title: A robust and reproducible animal serum-free culture method for clinical-grade bone marrow-derived mesenchymal stromal cells.
abstract: 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.
authors.affiliation: Research and Cell Therapy Services, Finnish Red Cross Blood Service, Kivihaantie 7, 00310, Helsinki, Finland. anita.laitinen@bloodservice.fi.
authors.forename: Anita
authors.initials: A
authors.lastname: Laitinen
authors.affiliation: Research and Cell Therapy Services, Finnish Red Cross Blood Service, Kivihaantie 7, 00310, Helsinki, Finland.
authors.forename: Sofia
authors.initials: S
authors.lastname: Oja
authors.affiliation: Research and Cell Therapy Services, Finnish Red Cross Blood Service, Kivihaantie 7, 00310, Helsinki, Finland.
authors.forename: Lotta
authors.initials: L
authors.lastname: Kilpinen
authors.affiliation: Research and Cell Therapy Services, Finnish Red Cross Blood Service, Kivihaantie 7, 00310, Helsinki, Finland.
authors.forename: Tanja
authors.initials: T
authors.lastname: Kaartinen
authors.affiliation: Research and Cell Therapy Services, Finnish Red Cross Blood Service, Kivihaantie 7, 00310, Helsinki, Finland.
authors.forename: Johanna
authors.initials: J
authors.lastname: Möller
authors.affiliation: Research and Cell Therapy Services, Finnish Red Cross Blood Service, Kivihaantie 7, 00310, Helsinki, Finland.
authors.forename: Saara
authors.initials: S
authors.lastname: Laitinen
authors.affiliation: Research and Cell Therapy Services, Finnish Red Cross Blood Service, Kivihaantie 7, 00310, Helsinki, Finland.
authors.forename: Matti
authors.initials: M
authors.lastname: Korhonen
authors.affiliation: Research and Cell Therapy Services, Finnish Red Cross Blood Service, Kivihaantie 7, 00310, Helsinki, Finland.
authors.forename: Johanna
authors.initials: J
authors.lastname: Nystedt
keywords: FBS; HLA-DR; Low oxygen; Mesenchymal stromal cell (MSC); Platelet lysate (PL)