A Microcarrier‐Based Cultivation System for Expansion of Primary Mesenchymal Stem Cells

Abstract

Microcarrier cultures have been shown to allow extensive cell expansion of tissue engineering relevant cells, such as chondrocytes, while maintaining their phenotype. Our aim was to investigate the in vitro three-dimensional expansion of porcine bone-marrow-derived primary mesenchymal stem cells (MSC) using commercially available Cytodex type 1, type 2, and type 3 microcarriers. In comparison, the Cytodex type 1 microcarriers showed the best results for adherence with over 80% adherent cells after 3 h of incubation, analyzed by the Poisson distribution. Different start cell densities ranging from 1 to 3 x 106 cells per 100 cm2 had only a minor influence on adhesion. The proliferation was examined on Cytodex type 1 microcarriers over a cultivation time of 28 days, which could reveal cell growth and proof of cells recolonizing freshly added microcarriers. Scanning electron microscopy displayed appropriate cell morphology and confirmed cell proliferation. After enzymatic harvest from microcarriers, the osteogenic and chondrogenic differentiation of these cells was induced and shown by relevant histochemistry, such as von Kossa and Alcian blue staining. Totaling the results, we have shown that the three-dimensional expansion of MSC on microcarriers represents a beneficial alternative to the conventional two-dimensional monolayer cultivation method.

Expansion of human mesenchymal stem cells on microcarriers

“…Indeed, expanding human stem cells in culture is currently more of an art than a science so much so that some laboratories can"t or don"t grow human stem cells at all. In fact it is only now that successful, yet empirical, protocols for expanding human stem cells in bioreactors are starting to appear in the literature (Abranches et al 2007;Frauenschuh et al 2007;Oh et al 2009), few of which deal with any form of systematic optimisation and none consider the engineering aspects in any depth. An attempt has been made here to understand some of these issues and it is important to note that some of the parameters measured were similar to those associated with other adherent cell lines.…”

“…2008). Thus whilst microcarriers have been shown to be appropriate for human embryonic and mesenchymal stem cells (Abranches et al 2007;Frauenschuh et al 2007;Oh et al 2009), little detailed work has been done on them to optimise cell microcarrier attachment protocols or cell proliferation and growth. Importantly, human stem cells can"t automatically be considered to behave in a similar way to other adherent cells lines.…”

“…In this work, these phenomenon were investigated using hMSCs isolated from placental tissue in 250 mL spinner flasks with two impeller geometries and a well-known commercially available microcarrier, Cytodex 3 (GE Healthcare, Sweden) shown to be suitable for the expansion of both murine embryonic and mesenchymal stem cells (Abranches et al 2006;Frauenschuh et al 2007). …”

hMSC Production in Disposable Bioreactors with Regards to GMP and PAT

“…Highest adhesion rates described in the literature were usually achieved by a cyclic procedure with transient stirring and resting phases [22, 25 -28]. Inoculation densities ranging from 1 to 3 · 10 4 cm -2 had only minor influence on the efficiency of cell adhesion [29]. Regarding a constant final cell density at the end of the process, the inoculation density has an influence on the process efficiency as the expansion factor is influenced.…”

Expansion and Harvesting of hMSC-TERT

“…This results in the need for large incubator capacities and extensive benchtop work to achieve high numbers of cells. Suspension cultures on the basis of microcarriers are an alternative to culture flasks because of their high growth surface per volume and therefore a greater achievable spatial yield [7]. Many investigations on microcarriers mainly with respect to their yields of secreted biomolecules or viruses as product of the cultivation process have been published [8][9][10][11].…”