ALTEX :: Alternatives to Animal Experiments

2001, VOLUME 1

Three-Dimensional Culture of Human Articular Chondrocytes in Rotating-Wall Vessels

Stefan Marlovits, Vilmos Vécsei, Michaela Truppe, Markus Dezfulian, and Brigitte Tichy

University of A-Vienna

INTRODUCTION

This study describes the culture and three-dimensional assembly of human articular chondrocytes under controlled oxygenation and low shear stress in the NASA-designed rotating-wall vessel (RWV). The bioreactor is a cell culture device that is able to integrate three-dimensional interactions with low-shear mass transfer of nutrients and wastes. Designed with no internal moving parts, the vessel operates in an unusually low shear regimen (0.2 dyn/cm2).

METHODS

Chondrocyte monolayer cultures were established from small cartilage samples from the macroscopic unchanged areas of femoral heads of patients after femoral neck fracture and implantation of hip hemiprosthesis. After serial subculture chondrocytes from the monolayer cultures were released and placed without any scaffold as a single cell suspension in the rotating-wall vessel.

The seeded cells showed a spontaneous aggregation and formation of a solid tissue during the culture time of 10 weeks. In histologic sections a cartilage-like formation with dense extracellular matrix is observed Immuncytochemical analysis reveals differentiated chondrocytes similar to fresh tissue. The phenotype of the cultivated chondrocytes was determined with immuncytochemical techniques using specific monoclonal antibodies against human collagen type I and type II, protein S-100, keratansulfat and vimentin. The staining of sulfated proteoglycans was performed with alcian blue and safranin O.

RESULTS

The conditions provided by the RWV promotes the differentiation of articular chondrocytes as well as the production of cartilage specific extracellular matrix. This culture model may be useful for the study of cartilage matrix metabolism, and may be a useful tool in understanding the process of matrix degradation during aging and osteoarthrits. The in vitro bioreactor may also provide useful, complementary information regarding the effect of specific growth factors and chemical substances on the process of cartilage regeneration in higher age.