Effect of chitin-architected spatiotemporal three-dimensional culture microenvironments on human umbilical cord-derived mesenchymal stem cells

Abstract

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.

Graphical abstract

This study utilized chitin-based nanoscaffolds to establish a three-dimensional (3D) culture of Human Umbilical Cord Mesenchymal Stem Cells (UCMSCs), comparing their characteristics with traditional two-dimensional (2D) cultures. The findings indicate that the 3D culture preserved the stemness and differentiation capabilities of the UCMSCs while enhancing their proliferative abilities. Additionally, an upregulation 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), was observed. The supernatant from UCMSCs in 3D culture significantly enhanced the lumen area, total length, and branching points of the tubular structures formed by Human Umbilical Vein Endothelial Cells (HUVECs). These findings suggest that chitosan-based nano-scaffolds show promise as biomaterials for the early in-vitro culture of artificial MSCs, positioning them as ideal candidates for regenerative medicine and therapeutic applications.

Highlights

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  3D culture with chitin materials preserves stemness, differentiation potential, proliferative capacity, and paracrine effects of human umbilical cord-derived mesenchymal stem cells (UCMSCs) better than 2D culture.

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  mRNA analysis of UCMSCs in 3D culture shows significant upregulation of expression, including genes such as CXCL12, MCM8, and MCM10.

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  3D culture with chitin material provides an in vivo-like microenvironment for culturing mesenchymal stem cells (MSCs).

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  High-quality methods and strategies are very useful for producing high-quality MSCs and expanding their clinical application.