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

Autumn: Status Check

So here it is, the end of summer. The beginning of Autumn. I’m leaving the NIH and moving back to Oxford November 1. This is a rather arbitrary date, but then again, as I’ve come to learn, many things in the life of a PhD student are arbitrary. For a long time the date was October 1st, but then a shipment of mice back in May was backordered by several weeks, so it got moved back a month.

November 1. 39 days.

So far, everything seems to be reasonably under control. I learnt how to do qPCR last week so there will be a lot qPCR stuff. And the last big surgery was done on Thursday of last week, with the DIVAA transplants coming out in 2 days. Oh, and my final regular transplants were embedded yesterday and will be sectioned tomorrow. Finally, everything seems to be coming together.

Except for the staining. In order for my grand PhD scheme to work I need to stain and quantify the amount of blood vessels in transplants. So far, I haven’t yet gotten this to work, and it’s imperative that it starts to work soon. Because I intend to retroactively stain all the slides I’ve made during my. This quantity to get through is really large, and will take time to stain, image, quantify, and analyze. The staining is the one huge hurdle to my success. I may have to take some of it to Oxford to finish there, which goes against my plan of just writing and not doing any bench work in Oxford – but hopefully I can finish before then.

So… I’ll keep updating the blog regularly – the next blog post is going to be about how to measure the velocity of cells. Please also check on me on Twitter (@makingbones) – starting October 1st I’ll be tweeting daily on progress in the countdown towards the big move!

News in Tissue Engineering: Growing Meat in a Petri Dish?

BBC: How growing meat in a petri dish may be the future

This is an excellent video made by the BBC in talks with Professor Post at Maastricht University, which addresses many of the key issues. Briefly, there are some very important issues to bring up.

Macro-impact:
o Growing meat in the current way has huge environmental effects based on the amount of food being fed to animals, and the amount of waste being put out
o Not discussed: the effects on the economy in transitioning from an agricultural system to a biological factory system.
o Also not discussed: what would happen to all the waste from a meat factory?

Taste:
o No one knows exactly why meat tastes the way it does.
o Fat, which is believed to give flavor to meat from an animal, would have to be grown separately and mixed in, and there is no saying that fat from a dish tastes like fat from an animal. Back to square one.
o Texture was not directly discussed in the video, though as it appears that Professor Post is applying mechanical pressure to cells (conjecture based on an apparatus I observed in the video); this would lead us to the conclusion that he is actually producing muscle tissue and not just layers of muscle cells – an important distinction.

Scale
o Professor Post rightly talks about the vascularization issue, which I talked about in my tissue engineering post on August 5 – it’s hard to grow a thick chunk of meat in a dish because of the inside-outside issue.
o Professor Post suggests bioreactor systems, but these still produce many thin layers that would have to be pressed together to form a steak (a bit like a really thick version of chicken slices seen at the deli counter).
o When Professor Post talks about the $250,000 burger, he’s almost certainly going to take many of these smaller pieces and put them together to get the final product
o Scale is also a problem because, although cells can be exponentially expanded in culture, as discussed in my August 11 post on passaging cells their characteristics change with each passage out of the animal. (I presume he is not talking about using immortalized cell lines because he intends to keep donor animals.)

Summary: Professor Post is working on a project that, for obvious reasons, gets a lot of attention from mainstream media. He is obviously well aware about the limitations of his project, but is taking a practical approach to getting it done right. It’s a great theory, and deserves investment, but we shouldn’t expect to be eating faux meat (“in vitro meat”) for years to come.

Technique: Slicing Bones

Have you ever read one of those stir-fry recipes that calls for freezing the meat for a few minutes to that you can easily slice it really thinly? As a cook, I see that advice, but then often ignore it because I’m too impatient. As a result, the meat tends to squish as I try to slice it (with my less than super-sharp knife), and I’m left with pieces which are uneven in thickness, and even at their best, are thicker than really should be used in a stir-fry. But how does one cut through engineered tissue? When one is talking about slicing one’s samples for analysis, those are the results you’ve worked long and hard to create, and no corners should be cut (pun intended).

You can’t just slice willy-nilly and eat the results. I don’t even want to think about eating tissue engineered meat – that’s a blog post for later this week.

One method is indeed to freeze it, that’s called “cryo-sectioning”, “cryo” actually being Greek for “icy cold”! Another technique is to embed the tissue in plastic and then cut it. Ever tried to cut plastic accurately? Only a small handful of technicians in the US are experts in that. But let us think about the reasons why the stir-fry recipe tells you to freeze the meat. As the meat freezes it becomes harder, and more stable; hence, easier to cut.

What we can do is embed the samples in wax. Wax infiltrates the entire tissue (if it’s soft) and stabilizes it, making it possible to cut thin slices without any squishing. And the slices that we can cut are super-thin! A microtome could slice a human hair. Length-wise. This means that we can very precisely see what is going on in the tissue that we have made.

 

To skip a lot of the science jargon, the process is basically thus:

1. The tissue is slowly transitioned to being in a wax solution.
2. The tissue can then be placed in a mold with more wax and let to cool.
3. The mold is then removed and the block can be cut

Ever seen a block of amber with a fly or something prehistoric in it in a natural history museum? That’s what this is like, except unfortunately the samples don’t look nearly as cool as flies. And they’re in wax, not amber; so aren’t worth much unless you’re the scientist that made them. My precious…….

So there you have it! The non-scientist’s version of how to slice bone.

Thesis Writing: Backing Up

Every PhD student, towards the end of her research, becomes just a tad (or very) paranoid that they’re going to lose all their research. Back in the day before computers, students had lab notebooks with all their results and doodlings, along with photocopies of relevant papers. If you go not too far back (only about 30 years ago!), we reach the realm of typewriters, and professional medical media artists. These days the student has word documents with all their plans and proposals, powerpoints from seminars and poster presentations, and countless PDF files of scientific papers. The medium may be different, but the concern is still the same.

What happens if I lose all my research?

What if the building catches on fire and my papers burn up?
What if the professional typewriter loses my copy?
What if my computer crashes?
What if someone deletes my files from the shared computer?

So what’s the solution? The solution is to back up.

We are often told that. But what does backing up mean?

I used to transfer my files between computers on an external hard drive. This meant I had all my files on both my work and home computer and the external hard drive. This worked until instead of working on the actual computer and then transferring files between computers I decided it was easier to just keep the most recent copy on the external. Soon I was only using the external and my computer files were a few months out of date. Then, one day, the external got knocked off a table and broke when it hit the floor. The files had to be restored by a technology company for $1600. This, obviously, was not what was meant by “backing up”.

After the incident when my lab paid $1600 to get a few months of data back, I started using “the cloud”. I started using Dropbox, because I realized I needed a way of syncing my files between computers, without relying on an external or USB. The bonus with Dropbox is that the synced files are also stored on the hard drive of computers with the program. This means I have the most up to date files on 2 different computers, should the online system go down for whatever reason. Additionally, I back up once a month to a “shock-proof” external hard drive; perfect, giving the precedent I set. The cloud is amazing. I can work on my files from multiple computers without the need to carry stacks of papers (the old-fashioned way) around with me, or even just a USB stick (the less-old-fashioned way).

I do have one special backup method for my thesis write-up. A USB necklace. If the internet dies, my hard-drive gets smashed by a bulldozer, and both of my computers go up in flames, I’ll still have my thesis around my neck.

That’s my thesis insurance.

Image from http://www.phdcomics.com