The Influence of Culture Medium Type on Cellular Phenotype of Canine Adipose Derived Stem Cells


Canine adipose derived stem cells (ASCs) hold a great promise for the therapy of osteoarthritis in veterinary medicine. Current therapy is an autologous, stromal vascular fraction. Allogeneic ASCs provide many advantages, including efficient, cost-effective treatments while eliminating a surgical procedure in a diseased animal. Cultured ASCs can be expanded and characterized, allowing selection of desirable qualities. Use of allogeneic ASCs requires selection of a culture medium that provides consistent, desirable cellular products. The supplements within a medium can greatly influence cellular phenotypes. We hypothesized that medium type influenced cellular phenotype, allowing selection of a specified cellular product for clinical applications. We evaluated ASCs derived from adipose tissue of six dogs, assessing mRNA expression of proinflammatory: interleukin-1b, cyclooxygenase-2, and anti-inflammatory mediators: tissue inhibitor metalloproteinase-2 and interleukin -1 receptor antagonist, via quantitative RT-PCR prior to, and following culture in five cell culture media: basic cell growth medium (BGM), Keratinocyte N acetyl-L-cysteine supplemented (KNAC) medium, Multipotent Adult Progenitor Cell (MAPC) medium, serum free medium (SFM) and xeno-free medium. Major histocompatability complex I (MHCI), major histocompatability complex II (MHCII), CD44 and CD90 immunophenotypes were assessed via flow cytometry analysis. Tri-lineage differentiation (bone, adipose and cartilage tissue) was utilized to verify multipotency. SFM and xeno-free culture conditions did not produce cell expansion sufficient to assess phenotype. ASCs prior to culture had wide variability in all mediator levels, while culturing in the remaining conditions resulted in more predictable expression levels of inflammatory mediators, with a decrease in all levels. Cultured ASCs retained expression of cell surface markers MHCI, CD44 and CD90, while decreasing MHCII expression levels. KNAC and MAPC medium conditions consistently produced tri-lineage differentation; BGM, SFM and xeno-free medium did not. Culture condition will influence phenotype of ASCs, and should be selected according to the intended therapeutic effect.

Share and Cite:

Kiefer, K. , Pluhar, G. , Conzemius, M. and O’Brien, T. (2014) The Influence of Culture Medium Type on Cellular Phenotype of Canine Adipose Derived Stem Cells. Open Journal of Regenerative Medicine, 3, 28-37. doi: 10.4236/ojrm.2014.31004.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Kang, B.J., Ryu, H.H., Park, S.S., et al. (2012) Comparing the Osteogenic Potential of Canine Mesenchymal Stem Cells Derived from Adipose Tissues, Bone Marrow, Umbilical Cord Blood, and Wharton’s Jelly for Treating Bone Defects. Journal of Veterinary Science, 13, 299-310.
[2] Kisiel, A.H., McDuffee, L.A., Masaoud, E., Bailey, T.R., Esparza Gonzalez, B.P. and Nino-Fong, R. (2012) Isolation, Characterization, and in Vitro Proliferation of Canine Mesenchymal Stem Cells Derived from Bone Marrow, Adipose Tissue, Muscle, and Periosteum. American Journal of Veterinary Research, 73, 1305-1317.
[3] Csaki, C., Matis, U., Mobasheri, A., Ye, H. and Shakibaei, M. (2007) Chondrogenesis, Osteogenesis and Adipogenesis of Canine Mesenchymal Stem Cells: A Biochemical, Morphological and Ultrastructural Study. Histochemistry and Cell Biology, 128, 507-520.
[4] Reich, C.M., Raabe, O., Wenisch, S., Bridger, P.S., Kramer, M. and Arnhold, S. (2012) Isolation, Culture and Chondrogenic Differentiation of Canine Adipose Tissue-and Bone Marrow-Derived Mesenchymal Stem Cells—A Comparative Study. Veterinary Research Communications, 36, 139-148.
[5] Black, L.L., Gaynor, J., Adams, C., et al. (2008) Effect of Intraarticular Injection of Autologous Adipose-Derived Mesenchymal Stem and Regenerative Cells on Clinical Signs of Chronic Osteoarthritis of the Elbow Joint in Dogs. Veterinary Therapeutics, 9, 192-200.
[6] Black, L.L., Gaynor, J., Gahring, D., et al. (2007) Effect of Adipose-Derived Mesenchymal Stem and Regenerative Cells on Lameness in Dogs with Chronic Osteoarthritis of the Coxofemoral Joints: A Randomized, Double-Blinded, Multicenter, Controlled Trial. Veterinary Therapeutics, 8, 272-284.
[7] Guercio, A., Di Marco, P., Casella, S., et al. (2012) Production of Canine Mesenchymal Stem Cells from Adipose Tissue and Their Application in Dogs with Chronic Osteoarthritis of the Humeroradial Joints. Cell Biology International, 36, 189-194.
[8] Arinzeh, T.L., Peter, S.J., Archambault, M.P., et al. (2003) Allogeneic Mesenchymal Stem Cells Regenerate Bone in a Critical-Sized Canine Segmental Defect. The Journal of Bone & Joint Surgery of America, 85-A, 1927-1935.
[9] Park, S.S., Lee, Y.J., Lee, S.H., et al. (2012) Functional Recovery after Spinal Cord Injury in Dogs Treated with a Combination of Matrigel and Neural-Induced Adipose-Derived Mesenchymal Stem Cells. Cytotherapy, 14, 584-597.
[10] Perin, E.C., Silva, G.V., Assad, J.A., et al. (2008) Comparison of Intracoronary and Transendocardial Delivery of Allogeneic Mesenchymal Cells in a Canine Model of Acute Myocardial Infarction. Journal of Molecular and Cellular Cardiology, 44, 486-495.
[11] Ryu, H.H., Lim, J.H., Byeon, Y.E., et al. (2009) Functional Recovery and Neural Differentiation after Transplantation of Allogenic Adipose-Derived Stem Cells in a Canine Model of Acute Spinal Cord Injury. Journal of Veterinary Science, 10, 273-284.
[12] Kang, J.W., Kang, K.S., Koo, H.C., Park, J.R., Choi, E.W. and Park, Y.H. (2008) Soluble Factors-Mediated Immunomodulatory Effects of Canine Adipose Tissue-Derived Mesenchymal Stem Cells. Stem Cells and Development, 17, 681-693.
[13] Neupane, M., Chang, C.C., Kiupel, M. and Yuzbasiyan-Gurkan, V. (2008) Isolation and Characterization of Canine Adipose-Derived Mesenchymal Stem Cells. Tissue Engineering Part A, 14, 1007-1015.
[14] Vieira, N.M., Brandalise, V., Zucconi, E., Secco, M., Strauss, B.E. and Zatz, M. (2010) Isolation, Characterization, and Differentiation Potential of Canine Adipose-Derived Stem Cells. Cell Transplantation, 19, 279-289.
[15] Alam, M.R., Ji, J.R., Kim, M.S. and Kim, N.S. (2011) Biomarkers for Identifying the Early Phases of Osteoarthritis Secondary to Medial Patellar Luxation in Dogs. Journal of Veterinary Science, 12, 273-280.
[16] Caron, J.P., Fernandes, J.C., Martel-Pelletier, J., et al. (1996) Chondroprotective Effect of Intraarticular Injections of Interleukin-1 Receptor Antagonist in Experimental Osteoarthritis. Suppression of Collagenase-1 Expression. Arthritis & Rheumatology, 39, 1535-1544.
[17] Clements, D.N., Carter, S.D., Innes, J.F., Ollier, W.E. and Day, P.J. (2006) Analysis of Normal and Osteoarthritic Canine Cartilage mRNA Expression by Quantitative Polymerase Chain Reaction. Arthritis Research & Therapy, 8, R158.
[18] Clements, D.N., Fitzpatrick, N., Carter, S.D. and Day, P.J. (2009) Cartilage Gene Expression Correlates with Radiographic Severity of Canine Elbow Osteoarthritis. The Veterinary Journal, 179, 211-218.
[19] Fernandes, J.C., Martel-Pelletier, J. and Pelletier, J.P. (2002) The Role of Cytokines in Osteoarthritis Pathophysiology. Biorheology, 39, 237-246.
[20] Fernandes, J., Tardif, G., Martel-Pelletier, J., et al. (1999) In Vivo Transfer of Interleukin-1 Receptor Antagonist Gene in Osteoarthritic Rabbit Knee Joints: Prevention of Osteoarthritis Progression. American Journal of Pathology, 154, 1159-1169.
[21] Ljunggren, H.G. and Karre, K. (1990) In Search of the “Missing Self”: MHC Molecules and NK Cell Recognition. Immunology Today, 11, 237-244.
[22] Jiang, Y., Vaessen, B., Lenvik, T., Blackstad, M., Reyes, M. and Verfaillie, C.M. (2002) Multipotent Progenitor Cells Can Be Isolated from Postnatal Murine Bone Marrow, Muscle, and Brain. Experimental Hematology, 30, 896-904.
[23] Livak, K.J. and Schmittgen, T.D. (2001) Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25, 402-408.
[24] Riordan, N.H., Ichim, T.E., Min, W.P., et al. (2009) Non-Expanded Adipose Stromal Vascular Fraction Cell Therapy for Multiple Sclerosis. Journal of Translational Medicine, 7, 29.
[25] Schwarz, C., Leicht, U., Rothe, C., et al. (2012) Effects of Different Media on Proliferation and Differentiation Capacity of Canine, Equine and Porcine Adipose Derived Stem Cells. Research in Veterinary Science, 93, 457-462.
[26] Gotherstrom, C., Ringden, O., Tammik, C., Zetterberg, E., Westgren, M. and Le Blanc, K. (2004) Immunologic Properties of Human Fetal Mesenchymal Stem Cells. American Journal of Obstetrics & Gynecology, 190, 239-245.
[27] Le Blanc, K., Tammik, C., Rosendahl, K., Zetterberg, E. and Ringden, O. (2003) HLA Expression and Immunologic Properties of Differentiated and Undifferentiated Mesenchymal Stem Cells. Experimental Hematology, 31, 890-896.

Copyright © 2021 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.