Getting Back on The Bike of Research: the Basics of Science Research

This week I moved back to the United States to work at my lab at the NIH for two months before my thesis defense in July. Not only was I in Oxford for 6 months, I was also concentrating on writing my thesis, and didn’t go into the lab except to meet with supervisors. So now I’m back in the lab on a daily basis and doing science. That said, I ‘m currently in the stage of simply accumulating supplies before I can do actually do any experiments.

Originally, the plan had been to simply write up, have my viva, and graduate, without returning back to the NIH. However, my examiners were busy, and couldn’t schedule by defense until July. This gives me some time now to return and try to generate more data. If the data is good, this means that I will have more work to support both my thesis defense and also to help make more coherent papers for publication. Since I’m only going to be back for 2 months, this really limits the scale of experiments that I can do, and certainly rules out completing any new transplant experiments since they take a minimum of 8 weeks to run, with the preparation and analysis times extra. However, I’ve come up with an experiment plan that closely matches what I’ve already done, but to take it a step further in some cases, or to simply do a more sophisticated study to glean better results.

I’m not concerned about coming back into the lab having been away for 6 months. With the exception of one major technique, all others are methods that I’ve done before. And I find that for planning and executing experiments, the logic and the process never change. It’s in line with belief that once you learn to ride a bike, you never really forget. I’ve been seriously studying science for 10 years now, 6 of which have been spent doing genuine research (instead of planned coursework experiments), and over time on learns the basics of experimental logic:

 

1)   How to choose an assay that will allow you to observe the specific factor you wish to

2)   How to minimize distraction by other factors by designing more simplistic experiments

3)   How to use proper controls to make sure that your experiment design is correct, even if the results you get are negligible or puzzling

4)   How to manage resources and time

5)   How to minimize human error (this involves knowing your personal limitations)

 

Over the next two months I’ll keep you informed about what I’m doing, the techniques and reasoning, and also my adventures into paper writing, along with updates on developments in tissue engineering and bone research.

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Thesis Writing: A Hectic March

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Image from http://www.phdcomics.com

I submitted my thesis on March 8th. Submitting the thesis actually much more stressful than I thought (discussed more in my next 2 posts: Tips for Submitting a Thesis). The final few weeks of thesis preparation were very stop and start as I waited for supervisors to get feedback to me and then rushed to get the newest version out, to ensure that they weren’t wasting their or my time by editing sections which had already been changed. Finally, however, I was able to print the thesis, get it bound, and officially submitted it to the university.

Throughout this process I had to file a lot of beaurocratic paperwork with the University including permission to receive dispensation for being in residence. Oxford requires that graduate students be “in residence” in Oxford for 6 trimesters of their DPhil. This generally does not pose a problem, because most students are here for the whole of their studies, but as I was traveling back and forth to  the NIH in DC, this meant that I was only in residence for 5 full academic trimesters (they don’t count all the time spent working outside of term time).  Additonally, I officially set who my examiners were to be, and officially requested that my viva (thesis defense) occur by the end of April (which I thought reasonable given that it was seven weeks after the thesis was to be submitted). However, it became clear that the examiners weren’t available within these time constraints, and neither were the back-up examiners, and therefore the thesis defense has now been set for July 8th – exactly four months after I submitted the thesis.

As an international student this posed a large problem because the question became one of what to do during the interim period. I discussed this with my supervisor at the NIH, and we came to the solution that I would stay and write papers/plan experiments in Oxford until the April (I’d already paid for the rent) and then move back to DC and work at the NIH for 2 months prior to returning to England for the viva.  I’m really lucky to have a supervisor who’s willing to pay for the extra months that I didn’t think I would require to get everything taken care of.

Right after I submitted my thesis my boyfriend came into town for a few days and we took a short vacation to Paris, where he proposed. So now I’m aflutter with wedding planning as well as viva preparation and continued science writing. Currently I’m still living in college in Oxford. I’ve just finished the first draft of my first paper, the topic of which is the syngeneic murine model of ectopic bone formation.

Technique: Chemotaxis Revisited – the Boyden Chamber Assay

Chemotaxis, introduced in my post on January 26, is the process by which cells migrate along a gradient of growth factor. Researchers study chemotaxis for many different reasons. First, there’s the biological study of how these cells move. Then, there’s the study of why these cells move. In my post from September 28, I describe how random movement of cells in response to an even distribution of growth factor can be measured. However, the next step is to measure whether this response can be seen in a directional manner in response to a gradient.

The easiest method, perhaps, for studying chemotaxis is the Boyden Chamber assay. The concept of the Boyden Chamber assay is straightforward. Known as a transwell system, a smaller well is suspended over the main well of a tissue culture plate. However, the bottom of this insert is not solid plastic, put a permeable membrane. Cells are added to the top of this membrane, in solution, and their migration across the membrane to the other side in response to stimuli can be measured simply by counting the cells that have passed through. A great product for this are the FluoroblokTM inserts from BD Biosciences. Cells can be fluorescently labeled, and light from cells on the top side of the membrane won’t shine through to the bottom. The layout of a basic Boyden Chamber Assay is shown (blue dots are cells, green dots are stimuli):

boyden blog

The Boyden Chamber Assay may be simple, but requires a high level of finesse. The first main concern is the use of proper controls. Not only are negative and positive controls important, but also we need to measure the directionality of response to control for random movement. I.e., although, for example, growth factor might be added to the bottom well, and cells to the top, we need to show that the a gradient is actually maintained and that it is not simply a case of growth factor being evenly distributed on either side of the membrane (equilibrium) and cell movement increased in general.

Also, there are several steps in which human inaccuracy can lead to deviation between wells, ultimately resulting in lack of statistical significance because of too much variability. The assay is indeed incredibly sensitive to both the number of cells and the amount of stimulus, both of which tend to be very low, meaning that everything must be incredibly accurate. The first year of my PhD I conducted a gigantic Boyden Chamber assay, but got no significant results, just trends, because the error bars were too large.

The third part of the assay which requires delicacy is the counting of the cells. As I mentioned, the cells are fluorescently stained. However, in order to calculate fluorescence, because the desired cells are on the bottom of the membrane, in order to use spectrophotometry, you’d have to have a bottom plate reader. Our spectrophotometer could not be reprogrammed to do this (although in theory it had the ability). This meant that the membrane had to be cut off and mounted upright before being imaged. The cutting of the membrane is tricky, because it becomes easy to crease, which can lead to problems when trying to mount it flat (it’s very difficult to image a crinkled sample due to focus issues).

The protocol for Boyden Chamber Assays:

  1. Trypsin cells, centrifuge, resuspend in desired medium at desired density
    1. Make sure this cell suspension is evenly mixed
    2. Place cells in the top of the transwell
    3. Place desired medium in the bottom chamber
      1. Make sure the volume of the bottom corresponds to the top volume so no fluid pressure is exerted across the membrane (manufacturer’s recommendations)
      2. Incubate for the desired amount of time (good idea to perform a time-course with controls as a preliminary study)
      3. Gently aspirate medium on top and bottom, wash 3x with PBS
      4. Incubate 15 minutes in 4% PFA in PBS in order to fix the cells
      5. Wash 3x with PBS
      6. Incubate with 1:10000 DAPI in TBS with 0.1% Tween for 5 minutes at 37°C.
      7. Wash 3x with PBS
      8. Carefully cut out membranes and place, bottom-up on slides
        1. Mount with fluorescence-stabilizing hard-set mounting medium
        2. Pressing down to eliminate any air bubbles and make sure membrane is flat
        3. Image at 4x  magnification
        4. Use ImageJ to increase brightness/contrast to max, and convert to binary for quantification

Ten Hundred Words of Science Challenge

I was recently told about the amazing Ten Hundred Words of Science challenge. The challenge is really simple. Try to explain what your scientific project entails, using only the ten hundred (thousand) most commonly used words in the English language.

That sounds easy enough, until you try to use the text editor, and realize that out of the paragraph above there are many many words that would not be allowed: recently, challenge, scientific, project, entails, thousand, commonly, English, language… and SCIENCE. Science is not one of the top 1000 words in English!

This is my project description I ended up writing:

My college paper is about a fix for when a human breaks a leg or other body part. Many people in the world are working on such things. The best way to do this is to come up with a thing, that turns into part of the leg when it is put into the person, instead of trying to make a leg outside of the person and then put it in. To do this we need to focus on having good blood roads quickly, and then good leg can form. We also need to give our pretend leg everything it needs so that it can become real leg over time.

Thank goodness leg and blood was allowed! Neither bone, nor transplant, nor vessels, were allowed, which are very key words in my thesis. In the current thesis draft I mention the word “bone” over 500 times.Above, “college paper” translates into PhD thesis, leg = bone, and blood roads = blood vessels. Oh, and “thing” and “pretend leg” translate to “transplant”.

Are you up for the challenge? Scientific or not, I dare you to attempt to explain what you do in only ten hundred words! The tool can be found here:

The Up-Goer Five Text Editor

And the website to view other people’s scientific projects is here:

Ten Hundred Words of Science

Technique: Chemotaxis concepts – Why did the chicken cross the road?

Why did the chicken cross the road? Well, frankly, who knows what induced the chicken to cross the road. Generally, we can assume that the chicken had a reason. Arguably everything we do in everyday life we do for a reason – whether voluntary or involuntary. Like most other creatures in the world, we respond to our environments.

If you show a dog a bone, and then place that on the far side of the room, the dog will run over to get it. But the stimulus doesn’t have to be food. If a person enters the room, a dog will typically rush over to greet it – though whether that greeting is friendly or threatening depends on many factors.  Lack of movement can also be due to the environment that we sense around us. For example, when I often wake up, I am tempted not to get out of bed, because it Is incredibly comfortable where I am, and, for the time being, there is no specific reason to move.  Therefore:

Movement = response to factors signalling that another place is desirable and/or lack of factors signalling that the current location is desirable

Lack of movement = response to factors signalling that the current location is desirable and/or lack of factors signalling another place is desirable

Like whole organisms, cells behave in response to the environments they sense around them. For example, consider cells in the circulation. While initially one thinks of these cells as being simply pushed around by the physical pressures exerted by fluid dynamics, these cells are actually constantly sensing their environments and can respond by clotting, leaving the leaving the blood system and moving into surrounding tissues, or continuing circulating. In contrast, think of skin cells. These cells stay in one place, because they are surrounded by similar cells and supporting layers of cells that provide the signals to stay in place. However, in response to injury, e.g. a paper-cut, the cells quickly change their entire behaviour and can both move into the wound site as well as signal to the blood and immunological cell types necessary for repair.

Outside of the body, in the laboratory setting, it’s easy to study the effect of each movement signal, also known as a chemotactic factor (chemotaxis meaning movement in response to a chemical signal) because we can add the factors in individually and in combinations to observe their exact effects. In this blog I have already discussed a way to measure random cell movement in response to growth factors (September 28: Calculating Cell Velocity). In my next post I will discuss the Boyden Chamber assay for studying directional cell movement in response to factors.

 

And now, my body is telling me I need to move to a location that provides caffeine. I’m off to get some coffee and get back to working on my thesis.

Technique: The Simplest Way to Make a Collagen Scaffold

Collagen scaffolds are very common within the field of tissue engineering. They are varied in composition as different types of collagen can be used, and virtually anything added s a supplement. In this post I’m going to discuss the simplest method of creating a collagen scaffold, a method that I employed for two out of the three materials I made for my PhD project. The first material was a plain fibril bovine collagen type I scaffold, and the second was the same except with an added hydroxyapatite component. I actually made the hydroxyapatite myself, through the standard precipitation method (topic for a future technique post) in order to eliminate any byproducts present in commercially available product.

Basically, all materials in the final scaffold are mixed together to form a homogenous aqueous solution, which is then centrifuged and vacuumed in order to remove any air bubbles. The solution is then placed in a mold (we used PVC) and frozen at the desired temperature (in my experiments I used -20 degrees Celsius, though in past experiments I have also used -80 degrees Celsius freezers). The samples are then lyophilized (a.k.a. freeze-dried) for a few hours, pushed out of their molds, and then lyophilized some more. Note: beware of removing the samples too early; they dry from the outside, so if moisture is still present in the center they can collapse later. Finally, before use, samples are trimmed and cut as desired. I found that some trimming was necessary as sem micrographs showed walls of collagen on the outer edges, hindering cell and nutrient penetration.

Like any other polymer scaffold, the size and shape of the pores can be easily modified for different desired pore sizes etc. Given a set amount of collagen in an area, pore size is directly controllable by modifying the freezing temperature. At lower temperatures, ice crystals nucleate faster, and therefore the crystals formed will be more numerous and smaller. Since final pore size is directly related to aqueous crystal size, this means that the lower the freezing temperature, the smaller the pores. If the pores are more numerous, this means that the number of walls between pores also rises; but, since the total amount of collagen remains the same, this means each individual wall is thinner. One of the advantages to collagen and this fabrication method is the variation of the scaffold in its micro-porosity under 100 micrometers. It has actually been shown that variation on this scale is more conducive to biological activity, a long as the larger pore structures average the same as an otherwise similar scaffold.

Technique: Calculating Cell Velocity

Most people tend to think of their bodies as being static, with the only moving cells being blood cells in circulation. This couldn’t be farther from the truth. We are truly very amazing dynamic individuals. Even our healthy organs are constantly turning over, with older cells dying and being replaced by new cells. This statement even includes bone. However, in no unique instance is cell movement more important than in the case of wound healing. Just think about your basic scrape. In no time it seems that the bleeding has stopped and the wound has healed. The body does this by immediately sending out signals as soon as the wound occurs, to recruit cells to the site of injury and these all work together in a spatial and temporal fashion to facilitate healing.

Similar processes occur in the process of distraction osteogenesis – a process I will interview my friend Cynthia Chang about in the next technique post, but also in normal bone fracture healing. In the case of my transplants I have seen firsthand the ability to transplant sponges completely devoid of any cells into a mouse, and harvest tissue weeks later that are not only full of cells, but have also created living tissue in these spaces. Where do these cells come from? And how to we get the right cells to come to our transplant?

In order to answer this question I’ve been studying the movement of human umbilical vein endothelial cells (HUVECs) because I want to encourage faster blood vessel formation in my scaffolds. The way that we do this is really simple. We grow cells on a petri dish in a special microscope that allows us to constantly monitor the cells while at the same time keeping them at the proper temperature and carbon dioxide level. Then, instead of taking a video, which would be enormous in file size, we take pictures every 5 minutes to see how far the cells have moved. This method, called “time-lapse” means that when we stitch all these photos together we essentially get a flip-book video of the cells movement – similar to the cartoon flip-books of childhood (I had a Wiley Coyote flipbook).

Once we have this flipbook of images, we can analyze it using the NIH free software ImageJ to individually track cells (Cell Tracking plugin). Then we can use another plugin (Chemotaxis Tool) to measure the directionality (i.e. whether movement is in one direction or truly random) as well as velocity. It’s very simple and gives a lot of data in a really short amount of time, so I would definitely recommend it to any PhD student out there who needs more in vitro data.

Basic Method:
1. Cells are plated at 0.075×106 per 35mm plate in normal medium and left overnight
2. The next day medium is changed to the desired conditions (I did growth factors in just basal medium vs. growth factors in addition to the full, serum-containing serum)
3. Dishes are placed in the time-lapse microscopes, and monitored over 48 hours, pictures every 5 minutes
4. Images are analyzed using ImageJ and statistics performed