Mesenchymal stem cell (MSC) transplantation has been explored for the clinical treatment of various diseases. However, the current two-dimensional (2D) culture method lacks a natural spatial microenvironment in vitro. This limitation restricts the stable establishment and adaptive maintenance of MSC stemness. Using natural polymers with biocompatibility for constructing stereoscopic MSC microenvironments may have significant application potential. This study used chitin-based nanoscaffolds to establish a novel MSC three-dimensional (3D) culture. We compared 2D and 3D cultured human umbilical cord-derived MSCs (UCMSCs), including differentiation assays, cell markers, proliferation, and angiogenesis. When UCMSCs are in 3D culture, they can differentiate into bone, cartilage, and fat. In 3D culture condition, cell proliferation is enhanced, accompanied by an elevation in the secretion of paracrine factors, including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), Interleukin-6 (IL-6), and Interleukin-8 (IL-8) by UCMSCs. Additionally, a 3D culture environment promotes angiogenesis and duct formation with HUVECs (Human Umbilical Vein Endothelial Cells), showing greater luminal area, total length, and branching points of tubule formation than a 2D culture. MSCs cultured in a 3D environment exhibit enhanced undifferentiated, as well as higher cell activity, making them a promising candidate for regenerative medicine and therapeutic applications.

Mesenchymal stem cell (MSC) transplantation, in particular allogeneic transplantation, is a promising therapy for a variety of diseases. However, before performing allograft treatment it is necessary to find suitable donors, establish culture methods that maintain cell quality, and reduce cell production costs. Here, we present a new method of producing allogeneic MSCs combining human umbilical cord-derived mesenchymal stem cells (UCMSCs) and chitin-based polysaccharide fibers (Cellhesion^® MS). UCMSC numbers significantly increased, and cells grew as dispersed spheres on Cellhesion^® MS. Subsequent biological analyses showed that the expression levels of stemness-related and migration-related genes were significantly upregulated, including octamer-binding transcription factor 4 (OCT4), Nanog homeobox (NANOG), and C-X-C chemokine receptor type 4 (CXCR4). The secretion levels of paracrine factors such as prostaglandin E2 (PGE₂), TNFα-stimulating gene (TSG)-6, fibroblast growth factor 2 (bFGF), and Angiogenin (Ang) from UCMSCs using Cellhesion^® MS were significantly higher than with microcarrier and U-bottom plate culture. In addition, culture supernatant from UCMSCs with Cellhesion^® MS had better angiogenic potential than that from monolayer cultured UCMSCs. Furthermore, we succeeded in a scaled-up culture of UCMSCs with Cellhesion^® MS using a closed culture bag. Therefore, Cellhesion^® MS is a key material for producing high-quality UCMSCs in a three-dimensional (3D) culture system.

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.

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