Histological validation of adipogenic differentiation potential of ASC on collagen-based 2D scaffolds
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Histological validation of adipogenic differentiation potential of ASC on collagen‑based 2D scaffolds Marta Gomarasca1 · Paolo Savadori2 · Sara Mariano3 · Laura Cipolla3 · Giovanni Lombardi1,4 Accepted: 8 July 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Determination of the adipogenic potential and behavior of adipose-derived mesenchymal stem/stromal cells (ASCs) is particularly relevant for their potential clinical application in regenerative medicine, especially when regeneration is supported by biomaterials or scaffolds. Scaffolds need to be able to induce tissue repair and limit undesired adipogenic differentiation. Depending on the scaffold employed, determination of cell behavior may be hindered by material interference with staining, which will limit either cells identification or dye quantification. Collagen is a promising biomaterial in regenerative medicine, however, histological analysis of cells cultured on collagen-based scaffolds is challenging. Here we describe a new histological method based on iron hematoxylin combined with Oil red O (ORO) staining, for the determination of the adipogenic differentiation of ASCs cultivated on a collagen-based 2D scaffold. ASCs were seeded on collagen films or plastic, differentiated into adipocytes for 14 days, and then stained with either ORO or iron hematoxylin and ORO combined. The collagen films avidly absorbed the ORO dye; conventional staining and quantification by dye extraction failed to discriminate between differentiated and undifferentiated cells on the films. On the contrary, the iron hematoxylin-ORO combination provided a quantitative and more reliable determination of adipocytes based on single cell count. This method is particularly recommended for determining the adipogenic differentiation potential of ASCs and other cell types grown on highly absorptive materials that need to be validated for their potential use in bioengineering and regenerative medicine. Keywords Adipose-derived stem cells (ASC) · Adipogenic differentiation · Collagen · Oil red O · Nile blue · Histological quantification
Introduction Adipose tissue, one of the most abundant tissues of human body, carries out essential endocrine and energy regulatory functions (Khandekar et al. 2011; Scherer 2006). In vitro * Marta Gomarasca [email protected] 1
Laboratory of Experimental Biochemistry and Molecular Biology, IRCCS Istituto Ortopedico Galeazzi, Via Riccardo Galeazzi, 4, 20161 Milan, Italy
2
Histology Unit, IRCCS Istituto Ortopedico Galeazzi, Via Riccardo Galeazzi, 4, 20161 Milan, Italy
3
Department of Biotechnology and Biosciences, University of Milano - Bicocca, Piazza della Scienza 2, 20126 Milan, Italy
4
Department of Athletics, Strength and Conditioning, Poznań University of Physical Education, Królowej Jadwigi 27/39, 61‑871 Poznań, Poland
adipogenesis is critically important in the determination of the differentiation potential of multipotent stem cells such as mesenchymal stem/str
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