Abstract
<p><strong> </strong>Bone marrow (BM) is an important source for isolating mesenchymal stem cells (MSC) useful in immunomodulation and tissue regeneration therapies. <strong>Objective. </strong>To isolate and characterize mesenchymal stem cells obtained from BM meeting the requirements of the International Society for Cellular Therapy. <strong>Materials and methods.</strong> BM samples were collected from volunteer donors attending the Orthopedics Service of the San Ignacio University Hospital (Bogotá, Colombia). Morphological characteristics were evaluated by inverted microscopy and the immunophenotype was determined by flow cytometry. Protocols were developed for adipogenic, osteogenic and chondrogenic differentiation using the Oil Red O, alkaline phosphatase and safranin stains, respectively. <strong>Results</strong>. We collected 24 samples of BM from patients with total hip replacement (volume of BM sample: 5-45 ml). Cells with a fibroblastoid morphology were isolated from 21 BM samples (isolation efficiency: 87.5%). No statistical significant differences were found between the hematopoyetic antigens (CD34 and CD45, p&gt;0.05) in the immunophenotypic evaluation (of MSC from BM); on the contrary, there were differences (p=0.006) between the hematopoyetic antigen CD45 and the mesenchymal antigens (CD13, CD44, CD73, CD90, CD105, HLA-I, and HLA-DR). Oil Red O stain revealed the presence of multilocular adipocytes, in the osteogenic induction we observed localized mineralization nodules, and chondrogenesis was positive as revealed by the safranin stain. <strong>Conclusion</strong>. MSC were satisfactorily isolated from BM and characterized according to the international standards.</p> <p><strong>Key words:</strong> stem cells, mesenchymal cells, flow cytometry, bone marrow, cell differentiation</p><br />
Highlights
Isolamento e caracterização de células-tronco mesenquimais de medula óssea humana de acordo com critérios da Sociedade Internacional de Terapia Celular
Isolation and characterization of mesenchymal stem cells from human bone marrow according to the criteria of the International Society for Cellular Therapy
To isolate and characterize mesenchymal stem cells obtained from Bone marrow (BM) meeting the requirements of the International Society for Cellular Therapy
Summary
Las muestras de MO se obtuvieron a partir de pacientes sometidos a reemplazos totales de cadera que cumplían con los siguientes criterios de inclusión: [1] Pacientes entre 18 y 80 años de edad; [2] Ausencia de neoplasias relacionadas con el sistema hematopoyético; [3] Serología negativa para los virus de inmunodeficiencia humana (VIH), virus de la hepatits B (VHB) y virus de la hepatitis C (VHC); [4] Pacientes sin tratamientos relacionados con medicamentos inmunosupresores. Las muestras de MO se recolectaron durante el proceso quirúrgico de reemplazo de cadera realizado por los médicos ortopedistas del Hospital Universitario San Ignacio. La muestra de rimado y/o MO se recolectó (volumen mínimo requerido 5 ml), se maceró y filtró con una gasa estéril y posteriormente se transfirió a tubos cónicos de 15 ml para centrifugación y obtención del “buffy coat”; luego se realizó la separación de células mononucleares (CMN) por gradiente de densidad con Histopaque®1077 Sigma-Aldrich), 1% (v/v) de piruvato de sodio (GIBCO-Invitrogen). El cultivo primario de células adherentes con morfología fibroblastoide se mantuvo hasta obtener una confluencia del 90%, luego se realizaron pases de las células mediante tripsinización (Incubación de las células durante 5 minutos a 37oC con una solución de Tripsina 0,25% (p/v) y EDTA 1mM.
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