Week 5: Loaded in the City

Tibra Wheeler

My weekend away from the city was relaxing, but I was ready to come back to the fast life. This week was filled with much time spent in lab.

Monday morning I was traveling back to NYC, but that afternoon I met with Tony to watch him and another student strip some cadaver knees. The knees must be stripped of the tissue and muscle so that they can be potted for the knee simulator. There were two knees that needed to be stripped that day. We really wanted to make sure that the knees that will be used on the knee simulator are healthy knees. This was the case for the first knee that was stripped. There was lots of synovial fluid in the joint space, the bone was smooth, and the cartilage was healthy (left picture). Unfortunately, this was not the case for the second knee. The second knee was very inflamed, there was blood in the synovial fluid, and much of the cartilage had become mineralized and was hard (right picture). It was really interesting to see this because I know what OA is supposed to look like in the knee but to actually see it in the cadaver really put it in perspective for me. We decided against using that knee for the knee simulator because as stated before, we want healthy knees.

That evening I went to get dinner from Pizza Park. I tried to change it up and not get pepperoni so I decided to get barbecue chicken pizza. Great decision on my part. We also got an order of mozzarella sticks so I definitely had my cheese fix that evening. After pizza, we walked to get Gelato from Grom. I got vanilla and strawberry. Of course, the vanilla tasted good, but the strawberry tasted amazing. It was so refreshing and a cool end to a very humid day.

We did a live/dead assay on Tuesday to make sure we had viable cells in the cartilage punches. Kirsty went through the protocol with me as well as showed me how to use the microscope and what images to captures to visualize the tissue. After we had our images, Tony overlaid them live and deal cell images so that we could see the live and dead cells in the same image. Our images looked great, as we only had a few dead cells in the superficial zone. This is expected as this is the part of the cartilage that our scalpels and tweezers touch when doing the dissections. The live/dead assay confirmed that we had living tissue so we could proceed with the loading on Wednesday.

Tuesday evening, I grabbed barbecue with some friends at Mighty Quinn’s. I got ribs and pulled pork and it felt like I was in the South for a little (they even had sweet tea!). After dinner, we got dessert at DŌ. This place is very popular so the line was on the other side of the street. But we waited because we had to get this experience. DŌ serves cookie dough like ice cream. They have many different flavors of cookie dough as well as different cones (or cups) that you can get it in. I decided to get the cake batter cookie dough in a cup. I got a small, but it was so much cookie dough that I had to take it home. Nonetheless, it was good and well worth the wait.

On Wednesday, we loaded the cartilage punches from last week in the bioreactor. We had a static condition and a loaded condition for each cow. After the samples were loaded in the bioreactor, they were left in the incubator for a few hours. That evening, we placed the loaded and unloaded samples in RNA later to prepare them for RNA extraction on Thursday and Friday.  

As usual, Thursday morning was filled with meetings. During lab meeting, we talked about some the qt-PCR results that had been analyzed from last week. There weren’t gene expression differences in the primers that we had checked for between the loaded and unloaded cartilage punches. For those results, the cartilage wasn’t prepped for RNA extraction until the 24-hour mark. We believe that some of these transcription factors are changing much earlier, so we have decided to prep the samples for RNA extraction earlier than 24 hours. We also have added an impact sample group because we know we should see changes in gene expression there so it will serve as a positive control. That afternoon, I met with Dr. Maher to catch her up on everything I had been working on in the lab with Kristy and Tony. We also discussed my segmentation project and talked about reasonable goals to have completed for the end of the immersion term.

After the meetings, I did a live/dead assay on the loaded, unloaded, and impact cartilage samples to compare them to the ones done on Tuesday. As expected, there was more cell death in those samples as compared to the ones on Tuesday. There were also more dead cells in the impact cartilage that diffused through the tissue in comparison to the loaded cartilage. Later that day, we began homogenizing some of the cartilage punches. I observed another student go through the process so that I could do the other half of the samples on Friday. To homogenize the samples, we use a tissue-ruptor. It is a handheld rotor that uses a probe to disrupt tissue.

Today, we had our weekly immersion meeting where we discussed project updates with Dr. Min and Dr. Prince. Later this afternoon, I tried my hand at crushing the cartilage samples with the tissue-ruptor in order to homogenize them. This process is very tedious even with a small number of samples, but I got it done. On Monday, we will continue with the RNA extraction.

This weekend we’re throwing it back to the 90’s and dancing the night away. Catch ya on the flip side!

*picture of tissue ruptor from https://www.qiagen.com/us/shop/automated-solutions/sample-disruption/tissueruptor/?clear=true#orderinginformation