Author Archives: Elizabeth Snyder-Mounts

Cells, Confluency/Counting, Contamination, Chores, Confidence and Career Choices: My Summer Research Experience

I have learned a lot this summer about a variety of different things, most of which began with the letter C (as you can probably tell from my title).


Cells

Learning about cell culture has been an interesting experience. I had the opportunity to work with stem cells as well as primary cells in both sterile (changing media and passaging) and unsterile (immunostaining and fixation) conditions. Everything requires specific techniques and careful practicing in order to avoid cross contamination between cell lines. It was definitely stressful at times, but was super exciting when we got results.


Confluency/Counting

I have come to recognize confluency as one of the most important factors in cell culture. If a plate is confluent, it basically is completely covered with cells. This can be a good thing, such as when we would grow cells for differentiation and end up with around 20 million cells. However, it can also be a bad thing, as contact between cells can sometimes cause weird differentiation of cells, which causes them to be unhealthy and unfit for use. It took a while for me to be able to gauge how confluent cells were, but it was one of the most prevalent things I remember from this experience. Along with confluency, the ability to count cells is an extremely important skill for plating and passaging cells. Getting used to the hemocytometer and being able to tell if a cell was really a cell or just debris, were a few of the challenges that came with this. It was one of the more time consuming activities in the lab, but was also one the ones I feel most confident about coming out of this summer.


Contamination

One of my biggest fears going into this summer was that I would do something to contaminate everyone’s cells and ruin weeks worth of research. Fortunately, that did not happen. There were a couple times I thought my cells were contaminated, but I was being over cautious (aka they were fine). It was good for me to see that while I failed (although not as much as my mentor would have liked), it didn’t always ruin everything.


Chores

During the summer the undergrads in the lab had the assigned lab chore of filling and autoclaving micropipette boxes every few weeks. This showed me that research is not always glamorous, and gave me a better appreciation for lab resources. It also was kind of cool to use the autoclave, even though it kinda smells.


Confidence

I went into this summer with very little confidence. My only background in working with cells was a bench top lab in my Bio201 class, and that was more about genetic and molecular assays than cell culture. I had no previous research experience. I was uninformed, and that scared me a little bit. However, as I was thrown into changing media, counting cells, and performing assays, I quickly became acclimated to the work environment and gained confidence in my abilities to care for cells. My confidence was probably boosted by the amazing support everyone in the lab gave me. I could always ask anyone when I was looking for something or unsure and they would try to help me.


Career Choices

I applied to the BSURF program with the idea in mind that I would try out biomedical engineering and research before I decided to pursue it completely. I can definitely say: Mission Accomplished. I loved being in the lab this summer, being able to work on a cutting edge project and watch my mentor work with 3D modeling systems. Although it was stressful and frustrating at times, the good most definitely outweighed the bad. My mentor also gave me great advice on the different routes you can take after graduation. As a result, I am now strongly considering going to graduate school post-undergrad. I think it would be incredibly rewarding and that I would enjoy it. From there, who knows? I’m just glad I don’t have to decide right now.

Dr. Brian Coggins—Radioastronomy meets Medicine

The series of seminars I have had the privilege of partaking in this summer have been very interesting and enlightening. The science was exciting and the presenters offered thought provoking ideas and concepts. It was very interesting to see people from different fields and explore areas of science I never would have sought out on my own.


One of my favorite things about this summer was having the opportunity to hear about people’s career pathways. One of the seminars that I found very interesting was by Dr. Brian Coggins who works in the department of biochemistry at Duke. The science was interesting, but one of the things I liked the most about what he said was that curiosity drove his career path. It was inspiring to hear a story of someone who was not entirely sure about their scientific path going in, but was shaped by their experiences during education. I also found it odd that I really enjoyed seeing the connections between different scientific fields. He connected physics, chemistry, and biology in ways that made all of the fields seem interesting and less divided. It was cool to see the scientific fields presented together, instead of in categories from which you can only like one and must hate the rest.


I think my favorite quote from his talk was the following:

“There’s more to life than science.”

Project Update: The Good, The Bad, and The Confluency

Overall, this project has taught me a lot. I have learned not only about the specifics of dealing with cell culture, but also about how a lab works in general. I have come across many things that have taken practice to get comfortable with. One of the most stressful things I have had to deal with in this project is dealing with two different cell lines at once. It is very important to avoid cross contamination between the two lines, so it takes a lot of attention when dealing with both at the same time.


My project has been going fairly well so far. We have had little to no trouble with the differentiation process. That is, until this past week. We were attempting to go through the process in a greater surface area than we had before, as this would allow us to culture more cells. As usually happens when trying something new, we hit a snag in middle. The cells became over confluent on day 5 out of 6 of the differentiation protocol and so we fixed them in PFA—so that we could salvage something out of this run of the protocol—and started over by culturing iPSCs. This set us back a bit in acquiring data about the differentiation process. However, this was one of the only set backs we have had. So, all in all, the project has been quite successful. My mentor was actually quite excited that I had finally failed at the protocol, as it is the best way to learn and grow.


To see an overview of my project click here → Project Overview

Chalk Talk Response: Viruses to Vaccines

This week I had the pleasure of hearing about the projects of my fellow researchers in the form of Chalk Talks. Everything was very interesting and it was exciting to hear about different types of research going on around campus. I found Griffin’s chalk talk to be very interesting.

His project, which you can read more about here, is about modifying poxviruses to create an HIV vaccine. I found it interesting that there were so many ways to modify the invasive measures that have been created by nature for a good cause, such as disease prevention. It was also impressed with the complexity of the issues of reengineering the viruses so that they are safe for use. For example, looking into the modification of certain genes to avoid pathogenic consequences.

Griffin also did a very good job presenting the information. His diagrams were easy to follow, which made the understanding of the process of his research project easy to follow as well. I enjoyed his talk very much.

Life in the Lab: A Cell Caretaker

A day for me in the lab differs from day to day. It all depends on where I am in a protocol or how the cells look that day. Some days I am changing media on cells or completing assays. Other days I am sitting down and reading papers or doing research on a procedure.


Rocker Example

An example of some lab work! These are cells on a rocker during the immunostaining process.


Most of the time I am taking care of cells. This includes changing the media on cells or passaging them to keep them healthy and viable for experiments. I also go through protocols to carry out experimentation, which involves a lot of different techniques.

There’s also a lot of down time in lab, whether it is during incubation periods or just waiting for cells to grow. This is when I usually read papers or work on designing experiments/protocols. I also look up protocols for techniques that we will be using or call companies to try and figure out what products to use.

Research Project: iPSCs Differentiation into vSMCs

My research project for this summer is to model the protocol as outlined in the publication Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4566857/) for differentiation of induced pluripotent stem cells (iPSCs) into vascular smooth muscle cells (vSMCs).


Differentiation Picture

This is an overview of the differentiation process.


iPSCs are stem cells that are derived from adult cells. They grow in abundant numbers and grow more quickly than primary cells (cells collected as differentiated cells). This makes them ideal for use in disease and drug modeling. There are two pathways of differentiation using this protocol: synthetic and contractile. The synthetic condition results in more round, confluent cells that are not very responsive to external signals. The contractile condition–which is ideal for our purposes–results in cells that are more responsive to external stimuli, but do not grow as confluent as the synthetic cells.

My tasks are to replicate the protocol and then modify it, in order to optimize the amount of cells produced and the functionality of the cells. We do this by changing out different media formulations at different time points during the differentiation process. At the end of protocol, we check using immunostaining to see if certain proteins are being expressed by the cells, which allows us to see if the differentiation protocol has been successful.


Synthetic Staining

This is an example of the Immunostaining that we have done so far.


There are various reasons for doing this project. For example, in optimizing the protocol the cells will be able to be cultured in greater volumes and be used for various disease models. These models could also be used to show the effects that certain drugs/medications have on the cells. The Truskey lab does a lot of disease/drug modeling, relating to the idea of microphysicological systems, so this type of project could also allow for more effective usage of lab resources.

Interview with Dr. George Truskey: Tissues and the Balancing Act

This week I had the pleasure of meeting with and interviewing the Primary Investigator (PI) of the lab I am working in this summer, Dr. George Truskey.


Dr. G. Truskey

Dr. George A. Truskey


Dr. Truskey is a professor of biomedical engineering and is the interim dean of the Pratt School of Engineering. I have summarized his answers to some questions I asked him during our meeting.


Q: What is your typical day like?

Being interim dean has made his days a little more atypical than that of what they were before he assumed this role. For the most part, his week consists of various kinds of meetings. He meets with staff to deal with issues and has collaboration meetings with administration. Sometimes he meets with alumni and donors as well. He also has a lot of student meetings. For example, he tries to meet with the graduate students in his lab once a week.


Q: What was your career path like?

Dr. Truskey did his undergrad at Penn in Bioengineering, which was relatively new at the time. He began to think about academia and being a professor in undergrad and he carried that idea with him into graduate school at MIT. He remembers graduate school being very hard, and that on his first day his class was told that 1/3 of them wouldn’t be there after that year. Fortunately, he wasn’t one of that 1/3 and upon graduation was offered both an industry job and a job at Duke in biomedical engineering.


Q: How did you get interested in tissue engineering?

It was mostly through collaborations with other faculty such as Dr. Reichert looking at grafts for cells and tissues. Dr. Truskey started off by saying that he wasn’t doing tissue engineering, but was just working on the edge of it. He also had the influence of grant opportunities. One of the biggest influences was a grant focused on microphysiological systems and the idea of “tissues on a chip.” One of the most exciting parts for Dr. Truskey is the disease and drug modeling aspect of creating microphysiological systems that would allow for more effective drug trials.


Q: What is your favorite part of being a PI?

Dr. Truskey really enjoys seeing how student’s ideas play out through out the course of the research. He likes seeing students succeed, whether it is making a discovery, getting an award, or coming up with ideas.


Q: Do you prefer Administration or Teaching?

Dr. Truskey explained that he likes them both and that they both have satisfying elements. In administration he gets to help build a better school. For example, he has helped launch collaboration with the hospital. In teaching, he gets to help people and get them excited about what they are doing. It’s a balancing act.

Expectations: Focusing on Techniques and Looking to the Future

This summer I am working the Truskey Lab in the Department of Biomedical Engineering under PhD Student, Leigh Atchison. This is my first time working in a research lab, and I am very excited for this opportunity. So far, the atmosphere has been great and the people are very helpful. One of the things I am hoping to get out of this summer is really trying to understand what it is I am doing. I am working with cell cultures—something that I have found to be very interesting so far. However, the cells and techniques are still a little mysterious to me at times.


TruskeyLabDoor

The Door of the Truskey Lab! Click on the picture to learn more about the lab!


On the technical side, I want to come out of this summer knowing the techniques that I need to know and having a better understanding of how to work with the different cell lines. I have already begun working with basic passaging of cells and changing media, as well as learning how to Immunostaining. My expectation is to be taught many things in this short amount of time, and to make mistakes that I will grow from. I do not have much experience, but I plan to catch on quickly and work hard to make up for that. I also expect to be tested. I know that I have a lot to learn and I’m looking forward to learning a lot more.


LabNotebook

My Laboratory Notebook!


The main reason I applied for this program was to assess the way I feel about working in biomedical engineering. As a result, one of my most lofty goals for the summer is to make a decision about my future. I wanted to get experience in the field and make sure that I will really enjoy what I plan on doing for my career. This is my biggest expectation. Even if I only get an inkling of how I feel about this field, I will have more of an idea about my future than I did before.