Investigation of effects of 3D spheroid culture and different cell culture media on the proliferation of chondrocytes in-vitro

Introduction Chondrocytes could be of central importance for the application of tissue engineering in the head and neck region for cartilage reconstruction. Since only a few cells are found in cartilage tissue, they must be multiplied. However, chondrocytes isolated from cartilage tissue dedifferentiate very quickly under in vitro culture conditions and the ability to redifferentiate is lost. The aim of this study was to determine whether chondrocytes can be proliferated over a longer period of time using a 3D cell culture model without loss of cartilage-specific properties.

Methods Chondrocytes were isolated from ear (n=2) or nasal cartilage (n=2) by outgrowth culture (CPC) and enzymatic digestion (CC) (Ethical vote No.: 2018-584N-MA). The cells were propagated for 2 to 3 passages in 2D culture and then transferred to 3D spheroid culture. Different cell numbers and cell culture media were analysed. A specific medium for chondrocytes (ChondroDiff Medium, Miltenyi Biotec) served as reference medium. Cell viability in the spheroid was followed for 21 days with fluorescent dyes for dead (Sytox®Green) and viable cells (ATP-Red). Cartilage-specific markers were analysed using IHC.

Results The viability in the spheroids was higher at lower initial cell numbers (5000, 10000, 25000) than at higher cell numbers (50000 and 100000), regardless of the source or processing method and regardless of the medium used. In the smaller spheroids, the addition of IGF-1 increased the proportion of viable cells and induced the expression of cartilage-specific components.

Conclusion The proliferation of chondrocytes in 3D spheroid culture over a longer period is possible. The addition of IGF-1 induces the ability to produce cartilage-specific matrix.

Poster-PDF A-1521.pdf

Publication History

Article published online:
13 May 2021