Osteoarthritic human chondrocytes proliferate in 3D co‐culture with mesenchymal stem cells in suspension bioreactors

Abstract

Osteoarthritis (OA) is a painful disease, characterized by progressive surface erosion of articular cartilage. The use of human articular chondrocytes (hACs) sourced from OA patients has been proposed as a potential therapy for cartilage repair, but this approach is limited by the lack of scalable methods to produce clinically relevant quantities of cartilage-generating cells. Previous studies in static culture have shown that hACs co-cultured with human mesenchymal stem cells (hMSCs) as 3D pellets can upregulate proliferation and generate neocartilage with enhanced functional matrix formation relative to that produced from either cell type alone. However, because static culture flasks are not readily amenable to scale up, scalable suspension bioreactors were investigated to determine if they could support the co-culture of hMSCs and OA hACs under serum-free conditions to facilitate clinical translation of this approach. When hACs and hMSCs (1:3 ratio) were inoculated at 20,000 cells/ml into 125-ml suspension bioreactors and fed weekly, they spontaneously formed 3D aggregates and proliferated, resulting in a 4.75-fold increase over 16 days. Whereas the apparent growth rate was lower than that achieved during co-culture as a 2D monolayer in static culture flasks, bioreactor co-culture as 3D aggregates resulted in a significantly lower collagen I to II mRNA expression ratio and more than double the glycosaminoglycan/DNA content (5.8 vs. 2.5 μg/μg). The proliferation of hMSCs and hACs as 3D aggregates in serum-free suspension culture demonstrates that scalable bioreactors represent an accessible platform capable of supporting the generation of clinical quantities of cells for use in cell-based cartilage repair.

Serum-Free Culture of Human Mesenchymal Stem Cell Aggregates in Suspension Bioreactors for Tissue Engineering Applications

“…Single cells were inoculated into a suspension bioreactor on culture day 0 after being harvested from culture flasks. Based on standard methods developed in our laboratory [29], the suspension bioreactors were placed on a magnetic stir plate set at 80 rpm inside a humidified 5% CO 2 incubator at 37°C. A 50% medium change was carried out every fourth day throughout the culture period by halting impeller motion, allowing the aggregates to settle briefly and carefully drawing off half of the spent medium.…”

Bioprocessing of Mesenchymal Stem Cells and Their Derivatives: Toward Cell-Free Therapeutics

“…MSCs cultured as 3D spheroids also exhibited an increased level of certain proteins and cytokines with more than purely immunomodulatory effects, such as antioncogenic proteins IL-24, TNF- α , CD82, vasculogenesis and angiogenesis promoter VEGF, and proteins involved in cell differentiation and survival such as TGF- β 3 [ 82 , 83 ]. Furthermore, MSCs grown in 3D spheroids cocultured with osteoarthritic chondrocytes exhibited higher potential for cartilage repair compared to those grown as a 2D monolayer [ 84 ]. Although there is limited research on the effect of spheroid culture on many aspects of the secretome, one would expect the increase in cell-cell interaction, altered cell morphology, and the potentially hypoxic nature of the spheroid's internal microenvironment to have a large effect on the composition of the resulting MSC secretome.…”

Mesenchymal Stem Cell Therapy for Ischemic Tissues

“…Moreover, other considerations related to the administration of cells include optimization of cell dosage, cell administration method (cell alone versus cell-laden scaffold), and timing. Although work to address some of these important concerns is already well underway [ 15 , 16 , 77 – 81 ], it would take a considerable amount of time before these cells can be widely implemented clinically to treat ischemic tissues.…”