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.

确定脂肪来源的间充质干/基质细胞 (ASC) 的脂肪生成潜力和行为与其在再生医学中的潜在临床应用特别相关，尤其是当再生得到生物材料或支架支持时。支架需要能够诱导组织修复并限制不需要的成脂分化。根据所使用的支架，细胞行为的确定可能会受到染色的材料干扰，这将限制细胞识别或染料定量。胶原蛋白是再生医学中一种很有前途的生物材料，但是，对在基于胶原蛋白的支架上培养的细胞进行组织学分析具有挑战性。在这里，我们描述了一种基于铁苏木精结合油红 O (ORO) 染色的新组织学方法，用于测定在基于胶原蛋白的 2D 支架上培养的 ASC 的成脂分化。将 ASC 接种在胶原膜或塑料上，分化为脂肪细胞 14 天，然后用 ORO 或铁苏木精和 ORO 染色。胶原膜强烈地吸收了 ORO 染料；通过染料提取进行的常规染色和定量无法区分薄膜上的分化细胞和未分化细胞。相反，铁苏木精-ORO 组合提供了基于单细胞计数的脂肪细胞的定量和更可靠的测定。