Isolation and characterization of human articular chondrocytes from surgical waste after total knee arthroplasty (TKA)

Abstract

BackgroundCartilage tissue engineering is a fast-evolving field of biomedical engineering, in which the chondrocytes represent the most commonly used cell type. Since research in tissue engineering always consumes a lot of cells, simple and cheap isolation methods could form a powerful basis to boost such studies and enable their faster progress to the clinics. Isolated chondrocytes can be used for autologous chondrocyte implantation in cartilage repair, and are the base for valuable models to investigate cartilage phenotype preservation, as well as enable studies of molecular features, nature and scales of cellular responses to alterations in the cartilage tissue.MethodsIsolation and consequent cultivation of primary human adult articular chondrocytes from the surgical waste obtained during total knee arthroplasty (TKA) was performed. To evaluate the chondrogenic potential of the isolated cells, gene expression of collagen type 2 (COL2), collagen 1 (COL1) and aggrecan (ACAN) was evaluated. Immunocytochemical staining of all mentioned proteins was performed to evaluate chondrocyte specific production.ResultsCartilage specific gene expression of COL2 and ACAN has been shown that the proposed protocol leads to isolation of cells with a high chondrogenic potential, possibly even specific phenotype preservation up to the second passage. COL1 expression has confirmed the tendency of the isolated cells dedifferentiation into a fibroblast-like phenotype already in the second passage, which confirms previous findings that higher passages should be used with care in cartilage tissue engineering. To evaluate the effectiveness of our approach, immunocytochemical staining of the evaluated chondrocyte specific products was performed as well.DiscussionIn this study, we developed a protocol for isolation and consequent cultivation of primary human adult articular chondrocytes with the desired phenotype from the surgical waste obtained during TKA. TKA is a common and very frequently performed orthopaedic surgery during which both femoral condyles are removed. The latter present the ideal source for a simple and relatively cheap isolation of chondrocytes as was confirmed in our study.

MicroRNA-197 regulates chondrocyte proliferation, migration, and inflammation in pathogenesis of osteoarthritis by targeting EIF4G2

“…The human OA cartilage tissues were treated and digested with 0.2% collagenase II (Thermo Fisher Scientific, Waltham, MA, U.S.A.), and primary chondrocytes were obtained as described previously [ 24 ]. The cells were cultured in the Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, U.S.A.) containing 15% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, U.S.A.), 100 units/ml of penicillin, and 100 units/ml of streptomycin for 24 h at 37°C with 95% air.…”

Biomedical grafts for tracheal tissue repairing and regeneration “Tracheal tissue engineering: an overview”

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Isolation and characterization of the first Slovenian human triple‐negative breast cancer cell line

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“…First, we removed all noncancer cell types from the cell culture (eg fibroblasts). To achieve this, we developed 2 procedures: (a) the cells were cultivated according to standard cultivation protocols established in our laboratories until the fourth passage (within every passage, cells exhibiting spontaneous epithelial cell growth were preserved; (b) after trypsinization of the primary culture, we removed individual cell groups by pipette and transferred them into a microtiter P24 plates. Both procedures were successful in raising TNBC CLs.…”

“…Cell isolation was carried out in a sterile laminar flow cabinet (Laminar Air Flow Chamber, ISKRA-Pio) and in general resembled our established isolation protocols. 4 The cells were placed into an incubator at 37°C and an atmosphere of 5 wt.% CO 2 . Three days after isolation, the cells began to adhere to the surface of the Petri dishes.…”