A serum free approach towards the conservation of chondrogenic phenotype during in vitro cell expansion.
OBJECTIVE
Functionally viable chondrocytes in sufficient quantity is crucial for the success of matrix associated autologous chondrocyte implantation. This is difficult with conventional methods as chondrocytes dedifferentiate during 2D expansion with the loss of their chondrogenic phenotype. Moreover, established protocols are dependent on the use of serum which is not without its drawbacks. This study sought to address the issue by evaluating the feasibility of serum free, growth factors supplemented chondrocyte media with extracellular matrix (ECM) coatings.


METHODS
Passage 2 human chondrocytes were cultured in serum supplemented media or serum free media with collagen I or fibronectin coatings. Cell attachment and proliferation were assessed in these conditions. The cells were redifferentiated via pellet cultures for 7 and 14 days before being subjected to histological and gene expression analysis.


RESULTS
The serum-free, growth factor cocktail supplemented with ECM coating improved long-term chondrocyte proliferation with enhanced basal Sox 9 expression. Upon induction, the redifferentiated chondrocytes expressed aggrecan and collagen II especially so for the cells plated on collagen coated surfaces. The chondrocytic phenotype was better conserved under the serum free conditions but the loss of the hyaline cartilage characteristics was not completely halted given the expression of collagen I. These essential cartilage markers were, however, reduced or absented for cells expanded with serum. Moreover, serum cultures displayed a higher tendency of undergoing hypertrophy given the stronger collagen X gene expression.


CONCLUSIONS
The advocated technique promoted cell expansion with respect to conventional serum supplemented cultures while reducing the loss of the chondrogenic phenotype. This demonstrates the feasibility and potential of the novel concomitant use of serum free media and ECM coatings in the expansion of chondrocytes for cartilage regenerative applications.
Ho
Saey Tuan Barnabas
ST
Department of Orthopaedic Surgery, Yong Loo Lin School of Medical, National University of Singapore, Singapore, Singapore.
Yang
Zheng
Z
Hui
Hoi Po James
HP
Oh
Kah Weng Steve
KW
Choo
Boon Hwa Andre
BH
Lee
Eng Hin
EH
D002358:Cartilage, Articular / Q000166:cytology; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D019902:Chondrocytes / Q000502:physiology*; D016895:Culture Media, Serum-Free; D005109:Extracellular Matrix; D006801:Humans; D010641:Phenotype; D055758:SOX9 Transcription Factor / Q000378:metabolism; D012692:Serial Passage
D016895:Culture Media, Serum-Free; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human