DNA copy number, immunohistochemistry and some other “mito things”. Cool stuff ahead!

 

Friday 19th July 2019

Hello! My second week in Salzburg is already over, and it went so fast! My human colon healthy and tumour organoids are happy growing in Salzburg, I think the beautiful sunny weather might be helping.

The main goal of my secondment here is to evaluate the metabolism of my healthy vs tumour colon organoids. It is reviewed in the literature how metabolism can affect cancer progression. In my project I want to develop a human model where changes in cancer metabolism are verified and what better model than “mini-organs”? (Imagine how cute a mini-organ is!)

I will evaluate the metabolism of my colon organoids using a few different techniques including mitochondrial DNA number, immunohistochemistry (for metabolic enzymes and respiration complexes), detection of mitochondrial respiration and glycolysis by a Seahorse XF Analyser and assays for secreted proteins, inflammatory markers and cytokines. This is not very easy because the organoid field is still so novel that the methods are not as straight forward as for an established cell line. But that’s where my challenge is!

My samples for mitochondrial DNA number have been collected and are waiting to be assayed – I cannot wait for the results! And next week I’ll be performing mitochondrial respiration and glycolysis analysis using the Seahorse Analyser. Let’s see what my organoids “eat”! 

 

Friday 26th July 2019

Hello! Yesterday I started immunohistochemistry with my colon tumoroids. Embedding organoids in paraffin can be very tricky and there are some different protocols that include mixing the pellet with agarose before paraffin embedding. However, I learned a super cool and easy method of making a clot with the cell pellet! First, you harvest your organoids and once you have your cell pellet you then add plasma and thrombin. This will induce the coagulation of the pellet just like normal coagulation in the human body and you have a clot! This clot can then easily be transferred to a histo-cassete and treated just like a tissue sample! How cool is that?

Below you can see the clot with my colon tumoroids! Can’t wait for next week to continue the process!

 

Friday 2nd August 2019

Good morning everyone! The embedding of colorectal cancer organoids was very successful and yesterday I H&E stained them.

It was very cool that the clot method worked as it saved me a lot of time in optimising this. I noticed these little white “dots” indicated by red arrows. They look like what you see in colon metaplasia so I must research this a bit more.

Next week, I’ll be doing some immunofluorescence with mitochondrial markers. And my healthy adjacent colonoids from the same donor are also being prepared for staining. Now I must run to the lab and prepare my plate for oxygen consumption assays!

 

Friday 9th August 2019

Hello! The time is going by really fast and there’s only 3 weeks until I leave Salzburg!

This week I have been trying to optimize an assay to assess the metabolic state of tumour colonoids versus healthy colon organoids but this has been quite difficult.

The assay is performed using a device called a Seahorse from Agilent® and is usually performed on cell lines grown in a monolayer. It is a very useful assay because it detects both oxygen consumption and proton release due to glycolysis and both these readouts can indicate the metabolic state of the cells. By understanding this we can develop and/or test candidate drugs that target specific tumour metabolic pathways to destroy the tumour cells.

Adapting assays to 3D cultures can be quite challenging and I have been experiencing that throughout my project! Many factors can influence the readout such as; cell density, culture medium components and incubation times. Yet, after some unsuccessful trials I think I am on the right path! Keep posted for next week’s blog to see how I get on.

 

Friday 16th August 2019

Good morning! I’m on the final “sprint” to finish all my work here in Salzburg within the 2 weeks I have left. I have mixed feelings because I am getting a bit home sick but at the same time, I’m learning a lot here.

Unfortunately, the trial last week with the Seahorse machine did not go perfectly. I have however realised more things that I can improve, and I will do another test today. Hopefully next week I will have some good results. We always learn even when things do not go as planned.

I have been seeding fully-grown organoids into my assay plate and running the assay one hour later but seeding organoids in an even ratio (as you do when seeding cells) is not possible. Organoids are not all the same size and are quite difficult to count, making normalisation very challenging. Because of how I am seeding my plates, I found that my assay wells are not as confluent as they should be and I do not have enough cells to run the assay. So instead today I will split and seed them into my assay plate and then run the assay next week when they are fully grown. This way I hope to have a full well and get a proper metabolic readout!

On another note, the immunostainings have been going well! Today I will slice my healthy organoids that are embedded in paraffin and next week do a comparison staining between healthy and cancer using mitochondrial markers.

Stay tuned!

 

Friday 23rd August 2019

Good morning! Finally, I was able to obtain some good results from the Seahorse metabolic assay! After a lot of trials and errors I got it running, but now it’s one week until I go home and I do not have time to do more experiments (sad face). Anyway, it was very good that I finally got some data using the tumoroids! And I can always come back later for more. I still have 2 plates to assay that will hopefully give me some insight into the metabolic phenotype of cryopreserved tumoroids versus non-frozen tumoroids. The in-plate cryopreserved plates with tumoroids that I brought with me to Salzburg allowed me to save a lot of culture time and were an extra value to my experiments here.

And because it is Friday and everyone is in a good mood, my organoids also send their love to everyone. Have a nice weekend.

 

Friday 30th August 2019

Good afternoon everyone! My secondment is finished and it’s time to think about everything that has been done, that could have been done and that can be done in the future. In these 2 months I learned how to characterise the metabolism of colon tumoroids by immunostaining for mitochondrial and OXPHOS markers, performing metabolic assays on the Seahorse machine from Agilent® and analysing the mitochondrial DNA content. I think the goals I had before coming here were very large for only 2 months, and some of the work I intended to do (such as the comparison between healthy and tumor organoids) was unfortunately not accomplished. However, this was just a matter of time and with all the protocols already optimised, I can achieve that soon. And speaking about time, the cryopreserved tumoroid plates (using CryotixTM technology) I brought with me were advantageous to optimising my time. Sending frozen plates already seeded with organoids saved me around 2 weeks of work and I’m very pleased I could experience that first-hand!

I already have an invitation to come back here to do more experiments, and I’m really hoping that will happen.

But for now, it’s Friday and time to go home to Scotland!

See you all soon!