Expansion of human mesenchymal stromal cells from fresh bone marrow in a 3D scaffold-based system under direct perfusion

PLoS One. 2014 Jul 14;9(7):e102359. doi: 10.1371/journal.pone.0102359. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bioreactors
  • Cell Culture Techniques / instrumentation
  • Cell Culture Techniques / methods*
  • Cell Proliferation
  • Cell Separation
  • Humans
  • Mesenchymal Stem Cells / cytology*
  • Oligonucleotide Array Sequence Analysis
  • Perfusion / instrumentation
  • Perfusion / methods*
  • Phenotype

Grant support

The authors would like to acknowledge Novartis AG, Basel, Switzerland and the European Union (OPHIS; #FP7-NMP-2009-SMALL-3-246373) for financial support. The funding organization (Novartis AG) provided financial support i) in the form of authors' salaries (SBrachat and CJacobi) where their specific roles have been articulated in the ‘author contributions’ section and ii) for the research materials.