Serum-Free Co-Culture of Human Mesenchymal Stem Cells and Articular Chondrocytes in Suspension Bioreactors for Cartilage Repair

Background:

Defects in articular cartilage can lead to a chronic and painful condition called osteoarthritis^1,2. A proposed cell therapy is the implantation of human articular chondrocytes (hACs) into the defect site. A major drawback of this technique is that hACs do not proliferate extensively in culture without losing their native phenotype^2,3. Recently, it has been discovered that when hACs and human mesenchymal stem cells (hMSCs) are co-expanded in static culture, hACs proliferate extensively and hMSCs exhibit enhanced chondrogenesis compared to monoculture of each cell type². Thus, the co-culture of hACs and MSCs represents a feasible method of generating cells capable of cartilage repair.

Static culture is an inefficient means of generating cells that lacks reproducibility and the ability to control key aspects of the culture environment. However, suspension bioreactors can reproducibly generate large quantities of cells under controlled conditions. Thus, the aim of this project was to determine if it is possible to co-expand populations of hACs and hMSCs in bioreactors as aggregates under serum-free conditions.

Methods:

First, two serum-free media (PPRF-msc6⁴ and TheraPEAK) and a serum-containing medium were screened to determine if they could support the proliferation of co-expanded cells in static culture. Next, bone marrow-derived hMSCs and hACs were inoculated in a 1:3 ratio² at 20,000 cells/mL in 125 mL stirred suspension bioreactors. Samples of aggregates were enzymatically dissociated and counted to determine the cell density. Cell and matrix characteristics, such as gene expression, the amount of matrix produced and the distribution of each cell type in the aggregates were also analyzed. We have examined several variables to improve cell proliferation in the bioreactors including: agitation rate, feeding schedule and oxygen tension.

Results:

The cells reached the highest cell densities in a serum-free medium, PPRF-msc6 (540,000±30,000 cells/mL on day 6), as compared to TheraPEAK (9,000±2,000 cells/mL), and serum-containing medium (36,000±2,000 cells/mL). Thus, from this point forward, all experiments were conducted with PPRF-msc6.

We have demonstrated, for the first time, that hACs and hMSCs can be co-expanded in suspension bioreactors as aggregates of cells as shown in Figure 1, producing more matrix (3.3 µg glycosaminoglycans (GAG)/µg DNA) than in static vessels (1.5 µg GAG/µg DNA). Agitation rate had a significant impact on cell proliferation. Specifically, the average cell density was greater in the 80 rpm condition, peaking at 89,000±1,000 cells/mL at day 12, as compared to the 60 rpm (48,000±1,700 cells/mL on day 10) and the 100 rpm conditions (68,000±0 cells/mL on day 12). Additionally, bioreactor operation as cyclic fed-batch, with 50% medium changes on day 8 and 12, resulted in higher cell densities (95,000±7,000 cells/mL on day 16) than batch operation (40,000±1,600 cells/mL). Also, hypoxic conditions (3% O₂ tension⁵) resulted in more than double the cell growth compared to normoxic conditions.

Significance:

We have developed protocols to rapidly and reproducibly co-culture human stem cells and chondrocytes to clinically relevant quantities. The large-scale production of hACs and hMSCs in bioreactors with serum free medium represents a novel strategy for the generation of cells for cartilage tissue-engineering.

References:

1.     Gao, J. et al. 2007. J Eng Med 221:441?450. 2. Acharya, C. et al. 2012. J Cell Physiol. 227:88?97. 3. Dhinsa, B. & Adesida, AB. 2012. Curr Stem Cell Res Ther. 7:143?148. 4. Jung, S. et al. 2011. J Tissue Eng Regen Med 6:391-403. 5. Adesida, A. et al. 2012. Stem Cell Res & Ther, 3:9.