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