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Expansion and preservation of multipotentiality of rabbit bone-marrow derived mesenchymal stem cells in dextran-based microcarrier spin culture

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Abstract

The use of mesenchymal stem cells (MSCs) in tissue repair and regeneration despite their multipotentiality has been limited by their cell source quantity and decelerating proliferative yield efficiency. A study was thus undertaken to determine the feasibility of using microcarrier beads in spinner flask cultures for MSCs expansion and compared to that of conventional monolayer cultures and static microcarrier cultures. Isolation and characterization of bone marrow derived MSCs were conducted from six adult New Zealand white rabbits. Analysis of cell morphology on microcarriers and culture plates at different time points (D0, D3, D10, D14) during cell culture were performed using scanning electron microscopy and bright field microscopy. Cell proliferation rates and cell number were measured over a period of 14 days, respectively followed by post-expansion characterization. MTT proliferation assay demonstrated a 3.20 fold increase in cell proliferation rates in MSCs cultured on microcarriers in spinner flask as compared to monolayer cultures (p < 0.05). Cell counts at day 14 were higher in those seeded on stirred microcarrier cultures (6.24 ± 0.0420 cells/ml) × 105 as compared to monolayer cultures (0.22 ± 0.004 cells/ml) × 105 and static microcarrier cultures (0.20 ± 0.002 cells/ml) × 105. Scanning electron microscopy demonstrated an increase in cell colonization of the cells on the microcarriers in stirred cultures. Bead-expanded MSCs were successfully differentiated into osteogenic and chondrogenic lineages. This system offers an improved and efficient alternative for culturing MSCs with preservation to their phenotype and multipotentiality.

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References

  1. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284(5411):143–7. doi:10.1126/science.284.5411.143.

    Article  CAS  Google Scholar 

  2. Caplan AI. Mesenchymal stem cells. J Orthop Res. 1991;9(5):641–50.

    Article  CAS  Google Scholar 

  3. Goshima J, Goldberg VM, Caplan AI. The osteogenic potential of culture-expanded rat marrow mesenchymal cells assayed in vivo in calcium phosphate ceramic blocks. Clin Orhtop Relat Res. 1991;262:298–311.

    Google Scholar 

  4. Haynesworth SE, Goshima J, Goldberg VM, Caplan AI. Characterisation of cells with osteogenic potential from human marrow. Bone. 1992;13(1):81–8.

    Article  CAS  Google Scholar 

  5. Mauney JR, Volloch V, Kaplan DL. Role of adult mesenchymal stem cells in bone tissue engineering applications: current status and future prospects. Tissue Eng. 2005;11(5–6):787–802. doi:10.1089/ten.2005.11.787.

    Article  CAS  Google Scholar 

  6. Horwitz EM, Prockop DJ, Fitzpatrick LA, Koo WW, Gordon PL, Neel M, et al. Transplantability and therapeutic effects of bone-marrow derived mesenchymal stem cells in children with osteogenesis imperfecta. Nat Med. 1999;5(3):309–13.

    Article  CAS  Google Scholar 

  7. Pereira RF, Halford KW, O’Hara MD, Leeper DB, Sokolov BP, Pollard MD, et al. Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice. Proc Natl Acad Sci USA. 1995;92(11):4857–61.

    Article  CAS  Google Scholar 

  8. Bruder SP, Fink DJ, Caplan AI. Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy. J Cell Biochem. 1994;56(3):283–94.

    Article  CAS  Google Scholar 

  9. Horwitz EM, Gordon PL, Koo WK, Marx JC, Neel MD, McNall RY, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone. Proc Natl Acad Sci USA. 2002;99(13):8932–7. doi:10.1073/pnas.132252399.99/13/8932[pii].

    Article  CAS  Google Scholar 

  10. Zhang XS, Linkhart TA, Chen ST, Peng H, Wergedal JE, Guttierez GG, et al. Local ex vivo gene therapy with bone marrow stromal cells expressing human BMP4 promotes endosteal bone formation in mice. J Gene Med. 2004;6(1):4–15. doi:10.1002/jgm.477.

    Article  CAS  Google Scholar 

  11. Jorgensen C, Noel D, Apparailly F, Sany J. Stem cells for repair of cartilage and bone: the next challenge in osteoarthritis and rheumatoid arthritis. Ann Rheum Dis. 2001;60(4):305–9.

    Article  CAS  Google Scholar 

  12. Redding PJ, Juliano RL. Clinging to life: cell to matrix adhesion and cell survival. Cancer Metastasis Rev. 2005;24(3):425–39.

    Article  Google Scholar 

  13. Carstanjen B, Desbois C, Hekmati M, Behr L. Successful engraftment of cultured autologous mesenchymal stem cells in a surgically repaired soft palate defect in an adult horse. Can J Vet Res. 2006;70(2):143–7.

    Google Scholar 

  14. Yan H, Yu C. Repair of full-thickness cartilage defects with cells of different origin in a rabbit model. Arthroscopy. 2007;23(2):178–87. doi:10.1016/j.arthro.2006.09.005.

    Article  Google Scholar 

  15. Lee KB, Hui JH, Song IC, Ardany L, Lee EH. Injectable mesenchymal stem cell therapy for large cartilage defects-a porcine model. Stem Cells. 2007;25(11):2964–71. doi:10.1634/stemcells.2006-0311.

    Article  Google Scholar 

  16. van Wezel AL. Growth of cell-strains and primary cells on micro-carriers in homogeneous culture. Nature. 1967;216(5110):64–5.

    Article  Google Scholar 

  17. Frondoza C, Sohrabi A, Hungerford D. Human chondrocytes proliferate and produce matrix components in microcarrier suspension culture. Biomaterials. 1996;17(9):879–88.

    Article  CAS  Google Scholar 

  18. Malda J, Frondoza CG. Microcarriers in the engineering of cartilage and bone. Trends Biotechnol. 2006;24(7):299–304.

    Article  CAS  Google Scholar 

  19. Malda J, Kreijveld E, Temenoff JS, van Blitterswijk CA, Riesle J. Expansion of human nasal chondrocytes on macroporous microcarriers enhances redifferentiation. Biomaterials. 2003;24(28):5153–61.

    Article  CAS  Google Scholar 

  20. Malda J, van Blitterswijk CA, Grojec M, Martens DE, Tramper J, Riesle J. Expansion of bovine chondrocytes on microcarriers enhances redifferentiation. Tissue Eng. 2003;9(5):939–48. doi:10.1089/107632703322495583.

    Article  CAS  Google Scholar 

  21. Barry FP, Murphy JM. Mesenchymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol. 2004;36(4):568–84.

    Article  CAS  Google Scholar 

  22. Chen Y, Shao J-Z, Xiang L-X, Dong X-J, Zhang G-R. Mesenchymal stem cells: a promising candidate in regenerative medicine. Int J Biochem Cell Biol. 2008;40(5):815–20.

    Article  CAS  Google Scholar 

  23. Muzlifah AH, Matthew PC, Christopher DB, Dazzi F. Mesenchymal stem cells: the fibroblasts’ new clothes? Haematologica. 2009;94(2):258–63.

    Article  Google Scholar 

  24. Caplan AI. Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. J Cell Physiol. 2007;213(2):341–7.

    Article  CAS  Google Scholar 

  25. Copland I, Sharma K, Lejeune L, Eliopoulos N, Stewart D, Liu P, et al. CD34 expression on murine marrow-derived mesenchymal stromal cells: impact on neovascularization. Exp Hematol. 2008;36(1):93–103.

    Article  CAS  Google Scholar 

  26. Neupane M, Chang C-C, Kiupel M, Yuzbasiyan-Gurkan V. Isolation and characterization of canine adipose derived mesenchymal stem cells. Tissue Eng A. 2008;14(6):1007–15. doi:10.1089/ten.tea.2007.0207.

    Article  CAS  Google Scholar 

  27. Kassem M, Kristiansen M, Abdallah BM. Mesenchymal stem cells: cell biology and potential use in therapy. Basic Clin Pharmacol Toxicol. 2004;95(5):209–14.

    Article  CAS  Google Scholar 

  28. da Silva Meirelles L, Nance BN. Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. Br J Haematol. 2003;123(4):702–11.

    Article  Google Scholar 

  29. da Silva Meirelles L, Chagastelles PC, Nardi NB. Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci. 2006;119(11):2204–13. doi:10.1242/jcs.02932.

    Article  Google Scholar 

  30. Boeuf S, Richter W. Chondrogenesis of mesenchymal stem cells: role of tissue source and inducing factors. Stem Cell Res Ther. 2010;1(4):31. doi:10.1186/scrt31.

    Article  Google Scholar 

  31. Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem. 1997;64(2):295–312.

    Article  CAS  Google Scholar 

  32. Caplan AI, Bruder SP. Mesenchymal stem cells: building blocks for molecular medicine in the 21st century. Trends Mol Med. 2001;7(6):259–64.

    Article  CAS  Google Scholar 

  33. Hopcroft D, Mason D, Scott R. Adult rat pancreatic islet cells adherent to microcarrier beads: evaluation of function and morphology. In Vitro Cell Dev B. 1985;21(9):485–7. doi:10.1007/bf02620838.

    Article  CAS  Google Scholar 

  34. Schop D, Janssen FW, Borgart E, de Bruijn JD, van Dijkhuizen-Radersma R. Expansion of mesenchymal stem cells using a microcarrier-based cultivation system: growth and metabolism. J Tissue Eng Regen Med. 2008;2(2–3):126–35.

    Article  CAS  Google Scholar 

  35. Park Y, Subramanian K, Verfaillie CM, Hu WS. Expansion and hepatic differentiation of rat multipotent adult progenitor cells in microcarrier suspension culture. J Biotechnol. 2010;150(1):131–9.

    Article  CAS  Google Scholar 

  36. Kwon J, Kim B-S, Kim M-J, Park H-W. Suspension culture of hematopoietic stem cells in stirred bioreactors. Biotechnol Lett. 2003;25(2):179–82. doi:10.1023/a:1021994026859.

    Article  CAS  Google Scholar 

  37. Sart S, Schneider YJ, Agathos SN. Ear mesenchymal stem cells: an efficient adult multipotent cell population fit for rapid and scalable expansion. J Biotechnol. 2009;139(4):291–9.

    Article  CAS  Google Scholar 

  38. Gigout A, Buschmann MD, Jolicoeur M. Chondrocytes cultured in stirred suspension with serum-free medium containing pluronic-68 aggregate and proliferate while maintaining their differentiated phenotype. Tissue Eng A. 2009;15(8):2237–48. doi:10.1089/ten.tea.2008.0256.

    Article  CAS  Google Scholar 

  39. Weber C, Pohl S, Pörtner R, Wallrapp C, Kassem M, Geigle P, et al. Expansion and harvesting of hMSC-TERT. Open Biomed Eng J. 2007;1:38–46.

    CAS  Google Scholar 

  40. Frauenschuh S, Reichmann E, Ibold Y, Goetz PM, Sittinger M, Ringe J. A microcarrier-based cultivation system for expansion of primary mesenchymal stem cells. Biotechnol Prog. 2007;23(1):187–93.

    Article  CAS  Google Scholar 

  41. Athari A, Unthan-Fecher K, Schwartz P, Probst I. Adult rat hepatocyte microcarrier culture. Comparison to the conventional dish culture system. In Vitro Cell Dev Biol. 1988;24(11):1085–91.

    Article  CAS  Google Scholar 

  42. Alves PM, Moreira JL, Rodrigues JM, Aunins JG, Carrondo MJ. Two-dimensional versus three-dimensional culture systems: effects on growth and productivity of BHK cells. Biotechnol Bioeng. 1996;52(3):429–32.

    Article  CAS  Google Scholar 

  43. Chun BH, Chung SI. Attachment characteristics of normal human cells and virus-infected cells on microcarriers. Cytotechnology. 2001;37(1):1–12.

    Article  CAS  Google Scholar 

  44. Mered B, Albrecht P, Hopps HE. Cell growth optimization in microcarrier culture. In Vitro. 1980;16(10):859–65.

    Article  CAS  Google Scholar 

  45. Melero-Martin JM, Dowling M-A, Smith M, Al-Rubeai M. Expansion of chondroprogenitor cells on macroporous microcarriers as an alternative to conventional monolayer systems. Biomaterials. 2006;27(15):2970–9.

    Article  CAS  Google Scholar 

  46. Yang Y, Rossi FMV, Putnins EE. Ex vivo expansion of rat bone marrow mesenchymal stromal cells on microcarrier beads in spin culture. Biomaterials. 2007;28(20):3110–20.

    Article  CAS  Google Scholar 

  47. Dürrschmid M, Landauer K, Simic G, Blüml G, Doblhoff-Dier O. Scalable inoculation strategies for microcarrier-based animal cell bioprocesses. Biotechnol Bioeng. 2003;83(6):681–6.

    Article  Google Scholar 

  48. Abranches E, Bekman E, Henrique D, Cabral JMS. Expansion of mouse embryonic stem cells on microcarriers. Biotechnol Bioeng. 2007;96(6):1211–21.

    Article  CAS  Google Scholar 

  49. Stiehler M, Bünger C, Baatrup A, Lind M, Kassem M, Mygind T. Effect of dynamic 3-D culture on proliferation, distribution, and osteogenic differentiation of human mesenchymal stem cells. J Biomed Mater Res A. 2009;89(1):96–107.

    Google Scholar 

  50. Chen X, Xu H, Wan C, McCaigue M, Li G. Bioreactor expansion of human adult bone marrow-derived mesenchymal stem cells. Stem Cells. 2006;24(9):2052–9. doi:10.1634/stemcells.2005-0591.

    Article  CAS  Google Scholar 

  51. Kuriyama S, Nakano T, Yoshimura N, Ohuchi T, Moritera T, Honda Y. Mass cultivation of human retinal pigment epithelial cells with microcarrier. Ophthalmologica. 1992;205(2):89–95.

    Article  CAS  Google Scholar 

  52. Qiu Q, Ducheyne P, Gao H, Ayyaswamy P. Formation and differentiation of three-dimensional rat marrow stromal cell culture on microcarriers in a rotating-wall vessel. Tissue Eng. 1998;4(1):19–34. doi:10.1089/ten.1998.4.19.

    Article  CAS  Google Scholar 

  53. da Silva Meirelles L, Caplan AI, Nardi NB. In search of the in vivo identity of mesenchymal stem cells. Stem Cells. 2008;26(9):2287–99. doi:10.1634/stemcells.2007-1122.

    Article  Google Scholar 

  54. Afizah H, Yang Z, Hui JH, Ouyang H-W, Lee E-H. A comparison between the chondrogenic potential of human bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs) taken from the same donors. Tissue Eng. 2007;13(4):659–66. doi:10.1089/ten.2006.0118.

    Article  CAS  Google Scholar 

  55. Bosnakovski D, Mizuno M, Kim G, Takagi S, Okumura M, Fujinaga T. Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: influence of collagen type II extracellular matrix on MSC chondrogenesis. Biotechnol Bioeng. 2006;93(6):1152–63.

    Article  CAS  Google Scholar 

  56. Eslaminejad MB, Mirzadeh H, Mohamadi Y, Nickmahzar A. Bone differentiation of marrow-derived mesenchymal stem cells using beta-tricalcium phosphate-alginate-gelatin hybrid scaffolds. J Tissue Eng Regen Med. 2007;1(6):417–24.

    Article  CAS  Google Scholar 

  57. Oh SK, Chen AK, Mok Y, Chen X, Lim UM, Chin A, et al. Long-term microcarrier suspension cultures of human embryonic stem cells. Stem Cell Res. 2009;2(3):219–30.

    Article  CAS  Google Scholar 

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Acknowledgments

We would like to thank to Puan Vijaya, Encik Roslee Halpi and staffs from the Scanning Electron Microscopy Unit, University of Malaya for their technical support and assistance. We are also grateful to Cik Noor Azera Bakar, Cik. Sahrinanah Mappiare, Cik. Hidaitul Masalaina bt Mohamed, and Dr. Haryanti Azura bt Hj Mohd Wali for their help in animal work. This work was funded by research grants from University of Malaya (Research grant number: PPP177/2009B & FS116/2008A).

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Authors and Affiliations

  1. Tissue Engineering Group, Department of Orthopaedic Surgery, Faculty of Medicine, National Orthopaedic Centre of Excellence for Research and Learning (NOCERAL), University of Malaya, 50603, Kuala Lumpur, Malaysia

    Lily Boo, Tunku Sara Ahmad & Tunku Kamarul

  2. School of Medicine and Health Sciences, Monash University, 46150, Bandar Sunway, Selangor Darul Ehsan, Malaysia

    Lakshmi Selvaratnam

  3. Sime Darby Medical Centre, 47500, Subang Jaya, Selangor Darul Ehsan, Malaysia

    Cheh Chin Tai

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  1. Lily Boo
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  2. Lakshmi Selvaratnam
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Correspondence to Lily Boo.

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Boo, L., Selvaratnam, L., Tai, C.C. et al. Expansion and preservation of multipotentiality of rabbit bone-marrow derived mesenchymal stem cells in dextran-based microcarrier spin culture. J Mater Sci: Mater Med 22, 1343–1356 (2011). https://doi.org/10.1007/s10856-011-4294-7

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