Optimization of the Cultivation of Donor Mesenchymal Stromal Cells for Clinical Use in Cellular Therapy

DOI: 10.4236/cellbio.2014.31003   PDF   HTML     5,587 Downloads   7,691 Views   Citations


Mesenchymal stromal cells(MSC)have shown their benefits in graft-versus-host disease(GVHD), with five unsettled matters: 1) MSCs expansion in medium with Fetal Bovine Serum(FBS)and its risk of xenoreaction; 2) The number of cells indicated for therapy that is relatively high, with the need to optimize the expansion, number and time wise; 3) The utilization of third party donors; 4) Culture passage number(P);and 5) Source of the cells. This study was designed to determine the superiority of the Platelet Lysates(PL)over FBS on the expansion of MSC, the optimal cell plating density and days between each pass, and to investigate if donor total nucleated cells(TNC)obtained from the washouts of discharged bags and filters of hematopoietic stem cell transplantation(HSCT)can be expanded to be used at clinical grade. TNC were removed, plated and after the first passage were cultivated in different concentrations with FBS or PL, and the number of days to reach 80% of confluence was observed. Next, cultures with the same plating density were fed either with PL or with FBS and after seven days counted to analyze how much they had grown in that period. The proliferation of mesenchymal stromal cells in the presence of PL and SFB was averaged 11.88 and 2.5 times, respectively, in a period of 7 days. The highest concentration of plating cells using PL took less time to reach confluence as compared with the three lower ones. This study suggests that the PL is the best choice as a supplement to expand MSC and to allow the proliferation of enough number of MSC at P2 for clinical use.

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Valim, V. , Amorin, B. , Pezzi, A. , Aparecida Lima da Silva, M. , Paula Alegretti, A. and Silla, L. (2014) Optimization of the Cultivation of Donor Mesenchymal Stromal Cells for Clinical Use in Cellular Therapy. CellBio, 3, 25-33. doi: 10.4236/cellbio.2014.31003.

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The authors declare no conflicts of interest.


[1] Amorin, B., Alegretti, A.P., Valim, V., Pezzi, A., Silva, M.A., Sehn, F. and Silla, L. (2013) Characteristics of Mesenchymal Stem Cells under Hypoxia. CellBio, 2, 9.
[2] Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., Deans, R., Keating, A., Prockop, D. and E. Horwitz, (2006) Minimal Criteria for Defining Multipotent Mesenchymal Stromal Cells. The International Society for Cellular Therapy Position Statement. Cytotherapy, 8, 315-317.
[3] Harichandan, A. and Buhring, H.J. (2011) Prospective Isolation of Human MSC. Best Practice & Research Clinical Haematology, 24, 25-36. http://dx.doi.org/10.1016/j.beha.2011.01.001
[4] Caplan, A.I. and Bruder, S.P. (2001) Mesenchymal Stem Cells: Building Blocks for Molecular Medicine in the 21st Century. Trends in Molecular Medicine, 7, 259-264. http://dx.doi.org/10.1016/S1471-4914(01)02016-0
[5] Kebriaei, P., Isola, L., Bahceci, E., Holland, K., Rowley, S., McGuirk, J., Devetten, M., Jansen, J., Herzig, R., Schuster, M., Monroy, R. andUberti, J. (2009) Adult Human Mesenchymal Stem Cells Added to Corticosteroid Therapy for the Treatment of Acute Graft-Versus-Host Disease. Biology of Blood and Marrow Transplantation, 15, 804-811.
[6] Le Blanc, K., Frassoni, F., Ball, L., Locatelli, F., Roelofs, H., Lewis, I., Lanino, E., Sundberg, B., Bernardo, M.E., Remberger, M., Dini, G., Egeler, R.M., Bacigalupo, A., Fibbe, W. and Ringden, O. (2008) Mesenchymal Stem Cells for Treatment of Steroid-Resistant, Severe, Acute Graft-Versus-Host Disease: A Phase II Study. Lancet, 371, 1579- 1586.
[7] Ringden, O. and Le Blanc, K. (2011) Mesenchymal Stem Cells for Treatment of Acute and Chronic Graft-Versus-Host Disease, Tissue Toxicity and Hemorrhages. Best Practice & Research Clinical Haematology, 24, 65-72.
[8] Fouillard, L., Chapel, A., Bories, D., Bouchet, S., Costa, J.M., Rouard, H., Herve, P., Gourmelon, P., Thierry, D., Lopez, M. and Gorin, N.C. (2007) Infusion of Allogeneic-Related HLA Mismatched Mesenchymal Stem Cells for the Treatment of Incomplete Engraftment Following Autologous Haematopoietic Stem Cell Transplantation. Leukemia, 21, 568-570.
[9] Le Blanc, K., Samuelsson, H., Gustafsson, B., Remberger, M., Sundberg, B., Arvidson, J., Ljungman, P., Lonnies, H., Nava, S. and Ringden, O. (2007) Transplantation of Mesenchymal Stem Cells to Enhance Engraftment of Hematopoietic Stem Cells. Leukemia, 21, 1733-1738.
[10] Macmillan, M.L., Blazar, B.R., DeFor, T.E. and Wagner, J.E. (2009) Transplantation of Ex-Vivo Culture-Expanded Parental Haploidentical Mesenchymal Stem Cells to Promote Engraftment in Pediatric Recipients of Unrelated Donor Umbilical Cord Blood: Results of a Phase I-II Clinical Trial. Bone Marrow Transplantation, 43, 447-454.
[11] Ringdén, O., Uzunel, M., Sundberg, B., Lonnies, L., Nava, S., Gustafsson, J., Henningsohn, L. and Le Blanc, K. (2007) Tissue Repair Using Allogeneic Mesenchymal Stem Cells for Hemorrhagic Cystitis, Pneumomediastinum and Perforated Colon. Leukemia, 21, 2271-2276.
[12] Fouillard, L., Bensidhoum, M., Bories, D., Bonte, H., Lopez, M., Moseley, A.M., Smith, A., Lesage, S., Beaujean, F., Thierry, D., Gourmelon, P., Najman, A. and Gorin, N.C. (2003) Engraftment of Allogeneic Mesenchymal Stem Cells in the Bone Marrow of a Patient with Severe Idiopathic Aplastic Anemia Improves Stroma. Leukemia, 17, 474-476.
[13] Bruder, S.P., Jaiswal, N. and Haynesworth, S.E. (1997) Growth Kinetics, Self-Renewal, and the Osteogenic Potential of Purified Human Mesenchymal Stem Cells during Extensive Subcultivation and Following Cryopreservation. Journal of Cellular Biochemistry, 64, 278-294.
[14] Caplan, A.I. (2007) Adult Mesenchymal Stem Cells for Tissue Engineering versus Regenerative Medicine. Journal of Cellular Physiology, 213, 341-347. http://dx.doi.org/10.1002/jcp.21200
[15] Owen, M. (1988) Marrow Stromal Stem Cells. Journal of Cell Science Supplement, 10, 63-76.
[16] Zuk, P.A., Zhu, M., Mizuno, H., Huang, J., Futrell, J.W., Katz, A.J., Benhaim, P., Lorenz, H.P. and Hedrick, M.H. (2001) Multilineage Cells from Human Adipose Tissue: Implications for Cell-Based Therapies. Tissue Engineering, 7, 211-228. http://dx.doi.org/10.1089/107632701300062859
[17] Capelli, C., Salvade, A., Pedrini, O., Barbui, V., Gotti, E., Borleri, G., Cabiati, B., Belotti, D., Perseghin, P., Bellavita, P., Biondi, A., Biagi, E., Rambaldi, A., Golay, J. and Introna, M. (2009) The Washouts of Discarded Bone Marrow Collection Bags and Filters Are a Very Abundant Source of hMSCs. Cytotherapy, 11, 403-413.
[18] Ahrens, N., Tormin, A., Paulus, M., Roosterman, D., Salama, A., Krenn, V., Neumann, U. and Scheding, S. (2004) Mesenchymal Stem Cell Content of Human Vertebral Bone Marrow. Transplantation, 78, 925-929.
[19] Young, H.E., Steele, T.A., Bray, R.A., Hudson, J., Floyd, J.A., Hawkins, K., Thomas, K., Austin, T., Edwards, C., Cuzzourt, J., Duenzl, M., Lucas, P.A. and Black Jr., A.C. (2001) Human Reserve Pluripotent Mesenchymal Stem Cells Are Present in the Connective Tissues of Skeletal Muscle and Dermis Derived from Fetal, Adult, and Geriatric Donors. The Anatomical Record, 264, 51-62. http://dx.doi.org/10.1002/ar.1128
[20] De Bari, C., Dell’Accio, F., Tylzanowski, P. and Luyten, F.P. (2001) Multipotent Mesenchymal Stem Cells from Adult Human Synovial Membrane. Arthritis & Rheumatology, 44, 1928-1942.
[21] Covas, D.T., Siufi, J.L., Silva, A.R. and Orellana, M.D. (2003) Isolation and Culture of Umbilical Vein Mesenchymal Stem Cells. Brazilian Journal of Medical and Biological Research, 36, 1179-1183.
[22] Covas, D.T., Piccinato, C.E., Orellana, M.D., Siufi, J.L., Silva Jr., W.A., Proto-Siqueira, R., Rizzatti, E.G., Neder, L., Silva, A.R., Rocha, V. and Zago, M.A. (2005) Mesenchymal Stem Cells Can Be Obtained From the Human Saphena vein. Experimental Cell Research, 309, 340-344.
[23] Almeida-Porada, G., Shabrawy, D.E., Porada, C. and Zanjani, E.D. (2002) Differentiative Potential of Human Metanephric Mesenchymal Cells. Experimental Hematology, 30, 1454-1462.
[24] Lee, M.W., Choi, J., Yang, M.S., Moon, Y.J., Park, J.S., Kim, H.C. and Kim, Y.J. (2004) Mesenchymal Stem Cells from Cryopreserved Human Umbilical Cord Blood. Biochemical and Biophysical Research Communications, 320, 273-278. http://dx.doi.org/10.1016/j.bbrc.2004.04.206
[25] Lee, O.K., Kuo, T.K., Chen, W.M., Lee, K.D., Hsieh, S.L. and Chen, T.H. (2004) Isolation of Multipotent Mesenchymal Stem Cells from Umbilical Cord Blood. Blood, 103, 1669-1675.
[26] Alsalameh, S., Amin, R., Gemba, T. and Lotz, M. (2004) Identification of Mesenchymal Progenitor Cells in Normal and Osteoarthritic Human Articular Cartilage. Arthritis & Rheumatism, 50, 1522-1532.
[27] Seo, B.M., Miura, M., Gronthos, S., Bartold, P.M., Batouli, S., Brahim, J., Young, M., Robey, P.G., Wang, C.Y.and Shi, S. (2004) Investigation of Multipotent Postnatal Stem Cells from Human Periodontal Ligament. The Lancet, 364, 149-155. http://dx.doi.org/10.1016/S0140-6736(04)16627-0
[28] Sabatini, F., Petecchia, L., Tavian, M., de Villeroche, V.J., Rossi, G.A. and Brouty-Boye, D. (2005) Human Bronchial Fibroblasts Exhibit a Mesenchymal Stem Cell Phenotype and Multilineage Differentiating Potentialities. Laboratory Investigation, 85, 962-971.
[29] Grisendi, G., Anneren, C., Cafarelli, L., Sternieri, R., Veronesi, E., Cervo, G.L., Luminari, S., Maur, M., Frassoldati, A., Palazzi, G., Otsuru, S., Bambi, F., Paolucci, P., Pierfranco, C., Horwitz, E. and Dominici, M. (2010) GMP-Manufactured Density Gradient Media for Optimized Mesenchymal Stromal/Stem Cell Isolation and Expansion. Cytotherapy, 12, 466-477. http://dx.doi.org/10.3109/14653241003649510
[30] Selvaggi, T.A., Walker, R.E. and Fleisher, T.A. (1997) Development of Antibodies to Fetal Calf Serum with Arthus-Like Reactions in Human Immunodeficiency Virus-Infected Patients Given Syngeneic Lymphocyte Infusions. Blood, 89, 776-779.
[31] Sundin, M., Ringden, O., Sundberg, B., Nava, S., Gotherstrom, C. and Blanc, K.L. (2007) No Alloantibodies against Mesenchymal Stromal Cells, but Presence of Anti-Fetal Calf Serum Antibodies, after Transplantation in Allogeneic Hematopoietic Stem Cell Recipients. Haematologica, 92, 1208-1215. http://dx.doi.org/10.3324/haematol.11446
[32] WHO (1997) Medicinal and Other Products and Human and Animal Transmissible Spongiform Encephalopathies: Memorandum from a WHO Meeting. Bulletin of the World Health Organization, 75, 505-513.
[33] Crespo-Diaz, R., Behfar, A., Butler, G.W., Padley, D.J., Sarr, M.G., Bartunek, J., Dietz, A.B. and Terzic, A. (2010) Platelet Lysate Consisting of a Natural Repair Proteome Supports Human Mesenchymal Stem Cell Proliferation and Chromosomal Stability. Cell Transplant, 20, 797-811.
[34] Bernardo, M.E., Avanzini, M.A., Perotti, C., Cometa, A.M., Moretta, A., Lenta, E., Del Fante, C., Novara, F., de Silvestri, A., Amendola, G., Zuffardi, O., Maccario, R. and Locatelli, F. (2007) Optimization of In vitro Expansion of Human Multipotent Mesenchymal Stromal Cells for Cell-Therapy Approaches: Further Insights in the Search for a Fetal Calf Serum Substitute. Journal of Cellular Physiology, 211, 121-130. http://dx.doi.org/10.1002/jcp.20911
[35] Blande, I.S., Bassaneze, V., Lavini-Ramos, C., Fae, K.C., Kalil, J., Miyakawa, A.A., Schettert, I.T. and Krieger, J.E. (2009) Adipose Tissue Mesenchymal Stem Cell Expansion in Animal Serum-Free Medium Supplemented with Autologous Human Platelet Lysate. Transfusion, 49, 2680-2685. http://dx.doi.org/10.1111/j.1537-2995.2009.02346.x
[36] Carrancio, S., Lopez-Holgado, N., Sanchez-Guijo, F.M., Villaron, E., Barbado, V., Tabera, S., Diez-Campelo, M., Blanco, J., Miguel, J.F.S. and Del Canizo, M.C. (2008) Optimization of Mesenchymal Stem Cell Expansion Procedures by Cell Separation and Culture Conditions Modification. Experimental Hematology, 36, 1014-1021.
[37] Muller, I., Kordowich, S., Holzwarth, C., Spano, C., Isensee, G., Staiber, A., Viebahn, S., Gieseke, F., Langer, H., Gawaz, M.P. Horwitz, E.M., Conte, P., Handgretinger, R. and Dominici, M. (2006) Animal Serum-Free Culture Conditions for Isolation and Expansion of Multipotent Mesenchymal Stromal Cells from Human BM. Cytotherapy, 8, 437-444. http://dx.doi.org/10.1080/14653240600920782
[38] Beyer, N.N. and da Silva M.L. (2006) Mesenchymal Stem Cells: Isolation, In vitro Expansion and Characterization. Handbook of Experimental pharmacology, 174, 249-282.
[39] Schallmoser, K., Bartmann, C., Rohde, E., Reinisch, A., Kashofer, K., Stadelmeyer, E., Drexler, C., Lanzer, G., Linkesch, W. and Strunk, D. (2007)Human Platelet Lysate Can Replace Fetal Bovine Serum for Clinical-Scale Expansion of Functional Mesenchymal Stromal Cells. Transfusion, 47, 1436-1446.
[40] Horn, P., Bokermann, G., Cholewa, D., Bork, S., Walenda, T., Koch, C., Drescher, W., Hutschenreuther, G., Zenke, M., Ho, A.D. and Wagner, W. (2010) Impact of Individual Platelet Lysates on Isolation and Growth of Human Mesenchymal Stromal Cells. Cytotherapy, 12, 888-898. http://dx.doi.org/10.3109/14653249.2010.501788
[41] Perez-Ilzarbe, M., Diez-Campelo, M., Aranda, P., Tabera, S., Lopez, T., del Canizo, C., Merino, J., Moreno, C., Andreu, E.J., Prosper, F. and Perez-Simon, J.A. (2009) Comparison of Ex vivo Expansion Culture Conditions of Mesenchymal Stem Cells for Human Cell Therapy. Transfusion, 49, 1901-1910.
[42] Samuelsson, H., Ringden, O., Lonnies, H. and Blanc, K.L. (2009) Optimizing In vitro Conditions for Immunomodulation and Expansion of Mesenchymal Stromal Cells. Cytotherapy, 11, 129-136.
[43] Sundin, M., Lindblom, A., Orvell, C., Barrett, A.J., Sundberg, B., Watz, E., Wikman, A., Broliden, K. and Blanc, K.L. (2008) Persistence of Human Parvovirus B19 in Multipotent Mesenchymal Stromal Cells Expressing the Erythrocyte P Antigen: Implications for Transplantation. Biology of Blood and Marrow Transplantation, 14, 1172-1179.
[44] Sundin, M., Orvell, C., Rasmusson, I., Sundberg, B., Ringden, O. and Blanc, K.L. (2006) Mesenchymal Stem Cells Are Susceptible to Human Herpesviruses, but Viral DNA Cannot Be Detected in the Healthy Seropositive Individual. Bone Marrow Transplant, 37, 1051-1059.
[45] Hertenstein, B., Hambach, L., Bacigalupo, A., Schmitz, N., McCann, S., Slavin, S., Gratwohl, A., Ferrant, A., Elmaagacli, A., Schwertfeger, R., Locasciulli, A., Zander, A., Bornhauser, M., Niederwieser, D. and Ruutu, T. (2005) Development of Leukemia in Donor Cells after Allogeneic Stem Cell Transplantation—A Survey of the European Group for Blood and Marrow Transplantation (EBMT). Haematologica, 90, 969-975.
[46] Reichard, K.K., Zhang, Q.Y., Sanchez, L., Hozier, J., Viswanatha, D. and Foucar, K. (2006) Acute Myeloid Leukemia of Donor Origin after Allogeneic Bone Marrow Transplantation for Precursor T-Cell Acute Lymphoblastic Leukemia: Case Report and Review of the Literature. American Journal of Hematology, 81, 178-185.
[47] Sundin, M., Remberger, M., Lonnies, H., Sundberg, B., Ringden, O. and Blanc, K.L. (2008) No Increased Trapping of Multipotent Mesenchymal Stromal Cells in Bone Marrow Filters Compared with Other Bone Marrow Cells. Cytotherapy, 10, 238-242. http://dx.doi.org/10.1080/14653240801965164
[48] Capelli, C., Domenghini, M., Borleri, G., Bellavita, P., Poma, R., Carobbio, A., Mico, C., Rambaldi, A., Golay, J. and Introna, M. (2007) Human Platelet Lysate Allows Expansion and Clinical Grade Production of Mesenchymal Stromal Cells from Small Samples Of Bone Marrow Aspirates or Marrow Filter Washouts. Bone Marrow Transplant, 40, 785-791. http://dx.doi.org/10.1038/sj.bmt.1705798

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