Martial Hervy

Long term expansion of bone marrow-derived hMSCs on novel synthetic microcarriers in xeno-free, defined conditions.

Human mesenchymal stem cells (hMSCs) present an attractive target for cell therapy given their wide availability, immunomodulatory properties, and multipotent nature for differentiation into chondrocytes, osteocytes, and adipocytes. With the progression of hMSC clinical studies, there is an increasing demand for development of technologies that enable efficient cell scale-up into clinically relevant quantities. Commercial scale manufacturing of hMSCs will require a large surface area which is not cost effective with available two-dimensional culture vessels. Recent studies showed that microcarriers provide a three-dimensional culture environment suitable for hMSC expansion. Traditionally, biological coatings and/or serum-containing medium are required to facilitate hMSC attachment and expansion in dynamic conditions. These limitations may hinder the use of microcarriers as a scale-up technology for hMSC therapeutics, where cell products, and therefore patient safety, are more controlled with the use of xeno-free, defined culture conditions. Here we report the long term culture of hMSCs on novel synthetic Synthemax II microcarriers in two different xeno-free media. Cells were maintained over 40 days on sterile, ready-to-use microcarriers in spinner flasks with programmed agitation. hMSC expansion was obtained by addition of fresh beads without the need for enzymatic dissociation. We achieved a cumulative cell expansion of >10,000 fold, and cells retained normal hMSC phenotype, karyotype, and tri-lineage differentiation potential. To our knowledge, this report is the first example of long term culture of hMSCs on synthetic microcarriers in xeno-free, defined conditions.

Influence of Substrate Thickness and Stiffness on Cell Behavior

It is known that various cell types can sense and respond to the mechanical properties of their microenvironment. Specifically, cells have been known to spread more when cultured on stiff substrates and are able to match their internal stiffness to that of the substrate. It has also been recently reported that even some cells are cultured on soft but thin coatings attached to stiff supports, they can perceive an effective stiffness that is much larger than that of the bulk coating. Building on earlier models [1,2], we present a model and invoke arguments based on energetics that explain why stiff and thin substrates encourage some cells to spread more easily and why these cells can match their internal stiffness to external stiffness more efficiently than others.Figure : Traction energy as a function of coating thickness for different cell stiffnessesReference:1. U. S. Schwarz et al, Biosystems, 83, 225 (2006).2. J. M. Maloney et al, Phys. Rev. E, 78, 041923 (2008).View Large Image | View Hi-Res Image | Download PowerPoint Slide