engleski

Histological evaluation of chondro-differentiated cAD-MSC spheroids

Adipose derived mesenchymal stem cells are multipotent cells that in vitro differentiate into adipocytes, chondrocytes and osteoblasts depending on the differentiation media used. We investigated properties of chondro-differentiated canine AD- MSCs (cAD-MSCs) cultured in three dimensional structures, spheroids, using routine haematoxylin- eosin stain (HE) as well as three special stains. AD-MSCs from four young healthy female donors were incubated for 21 days in chondrodifferentiation media at 37°C, with 5% CO2 and 95% humidity. Formalin-fixed paraffin embedded (FFPE) spheroids were sectioned and stained with HE. After histological evaluation of slides, special stains Alcian Blue 8GX, Masson`s trichrome and Von Kossa were performed. Extracellular matrix (ECM) in spheroids stained positively for proteoglycan aggrecan and collagen fibers with Alcian Blue and Masson`s trichrome stain, respectively. Multifocal areas of mineralization were observed in HE stained sections which stained positive with Von Kossa stain. Positive staining for aggrecan showed successful chondro-differentiation and viability of cAD-MSCs in vitro. Since Masson`s trichrome stains mainly collagen type 1, we can presume that the predominant type of collagen in spheroids is type 1 which is the most common ECM protein produced by mesenchymal cells. Further immunohistochemical analysis of collagen types should be carried out. Areas of mineralization indicate ossification of early cartilaginous tissue which could be related to cultivation duration. Routine and special histological staining of FFPE spheroids enabled a more detailed and morphologically accurate representation of cells and their products inside a 3D cell culture. It is a useful method for evaluating differentiation efficacy and also opens new questions such as types of collagen present which should be further investigated.

spheroids, special stain, canine stem cells

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