Counting of mesenchymal stem cells
in regenerative medicine
The importance of mesenchymal stem cells in regenerative therapies
MSCs are used for the treatment of different diseases, mostly focused on bone diseases, cardiac diseases, musculoskeletal disorders and disorders affecting the blood system. These cells can be generated from a treated patient (autologous) or from genetically distinct individuals (allogeneic). Massive interests in studying MSCs stems from the discoveries that these cells can be differentiated into bone, fat, cartilage, muscle and neurons1-3 and are therefore interesting candidates for regenerative therapies. Potential clinical applications for MSCs transplantation range from tissue regeneration4, 5 to the therapy of immune disease6 such as graft-versus-host disease. Furthermore, MSCs secrete anti-apoptotic and angiogenic cytokines and growth factors which can support the healing process after myocardial infarction7 or in wound healing8. A valuable source of MSCs comes from bone marrow aspirates, liposuction of adipose tissue, umbilical cord blood, placenta and Wharton´s Jelly (Figure 1).
Figure 1. MSCs in allogeneic and autologous cell therapies. Adipose-derived MSCs can be isolated from the stromal vascular fraction (SVF) after liposuction, whereas another valuable source of MSCs stems from bone marrow aspirates. MSCs may be directly used or further expanded and used after long-term storage for autologous or allogeneic therapies. During all these different steps of production, cell count and viability can be reliably determined using the NucleoCounter® instruments.
Accurate counting of adipose-derived mesenchymal stem cells in lipoaspirates
Adipose-derived MSCs are highly proliferative and can be easily isolated from the stromal vascular fraction (SVF) after fat biopsies and liposuctions9-11. This fraction contains a bunch of different cells, e.g. MSCs, stromal cells, endothelial cells, adipocytes, erythrocytes as well as lipid droplets and micelles12. For cell therapeutic applications, the SVF can be directly used for patient treatment. It is crucial to precisely quantify the total cell count and viability for further applications as e.g. seeding the culture. Many counting techniques fail to discriminate between cells and artifacts or will give high variations between different users. To overcome this problem, the NucleoCounter® instruments offer an effective protocol to determine cell count and viability of adipose-derived MSCs13-18.
For determining the total cell population of the SVF with the NucleoCounter® instruments, the cell suspension is treated with Solution 10 (optional: reagents A100 and B), lysing cells and other membrane enclosed particles for cell nuclei staining with DAPI e.g. using the Via1-Cassette™ (Figure 2). Due to the specificity of DAPI binding to DNA, NucleoCounter® instruments only count cells containing nuclear DNA and thereby avoid counting artifacts often observed in the SVF, such as cell fragments, micelles, microvesicles, fat droplets as well as cells without DNA content like erythrocytes and platelets.
Figure 2. Precise determination of cell count and viability from the stromal vascular fraction (SVF). Beside different cell types, the SVF contains artifacts such as micelles, fat droplets, erythrocytes and microvesicles which interfere with the measurement. The addition of Solution 10 (optional: reagents A100 and B) lyses the cells, bringing the nuclei into suspension. The total number of nuclei will be stained with DAPI and detected by the NucleoCounter® instruments, resulting in the total cell count. In the second step, the non-viable cells are stained with DAPI without any pretreatment.
Precise counting of bone marrow-derived mesenchymal stem cells
For isolation of bone marrow-derived MSCs, bone marrow biopsies or aspirates are taken19, 20. The determination of cell count and viability of MSCs directly from bone marrow samples is especially challenging, as these contain great levels of erythrocytes, which interfere with conventional automated and manual counting methods. For easy and reliable counting of MSCs directly in bone marrow aspirates21, 22 the NucleoCounter® instruments offer a fast protocol to cope with erythrocytes in the sample (Figure 3). By adding a blood lysis buffer to the diluted bone marrow aspirate, erythrocytes will be lyzed and the hemoglobin diluted. Afterwards, the sample is stained with the two fluorescent dyes Acridine Orange and DAPI for analysis.
Figure 3: Easy counting of nucleated cells in bone marrow aspirates. By adding Solution 17 to the bone marrow aspirate and allowing for a short incubation, erythrocytes will be lyzed, leaving only the nucleated cells to be stained with Acridine Orange (all cells) and DAPI (dead cells). Afterwards, the nucleated cells can be detected reliably using the NucleoCounter® instruments.
Application Note (.PDF):
Simple data management and ready to use in GMP environments
The user-friendly accompanying NucleoView™ software automatically reports the viability, total count, live count and dead count. NucleoView™ also allows visual inspection of the fluorescence image and the opportunity to verify the counting. The NucleoCounter® instruments are ready to use within GMP facilities applying 21 CFR part 11-related regulations (Figure 4). The simple workflow, the calibrated volume and the build-in pipette of the unique Via1-Cassette™ ensure consistent cell sampling and eliminate production variation between users. Additionally, the Via1-Cassettes™ are volume-calibrated and avoid user-introduced errors in reagent mixing or loading, all of which translate into a precise and reproducible cell count which exceeds by far the trypan blue exclusion method.
Figure 4. The NucleoCounter® NC-200TM implemented in 21 CFR part 11. Different documentation steps e.g. electronic signatures and approvals as well as IQ/OQ/PQ protocols, the use of calibrated instruments and adapted protocols ensure reliable and reproducible results in cell count and viability determinations.
- Dominici, M., et al., Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006. 8(4): p. 315-317.
- Makino, S., et al., Cardiomyocytes can be generated from marrow stromal cells in vitro. Journal of Clinical Investigation, 1999. 103(5): p. 697-705.
- Arthur, A., et al., Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues. Stem Cells, 2008. 26(7): p. 1787-1795.
- Horwitz, E.M., et al., Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proceedings of the National Academy of Sciences of the United States of America, 2002. 99(13): p. 8932-8937.
- Kawada, H., et al., Nonhematopoietic mesenchymal stem cells can be mobilized and differentiate into cardiomyocytes after myocardial infarction. Blood, 2004. 104(12): p. 3581-3587.
- Zhao, K., et al., Immunomodulation effects of mesenchymal stromal cells on acute graft-versus-host disease after hematopoietic stem cell transplantation. Biology of Blood and Marrow Transplantation, 2015. 21(1): p. 97-104.
- Hahn, J.-Y., et al., Pre-treatment of mesenchymal stem cells with a combination of growth factors enhances gap junction formation, cytoprotective effect on cardiomyocytes and therapeutic efficacy for myocardial infarction. Journal of the American College of Cardiology, 2008. 51(9): p. 933-943.
- Schnabel, L.V., et al., Mesenchymal stem cells and insulin-like growth factor-I gene-enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons. Journal of Orthopaedic Research, 2009. 27(10): p. 1392-1398.
- Zuk, P.A., et al., Human adipose tissue is a source of multipotent stem cells. Molecular Biology of the Cell, 2002. 13(12): p. 4279-4295.
- Mizuno, H., M. Tobita, and A.C. Uysal, Concise review: Adipose-derived stem cells as a novel tool for future regenerative medicine. Stem Cells, 2012. 30(5): p. 804-810.
- Gimble, J.M., A.J. Katz, and B.A. Bunnell, Adipose-derived stem cells for regenerative medicine. Circulation Research, 2007. 100(9): p. 1249-1260.
- Astori, G., et al., “In vitro” and multicolor phenotypic characterization of cell subpopulations identified in fresh human adipose tissue stromal vascular fraction and in the derived mesenchymal stem cells. Journal of Translational Medicine, 2007. 5: p. 55-55.
- Kølle, S.-F.T., et al., Enrichment of autologous fat grafts with ex-vivo expanded adipose tissue-derived stem cells for graft survival: a randomised placebo-controlled trial. The Lancet, 2013. 382(9898): p. 1113-1120.
- Araña, M., et al., Preparation and characterization of collagen-based ADSC-carrier sheets for cardiovascular application. Acta Biomaterialia, 2013. 9(4): p. 6075-6083.
- Kazantseva, J., et al., Alternative splicing targeting the hTAF4-TAFH domain of TAF4 represses proliferation and accelerates chondrogenic differentiation of human mesenchymal stem cells. PLoS ONE, 2013. 8(10): p. e74799.
- Choi, J.S., et al., In vitro expansion of human adipose-derived stem cells in a spinner culture system using human extracellular matrix powders. Cell and Tissue Research, 2011. 345(3): p. 415-423.
- Suga, H., et al., IFATS collection: Fibroblast growth factor-2-induced hepatocyte growth factor secretion by adipose-derived stromal cells inhibits postinjury fibrogenesis through a c-Jun N-terminal kinase-dependent mechanism. Stem Cells, 2009. 27(1): p. 238-249.
- Miyazaki, T., et al., Isolation of two human fibroblastic cell populations with multiple but distinct potential of mesenchymal differentiation by ceiling culture of mature fat cells from subcutaneous adipose tissue. Differentiation, 2005. 73(2): p. 69-78.
- Meirelles, L.d.S., P.C. Chagastelles, and N.B. Nardi, Mesenchymal stem cells reside in virtually all post-natal organs and tissues. Journal of Cell Science, 2006. 119(11): p. 2204-2213.
- Kuznetsov, S.A., et al., Circulating skeletal stem cells. The Journal of Cell Biology, 2001. 153(5): p. 1133-1140.
- Heathman, T.R.J., et al., Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum-free microcarrier process. Biotechnology and Bioengineering, 2015. 112(8): p. 1696-1707.
- Heathman, T.R.J., et al., Serum-free process development: improving the yield and consistency of human mesenchymal stromal cell production. Cytotherapy, 2015. 17(11): p. 1524-1535.