Principios en el aislamiento y cultivo de condrocitos de acuerdo a buenas prácticas de laboratorio

OBJECTIVES: The objective of the present study was to determine if chondrocytes isolated from human cartilage of five elderly patients (mean 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 of chondrocytes were observed, but spindle/fibroblast like morphology was also detected in cultured cells. 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

Principios en el aislamiento y cultivo de condrocitos de acuerdo a buenas prácticas de laboratorio

Objectives: 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.Objetivos: El objetivo del presente estudio era determinar si los condrocitos aislados de cinco pacientes ancianos (edad media 63 años) con artrosis (grado 3) mantienen su proliferación y potencial condrogénico. El aislamiento y cultivo de condrocitos fueron llevados a cabo de acuerdo a los estándares de buenas prácticas de laboratorio. Métodos: Los condrocitos fueron aislados de una biopsia de cartílago mediante digestión enzimática. El cultivo fue llevado a cabo en un ambiente controlado (sala blanca). La caracterización del fenotipo de los condrocitos se logró mediante análisis de citometría de flujo. Resultados: Tras tres semanas de cultivo se podían observar estructuras poligonales propias de los condrocitos, pero también se observaba morfología de tipo fibroblasto en el cultivo. El análisis de la citometría de flujo reveló 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 hematopoyético CD45 (0,10%). Conclusiones: Los condrocitos articulares humanos obtenidos de cinco pacientes ancianos con artrosis mantenían un fenotipo condrocitario y podrían ser potencialmente utilizados para la implantación autóloga. Las condiciones para el cultivo fueron establecidas de acuerdo a los estándares de buenas prácticas de laboratorio para así minimizar el riesgo de contaminación celular in vitro.Slovak Research and Development Agency (APVV 0684-12); VEGA grant No.1/0772/13; Centre of Excellence for Neuroregenerative Research (project ITMS No. 26220120063)

Reparacija defekata hrskavice kunića autolognim i alogenim hondrocitima zasejanim na kolagen/ hijaluronan osnovu ili suspendovanim u fibrinskom lepku

The topic of the study was to verify in vivo survival of in vitro cultured autologous and allogenous chondrocytes suspended in a fi brin glue Beriplast® or seeded on Collagen type I-Hyaluronan (Col type I-HYA) scaffolds for the regeneration of articular cartilage defects in rabbits. The study was carried out on 15 domestic rabbits randomly assigned to fi ve groups (n = 3 in each) with different treatments of artifi cially created chondral defects (ChD ́s). These defects were made in a non-load-bearing area of medial condyle of the distal femur, and were treated as follows: 1st and 3rd group: the ChD ́s were fi lled with autologous or allogenous chondrocytes seeded on Col type I-HYA scaffolds, respectively. The scaffolds were fi xed to the ChD ́s by fi brin glue Beriplast®; 2nd and 4th group: the ChD ́s were fi lled with a suspension of autologous or allogenous chondrocytes in fi brin glue Beriplast®, respectively, and they were immediately covered by unseeded Col type I-HYA scaffolds; Control group: the ChD ́s were left to heal spontaneously without any treatment. Macroscopical, histological and immunohistochemical analyses of the ChD ́s were performed 12 months after the treatment. In all treated groups, the chondrocytes were capable to proliferate and produce the cartilage extracellular matrix, including proteoglycans and type II collagen, as compared to the control “untreated” group. On the other hand, the production of hyaline-like cartilage tissue confi rmed that both therapeutic methods using autologous chondrocytes can be applied successfully for the treatment of chondral defects in rabbits.Cilj studije je bio da se verifi kuje in vivo preživljavanje in vitro kultivisanih autolognih i alogenih hondrocita suspendovanih u fi brinski Beriplast® lepak ili na kolagen Hyaluronan tip I (Col type I-HYA) osnovu radi regeneracije oštećenja zglobne hrskavice kod kunića. Ispitivanje je obavljeno na 15 domaćih kunića, odabranih slučajnim izborom koji su podeljeni u pet grupa (3 po grupi) kod kojih su veštački izazvana oštećenja hrskavice (ChD), tretirana na različite načine. Oštećenja su napravljena na površinama koje nisu noseće na medijalnom kondilusu distalnog femura, a tretman je podrazumevao: prva i treća grupa: ChD su punjene sa autolognim ili alogenim hondrocitima zasejanim na Col tip I-HYA osnovu. Osnove su fi ksirane na ChD pomoću fi brinskog lepka Beriplast®; druga i četvrta grupa: oštećenja su punjena suspenzijom autolognih ili alogenih hondrocita u fi brinskom Beriplast® lepku, a neposredno posle toga, prekriveni su sa nezasejanom Col tip I-HYA osnovom; kontrolna grupa: oštećenja su ostavljena da spontano zarastu, bez tretmana. Dvanaest meseci posle tretmana obavljene su makroskopske, histološke i imunohistološke analize promena. Kod svih životinja tretiranih grupa, hondrociti su bili sposobni da proliferišu i proizvode ekstracelularni matriks hrskavice, uključujući proteoglikane i II tip kolagena u poređenju sa kontrolnom “netretriranom” grupom. Sa druge strane, proizvodnja hrskavičavog tkiva nalik na hijalin potvrđuje da se oba terapijska metoda koji koriste autologne hondrocite mogu da uspešno primene u tretiranju defekata hrskavice kod kunića