FP003B suppresses microcarrier aggregation and enhances cell yield in microcarrier-based mesenchymal stromal cell culture

Abstract

Background Allogeneic transplantation of mesenchymal stromal cells (MSCs) is a promising therapeutic strategy for various diseases. Large-scale expansion is required for allogeneic applications; however, conventional two-dimensional culture systems have limited scalability. Therefore, three-dimensional cultures using a microcarrier (MC) have become increasingly important. A major challenge in MC-based MSC cultures is excessive MC/MSC aggregation, which can compromise cell quality and yields. Here, we present a new approach using functional polymer 003B (FP003B) to suppress excessive aggregation during MC-based MSC cultures. Methods Adipose-derived MSCs (ADMSCs) were cultured on multiple MC types using various vessel formats, including 125 mL and 500 mL spinner flasks and a 30 mL disposable bioreactor. FP003B was added either during medium changes or by direct supplementation with concentrated FP003B in D-PBS (+). Aggregation size, cell yield, metabolite profiles, surface marker expression, trilineage differentiation, and paracrine factors were evaluated. Mechanistic studies included particle image velocimetry (PIV) to assess FP003B distribution and confocal microscopy using FITC-labeled FP003B to examine its localization on MC/MSC surfaces. Collagen type I deposition was quantified by enzyme-linked immunosorbent assay. Results FP003B significantly reduced the average diameter and area of MC/MSC aggregates and increased the number of harvested viable ADMSCs across multiple MC types and vessel formats. The expression of positive (CD73, CD90, and CD105) and negative (CD11b, CD34, and CD45) surface markers, trilineage differentiation and secretion of prostaglandin E2 and vascular endothelial growth factor from ADMSCs were maintained. In bead-to-bead transfer, FP003B significantly enhanced the expansion ratio and increased cell yields. Mechanistic analyses demonstrated that FP003B localized to the surface of MCs and MSCs, and under agitation, it remained dispersed in the culture medium. Furthermore, collagen type I deposition was significantly reduced in the presence of FP003B, suggesting that suppression of excessive aggregation may contribute to improved cell recovery. Conclusion FP003B is a versatile and scalable additive that stabilizes MC-based MSC manufacturing processes. These findings support FP003B as a promising tool for robust and efficient pre-clinical proof-of-concept MSC production.