Abstract
Equine mesenchymal stromal cells (MSCs) are increasingly investigated for their clinical therapeutic utility. Such cell-based treatments can require cell numbers in the millions or billions, with conventional expansion methods using static T-flasks typically inefficient in achieving these cell numbers. Equine cord blood-derived MSCs (eCB-MSCs), are promising cell candidates owing to their capacity for chondrogenic differentiation and immunomodulation. Expansion of eCB-MSCs in stirred suspension bioreactors with microcarriers as an attachment surface has the potential to generate clinically relevant numbers of cells while decreasing cost, time and labour requirements and increasing reproducibility and yield when compared to static expansion. As eCB-MSCs have not yet been expanded in stirred suspension bioreactors, a robust protocol was required to expand these cells using this method. This study outlines the development of an expansion bioprocess, detailing the inoculation phase, expansion phase, and harvesting phase, followed by phenotypic and trilineage differentiation characterization of two eCB-MSC donors. The process achieved maximum cell densities up to 75,000 cells/cm2 corresponding to 40 million cells in a 100 mL bioreactor, with a harvesting efficiency of up to 80%, corresponding to a yield of 32 million cells from a 100 mL bioreactor. When compared to cells grown in static T-flasks, bioreactor-expanded eCB-MSC cultures did not change in surface marker expression or trilineage differentiation capacity. This indicates that the bioreactor expansion process yields large quantities of eCB-MSCs with similar characteristics to conventionally grown eCB-MSCs.
Highlights
With nearly one million domestic horses in Canada, the horse industry contributes $19 billion annually to the Canadian economy [1]
A variety of different commercially manufactured microcarriers have been tested for the expansion of Mesenchymal stromal cell (MSC), both porous and non-porous, made from a variety of different materials, with different coatings [8,9,10,11]
Osteogenesis and adipogenesis: 6-well plates were seeded at 5000 cells/cm2 in MSC expansion medium containing Basic fibroblast growth factor (bFGF) and grown to 80% confluency
Summary
With nearly one million domestic horses in Canada, the horse industry contributes $19 billion annually to the Canadian economy [1]. $259 million is spent annually in Canada on equine veterinary services [1], with orthopedic injuries being the leading cause of loss of performance in horses [2]. Mesenchymal stromal cell (MSC) injections have been found to be a promising treatment. The type of microcarrier used is critical in a bioreactor process to ensure adequate attachment and expansion of the cells. A variety of different commercially manufactured microcarriers have been tested for the expansion of MSCs, both porous and non-porous, made from a variety of different materials, with different coatings [8,9,10,11]. The choice of microcarrier should be optimized for a given application [13]
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