Step into Our Research Lab: What Is a CFU?

By Chris Centeno, MD /

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You’ve occasionally seen me mention colony-forming units (CFUs) here on the blog. You’ve also likely seen the acronym in articles and while browsing the Internet, especially if you have an interest in stem cells, as the term “CFU” is thrown around a lot these days. But, what is a CFU? Broadly speaking, a CFU is one method that can be used for research purposes to roughly count the number of viable, or living, mesenchymal stem cells (MSCs) in a sample.

While you can grasp the surface concept by reading about it, seeing it in action really brings it to life and provides a deeper level of understanding. So step into our research lab (watch the brief video above) and meet one of our research scientists, Dustin, as he explains and demonstrates, what is a CFU?

The Lab Technique to Measure CFUs

There are a few observations you will make in our lab as you watch the video. You will see two culture plates, each holding six “wells,” or culture areas. Perhaps the most obvious visual will be the purple stuff in the culture dishes. We’ll explain what this is in a moment.

The technique for culturing cells begins with seeding the culture plates with a low density of bone marrow concentrate (i.e. highly diluted BMC), which simply means placing samples of BMC into the culture dishes. The BMC is the result of isolating the stem cell fraction from the bone marrow samples drawn from the patient. The cells are then placed in an incubator and cultured, or grown, in the dishes for ten days.

As the video above begins, Dustin has just added the purple stuff to the cultures—a dye called crystal violet that will stain viable colonies that dark-purple color. He allows the stain to sit for 10 minutes and then washes the plate surfaces with water. Purple dots will be left in the dishes, and each dot represents a colony of mesenchymal stem cells that are growing on the plastic surfaces—a CFU­ or Colony Forming Unit. The assumption is that each colony corresponded to one healthy MSC in the original sample.

Once we have our CFUs, we can then count the number of dots, and using image-based software, we can get an idea of how densely packed together our colonies are, which will, again, give us a very rough idea of how many stem cells we started with in our sample. So the more CFUs you have, the higher the original stem cell content of the tissue.

CFUs are for Research, Not for Dosing

While the CFU method that estimates the stem cell content is good for research purposes, it wouldn’t be a practical method for dosing same-day stem cell procedures. Why? The most obvious reason is that the sample has to incubate for 10 days, so clearly we wouldn’t be able to draw, process, and reinject on the same day. You also can’t link back to a direct stem cell quantity because only a certain percentage of the cells form CFUs. When dosing for use the same day, we don’t want an average or an estimate of what’s available; we want the most reliable stem cell count possible.

What is a better way to count stem cells for dosing? Flow cytometry is a method that doesn’t require culturing. The cells are instead stained with antibodies that attach to certain markers on their surfaces. These antibodies have a fluorescent dye which is excited by a certain wavelength of LASER light. When these dyed cells are run through a flow cytometry machine, it lines them up like school kids and counts them one by one using a counter. While this could allow for faster counting for a same-day procedure, it takes a Ph.D. or highly trained provider to run the machinery and interpret the results. Hence, it’s not practical for day to day clinic use.

Looking at the Total Nucleated Cell Count (TNCC) is another method of quantifying cellular content in a sample. The TNCC, however, is not the stem cell number itself, as only a small fraction of this number are stem cells. However, the TNCC is a surrogate measure for stem cells. This means that the number of stem cells roughly follows the TNCC number (i.e. the higher the TNCC, the higher the stem cell count). We published a scientific paper a few years back that showed a good correlation between the TNCC and outcome in knee arthritis patients treated with Bone Marrow Concentrate.

It’s imperative that every doctor performing same-day stem cell procedures knows how to count and dose stem cells. All available methods have their pluses and minuses, and you can read more about them and counting and dosing stem cells in depth at this link.

The upshot? What is a CFU? Hopefully we’ve answered that question and now when you see the term “CFU” on this blog, or anywhere else, you have the visual of the method (think dark purple) to associate with it. I hope you enjoyed stepping into our research lab with Dustin and learning more about CFUs and how we use them.

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Chris Centeno, MD

Regenexx Founder

Chris Centeno, MD is a specialist in regenerative medicine and the new field of Interventional Orthopedics. Centeno pioneered orthopedic stem cell procedures in 2005 and is responsible for a large amount of the published research on stem cell use for orthopedic applications.
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