Isolation, expansion and differentiation of mesenchymal stromal cells from rabbits' bone marrow

Abstract

RESUMO.-[Isolamento, expansão e diferenciação de celulas tronco mesenquimais oriundas da medula óssea de coelhos.] A engenharia de tecidos tem sido uma técnica fundamental no campo da medicina regenerativa, uma vez que permite a criação de peças teciduais tri-dimensionais por meio da associação de células mesenquimais indiferenciadas (ou células estromais mesenquimais -CEMs) e moldes de biomateriais in vitro. Assim, muitos estudos têm sido realizados utilizando estas células oriundas de diferentes espécies animais, e os coelhos são frequentemente utilizados como um modelo animal para estudos in vivo de reparação tecidual. No entanto, a maioria das informações disponíveis sobre a coleta e caracterização de CEMs referem-se às células humanas e murinas, o que traz algumas dúvidas para pesquisadores que desejam trabalhar com coelhos em estudos de reparação de tecidos baseados em CEMs. Neste contexto, o presente estudo objetivou contribuir e aprimorar as informações disponíveis na literatura científica fornecendo uma técnica completa para o isolamento, expansão e diferenciação das MSCs de coelhos. Cé-lulas mononucleares da medula óssea (CMMOs) do úmero e fêmur de coelhos foram obtidas e, para avaliar sua taxa de proliferação, três meios de cultura diferentes foram testadas, aqui referidos como DMEM-P, DMEM'S e α-MEM. As CMMOs também foram cultivadas em meios de indução osteogênico, condrogênico, e linhagens adipogênico para Tissue engineering has been a fundamental technique in the regenerative medicine field, once it permits to build tri-dimensional tissue constructs associating undifferentiated mesenchymal cells (or mesenchymal stromal cells -MSCs) and scaffolds in vitro. Therefore, many studies have been carried out using these cells from different animal species, and rabbits are often used as animal model for in vivo tissue repair studies. However, most of the information available about MSCs harvesting and characterization is about human and murine cells, which brings some doubts to researchers who desire to work with a rabbit model in tissue repair studies based on MSCs. In this context, this study aimed to add and improve the information available in the scientific literature providing a complete technique for isolation, expansion and differentiation of MSCs from rabbits. Bone marrow mononuclear cells (BMMCs) from humerus and femur of rabbits were obtained and to evaluate their proliferation rate, three different culture media were tested, here referred as DMEM-P, DMEM´S and α-MEM. The BMMCs were also cultured in osteogenic, chondrogenic and adipogenic induction media to prove their multipotentiality. It was concluded that the techniques suggested in this study can provide a guideline to harvest and isolate MSCs from bone marrow of rabbits in enough amount to allow their expansion and, based on the laboratory experience where the study was developed, it is also suggested a culture media formulation to provide a better cell proliferation rate with multipotentiality preservation. provar a sua multipotencialidade...

Comparison of biotechnological culture of hypoxia-conditioned rat mesenchymal stem cells with conventional in vitro culture of normoxia-conditioned rat mesenchymal stem cells for testicular failure therapy with low libido in rats

“…The purity of MSCs derived bone marrow was determined by immunofluorescence through the identification of CD105 + and CD45 − cells [17]. Immunofluorescence identification for CD105 was positive expression and CD45 was negative expression.…”

“…MSCs were collected from the bone marrow of iliac crest of male Wistar rats ( R. norvegicus ) at 3 months of age. The aspirate was collected in heparinized tubes and stored at 4°C for transportation to the laboratory at the Institute of Tropical Diseases, Universitas Airlangga, for in vitro culturing [17].…”

“…The cells were cultured continuously until approximately 75%–80% confluence was attained. The cells were then passaged into several dishes for subculture [17].…”

Bone Tissue Engineering Using Human Cells: A Comprehensive Review on Recent Trends, Current Prospects, and Recommendations

“…However, the isolation procedure of these cells is different in other animals. For example, the aspiration of tibia and femur has been standardized to isolate BM-MSCs from mouse and rabbit laboratory animals [154,155]. BM-MSCs as the most universal source of MSCs are identified through some criteria including the formation of clonogenic adherent cell clusters, fibroblastic morphology, and the potential to differentiate into all the three lineages including ectoderm [156,157], mesoderm [158,159], and endoderm [160,161].…”

Bone Marrow Mesenchymal Stromal Cells (BMMSCs) Augment Osteointegration of Dental Implants in Type 1 Diabetic Rabbits: An X-Ray Micro-Computed Tomographic Evaluation

“…FBS25-10 CAP (Sigma)), Naphthol AS-MX Phosphate Alkaline Solution (Sigma-Aldrich, Cat # 85L-2) mixture for ±20 min at room temperature. The excess stains were removed with successive washing with de-ionized water and documented for osteogenic induction through microscopic monitoring [30].…”