Clinical demand for mesenchymal stem cells (MSCs) drives the need for development of robust large-scale production. This study demonstrates the utility of a 3-L single-use bioreactor and collagen-coated microcarriers for the expansion of human bone marrow derived MSCs. This proof of principle study is a demonstration of the potential for large-scale stem-cell scale-up using stirred bioreactors.
Mesenchymal stem cells (MSCs) are multipotent cells with the ability to differentiate into a variety of cell types including osteoblasts, chondrocytes, and adipocytes. These cells have been explored for the repair and regeneration of connective tissues such as cartilage and bone, and for transfusion therapy in patients following bone marrow or peripheral blood stem cell transplants to reduce complications from life-threatening graft-versus-host disease (1, 2).As demand for stem cells for both drug discovery and clinical applications grows, effectively translating the promise of stem cells into therapeutic reality will require large-scale industrialized production under tightly controlled conditions. Achieving this level of production while meeting rigorous quality and regulatory standards will depend on further progress in the areas of cell culture and scale-up, characterization, enrichment, purification, and process control to deliver a consistent and reproducible supply of cells in a safe and cost-effective manner.
To meet the market needs and clinical demand for MSCs, rapid, robust expansion methods are required. To date, large-scale production is typically achieved using two dimensional (2D) tissue culture vessels—an expensive and time-consuming process. The research presented here examines the utility of a single-use, stirred-tank bioreactor in combination with microcarriers for mesenchymal stem-cell expansion, and comprehensively compares the characteristics of the product cells with those grown in standard 2D cultures.
MATERIALS AND METHODS
MSCs (EMD Millipore SCR108) were cultured under static conditions with low glucose DMEM (Invitrogen 11054), 10% FBS (HyClone SH30070.03), Pen/Strep (EMD Millipore TMS-AB2-C), L-Glutamine (EMD Millipore TMS-002-C), and 8 ng/mL human recombinant β fibroblast growth factor (EMD Millipore GF003AF-MG) in T-150 flasks coated with gelatin (EMD Millipore ES-006B). Low oxygen conditions were used during 2D propagation as well as during the attachment phase in which the MSCs were attached to the collagen-coated microcarriers (Solohill C102-1521) in Petri dishes.
For agitated culture, the growth medium was supplemented with pluronic acid (Sigma P5556) and antifoam C emulsion (Sigma A8011-500ML). Spinner flasks (Corning 3152) were pre-coated with Sigmacote (Sigma SL2-100ML) and operated at 30 RPM. The impeller speed in the the Mobius CellReady 3-L bioreactor (EMD Millipore CR0003L200) was set to 25 RPM at low volume (1 L) and then increased to 40 RPM at the larger volume (2 L). The cell concentrations were measured daily using a NucleoCounter (Eppendorf M1293-0000) after lysing the cells off the microcarriers. Supernatant was analyzed daily on a BioProfile Flex (Nova Biomedical) to generate the profiles of metabolite accumulation and nutrient consumption. Cells on microcarriers were fixed with 4% paraformaldehyde (USB 19943 1 LT) and stained with DAPI (Invitrogen D1306) to fluorescence the nuclei for images.
RNA was analyzed by reverse transcriptase–polymerase chain reaction (RT–PCR, Invitrogen 10928-042 kit) following isolation on glass fiber filters post guanidinium thiocyanate treatment (Ambion AM1912 kit). Custom DNA oligonucleotide primers (Invitrogen) were used for the PCR at 200 nM. Expression of cell surface markers on the MSCs (e.g. positive markers CD44, CD105, and CD90, and negative marker CD19), were measured using corresponding antibodies (EMD Millipore SCR067). An adipocyte differentiation kit (EMD Millipore SCR020) was used to identify cells containing lipid vacuoles that stained positive with oil red stain, indicative of cells that have undergone adipogenic differentiation.