Diferentes hemocomponentes como substitutos do soro fetal bovino no cultivo de células-tronco mesenquimais equinas derivadas de membrana sinovial

Abstract

Athlete horses can develop a series of musculoskeletal injuries that are a common consequence of physical overload, which negatively affects the quality of life and athletic performance of the animal. Stem cell therapy for tissue regeneration has rapidly gained ground as the treatment of choice for equine orthopedic injuries. Fetal bovine serum (FBS) is used as a universal supplement for the cultivation of stem cells, and has been the subject of ethical, scientific and safety questions. In order to address the possible shortcomings of FBS as a nutrient for the clinical expansion of human and animal mesenchymal stem cells (MSCs), researchers have focused on blood products as alternatives. In recent years, platelet-rich plasma and/or platelet lysate have been proposed as substitutes for FBS, by sustaining cellular expansion, mainly of human cells. Thus, the present study sought to compare different blood products as supplements to the equine synovial membrane-derived mesenchymal stem cell (MSCTM) culture medium, seeking to elect a blood component equivalent or superior to FBS, in order to remove xenogeneic components potentially causing reactions after transplantation of equine MSCs. Allogeneic platelet lysate (PL) and equine serum (SE) have been proposed as possible substitutes for FBS, which were evaluated in SmMSC culture regarding cell morphology; adherence to plastic; ability to differentiate into trilineages (adipogenic, osteogenic and chondrogenic); immunophenotypic characterization by expression of surface receptors CD13-, CD34-, CD44+, CD45-, CD90+, CD105+, MHC-II; expansion capacity by cell proliferation rate and cell population doubling time; and cellular metabolism by the MTT assay. All groups showed adhesion to the plastic and morphology as expected for stem cells. Tri-lineage differentiation was positive in all groups, with emphasis on spontaneous adipogenesis in the PL and SE groups. The expression of surface receptors was similar between the groups, maintaining the immunophenotypic pattern of stem cells. The FBS group had higher proliferation and duplication rates than the others, showing better clonal expansion capacity, followed by the PL group, whose concentration at the end of cultivation was similar to that of the FBS group. Cellular metabolism fluctuated between groups, with superiority of the FBS group, with a statistical difference at the 24h moment in the PL and SE groups and at the 48h moment in the PL group. The results of the study allowed us to affirm that the MsCTM cultivated with platelet lysate and equine serum were able to maintain the essential characteristics to be identified as stem cells. However, the spontaneous adipogenesis by the cells of the groups with equine blood components stands out, which justifies in-depth studies so that the clinical application of cell therapy is not impaired by the early and unexpected behavior of the cells. Although the proliferation and cell viability rates found with SE and PL were insufficient to maintain cell expansion at similar or higher levels than FBS, it is necessary to continue studies related to the subject in order to explore the composition of each blood product and its influence on cell culture, in addition to comparing different concentrations that can generate satisfactory results for serum-free culture.