Basics of isolation and cultivation of chondrocytes according to good laboratory practice

espanolObjetivos: El objetivo del presente estudio era determinar si los condrocitos aislados de cinco pacientes ancianos (edad media 63 anos) con artrosis (grado 3) mantienen su proliferacion y potencial condrogenico. El aislamiento y cultivo de condrocitos fueron llevados a cabo de acuerdo a los estandares de buenas practicas de laboratorio. Metodos: Los condrocitos fueron aislados de una biopsia de cartilago mediante digestion enzimatica. El cultivo fue llevado a cabo en un ambiente controlado (sala blanca). La caracterizacion del fenotipo de los condrocitos se logro mediante analisis de citometria de flujo. Resultados: Tras tres semanas de cultivo se podian observar estructuras poligonales propias de los condrocitos, pero tambien se observaba morfologia de tipo fibroblasto en el cultivo. El analisis de la citometria de flujo revelo que el fenotipo de los condrocitos cultivados tras el primer pasaje era positivo para CD44 (98,92%), CD90 (97,11%) y negativo para el marcador hematopoyetico CD45 (0,10%). Conclusiones: Los condrocitos articulares humanos obtenidos de cinco pacientes ancianos con artrosis mantenian un fenotipo condrocitario y podrian ser potencialmente utilizados para la implantacion autologa. Las condiciones para el cultivo fueron establecidas de acuerdo a los estandares de buenas practicas de laboratorio para asi minimizar el riesgo de contaminacion celular in vitro. EnglishObjectives: The objective of the present study was to determine if chondrocytes isolated from human cartilage of five elderly patients (middle age 63) with osteoarthritis (stage 3) maintain their proliferation and chondrogenic potential. Isolation and cultivation of chondrocytes was performed according to good laboratory practice (GLP) standards. Methods: Chondrocytes were isolated from cartilage biopsy by enzymatic digestion. Cultivation of cells was performed in a controlled environment (cleanroom). Phenotype characterization of chondrocytes was achieved by flow cytometry analysis. Results: Three weeks after cultivation polygonal structures typical for chondrocytes were observed, but spindle/fibroblast like morphology was also detected in culture. Flow cytometric analysis showed that chondrocytes were positive for CD44 (98,35% ± 0,50), CD90 (97,15% ± 0,13) after first passage (P1) and the cells were negative for hematopoietic marker CD45 (0,21% ± 0,11). Conclusions: Human articular chondrocytes obtained from five elderly patients with osteoarthritis maintained a chondrocyte phenotype and could be potentially used for autologous implantation. We have standardized the conditions for cultivation according to GLP standards to minimize the risk of in vitro cell contamination.