A novel isolation method for chondrocytes differentiated from synovial fluid-derived mesenchymal stem cell

Abstract

Mesenchymal stem cells (MSCs) in synovial fluid (SF) actively participate in the regeneration process of healthy joints and have been defined as a good source of chondrocyte cells, which can be obtained by in vitro differentiation of stem cells. This cell population derived from synovial fluid shares cellular characteristics with bone marrow and synovial membrane MSCs with respect to their differentiation potency. Current cell isolation protocols for cartilage therapy mainly focus on the isolation of chondrocytes or MSCs from biological samples. However, the isolation of chondrocytes after the in vitro differentiation from MSCs has not been described in the literature. In this context, we defined a novel method based on Ficoll-Paque density gradient centrifugation for high-throughput isolation of differentiated chondrocytes from human synovial fluid mesenchymal stem cells (hSF-MSCs) without the requirement for cell labeling. In this study, terminally differentiated chondrocytes were obtained after 21 days of chondrogenic differentiation from hSF-MSCs and were isolated using this novel protocol. The isolated chondrocytes were later analyzed for cell viability and functionality by staining with Alcian Blue and by gene expression analysis for chondrocyte markers. In conclusion, the novel chondrocyte isolation method described here is capable of achieving low-cost efficiency. According to the minimal process principles, we hope that this protocol will find use in both translational research and routine clinical applications involving the differentiation and isolation of chondrocytes in vitro.

Keywords

• Synovial fluid-derived mesenchymal stem cells
• Chondrocyte
• Differentiation
• Isolation

Supporting Institution

KOCAELİ ÜNİVERSİTY

Project Number

This reseach was supported by the Kocaeli University Scientific Research Project Department [grant no. BAP-2022-TDK-2948] and The Scientific and Technological Research Council of Türkiye (TÜBİTAK) [grant no. 123S367]

Ethical Statement

Ethical approval for this thesis study was obtained from the Non-Interventional Clinical Research Ethics Committee of Kocaeli University under the reference number GOKAEK-2021/22.17 on December 21, 2021.

Sinoviyal sıvı kaynaklı mezenkimal kök hücrelerden farklılaşan kondrositlerin izolasyonu için yeni bir yöntem

Abstract

Sinovyal sıvıdaki (SS) mezenkimal kök hücreler (MKH’ler), sağlıklı eklemlerin rejenerasyon sürecine aktif olarak katılır ve in vitro farklılaşma yoluyla elde edilebilen kondrosit hücrelerinin iyi bir kaynağı olarak tanımlanmıştır. SS'dan elde edilen bu hücre popülasyonu, kemik iliği ve sinovyal membran MKH'leriyle benzer şekilde farklılaşma potansiyeli açısından hücresel özellikler paylaşmaktadır. Mevcut hücre izolasyon protokolleri, genellikle biyolojik örneklerden kondrosit veya MKH izolasyonuna odaklanmaktadır. Ancak, MKH'lerden in vitro farklılaştırma yoluyla elde edilen kondrositlerin izolasyonu literatürde henüz tanımlanmamıştır. Bu bağlamda, hücre etiketleme gerektirmeksizin Ficoll-Paque yoğunluk gradyanı santrifüjleme temelli yeni bir yöntem tanımladık. Bu yöntem, insan SS-MKH'lerinden farklılaşmış kondrositlerin yüksek verimli izolasyonunu sağlamaktadır. Bu çalışmada, iSS-MKH'lerinden 21 günlük kondrojenik farklılaşma sürecinin ardından terminal farklılaşmış kondrositler bu yeni protokol kullanılarak elde edilmiştir. İzole edilen kondrositler, Alcian Blue boyaması ile hücre canlılığı ve fonksiyonelliği açısından incelenmiş ve kondrositlere özgü gen ekspresyonu analizi yapılmıştır. Sonuç olarak, burada tanımlanan yeni kondrosit izolasyon yöntemi düşük maliyetli ve verimli bir çözüm sunmaktadır. Minimal işlem ilkelerine uygun olarak bu protokolün, hem translasyonel araştırmalar hem de in vitro kondrosit farklılaşması ve izolasyonuna yönelik rutin klinik uygulamalarda kullanılması beklenmektedir.

Keywords

• Sinovyal sıvı kaynaklı mezenkimal kök hücreler
• kondrosit
• farklılaşma
• izolasyon

Project Number

This reseach was supported by the Kocaeli University Scientific Research Project Department [grant no. BAP-2022-TDK-2948] and The Scientific and Technological Research Council of Türkiye (TÜBİTAK) [grant no. 123S367]

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