A robust and reproducible animal serum-free culture method for clinical-grade bone marrow-derived mesenchymal stromal cells

• Published in: Cytotechnology (Dordrecht) Vol. 68; no. 4; pp. 891 - 906
• Main Authors: Laitinen, Anita, Oja, Sofia, Kilpinen, Lotta, Kaartinen, Tanja, Möller, Johanna, Laitinen, Saara, Korhonen, Matti, Nystedt, Johanna
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01.08.2016; Springer Nature B.V
• Subjects: Antibiotics; Biochemistry; Biomedicine; Biotechnology; Blood & organ donations; Blood platelets; Bone marrow; Cell culture; Cell proliferation; Cells; Chemistry; Chemistry and Materials Science; Culture media; Fetal calf serum; Freeze-thawing; Growth factors; Histocompatibility antigen HLA; Immunomodulation; Karyotypes; Leukocytes; Lymphocytes T; Manufacturing; Mesenchymal stem cells; Original Research; Phenotypes; Plasma; Platelets; Stromal cells; Supplements; Mesenchymal stromal cell (MSC); HLA-DR; Low oxygen; FBS; Platelet lysate (PL)
• ISSN: 0920-9069; 1573-0778
• DOI: 10.1007/s10616-014-9841-x

Efficient xenofree expansion methods to replace fetal bovine serum (FBS)-based culture methods are strongly encouraged by the regulators and are needed to facilitate the adoption of mesenchymal stromal cell (MSC)-based therapies. In the current study we established a clinically-compliant and reproducible animal serum-free culture protocol for bone marrow-(BM-) MSCs based on an optimized platelet-derived supplement. Our study compared two different platelet-derived supplements, platelet lysate PL1 versus PL2, produced by two different methods and lysed with different amounts of freeze–thaw cycles. Our study also explored the effect of a low oxygen concentration on BM-MSCs. FBS-supplemented BM-MSC culture served as control. Growth kinetics, differentiation and immunomodulatory potential, morphology, karyotype and immunophenotype was analysed. Growth kinetics in long-term culture was also studied. Based on the initial results, we chose to further process develop the PL1-supplemented culture protocol at 20 % oxygen. The results from 11 individual BM-MSC batches expanded in the chosen condition were consistent, yielding 6.60 × 10 9  ± 4.74 × 10 9 cells from only 20 ml of bone marrow. The cells suppressed T-cell proliferation, displayed normal karyotype and typical MSC differentiation potential and phenotype. The BM-MSCs were, however, consistently HLA-DR positive when cultured in platelet lysate (7.5–66.1 %). We additionally show that culture media antibiotics and sterile filtration of the platelet lysate can be successfully omitted. We present a robust and reproducible clinically-compliant culture method for BM-MSCs based on platelet lysate, which enables high quantities of HLA-DR positive MSCs at a low passage number (p2) and suitable for clinical use.