Large scale in vitro expansion of mesenchymal stem cells.

Abstract

Mesenchymal stem cells (MSCs) can undergo self-renewal and differentiation into a variety of mature cell types. Thus, using MSCs for tissue engineering and other medical applications holds many promising advantages over normal somatic cells. However, exactly these characteristics make MSCs more difficult to grow and control in vitro. The aim of this research project was to investigate different culture systems for their utility to expand bone marrow derived MSCs in large quantities. A large scale expansion of MSCs is especially of interest since only a small number of bone marrow derived MSCs are present in donor derived samples, which do not meet the demand for medical applications. In this thesis three different culture systems, static monolayer cultures, stirred suspension cultures, and pour-off cultures, were compared with each other for their ability to support MSCs proliferation while allowing them to keep their full differentiation capacity. Cell samples derived from these cultures were used for cell count, to start a CFU-f assay and to start osteogenic and adipogenic differentiation assays. The highest MSC numbers obtained from the static monolayer cultures were about 460% of the initial cells. The differentiation capacity of these cells was restricted, so they only formed osteoblasts. Furthermore, MSC samples obtained from this culture system were used for proteomic analysis on an electrospray ionisation quadrupol (ESI-qQ-STAR) mass spectrometer with the isobaric tag for relative and absolute quantitation (iTRAQ) method. This analysis revealed a difference in the proteome of MSCs from different passage levels which is involved in a changing proliferation and differentiation behaviour. In stirred suspension cultures, the increase in MSC number varied for the different culture media. The best result was achieved in MyeloCult® medium with Pluronic F- 68, IL-3 and SCF, however, reaching only 140% of the initial cell density, this result was significantly worse than in the control monolayer cultures. The pour-off cultures supported an increase in MSC number, which resulted in 860% of the initial cell number. In addition, MSCs expanded in this culture system were able to differentiate into ostoblasts and adipocytes. Thus, pour-off cultures are the most promising culture system for large scale expansion of MSCs with high differentiation potential