Stem Cell Counting Methods Can Lead To Inaccurate Dosing

By David G. Morrison, Dirk A. Hunt, Isaac Garza, Robbie A. Johnson and Mary Pat Moyer

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.

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).

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.

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.

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.

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.

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.

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