Novel Technique for Suspension Culture of Autologous Chondrocytes Improves Cell Proliferation and Tissue Architecture

Abstract

We have developed a new and simple method of chondrocyte suspension culture using a spinner bottle with rotation of the matrices. We compared the characteristics of chondrocytes cultured by this method with those grown in standard monolayer cultures. We also determined the optimal nutritional medium for suspension cultures. Periosteum explants seeded with chondrocytes were grown in monolayer and suspension cultures under three conditions: in medium with no additive (control), with 10% fetal bovine serum (FBS), or with 10% autologous serum (AS). After culturing, the explants were harvested, processed for histology, and stained with hematoxylin-eosin or TUNEL, or immunostained for type I, II, and III collagen, and Ki-67 antigen. In monolayer cultures, the attachment of the chondrocytes to the periosteum was weak and the superficial layer consisted of fibrotic tissue and few nucleated cells. Collagen type II staining was strong, but types I and III were weak. Among the suspension cultures the AS group produced the thickest layer of chondrocytes with the fewest apoptotic cells. The superficial layer of cartilage in these cultures stained positive for type I and III collagen and Ki-67 antigen. Among the suspension cultures, total chondroitin and chondroitin-4 sulfate (C-4S) concentration was highest in the AS group, while prostaglandin E₂ (PGE₂) was highest in the FBS group. In summary, our new method of suspension culture of periosteal explants using rotational matrices combined with AS nutritional media was the most effective method for maintaining the bond between the chondrocyte layer and periosteum, as well as the production of type I and III collagen in the superficial layer.