Development Of A Scalable Manufacturing Process For Bone-Marrow Derived hMSCs In A Low-Shear Single-Use Bioreactor System

Bone-Marrow derived Mesenchymal Stem Cells (BM-MSCs) are multi-potent plastic adherent non-hematopoietic cells found in bone marrow and stroma.  They have been widely studied for 40 years and although a rare cell population, only 0.01-0.001%, they are relatively easy to isolate, grow well in vitro, and possess both immunomodulatory and low immune reactivity properties.  For these reasons MSCs are thought by many to be an ideal cell for the development of “off the shelf” allogeneic cellular therapeutics for the treatment of a variety of diseases such as graft-vs-host disease, Crohn’s, stroke, and some cardiomyopathies. MSCs have also been isolated from adipose and other tissues, expanding their therapeutic potential.  A recent search of www.clinicaltrials.gov returns a list of more than 300 trials using mesenchymal stem cells.  However, in order to move from these small trials to commercialization, cells must be manufactured at large scale while remaining potent and safe. 

To address this manufacturing bottleneck, we have developed a unique MSC manufacturing process where BM-MSCs are cultured on microcarriers with low shear mixing using the Air-Wheel Bioreactor.  Microcarrier culture was chosen for its productivity and scalability advantage over 2D culture.  Successful microcarrier culture, particularly in large vessels, requires a judicious choice of bioreactor and impeller.  The PBS single-use bioreactors are able to gently suspend microcarrier cultures at a Kolmogorov scale of the order of 130 um at all vessel sizes (3, 15, 80 and  500 L.)

BM-MSCs (StemCell Technologies) were cultured in xeno-free Mesencult medium (StemCell Technologies) on Synthemax II microcarriers (Corning) in a PBS 3 Air-Wheel Bioreactor.  Processes for microcarrier seeding and expansion via bead-to-bead transfer took place within the Bioreactor.  Cell growth and metabolic rates were monitored, and a traditional stirred-tank bioreactor was run in parallel as control.  In the Air-Wheel Bioreactor, the cells attached to the microcarriers faster and reached confluence more quickly than a stirred-tank bioreactor.  MSCs cultured as described met the International Society of Cellular Therapy (ISCT) minimum criteria for BM-MSC (> 95% CD73, CD105, CD90 and < 2% CD34, CD45, CD14, CD19, HLA-DR) and tri-lineage differentiation.  In this study we show that BM-MSCs can be expanded on microcarriers using a low-shear Air-Wheel Bioreactor with bead-to-bead transfer that is amenable to scale up to commercial manufacturing.  When BM-MSCs are cultured using this novel expansion system, microcarrier loading is fast and uniform, the MSCs are highly proliferative, and they retain the characteristic markers of BM-MSC.