Over time homogeneity and stability of mesenchymal stromal cells 3D spheroids built using base-level laboratory equipment

Mesenchymal Stromal Cells (MSC) are widely used for tissue engineering applications because of their capacity to promote tissue regeneration while modulating immune responses. Three-dimensional (3D) MSC aggregates, also known as multicellular spheroids, better preserve phenotype and innate properties with respect to cells cultured in monolayer. They are the preferred format for many cell therapy and tissue engineering applications, and for bioprinting uses. Many approaches have been proposed in literature to generate spheroids composed of cancer cells, and most of them can be applied when constructing MSC spheroids. Efficiency, cost, and size of the produced spheroids are typically considered to compare different generation approaches. The pellet culture method provides a cost-effective and extremely rapid method to generate 3D MSC spheroids. Only a benchtop centrifuge and sterile polypropylene conical tubes, typically available in every biological laboratory, are required. However, although the pellet culture method is widely used to generate MSC spheroids, few studies have been performed to analyse the morpho-biological homogeneity and stability during time in culture of the obtained spheroids. In this study we monitored for two months changes in size and morphology of a set of spheroids generated by the pellet culture method. Each spheroid was imaged with brightfield microscopy twice a week. AnaSP (open-source software freely available at: http://sourceforge.net/p/anasp) was used to compute volume, sphericity and jagging (i.e. indentation degree of the spheroid's border) of each spheroid over time. The volume of the MSC spheroids showed a decreasing trend. However, just fifteen days from spheroid generation, all the values remained constant and similar, demonstrating good volume stability and homogeneity after an initial settling time. Sphericity of the MSC spheroids kept always high values, thus indicating that the shape is generally close to a perfect sphere. Jagging degree values were always very low, proving that MSC spheroids are characterized by a compact border. We also performed confocal and histological analyses to better investigate their stability. Hematoxylin&Eosin stained cryosections were analysed at different time points to evaluate the internal cellular architecture. Then, the cell viability and distribution on the spheroid surface was evaluated with Live&Dead staining and confocal imaging. No significant changes in internal distribution and spheroid compactness were observed up to 30 days from the spheroids generation. In conclusion, we can assume that the MSC spheroids generated are homogenous and stable for a long time interval. Accordingly, the proposed enhanced pellet culture method can be used as a reference system for laboratories interested in easily obtaining, without need for specialized equipment, stable homogeneous populations of MSC spheroids to be used in long term studies and applications