Kabir Grewal, UPitt Lab, Weeks 1,2,3, and some of 4

As it turns out, my previous blogs didn't save/upload correctly, so here I am writing my first blog going into my fifth week in the lab. The first two weeks in the lab involved a lot of basic training, so there is not too much to write about there; I will mainly focus on the third and fourth week. Because of this, this post is going to be a bit lengthy, so bear with me.   Fortunately, however, this bad luck is not at all representative of how my first four weeks in the Du lab at the University of Pittsburgh have gone thus far.

 The Du Lab is a small lab (8 people total including myself) in which there are two main focal points; the first is harnessing the regenerative capabilities of stem cells to try and treat the dysfunctional trabecular mesh work that causes Glaucoma, and the second being trying to find an ethical yet effective way to regenerate lost corneal endothelial cells. For the first 1-2 weeks, most of my days were spent learning my way around the lab/finding all the equipment rooms, and shadowing some of my other lab mates to learn how to do some basic procedures like making cell medium, changing cell medium,  and extracting DNA/RNA.  As it turned out, 2 of my current lab mates, Ye and MinWen arrived to the lab the same day I did, and so the three of us went through the process of learning the basics of the lab together, which was nice.  One of the highlights of the first two weeks was shadowing an eye surgery on a mouse. The mouse was under anesthesia so it was cooperative, and we proceeded to inject fluorescent protein markers into the eye of the mouse and look at the eye closely under a microscope.

One of my lab-mates, Ye, working with the confocal laser microscope

At the end of week two, Ye and I started to work on one of the projects I am currently assigned. This project involves studying characteristics of Schlemm's Canal Endothelial Cells (SCE) which are specialized cells that are found in a canal-like structure within the eye. We are studying the characteristics of these cells when cultured with other types of cells, mainly Trabecular Meshwork Cells (TM cells) and Trabecular Meshwork Stem Cells (TMSC's).  To study these cells, we set up a dish in which we had SCE cells alone for the control, TMSCs, TM cells, SCE cells cultered with TMSC's, SCE cultured with TM cells, and finally SCE cultured with an inhibitor. After allowing these cells to grow for a couple days, we proceeded to stain the samples of cells for certain protein markers, and we used a type of laser miscrscope called a confocal microscope to find these.  Unfortunately, there was a slight problem with the staining, so it took us around 2 days to actually locate the protein markers with the miscroscope and take good pictures of them to give to Dr. Du.  

My set up for when I extracted stem cells

By this point, at the end of the second week, Dr. Du assigned me another project that I would be working on alone.  The goal of my project is to determine if two types of stem cells, Trabecular Meshwork Stem Cells and Corneal Stromal Stem Cells (CSSC) are able to differentiate into Corneal Endothelial Cells (the cells that comprise the cornea within the eye). I must say, when I learned that I would be doing this project alone, I was nervous, yet very excited. Dr. Du handed me a long protocol of what I should be doing each day for the project, and when I first received it, I just kept looking it over and over to try and understand everything.  A couple hours and many, many questions later, I figured out what I needed to do, and wrote down detailed notes in my lab book. The first steps of this project involved making two types of cell medium that I would use to culture my cells.  I cultured the TMSC's and the CSSC's in their own well plate, and bovine corneal endothelial cells in another well plate. Then, I waited a couple days for the cells to grow, changing the medium and monitoring them closely under the microscope.   Four days later, I proceeded to lyse the corneal endothelial cells so that only the extracellular matrix was present, and then I injected the TMSC's/CSSC's from the other well plate in with the corneal endothelial cells. This was a rather tedious process, as I had to work under the micrscope to find the cells and gather them. Once I got the hang of it, it was actually pretty fun. In between working on this project, I read up more on the type of cells I am using in my project and helped some of my other lab mates with their projects. In the next week, I will continue this project and continue to work with Ye on another project that Dr. Du will assign us.

Living in Pittsburgh has been far easier than I expected.  It is very hilly, but I find that it makes for beautiful scenery as it is literally a city built on a series of hills. I am living with my Aunt and Uncle who live a 20 minutes walk away from my lab, so walking there is very easy.  There is so much that I have done in the city so far; some of my favorites have been attending a Pittsburgh Penguins game and Stanley Cup Victory parade, bike riding along the waterfront, and going to many of the museums Pittsburgh has to offer.  I am more excited than ever to go to work each day now that I have my own project, and I really look forward to having a great next two weeks in the lab.