Abstract
Human chondrocytes are expanded and used in autologous chondrocyte implantation techniques and are known to rapidly de-differentiate in culture. These chondrocytes, when cultured on tissue culture plastic (TCP), undergo both phenotypical and morphological changes and quickly lose the ability to re-differentiate to produce hyaline-like matrix. Growth on synoviocyte-derived extracellular matrix (SDECM) reduces this de-differentiation, allowing for more than twice the number of population doublings (PD) whilst retaining chondrogenic capacity. The goal of this study was to apply RNA sequencing (RNA-Seq) analysis to examine the differences between TCP-expanded and SDECM-expanded human chondrocytes. Human chondrocytes from three donors were thawed from primary stocks and cultured on TCP flasks or on SDECM-coated flasks at physiological oxygen tension (5%) for 4 passages. During log expansion, RNA was extracted from the cell layer (70–90% confluence) at passages 1 and 4. Total RNA was column-purified and DNAse-treated before quality control analysis and next-generation RNA sequencing. Significant effects on gene expression were observed due to both culture surface and passage number. These results offer insight into the mechanism of how SDECM provides a more chondrogenesis-preserving environment for cell expansion, the transcriptome-wide changes that occur with culture, and potential mechanisms for further enhancement of chondrogenesis-preserving growth.
Highlights
Arthritis is a debilitating disease, but an expensive one, with total arthritis-attributable medical expenditures and lost earnings surpassing $300 billion USD in 2013 [1]
Chondrocytes grown on tissue culture plastic (TCP) typically expanded for 1.8 population doublings (PD) in P1 and 1.4
At the end of P4, P4, cells grown on TCP had undergone 7.6 PDs and those on synoviocyte-derived extracellular matrix (SDECM) had undergone 16.0 PDs
Summary
Arthritis is a debilitating disease, but an expensive one, with total arthritis-attributable medical expenditures and lost earnings surpassing $300 billion USD in 2013 [1]. Human chondrocytes, expanded and used in autologous chondrocyte implantation techniques, are known to rapidly de-differentiate in culture [2], which has a detrimental impact on their utility for tissue engineering applications. The use of the term de-differentiation is distinct from the use of the term in re-programming or stem cell literature, as it indicates that the cells no longer have the ability to form hyaline-like cartilage tissue. Culture-expanded chondrocytes undergo both morphological and phenotypical changes and, eventually, lose the ability to produce hyaline like matrix. By passage 4, chondrogenic potential is essentially absent when tested in re-differentiation culture. This loss of differentiation potential limits their efficacy in the clinic and seriously impedes our ability to produce clinical-scale tissue engineering of human cartilage with suitable biomechanical properties
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