Browsing by Author "Jardim, Filipa Raquel Nunes"
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- Spheroid development for in vitro Chondrogenesis: preliminary studiesPublication . Jardim, Filipa Raquel Nunes; Tomás, Helena Maria Pires Gaspar; Gonçalves, Mara Isabel JesusThis thesis presents the first set of experimental work done at CQM – Centro de Química da Madeira aimed at the implementation of a new research line focused on spheroids for chondrogenesis study. Three different techniques for spheroid preparation using human mesenchymal stem cells (hMSCs) were tested. The preparation of spheroids by cell self-aggregation in ultra-low adhesion (ULA) cell culture plates was shown to be easy to use, and to lead to stable spheroids. The addition of 50 ng/mL carboxymethyl cellulose (CMC) to the cell culture medium helped to control spheroid’s size and spheroidicity, improving cell viability as well. The influence of different culture medium compositions on cell metabolic activity and on cell differentiation towards the chondrogenic lineage was evaluated in 2D cell cultures. The addition of TGF-β1, as well as other supplements, to the chondrogenic medium (CM) increased cell proliferation, and induced chondrogenesis (revealed through histochemical staining). For the assessment of the chondrogenesis in 3D constructs, spheroids were initially prepared and matured for 14 days (50 ng/mL of CMC was added to the medium at day 5) in ULA plates (poly-HEMA coating) in BM with high glucose content, at a high cell seeding density. Then, a spheroid group was exposed to CM; and another maintained in Basal medium (BM), as a control (CTRL) group for more 28 days (chondrogenesis induction). Spheroid’s growth was observed in the CM group, whereas their shrinking was evident in CTRL group. Also, CM led to higher cell viability values in spheroids. Histochemistry staining experiments were only successful using the Fast Green/Safranin O system, revealing the occurrence of chondrogenesis in spheroids exposed to CM. ALP activity measurements showed the absence of the hypertrophic phenotype in the differentiated spheroids (chondrospheres). Finally, ELISA assays allowed the detection of collagen type II and aggrecan in the chondrospheres cultured in CM.