Developing efficient bioreactor microcarrier cell culture system for large scale production of mesenchymal stem cells (MSCs)

Abstract

Background & Aim A large-scale production of clinical-grade MSCs and their derived products is essential due to their immense therapeutic potential. Even though 3D bioreactors are cost efficient in scale up, the plastic adherence of MSCs, makes expansion in suspension cultures challenging. A variety of microcarriers (MCs) allow plastic adherent cells to grow on their surface while maintaining cells in suspension within a bioreactor for expansion. However, this leads to loss of cells, particularly during separation of cells from the carriers. To overcome this, we identified “dissolvable microcarriers” such as Corning Synthemax II dissolvable MCs, which removes the filtration step. Our aim was to develop an efficient bioreactor microcarrier cell culture system to improve the yield of MSCs. Methods, Results & Conclusion Cells from primary human Adipose derived cell line at passage 4 were seeded into a T175 flask (media: aMEM + 10% Human platelet lysate). The cells were expanded for 3 days, harvested by TryplE dissociation and seeded onto a Corning Dissolvable MCs and the control solid core MCs at 8000 cells/cm2, in two separate PBS mini 0.5L MagDrive bioreactors containing 350ml of pre-warmed media. Bioreactors were operated at 16 rpm for the first 12 hours, and 22 rpm for the remainder, in an incubator (37C, 5%CO2). Cells were harvested at 7 days, dissociated from the control MCs using TryplE (15-20 min) followed by passing through a 100um filter, to separate the cells from MCs. The Corning dissolvable MCs were digested with a manufacturer recommended Pectinase/EDTA/TryplE solution, without filtration. Cells from both cultures were washed in DPBS and analysed. A total of 3 replicate runs were performed. The post-harvest cell count (median± range) for dissolvable MCs was 104.6±39.6 million cells and for the control MCs was 76.04±6.28 million cells. The culture supernatant was assayed for pH and osmolality and were found to be similar for both MCs. Analysis of vascular endothelial growth factor(VEGF), showed that dissolvable MCs had a lower level of secreted VEGF (5690±476.4 pg/ml/million cells) compared to control MCs (10103±5384 pg/ml/million cells). Similar trend was noted for macrophage chemoattractant protein-1; 6302±2094 pg/ml/million cells in dissolvable MCs vs 9047±2621 pg/ml/million cells in control MCs. These results indicate that dissolvable MCs could lead to a higher yield from bioreactors (36% increase). However, it is important to further characterise cells and assess their therapeutic potential. A large-scale production of clinical-grade MSCs and their derived products is essential due to their immense therapeutic potential. Even though 3D bioreactors are cost efficient in scale up, the plastic adherence of MSCs, makes expansion in suspension cultures challenging. A variety of microcarriers (MCs) allow plastic adherent cells to grow on their surface while maintaining cells in suspension within a bioreactor for expansion. However, this leads to loss of cells, particularly during separation of cells from the carriers. To overcome this, we identified “dissolvable microcarriers” such as Corning Synthemax II dissolvable MCs, which removes the filtration step. Our aim was to develop an efficient bioreactor microcarrier cell culture system to improve the yield of MSCs. Cells from primary human Adipose derived cell line at passage 4 were seeded into a T175 flask (media: aMEM + 10% Human platelet lysate). The cells were expanded for 3 days, harvested by TryplE dissociation and seeded onto a Corning Dissolvable MCs and the control solid core MCs at 8000 cells/cm2, in two separate PBS mini 0.5L MagDrive bioreactors containing 350ml of pre-warmed media. Bioreactors were operated at 16 rpm for the first 12 hours, and 22 rpm for the remainder, in an incubator (37C, 5%CO2). Cells were harvested at 7 days, dissociated from the control MCs using TryplE (15-20 min) followed by passing through a 100um filter, to separate the cells from MCs. The Corning dissolvable MCs were digested with a manufacturer recommended Pectinase/EDTA/TryplE solution, without filtration. Cells from both cultures were washed in DPBS and analysed. A total of 3 replicate runs were performed.