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Stem Cell Counting Methods Can Lead To Inaccurate Dosing

Considerable interest in stem cell treatment of humans and animals for osteoarthritis has occurred in recent years.

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There has been considerable interest in stem cell treatment of humans and animals for osteoarthritis in recent years. Quality and accuracy of the methods of isolation and counting of cells for therapeutic dosing is of great concern to practitioners whether their patients have two or four legs.

INCELL Corporation is a GMP cell therapy manufacturer of human and animal stem cells with extensive experience isolating stem and stromal vascular cells from fat removed from human and many animal species.

Intrigued by the high cell numbers reported by kit/device manufacturers such as MediVet-America (Nicholasville, Ky.), Intellicell Biosciences (New York, NY), and Adistem Ltd. (Hong Kong) in adipose stem cell therapy (5 to 20 million cells/gram1-3) compared to other methods, INCELL staff conducted an independent study to investigate the high apparent yield of stem cells isolated using the MediVet Kit as a representative example.

The cell yields reported for these kits are five to more than 10 times higher than the yields routinely obtained by INCELL from freshly harvested human or animal adipose tissue. These yields are an order of magnitude higher than reported in the literature by most academic researchers (Chung-canine4, Vidal–equine5, Yoshimura–human6). 

Because these high yields are used to support stem cell dosing recommendations and cell banking, there is a risk of improper dosing and/or storage by the practitioner. Furthermore, it is difficult to compare reports on dosing cell numbers when evaluating the literature without understanding the counting methodology.

INCELL scientists designed a comparative analytical study of three canines to evaluate the kit yields using the MediVet Kit as an example of this class of isolation system. A brief overview of the different available cell counting methods, and the resultant cell counts from each follow:

The Cellometer (Nexcelom Biosciences, Lawrence, Mass.) is cited commonly for cell counting. It uses a two-part dye that counts by staining DNA in live cells green with acridine orange (AO) and dead cells red with propidium iodide (PI) at the same time. The default settings on the Cellometer grossly overestimated mean SVF counts compared to all other methods we investigated (Figure 1).

Adistem and MediVet also state they add an emulsifying agent to the kits to assist in cell release and Intellicell uses ultrasound energy for cell release. Emulsifying agents, when mixed with water, can form myriad small fat droplets, often called micelles or liposomes and the Intellicell system actually uses a commercial liposome creation device.

 

Stem Cell Lab vs. In-house Systems
By Dr. Tom MacPhail, Deland Animal Hospital

Owners ask for stem cell therapy. You don’t want to send them to someone else. This leaves you with the task of deciding whether to do the processing inside your clinic or send it outside to a dedicated stem cell processing lab.

Here are some things I learned as I made my way through the task of comparing the different methods to make the decision that was right for my practice.

In-house systems would seem to be convenient and possibly more economical. The reality, however, is that in-house systems require a lot of money up front for special equipment and then more money to purchase the actual kits. They also require a lot of time from a dedicated technician (usually your best one) and a dedicated clean space in your clinic.

Minimal training is provided and once you have purchased the equipment, you do not have much contact or support to back you up as you practice stem cell therapy.

With the in-house systems, there is no way to accurately assess the cell solution to know what is being injected into the patient. Arthritis is a progressive disease. It is not cured by stem cell therapy, but many dogs only need injections every one to two years.

The in-house kit systems do not provide a way to know how many cells to send to their storage lab, a process that is quite time consuming and costly. Otherwise you have to re-collect fat every time you want to retreat a patient.

The INCELL study was performed using an in-house system touted as providing cell counts that are much higher than any other reported cell numbers. Itshowed that the reported numbers are not actual nucleated cells. In addition, the “activation” actually reduced the number of viable stem cells.

In comparison, I have used the Vet-Stem central lab in Poway, Calif. It provided a no-cost credentialing program (RACE approved) to educate me and my staff, so that I, in turn, can educate my pet parents.

There are no upfront fees. I merely pay for a case when I do a case. I don’t have to use my practice budget to invest in anything! I am charged at the point of service, exactly when I charge my clients, not before I have even identified a case or how many I may do in this economy in my location. Including the cost of the in-house equipment, the cost of each kit and the cost of my technician time, Vet-Stem’s charge to me is very competitive.

Vet-Stem has a state-of-the-art facility with technicians dedicated to processing adipose for stem cell isolation and cryopreservation, all day every day. Cell counts are done using the Nucleocounter, the instrument recommended in the aforementioned study. Cells that are not required for the initial treatment are stored for future use. Vet-Stem can also process up to 10 times the amount of fat, leading to a much larger yield for use now, and for storage for the future.

Vet-Stem has authored or coauthored 10 peer-reviewed papers: three in dogs, three in horses, one with dolphins (a Navy project) and three in conjunction with human immune-mediated diseases. It has experienced technical veterinarians to consult on my tricky cases, a knowledgeable customer service team who answers the phone (no phone tag), a dedicated marketing department that gives me counter cards, posters, owner brochures and helps me with press releases (all at no cost) and even helped me develop a webpage!

In the end, I found that Vet-Stem was right for my practice. The doctors and owners at this clinic expect the best science and treatment modalities; we have found that in Vet-Stem’s central processing lab. It was a matter of good science and knowing what I was injecting into my patients. I wanted to provide stem cell therapy to my clientele but I didn’t want to be (or fund) a stem cell lab. I prefer to leave that to the experts!

Vet-Stem has provided stem cell services for over 8,000 animals to date. It makes me feel like it can really be a partner in my practice, rather than just selling me some equipment and leaving me on my own.

The problem with using AO staining as an indicator of living cells is that background lipid micelles autofluoresce green and are detected as AO stained cells. Using recommended settings, the Cellometer counts particles much smaller than the average SVF cell as “cells,” including these micelles. As part of the reagents cross-check, Solution E (the presumed emulsifying agent) in the tested kit was evaluated without adding any adipose tissue. The Cellometer counted the lipid droplets as “cells” (Figure 1), leading us to conclude that the source of the highly exaggerated counts is the Solution E micellular structures generated by emulsification. In order to completely differentiate these background lipid particles from live cells in a stem cell product, methods to identify cell nuclei are required.
 
Coulter-type counters: An alternative method often recommended to users of these kits/devices is cell counting on a clinical hematology analyzer. Most use a Coulter counter method that measures the impedance as cells pass between electrodes. Cells and micelles (e.g., from Solution E) have a similar impedance, leading to the high cell count seen in clinical Coulter type counters. A Heska HemaTrue counter was evaluated in this study (Figure 1).

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Nucleocounter: The most common automated counter used with SVF cells is the Nucleocounter (ChemoMetec; Denmark).  This counting is based on staining cell nuclei with Propidium iodide (PI) dye. The Nucleocounter uses a two-stage process to achieve a viable SVF cell count.  Nucleocounter counts are not greatly affected by the presence of micelles from Solution E (Figure 1).
 
The cell counts from the Nucleocounter are more in line with the manual hemocytometer counts and more closely reflect the outgrowth of live cells in culture as colony-forming units (CFU below). The Nucleocounter method has been reported in the literature by human adipose stem cell companies (e.g. Cytori, San Diego, Calif.) and by at least one veterinary stem cell company (Vet-Stem Inc., Poway, Calif.) as being an accurate automated counting method for SVF cells.

Hemocytometer: SVF cells were also counted manually using a hemocytometer with a combination of DAPI (nuclear stain) and Trypan Blue (dye exclusion by viable cells) staining by overlaying the images on a fluorescent microscope and counting the DAPI stained nuclei in cells that excluded Trypan Blue (Figure 1 and Figure 2). This provides an approximation of the true nucleated, live cells in a population. 

This is the standard manual method to visually reduce the errors of counting non-cellular materials as cells.  However, it requires considerable training and experience to clearly distinguish cells and takes considerable time for each sample.

Colony-Forming Unit Assay (CFU): Comparative CFU assays were also done as a measure of the number of stem cells in the population. In this assay, SVF cells are placed in culture plates and allowed to attach and grow into visual colonies (also known as CFU-F assay). While only a subset of viable cells in the SVF will attach to the plastic dishes and form colonies, the CFU assay provides a good index for growth capacity.

The outgrowth of “renewable stem cells” measured by CFUs at about 1 percent clearly showed that the Cellometer counts were greatly exaggerated (Figure 3). Since the Nucleocounter and DAPI cell counting methods showed, respectively, 5X and 16X higher CFU counts than the Cellometer, the Cellometer counting method significantly overestimates the number of cells compared to counting methods based on the presence of cell nuclei.

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This error may result in improper doses calculated from drastically overestimated cell numbers and would result in a very low cell count for cryobanking.
 
The authors recommend the Nucleocounter, or similar equipment, as more acceptable for automated counting. The manual hemocytometer method is the most accurate, but requires a highly experienced cell biologist or technician to make accurate counts and would not be recommended for routine clinical use. 

Additionally, INCELL scientists were curious about whether the light activation system, reported by Adistem2 and MediVet1 to increase cell growth, actually had that effect. Figure 4 clearly shows that the light activation did not increase the number of stem cells or their comparative growth as measured by CFU% assay.

In fact, all three animals showed a reduction in CFU% after the exposure to the light activation system (Figure 4).  Whether this treatment is initiating cell death pathways cannot be determined from this study.

In summary, the authors caution that great care must be taken when using kits and automated cell counting for dosing and cryobanking. In this study, the Cellometer overstated the real cell count by 5 to 16 fold. Hopefully, this study will lead to more accurate counting being reported in the literature and proper counting for dosing stem cell therapy patients.

 

 

Figure 1.
Mean Nucleated Cell Counts per gram of adipose tissue are shown for four different counting methods. Additionally, the Cellometer count for Solution E without any cells is shown. The Solution E count accounts for nearly 75 percent of the inflated Cellometer SVF counts and demonstrates that the majority of the counts shown by the Cellometer are not cells. When the NucleoCounter was used to count Solution E (data not shown), it found no cells because of a lack of any nuclei to stain with PI. Using the Cellometer with the default settings, the machine was fooled into counting the micellular bodies as cells because it was unable to differentiate between the green autofluorescence of the micelles and the green AO staining that would have been present if there were any cells in the sample. Overall, the Cellometer reported a cell count approximately 5 to 16 times higher than the real viable cell count.

 

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Stem Cell Counting Figure 1
Figure 1.

 

 

Figure 2.
Micrograph of isolated SVF cells by one of the aforementioned methods shows what looks like a dense covering of cells. However, most of the small cell-like structures are lipid droplets from the added Solution E and these are carried into the cell preparation. It is important to note that these micelle bodies also have green autofluorescence and would appear to the Cellometer as countable units, which explains why this cell count is higher than industry or academic reports.

 

Micrograph of isolated SVF cells
Figure 2.

 

 

Figure 3.
Nucleated cell count per gram of adipose tissue comparison of the Cellometer counts and the actual colony-forming unit assay counts. This graph demonstrates that the vast majority of the particles counted by the Cellometer were not able to grow in the CFU assay.

 

Nucleated cell comparison
Figure 3.

 

Figure 4.
Colony Forming Unit assays showing the estimates of stem cell content/activity from a sample taken before and a sample taken after the MediVet light activation step. Data show that exposure to the light activation system resulted in a reduction in the number of colonies growing in all three canine samples. The average reduction was approximately 34 percent.

 

Colony Forming Unit
Figure 4.

 

REFERENCES
1. http://www.MediVetlabs.com/cellcounts.html; accessed June 21, 2012.
2. http://www.adistem.com/science-and-technology.htm
3. http://www.intellicellbiosciences.com/intellicell-facts.html
4. Chung D, Hayashi K, Toupadakis A, et al.  Osteogenic proliferation and differentiation of canine bone marrow and adipose tissue derived mesenchymal stromal cells and the influence of hypoxia.  Res Vet Sci, 2010; 92(1):66-75.
5. Vidal MA, Kilroy GE, Lopez MJ, Johnson JR, Moore RM, Gimble JM. Characterization of equine adipose tissue-derived stromal cells: adipogenic and osteogenic capacity and comparison with bone marrow-derived mesenchymal stromal cells. Vet Surg, 2007; 36:613–622
6. Yoshimura K, Shigeura T, Matsumoto D, et al:  Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirate.  J Cell Phys, 2006; 205:64-76.

Disclosures:  This study was funded in part by a grant from Personalized Stem Cells Inc., Ramona, Calif. INCELL is not involved in the veterinary stem cell business, but does provide stem cell services to the human stem cell industry.

This Education Series article was underwritten by Vet-Stem Inc. of Poway, Calif.

 8/3/2012 4:33 PM

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