A closed bag system for human mesenchymal stem cell adhesion, expansion, differentiation and cryopreservation

To ensure quality and safety of human mesenchymal stem cell expansion for advanced cell therapeutic purposes, a disposable cell culture bag system for GMP compliant adherent cell cultivation was developed. We have previously shown that the closed cell culture bag is suitable for the cultivation of suspension cells but did not support growth growth of adherent cells. To enhance adherent cell growth, the polymer surface was functionalized by an atmospheric-pressure plasma process whereby the inner bag surface is selectively modified through film forming agents to create an amino group (APTMS, DACH) or silane (TMOS) containing coating. Biocompatibility of the surface treated cell culture bag was assessed using human bone derived mesenchymal cells and SAOS-2 cells. Cellular responses were evaluated in terms of cell morphology, attachment (fluorescence microscopy, scanning electron microscopy), cell proliferation (WST-1) and osteogenic (Alizarin red staining) and adipogenic differentiation (Nile red staining). All modified surfaces showed initial cell adhesion in contrast to the cell repellent properties of the unmodified cell culture bag. Proliferation on the other hand was only observed in case of APTMS- and DACH- in contrast to TMOS-treated surfaces where after initial cell adhesion took place, no further increase in cell number could be observed. Both osteogenesis and adipogenesis of the MSC could be induced on the modified surfaces, comparable to classic polystyrene culture vessels. Further, we successfully cryopreserved adherent cell layers in situ on the modified culture bag surface whereby the cells retained their proliferative potential after thawing.