David G. Morrison, Dirk A Hunt, Isaac Garza, Robbie A. Johnson, Mary Pat Moyer*
INCELL Corporation LLC www.incell.com
12734 Cimarron Path, San Antonio, TX 78249
*Corresponding Author Contact information: mpmoyer@incell.com; 210.877.0100
INTRODUCTION
There has been considerable interest in stem cell treatment of humans and animals for osteoarthritis and other conditions 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 tissue derived cells and has extensive experience isolating stem and stromal vascular cells from fat removed from humans and many animal species.
INCELL is actively evaluating process methods to improve quality and quantity of cell therapy preparations, such as mammalian stromal vascular fraction (SVF) including stem cells isolated from adipose tissues. Intrigued by the high cell numbers (5 to 20 million cells/gram1-3) reported by kit/device manufacturers such as MediVet-America (Lexington, KY), Intellicell Biosciences (New York, NY), and Adistem, Ltd. (Hong Kong) in adipose stem cell therapy compared to other methods (e.g., 4-6), INCELL staff conducted a research study to investigate the high apparent yield of stem cells. This initial work was focused on SVF cells from the MediVet Kit, which is marketed to isolate adipose-derived canine SVF and stem cells.
The cell yields reported for the Medivet Kits are five to more than ten times higher than the yields routinely obtained by INCELL from freshly harvested human or animal adipose tissue using our adipose tissue processing methods. These yields are also tenfold or higher than those reported in the literature by most academic researchers (Chung-canine4, Vidal–equine5, Yoshimura–human6). Since these cell counts are used to support stem cell dosing recommendations and cell banking, it is important to better understand why the cell numbers are higher. If the numbers are accurate, this would be process improvement, but if they are inaccurate, then there is a risk of incorrect cell numbers that will impact therapeutic dosing by the practitioner and/or biorepository storage. Particularly important are the numbers of renewable, replicating cells that grow out in culture as attached fibroblastoid (F) cells and are quantified colony-forming units (CFU-F). When recommended processing methods and commonly used counting methodologies are compared to CFU-F results from the same processed tissue counting errors are evidenced as differences in CFU-F numbers.
A comparative analytical study of three dog donors of adipose tissue was designed to evaluate the cell yields using the MediVet Kit as an example of this class of isolation system. All kit procedures were followed as per the instructions provided. A brief overview of the different cell counting methods used, and the resultant cell counts, observations and explanations of the results observed, are described below.
INCELL White Paper: Stem Cell Counting Methods Can Lead to Inaccurate Dosing
METHODS AND RESULTS
The Cellometer (Nexcelom Biosciences, Lawrence, MA) is commonly used for cell counting and is recommended for use by MediVet. 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 problem with using AO staining as an indicator of living cells is that background lipid micelles auto-fluoresce green and would be detected as AO stained cells. Emulsifying agents used in these methods (such as Solution E in the kit), when mixed with water, can form a myriad of small fat droplets, called micelles or liposomes. Figure 1 shows that kit-recommended settings on the Cellometer overestimated mean SVF live cell counts compared to other methods investigated. The higher Cellometer counts are at least partially explained by the machine counting the micelles. As part of the reagent cross-check, Solution E (emulsifying agent) from the tested kits was evaluated without adding any adipose tissue or SVF. The Cellometer counted the micelles as “cells” (Figures 1 and 2), leading to erroneous cell counts due to the Solution E micellular structures. In order to completely differentiate these background lipid particles from live cells in any cell therapy product, methods to specifically identify cell nuclei are required.
Figure 1. Mean Nucleated Cell Counts per Gram of Adipose Tissue
Legend: Mean (+ SD) nucleated cell counts per gram of adipose tissue are shown for four different counting methods from 3 dogs, with counts in triplicate. Additionally, the Cellometer count for Solution E without any cells is shown. The Solution E count accounts for nearly 75% of the inflated Cellometer SVF counts and demonstrate that most 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 AO dead/alive program recommended by MediVet, the machine counted micelle 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 manual cell counts.
Coulter-type Counters. An alternative method recommended in the instructions to the kit is cell counting on a clinical hematology analyzer. Coulter counter methods measure the electrical impedance as cells pass between electrodes. Cells and micelles would have 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). That work was done off-site and immediately at a local veterinary practice within 0.2 miles of the INCELL offices.
NucleoCounter. A common automated counter used with SVF cells is the NucleoCounter (ChemoMetec; Denmark). This counting machine and method is based on staining cell nuclei with PI dye. The NucleoCounter uses a two-stage process to achieve a viable SVF cell count, and counts are not greatly affected by the presence of micelles. 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, CA) and by at least one veterinary stem cell company (Vet-Stem, Inc., Poway, CA) 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 (blue wavelength fluorescent nuclear stain) and trypan Blue (dye excluded by viable cells) staining by overlaying the digital light images and DAPI fluorescent images taken on a fluorescent microscope and counting the DAPI-stained nuclei in cells that excluded Trypan Blue (Figures 1 and 2). This provides an approximation of the “true” nucleated, live cells in a population. This is the method used to visually reduce errors of counting non-cellular materials as cells. However, it requires specialized expertise and training, and an imaging fluorescence microscope to clearly distinguish cells. It also takes considerable time for each sample.
Figure 2. Micrograph of Isolated SVF cells by the MediVet Method
Legend: This photomicrograph (bar=200 microns) shows the final SVF preparation from the MediVet kit process and what looks like a dense covering of cells. most of the small cell-like structures, however, are lipid droplets or micelles. These are carried into the cell preparation, have green autofluorescence and appear to the Cellometer as countable units, which may explain why the reported cell numbers from this method are higher than other industry or academic reports.
Colony-Forming Unit Assay (CFU). assays were also done as a measure of the number of stem cells in the population. In this assay, a defined number of SVF cells were placed in culture plates and allowed to attach and grow into visual colonies (also known as a “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 proliferative potential. Thus, the outgrowth of renewable stem cells as measured by CFUs comprises a fraction of the population. As expected, there was variability between animals and counting methods. The highest percentage was 8 to 10% of the cells seeded in the NucleoCounter counts and in all cases the Cellometer CFUs were lower for each dog tissue donor and overall (Figure 3). The differences in CFUs between methods were statistically significantly (p<0.05) in all animals and in the overall composite results comparing the NucleoCounter and the Cellometer. The lower CFU observations did not correlate with higher numbers of cells (Figure 1). This led to the conclusion that the cell numbers in the Cellometer are an overestimate, since the source tissue processed SVFs which were used for the counts are the same.
Figure 3. Cell Counting Methods and CFU-F of Seeded Cells
Legend: SVF cells were obtained for dog donor fat samples (N=3; 3 replicates) processed according to Medivet kit instructions. The resultant SVF cells were counted by a variety of methods. Cells were seeded into complete MSC culture medium for CFU studies with cell numbers for seeding designated according to two of the counting methods used: NucleoCounter (blue) and Cellometer (brown). After the colonies formed, they were counted and calculations were done to determine the Mean +/- SEM numbers of CFU-F per 200,000 cells seeded. These data were calculated for each individual dog and for All pooled data with each counting method. Statistical analyses (Students t tests for NucleoCounter vs. Cellometer) between the 2 counting methods showed statistical significance at p<0.05 for the individual dogs and overall.
Light Activation. As part of evaluating process improvements, Platelet Rich Plasma (PRP) in concert with the Medivet light activation, as per the kit instructions, was used to assess the increase of cell viability and the ability to form colonies in CFU assays as reported by MediVet1. Figure 4 results clearly show that light activation did not increase the number of stem cells or increase their ability to proliferate as measured by CFU assays in this study. In fact, 2/3 dog donors showed significant reductions in percent CFU after the exposure to the light activation system, whereas Dog #2 was essentially unchanged (Figure 4). Reasons for the variability and this unexpected result were not investigated further.
Figure 4. Colony Forming Units and Light Activation
Legend: Percent CFU-F assays show estimates of stem cell content/activity from a sample taken before (left columns) and a sample taken after (right columns) the MediVet light activation step. Data show that exposure to the light activation system resulted in a reduction in the mean number of colonies in Dogs #1 and #3 and no real difference in Dog #2. The average reduction in %CFU across all groups was approximately 34%.
SUMMARY
This study shows that incorrect counting of adipose derived SVF cells and the subset of regenerative stem cells can subsequently result in inaccurate dosing, both in direct therapeutic applications and in cryostorage of cells for future use. The DAPI-hemocytometer cell count (manual) was considered the most accurate, but there are various sources of technical difficulties that can lead to incorrect cell numbers. The nature of adipose tissue itself with variability in dissociation by enzymatic digestion can all contribute to the outcomes. Fat tissue has a propensity to form acellular micelles and oils upon tissue disruption. Processing methods or reagents (e.g., Solution E or lecithins) can generate micelles that may be erroneously counted as cells. Autofluorescence and dye trapping or uptake by the micelles can lead to very high inaccurate cell counts when automated cell counting is used.
In this study the most inaccurate counting came from the Cellometer. When used according to kit-recommended guidelines and on-site training provided by Nexelcom for counting cells by the MediVet procedure, the Cellometer overstated the DAPI-hemocytometer cell count by up to 20X or more. The Coulter Counter protocols also led to incorrect, high cell numbers. Although the cell counts were still a bit high, the authors recommend the NucleoCounter, or similar equipment, as more acceptable for automated counting. The manual hemocytometer-DAPI method is the most accurate, but requires a highly experienced cell biologist or technician to make accurate counts and is not suitable for routine clinical use.
The SVF was shared among the counting and test methods. Thus, the post-processing numbers of stem cells would be the same and cell numbers should directly correlate with CFU-F per cell numbers seeded if the cell counts are accurate. Lower CFU numbers than expected from the cell counts would directly demonstrate an error in the cell counting method. To that end, the CFU-F results for the NucleoCounter showed 2.5X to about 20X higher CFU-F than the Cellometer leading to the conclusion that the Cellometer counts are high and incorrect. Also, in this study the percent CFU was reduced or there was no change after light activation, demonstrating no benefit or a detrimental effect of this step. Significantly fewer cells suggested that a death pathway may have been induced by the light treatment in 2 of 3 dog donors, but further studies are needed to clarify the proposed mechanisms of action and the controlling factors of the outcomes. These might include clinically relevant features of the donor dogs, or equipment or technical issues.
Other companies also have claims of very high cell numbers when their processes are used. Adistem2, like MediVet1, states they add an emulsifying agent to their kits to assist in cell release, and they also use a light activation system. Their kits were not tested in this study but it is possible that the high cell numbers reported by Adistem are also incorrect and result from the same problems highlighted in this paper for the MediVet procedure. Ultrasonic energy, which is commonly used to manufacture micellular liposome structures and to disrupt and lyse cells, is another potentially problematic procedure for counting and verifying viable, regenerative cells. Intellicell3 uses ultrasonic energy to release cells from adipose tissue, and it is possible that resultant micelles or cell fragments contribute to the higher than expected cell numbers. This assumption could be verified with additional studies.
In summary, the authors caution that great care must be taken when using kits and automated cell counting for stem cell dosing and cryobanking of cells intended for clinical use. Overestimated cell numbers would be a major confounding source of variation when efficacy of stem cells injected are compared as doses based on cell number and when cryostored cells are aliquoted for use based on specific cell numbers as a treatment dose. Hopefully, this study will lead to more reproducible counting and processing methods being reported in the literature, more inter-study comparability of cell doses to clinical outcomes, more industry diligence to support claims, and more accurate counting for dosing stem cell therapies to patients.
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 performed as independent research by INCELL staff, but was funded in part by Personalized Stem Cells, Inc., Ramona, CA and VetStem, Inc. INCELL is not involved in the clinical veterinary stem cell business, but does provide services for veterinary and human R&D, contract manufacturing, and stem cell services to the human stem cell industry.
