title: Expansion of human mesenchymal stromal cells from fresh bone marrow in a 3D scaffold-based system under direct perfusion.
abstract: Mesenchymal stromal/stem cell (MSC) expansion in conventional monolayer culture on plastic dishes (2D) leads to progressive loss of functionality and thus challenges fundamental studies on the physiology of skeletal progenitors, as well as translational applications for cellular therapy and molecular medicine. Here we demonstrate that 2D MSC expansion can be entirely bypassed by culturing freshly isolated bone marrow nucleated cells within 3D porous scaffolds in a perfusion-based bioreactor system. The 3D-perfusion system generated a stromal tissue that could be enzymatically treated to yield CD45- MSC. As compared to 2D-expanded MSC (control), those derived from 3D-perfusion culture after the same time (3 weeks) or a similar extent of proliferation (7-8 doublings) better maintained their progenitor properties, as assessed by a 4.3-fold higher clonogenicity and the superior differentiation capacity towards all typical mesenchymal lineages. Transcriptomic analysis of MSC from 5 donors validated the robustness of the process and indicated a reduced inter-donor variability and a significant upregulation of multipotency-related gene clusters following 3D-perfusion--as compared to 2D-expansion. Interestingly, the differences in functionality and transcriptomics between MSC expanded in 2D or under 3D-perfusion were only partially captured by cytofluorimetric analysis using conventional surface markers. The described system offers a multidisciplinary approach to study how factors of a 3D engineered niche regulate MSC function and, by streamlining conventional labor-intensive processes, is prone to automation and scalability within closed bioreactor systems.
authors.affiliation: Departments of Surgery and of Biomedicine, Institute for Surgical Research and Hospital Management, University Hospital Basel, University of Basel, Basel, Switzerland.
authors.forename: Adam
authors.initials: A
authors.lastname: Papadimitropoulos
authors.affiliation: Departments of Surgery and of Biomedicine, Institute for Surgical Research and Hospital Management, University Hospital Basel, University of Basel, Basel, Switzerland.
authors.forename: Elia
authors.initials: E
authors.lastname: Piccinini
authors.affiliation: MusculoSkeletal Diseases, Novartis Institutes for Biomedical Research, Basel, Switzerland.
authors.forename: Sophie
authors.initials: S
authors.lastname: Brachat
authors.affiliation: Departments of Surgery and of Biomedicine, Institute for Surgical Research and Hospital Management, University Hospital Basel, University of Basel, Basel, Switzerland.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Braccini
authors.affiliation: Departments of Surgery and of Biomedicine, Institute for Surgical Research and Hospital Management, University Hospital Basel, University of Basel, Basel, Switzerland.
authors.forename: David
authors.initials: D
authors.lastname: Wendt
authors.affiliation: Departments of Surgery and of Biomedicine, Institute for Surgical Research and Hospital Management, University Hospital Basel, University of Basel, Basel, Switzerland.
authors.forename: Andrea
authors.initials: A
authors.lastname: Barbero
authors.affiliation: MusculoSkeletal Diseases, Novartis Institutes for Biomedical Research, Basel, Switzerland.
authors.forename: Carsten
authors.initials: C
authors.lastname: Jacobi
authors.affiliation: Departments of Surgery and of Biomedicine, Institute for Surgical Research and Hospital Management, University Hospital Basel, University of Basel, Basel, Switzerland.
authors.forename: Ivan
authors.initials: I
authors.lastname: Martin
mesh_terms: D019149:Bioreactors; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D049109:Cell Proliferation; D002469:Cell Separation; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D020411:Oligonucleotide Array Sequence Analysis; D010477:Perfusion / Q000295:instrumentation / Q000379:methods*; D010641:Phenotype