Regenerative Medicine

Bone Marrow Centrifugation: Is it Important?

IS THERE A DIFFERENCE WHEN BONE MARROW IS CENTRIFUGED?Recently I was reading some blogs at a site designed for physicians in the Regenerative Medicine field. The dispute in the blogs was that doctors were trying to compare bone marrows processed in two separate manners. On one side was a group that would take about 60 CCs of bone marrow aspirate from a few sites and than process the marrow by concentrating it by centrifugation. The other side would use bone marrow obtained by a special needle that takes the sample from multiple depths thru the same puncture area. Both sides tried to say their method was the best. There were a number of points made by both groups as to why their data was the correct data and why their technique was the best. I have no special interest in this argument but find it somewhat amusing.
I find that some important points are being missed in these discussions. First off we must realize that what is done in the lab may not hold water as to what is found in real life. In scientific circles we call these conditions in vitro (the lab) and in vivo (the body). We know that any attempts to analyze a cell alters the nature of the cell and thus alters various outcomes of the cells. This information comes from the work of Theise, N.D. (2010Exp. Hematology). Dr. Theise points out that any attempt to analyze a cell alters the nature of the cell at the time of isolation thereby altering any subsequent differentiation events. What one group was discussing was the number of CFUs. CFUs means fibroblast like colony forming units. This is a measure of viable stem cells. But this is not necessarily an accurate measure. Realize this is a procedure that is done in a lab in very favorable conditions for the cells to grow. There are a number of variables that that effect the results. This includes other cells present such as red blood cells. They can inhibit growth in the lab. This is nothing like what we encounter in the real world. So I have to ask myself if this is a valid method of measuring the effectiveness of one type of bone marrow procedure over another. If we just look at numbers this might be an accurate assessment but other factors will surely come into play.
The real test is to see what results we find on patients. I have done all types of bone marrow preparations. I have done multiple bone marrow aspirations with different kits using centrifugation. I have used multiple aspiration sites with centrifugation. I have also done bone aspirations with one stick but getting the bone marrow from different geographic areas in another words different depths. When I did the bone marrow from different depths I would not centrifuge the bone marrow aspirate but use it as is. I would have to say that my results have been better doing the aspiration from different depths with no centrifugation. I have not centrifuged bone marrow in a few years. I put my thinking cap on and tried to deduce why my results might be better. This is not just my observation but that of a number of physicians who have great experience in the bone marrow aspiration field. They have come to the same conclusion that their non-centrifuged bone marrow aspirate seemed to give better results clinically.
Centrifugation has been a concept that has bothered me for many years. By centrifugation we spin a product such as blood or bone marrow aspirate at a high speed to separate out different components. I still do this with the blood to obtain a Platelet Rich Plasma (PRP) product. When we centrifuge the blood we are looking for a Buffy coat which contains the platelets and a few other components. But we are throwing a number of components away. How important these components are is the magic question. I am working on a system to produce a PRP product without centrifugation but it is still on the drawing board.
When it comes to bone marrow this is a different story. It appears that the physicians that are doing centrifugation are taking a very elementary view of things. I suspect there could be some importance to CFU counts but I also suspect that this may not be a major factor. What else could explain why there is a difference between centrifugation and non-centrifugation?
Centrifugation as I previously stated involves separating a liquid into various parts. We must remember one extremely important fact. The body does not centrifuge its components when it achieves repair. In Regenerative Medicine we are attempting to mimic the body’s natural healing process. Yet we use a process namely centrifugation which the body never uses. The question becomes what might we be throwing away when we centrifuge? I suspect we are throwing away some very important regenerative cells.
One cell that I suspect is thrown away might be a Muse Cell. Muse cells were described by Dr. Gregorio Chazenbach and his group from UCLA. Muse cells are “natural cells” present in all connective tissues of the body. They are preexisting pluripotent stem cells that normally reside in mesenchymal tissues such as the bone marrow, dermis and adipose tissue. In the bone marrow, they represent one out of 3000 mono-nucleated cells. Muse cells have unique characteristics that distinguish them from other multi-potent/pluripotent stem cells. They migrate to and integrate into damaged tissues to replenish cells and restore tissue function with high efficiency. Muse cells have a tremendous capacity to function as restoring cells for a wide range of tissues and organs. Muse cells are unique from other types of stem cells due to their high capacity to home into injured/damaged tissue. Perhaps the most important aspect of the Muse cell is that it is resistant to harsh conditions. There are few harsher conditions than a joint in the human body. Muse Cells thrive in these conditions. We have done some flow cytometry studies and found Muse cells present in non-centrifuged bone marrow aspirate in much higher numbers than centrifuged bone marrow aspirate.
What else might be missing in centrifuged bone marrow? It is very possible that exosomes may be thrown away. Exosomes are now coming into their own. The slides below are from one of my talks.

We can see that exosomes are very important in cellular communication which is paramount for stem cell function.

Several studies have reported that mesenchymal stem cells (MSC)-derived exosomes have functions similar to those of MSCs, such as repairing tissue damage, suppressing inflammatory responses, and modulating the immune system. However, the mechanisms are still not fully understood. Compared with cells, exosomes are more stable and reservable and may provide an alternative therapy for various diseases. Research showed that MSCs engraftment and differentiation at injury sites are very low and transient. Currently, it is thought that MSCs exert their therapeutic effects mainly through secreted growth factors. Because exosomes are involved in cell-to-cell communication, some researchers hypothesize that they are the paracrine (paracrine means it effects nearby cells) effectors of MSCs. There is some talk in the future that exosomes themselves may be used to treat disease. I like to describe exosomes as the body’s Fed Ex System. We were throwing away exosomes from the PRP preparation but luckily we have found a way to recover them from the Platelet Poor Plasma. By not centrifuging bone marrow aspirate we are also preserving the exosomes!
The next possible missing link in centrifuged marrow is V cells. These are actually called Very Small Embryonic Like Stem Cells. These were for a number of years thought to not exist. This is still a controversial cell. This was a controversy in medicine which is now waning. V cells were first described in detail by Dr. Ratajczak and his group in 2008. There still is some controversy over these cells but what appears to be the problem is that some of the researchers may not be looking for the proper cell surface marker. By not finding this marker they feel these cells do not exist. A few weeks ago (May 2017) there was an article in the journal “Cell” concerning these cells and the effect a Vitamin A deficiency has upon them. I am a firm believer in these cells. We have used them clinically for a number of years. We have tricks to stimulate them. One thing we know for sure about these cells is that they have a specific parathyroid marker on the cell membrane. Parathyroid hormone has a very significant effect on cartilage. A classic article from Dr. ER Sampson and his group was published in 2011 showed the effects of parathyroid hormone on articular cartilage it is chondroregenerative. It can actually help repair cartilage. A derivative of parathyroid hormone has been an integral part of our treatment for years. Getting back to basics about discarding V cells here is an article that confirms this suspicion. It is from the Journal Stem Cell Developement Jan . 2012
“Very small embryonic-like stem cells with maximum regenerative potential get discarded during cord blood banking and bone marrow processing for autologous stem cell therapy”.
Bhartiya D1, Shaikh A, Nagvenkar P, Kasiviswanathan S, Pethe P, Pawani H, Mohanty S, Rao SG, Zaveri K, Hinduja I.

What can we take away fro the above study I will quote the authors. “The results of the present study may help explain low efficacy reported during adult autologous stem cell trials, wherein unknowingly progenitor stem cells are injected rather than the pluripotent stem cells with maximum regenerative potential”.
What else may be a problem with centrifugation? We must realize that we may be discarding some very important cells that are involved in our immune system. These include macrophages. If macrophages are put into an environment this can make a difference between success and failure. The above slides from a talk concerning macrophages. We can see that they are very important in getting the inflammatory response under control. We also know that macrophages are critical to stem cell homing to the area. Stem cell homing involves calling stem cells to the area. I COULD KEEP GOING ON WITH MY LIST BUT I THINK THE POINT HAS BEEN MADE WE ARE THROWING AWAY VERY VALUABLE CELLS WHEN WE CENTRIFUGE. As time goes on we will find even more valuable cells that are lost with centrifugation.

The bottom line is I will always take a non-centrifuged bone marrow aspirate done with good technique over a centrifuged one until something better comes along. Thanks Dr. P


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