Injectable, cryopreservable mesenchymal stromal cell-loaded microbeads for pro-angiogenic therapy: in vitro proof-of-concept

Abstract

Despite their recognized potential for ischemic tissue repair, the clinical use of human mesenchymal stromal cells (hMSC) is limited by the poor viability of cells after injection and the variability of their paracrine function. In this study, we show how the choice of biomaterial scaffolds and the addition of cell preconditioning treatment can address these limitations and establish a proof-of-concept for cryopreservable hMSC-loaded microbeads. Injectable microbeads in chitosan, chitosan-gelatin, and alginate were produced using stirred emulsification to obtain a similar volume moment mean diameter (D[4,3] ∼ 500 µm). Cell viability was determined through live/dead assays, and vascular endothelial growth factor (VEGF) release was measured by ELISA. Proangiogenic function was studied by measuring the wound closure velocity of human umbilical vein endothelial cells (HUVEC) co-cultured with MSC-loaded microbeads. The effect of freeze-thawing on microbeads morphology, porosity, injectability and encapsulated MSC was also studied. hMSC-loaded chitosan-based microbeads were found to release 11-fold more VEGF than alginate microbeads (p< 0.0001) and chitosan-gelatin was chosen for further studies because it presented the best cell viability. Preconditioning with celastrol significantly enhanced the viability (1.12-fold) and VEGF release (1.40-fold) of MSC-loaded in chitosan-gelatin microbeads, as well as their proangiogenic paracrine function (1.2-fold;p< 0.05). In addition, preconditioning significantly enhanced the viability of hMSC after 1 and 3 d in low-serum medium after cryopreservation (p< 0.05). Cryopreserved hMSC-loaded microbeads maintained their mechanical properties, were easily injectable through a 23G needle, and kept their paracrine function, enhancing the proliferation and migration of scratched HUVEC. This study shows the advantage of chitosan as a scaffold material and concludes that chitosan-gelatin microbeads with celastrol-preconditioned cells form a promising off-the-shelf, cryopreservable allogenic MSC product.In vivotesting is required to confirm their potential in treating ischemic diseases or other clinical applications.

Compression

The mechanical properties of each hydrogel were studied by unconfined compression tests using a MACH-1™ Micromechanical System (Biomomentum, Laval, Canada) equipped with a 100 N load cell. In brief, 300 µl of hydrogel was molded into cylinders (diameter of 8.6 mm and 7 mm in height) and underwent compression until reaching a nominal strain of 80% at a rate of 100% per minute. The nominal stress was calculated based on the applied force and initial cross-sectional area of the sample and then plotted as a function of the nominal strain. The elastic modulus was determined from the slope of the linear regression within 0%–20% of strain.

Microbeads hydrogel, human mesenchymal stromal cells, pro-angiogenic function, cryopreservation, pharmacological preconditioning, cell therapy.