3D morphological characterization of polymeric microcarriers for stem cell expansion using contrast-enhanced microCT

Aims Tissue Engineering (TE) is an interdisciplinary field aiming to provide solutions for the regeneration of organs and tissues. Many typical TE processes make use of stem cells due to their pluripotent behavior and their self-renewal capacities. The latter allows scientists to minimize the number of stem cells harvested from a donor and expand them at large scale to reach a desired amount of cells for a given therapy. In the field of large scale stem cell expansion, micro-carriers are commonly used. These are typically degradable porous or non-porous beads on which cells are seeded and expanded during culture in a spinner flask. However, one of the remaining challenges related to the use of micro-carriers for cell expansion is the limited information on their morphological characteristics provided by the manufacturers. This information is, however, crucial to improve process parameters for cell expansion, such as the cell seeding density, culture time, etc. For instance, the available surface area, on which the cells can attach, is an important property influencing the initial cell seeding density, and is highly dependent on the micro-carriers morphology (porosity, pore size, roughness, etc.), but it is rarely provided by the manufacturers. Therefore, it has been highlighted how useful a database of 3D morphological characteristics of different micro-carriers could be1. X-ray microfocus computed tomography (microCT) could provide a solution, as it allows for non-invasive analysis of the 3D morphology of porous materials. However, most of the micro-carriers we currently use are polymeric and they should be characterized in wet state to obtain the proper morphometric characteristics. Since there is no or negligible image contrast difference between the micro-carriers and the surrounding liquid when they are in wet state, in this study we propose the use of contrast-enhanced X-ray computed tomography (CE-CT), combined with detailed image processing and analysis, for 3D morphological characterization of polymeric micro-carriers. We will present the results for one specific type of porous micro-carrier, namely CultiSpher S, by which we highlight the added value of CE-CT combined with morphological quantification for in-depth characterization of polymeric micro-carriers. Methods Micro-carriers CultiSpher STM (HyCloneTM) micro-carriers were used. These micro-carriers are based on gelatin derived from collagen to enhance cell attachment and proliferation. From