As I reflect upon the past 7 weeks, I realize just how fast time has progressed. I feel like the first two weeks I was thrown into the deep end, immersing myself in the exhausting cycle of learning and digesting the various novel laboratory techniques I’d been exposed to. As I finally found my bearings within this cycle, I noticed my vision had broadened and I could see much farther regarding why I was doing the steps I was and how they impacted the growth of my cells. And within these processes I felt supported by the indelible questions and answers that led to the development and creation of the tools I used. The culmination of question asking and answering: this was science. I realized the the purpose of researchers was not to bombastically pierce into the unknown but to elevate the current understanding piece by piece to leave no gaps in our knowledge. And this Summer I was given the opportunity to leave my mark on this foundation.
In the future, I take away with me not just the techniques I learned, but the approach to questioning I learned from my amazing mentor Torie this Summer. Asking the right questions and knowing which directions to pursue is one of the most critical abilities and although I’m just a neophyte at it, it’s a skill I’ll definitely continue practicing in my future.
For me, BSURF has opened a door to the possibilities of a career in research and although I know not where life will take me I see this experience as the beginning of something great to come.
Tissue-engineered skeletal muscle is a promising platform for in vitro modeling of human muscle diseases and pharmacological testing. However, most engineered skeletal muscle tissues contain only muscle and fibroblasts, lacking the complexity of native muscle, which also includes motorneurons, macrophages, vasculature, etc. The Bursac Lab has been developing a pre-vascularizing engineered skeletal muscle with contractile function comparable to that of avascular, muscle-only engineered tissues. In an effort to improve this platform, we have started to investigate the effects of Apelin-13, previously shown to improve skeletal muscle function in vivo and angiogenesis of endothelial cells in vitro. The main hypothesis of this work has been that Apelin-13 will simultaneously improve both angiogenesis and contractile function of pre-vascularized engineered skeletal muscle. To test this hypothesis, I have been characterizing angiogenesis of human endothelial progenitor cells (EPCs) using a 16-hour Tube Formation Assay with varying concentrations of Apelin-13. By imaging the resulting endothelial networks and quantifying total tube length and area, I expect to show that supplementation of Apelin-13 can promote angiogenesis of EPCs. If successful, I will continue these studies by treating muscle-EPC co-cultures with Apelin-13 and quantifying the effects on muscle structure, function, and formation and stability of vascular networks.
The past week’s Chalk Talks carried lots of interesting information about the amazing research being conducted here at Duke. One of the presentations that caught my eye was by Nico Rey, who talked about his experiences at the Asokan Lab where they are designing a novel gene therapy for Muscular Dystrophy using viral vectors. The basis of Nico’s research is the Central Dogma, DNA makes RNA makes proteins. In many diseases, there is some error or mutation which occurs in one of these steps which causes the product to become unusable. For Muscular Dystrophy, this occurs in the first step, aka, there is a mutation within the DNA that causes the entire process to result in a failed protein.
Nico’s viral vectors will insert RNA into the cell which will complementary bind to regions of introns preceding a mutated exon which causes an alternative path for polymerase enzymes to take when producing mRNA. These inserts will contain the complement to the correct mRNA exon sequence that we wish to translate into protein. However, Nico noted that the current method of inserting the RNA and leaving it up to chance was relatively inefficient and up to chance. Therefore, his lab is focusing on leveraging novel molecular machinery to increase the odds of forcing enzymes to choose the insert rather than the original mutated DNA.
I remember after hearing Nico’s Chalk Talk that this type of procedure sounded rather familiar, and it seems to resemble a bypass surgery, where a vascular graft is taken to bypass a clotted region to provide blood to tissue. In a similar sense, here Nico’s lab is bypassing the mutated and ‘diseased’ DNA to allow for the cell to exhibit the healthy phenotype. I think that this technology is a really good way to tackle disease as it leverages viral vectors which are known to work, inserts a small amount of specific RNA which we know will only bind to the regions we want, and has the potential to greatly alleviate symptoms of debilitating diseases.
As one would expect, working in a tissue engineering lab revolves around one main thing: cells! In the lab there are myriads of ways to manipulate and care for cells and ensure that they are happy. In a sense, every day in the lab is like cell daycare where we have to cater to their every need. We also have to use procedures to analyze the cells and tissues that we culture to derive scientific insight from them.
Typically I arrive at the lab around 11 after getting a hearty breakfast from McDonalds and a fruit-flavored drink. As soon as I set my bag down, it’s go-time! Usually there will be cells in the incubator that I need to passage; a procedure which lifts them off the bottom of the flask and allows me to move them into 3 new flasks so that the cells have enough room to grow. I also might need to change their growth media, which just involves aspirating out the old media they’re in (which has accumulated cellular wastes) and replacing it with a new media, before putting the cells back in the incubator to grow. These are the most quintessential daily chores as we need cells in order to do research. There are also lots of side-projects that I will have to work on throughout the day. Usually there will be samples of cross-sections or whole bundles that I need to immunostain. This process sometimes takes 4 days, so if there’s a whole bundle stain I will start on Monday and replace them in blocking, Tuesday to add primaries, Wednesday to add secondaries, and Thursday to mount the samples to a slide. This process sometimes takes from 45 minutes to an hour, but is mainly washing steps which just require waiting. Sometimes I’ll also need to create cross-sections which involves putting a whole bundle in OCT (a compound which can be frozen quickly in liquid nitrogen with tissue embedded within). I then use the CryoStat to cut sections of the whole bundle and can immunostain these samples as well. Recently I’ve been working on practicing Tube Formation Assays, a process which allows one to evaluate endothelial vasculature formation. I’ve also been making molds by synthesizing a polymer so that we can grow whole bundles in them. Usually I will finish in the lab at 4:30 – 5:30, and may read a few papers to further my understanding afterwards.
This week I had the fortune of interviewing Dr. Bursac and asking him questions regarding his upbringing and exploration of his academic interests. Growing up, Dr. Bursac loved math, physics, biology, and sports which led him to pursuing Electrical Engineering at Belgrade University in Serbia. He later completed his PhD at Boston University and researched at MIT under Dr. Robert Langer. Afterwards, he completed postdoctoral work at JHU under Dr. Leslie Tung and later became a professor here at Duke, where he primarily researches cardiac disease and heart electric phenomena. When asked about what he liked about scientific research, he commented on how it has a greater cause, requires constant learning, and allows him to interact with other researchers; “It’s super exciting and that excitement is still with me after 25 years”. Dr. Bursac told me that as he spent more time in academia he started appreciating teaching and mentoring more, and how he realized that research is more complex than just science and knowledge and requires good management skills, PR, and networking as well.
As I finished up the interview, Dr. Bursac imparted some timely advice: “Follow this journey with passion and always think about the big picture. Be courageous to try something new and patient with failures as scientific breakthroughs never happen over night. Mentor your students thoughtfully. Work hard and try to have fun!”
In 2006, a Japanese researcher named Yamanaka discovered a chemical concoction which would allow the dedifferentiation of fated cells to once again become induced pluripotent stem cells (iPSCs). This discovery had significant impact in the world of regenerative tissue engineering, as it allowed for the indefinite creation and proliferation of complex cell types cultured from the patient’s own cells. In particular, the Bursac lab under which I am currently researching studies how iPSCs can be used to specify functional synthetic myocytes, both skeletal and cardiac. However, the large roadblock in tissue engineering research currently is that tissue fated from iPSCS are not able to generate as much force and muscular volume as primary myocytes.
The projects I am working on seek to better understand the roles that vascularization and innervation play in the development of functional skeletal muscle tissue, and more importantly, how co-culturing endothelial cells or neurons can allow muscles to exhibit greater forces. As one may expect, working with cell cultures are very demanding and requires a comprehensive set of procedures to ensure the growth and differentiation of each tissue type, as well as the necessary processing in order to understand the capabilities or functionality of the cells.
Although I’m only one week in, I’ve definitely picked up a plethora of laboratory techniques which were necessary in order to cover all aspects of an experiment. I learned how to create 2D and 3D growth media which facilitates the growth of cells within the first week of culture. After this, the cells will spend 2-3 weeks in a differentiation media which causes them to fuse and interact with each other and form tissue. It is during this step where endothelial/neuron cells will interact closely with the formed myofibers and form supporting vasculature or neuromuscular junctions. To understand the state of the muscle fibers at different points of the experiment, the tissue can be used in many ways. One of the most important of course is force testing, in which we rig bundles to a sensitive force guage and electrically stimulate them to understand their impulse responses or tetanus response. Another common way to understand the bundles is to do cross section stains, which requires the use of the cryostat machine I described last week, or whole bundle stains under which the entire skeletal muscle tissue will be imaged. Another procedure would be to use whole bundle RNA isolation which allows for the analysis of RNA expression at some week to better understand the genetic effects the presence of supporting cell types may have on muscle development. Additionally, we may use qPCR to quantify the relative amounts of certain genes and which ones are expressed more than others.
Although it sounds simple, there are inherent limitations to such research. For one, cells are fickle and it is difficult to understand why an experiment may go awry or why cells may behave differently, and these fluctuations can only be mitigated through strict sterilization practices. Additionally, the growth cycles for engineered tissue take weeks to months to produce tissue, which is currently ineffective for clinical application. However, I am optimistic that the research I am doing will allow for us to take one step closer to the goal of quick tissue regeneration and integration to save victims suffering from dire wounds.
I’ve always looked forward to applying the knowledge I’ve gained regarding Biomedical Engineering towards research, and this past week I was finally given the opportunity to do so! At first I was very nervous; was anything I learned in class useful or relevant? However, as I walked in on my first day to the Bursac Lab and began talking with one of my mentors, Ethan, it was like the gears fell into place. Knowledge from classes I’d taken before helped me understand the concepts and topics that I was tasked with knowing, ranging from BIO 221 to BME 221, I realized that many aspects of the classes I’d been spending two years taking were applicable towards the research I would be immersing myself in this Summer. This revelation was both exciting and encouraging to me, as it motivated me to continue on my path to become a Biomedical researcher.
Another aspiration I had for this Summer was to gain hands-on experience with laboratory techniques that I never would in class, and I can definitely say that this first week has more than sufficed. This week I was tasked with creating thin cuts of frozen tissue on a machine called a Cryostat. For about 8 hours this week, my left hand was perpetually maintained at about -20 degrees Celsius, while my right hand turned the wheel to make cuts (I should clarify: I was not cutting my own tissue, just had to handle some within the machine). I quickly learned how difficult it was to work some samples and what techniques to employ to acquire the best imaging results. From starting as a complete rookie, I can now confidently say I’m proficient in the ways of the Cryostat. I surely didn’t expect my first week of Summer to involve sticking my hand into a literal freezer, but it’s definitely better than the sweltering heat of the outdoors, and I’m looking forwards to the other techniques and machines I’ll be exposed to this Summer.