Expansion and Characterization of Bone Marrow Derived Human Mesenchymal Stromal Cells in Serum-free Conditions

Overview

Journal Sci Rep

Specialty Science

Date 2021 Feb 10

PMID 33564114

Citations 55

Authors

Samatha Bhat

Pachaiyappan Viswanathan

Shashank Chandanala

S Jyothi Prasanna

Raviraja N Seetharam

Affiliations

Soon will be listed here.

Abstract

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are gaining increasing importance in the field of regenerative medicine. Although therapeutic value of MSCs is now being established through many clinical trials, issues have been raised regarding their expansion as per regulatory guidelines. Fetal bovine serum usage in cell therapy poses difficulties due to its less-defined, highly variable composition and safety issues. Hence, there is a need for transition from serum-based to serum-free media (SFM). Since SFM are cell type-specific, a precise analysis of the properties of MSCs cultured in SFM is required to determine the most suitable one. Six different commercially available low serum/SFM with two different seeding densities were evaluated to explore their ability to support the growth and expansion of BM-MSCs and assess the characteristics of BM-MSCs cultured in these media. Except for one of the SFM, all other media tested supported the growth of BM-MSCs at a low seeding density. No significant differences were observed in the expression of MSC specific markers among the various media tested. In contrary, the population doubling time, cell yield, potency, colony-forming ability, differentiation potential, and immunosuppressive properties of MSCs varied with one another. We show that SFM tested supports the growth and expansion of BM-MSCs even at low seeding density and may serve as possible replacement for animal-derived serum.

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References

Bakopoulou A, Apatzidou D, Aggelidou E, Gousopoulou E, Leyhausen G, Volk J . Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects "stemness" properties. Stem Cell Res Ther. 2017; 8(1):247. PMC: 5667471. DOI: 10.1186/s13287-017-0705-0. View

Jung S, Sen A, Rosenberg L, Behie L . Identification of growth and attachment factors for the serum-free isolation and expansion of human mesenchymal stromal cells. Cytotherapy. 2010; 12(5):637-57. DOI: 10.3109/14653249.2010.495113. View

Sekiya I, Larson B, Smith J, Pochampally R, Cui J, Prockop D . Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells. 2002; 20(6):530-41. DOI: 10.1634/stemcells.20-6-530. View

Gottipamula S, Muttigi M, Chaansa S, Ashwin K, Priya N, Kolkundkar U . Large-scale expansion of pre-isolated bone marrow mesenchymal stromal cells in serum-free conditions. J Tissue Eng Regen Med. 2013; 10(2):108-19. DOI: 10.1002/term.1713. View

Tan K, Teo K, Lim J, Chen A, Choolani M, Reuveny S . Serum-free media formulations are cell line-specific and require optimization for microcarrier culture. Cytotherapy. 2015; 17(8):1152-65. DOI: 10.1016/j.jcyt.2015.05.001. View

Gstraunthaler G . Alternatives to the use of fetal bovine serum: serum-free cell culture. ALTEX. 2003; 20(4):275-81. View

Wappler J, Rath B, Laufer T, Heidenreich A, Montzka K . Eliminating the need of serum testing using low serum culture conditions for human bone marrow-derived mesenchymal stromal cell expansion. Biomed Eng Online. 2013; 12:15. PMC: 3599911. DOI: 10.1186/1475-925X-12-15. View

Martin P, Martinez A, Lara V, Naveros B . Regulatory considerations in production of a cell therapy medicinal product in Europe to clinical research. Clin Exp Med. 2012; 14(1):25-33. DOI: 10.1007/s10238-012-0213-6. View

Wu W, Zhou Y, Tan W . [In vitro culture of bone marrow-derived mesenchymal stem cells in a chemically-defined serum-free medium]. Sheng Wu Gong Cheng Xue Bao. 2009; 25(1):121-8. View

10.

Solchaga L, Penick K, Porter J, Goldberg V, Caplan A, Welter J . FGF-2 enhances the mitotic and chondrogenic potentials of human adult bone marrow-derived mesenchymal stem cells. J Cell Physiol. 2004; 203(2):398-409. DOI: 10.1002/jcp.20238. View

11.

Chase L, Yang S, Zachar V, Yang Z, Lakshmipathy U, Bradford J . Development and characterization of a clinically compliant xeno-free culture medium in good manufacturing practice for human multipotent mesenchymal stem cells. Stem Cells Transl Med. 2012; 1(10):750-8. PMC: 3659659. DOI: 10.5966/sctm.2012-0072. View

12.

Kotobuki N, Hirose M, Takakura Y, Ohgushi H . Cultured autologous human cells for hard tissue regeneration: preparation and characterization of mesenchymal stem cells from bone marrow. Artif Organs. 2004; 28(1):33-9. DOI: 10.1111/j.1525-1594.2004.07320.x. View

13.

Ullah I, Subbarao R, Rho G . Human mesenchymal stem cells - current trends and future prospective. Biosci Rep. 2015; 35(2). PMC: 4413017. DOI: 10.1042/BSR20150025. View

14.

Kinzebach S, Bieback K . Expansion of Mesenchymal Stem/Stromal cells under xenogenic-free culture conditions. Adv Biochem Eng Biotechnol. 2012; 129:33-57. DOI: 10.1007/10_2012_134. View

15.

Juhl M, Tratwal J, Follin B, Sondergaard R, Kirchhoff M, Ekblond A . Comparison of clinical grade human platelet lysates for cultivation of mesenchymal stromal cells from bone marrow and adipose tissue. Scand J Clin Lab Invest. 2016; 76(2):93-104. DOI: 10.3109/00365513.2015.1099723. View

16.

Wuchter P, Vetter M, Saffrich R, Diehlmann A, Bieback K, Ho A . Evaluation of GMP-compliant culture media for in vitro expansion of human bone marrow mesenchymal stromal cells. Exp Hematol. 2016; 44(6):508-18. DOI: 10.1016/j.exphem.2016.02.004. View

17.

Bocelli-Tyndall C, Zajac P, Di Maggio N, Trella E, Benvenuto F, Iezzi G . Fibroblast growth factor 2 and platelet-derived growth factor, but not platelet lysate, induce proliferation-dependent, functional class II major histocompatibility complex antigen in human mesenchymal stem cells. Arthritis Rheum. 2010; 62(12):3815-25. DOI: 10.1002/art.27736. View

18.

Devireddy L, Myers M, Screven R, Liu Z, Boxer L . A serum-free medium formulation efficiently supports isolation and propagation of canine adipose-derived mesenchymal stem/stromal cells. PLoS One. 2019; 14(2):e0210250. PMC: 6392232. DOI: 10.1371/journal.pone.0210250. View

19.

Bobis-Wozowicz S, Kmiotek K, Kania K, Karnas E, Labedz-Maslowska A, Sekula M . Diverse impact of xeno-free conditions on biological and regenerative properties of hUC-MSCs and their extracellular vesicles. J Mol Med (Berl). 2016; 95(2):205-220. PMC: 5239805. DOI: 10.1007/s00109-016-1471-7. View

20.

Schubert S, Brehm W, Hillmann A, Burk J . Serum-free human MSC medium supports consistency in human but not in equine adipose-derived multipotent mesenchymal stromal cell culture. Cytometry A. 2017; 93(1):60-72. DOI: 10.1002/cyto.a.23240. View