Large-scale expansion of human umbilical cord-derived mesenchymal stem cells using PLGA@PLL scaffold

Abstract

Mesenchymal stem cells (MSCs) are highly important in biomedicine and hold great potential in clinical treatment for various diseases. In recent years, the capabilities of MSCs have been under extensive investigation for practical application. Regarding therapy, the efficacy usually depends on the amount of MSCs. Nevertheless, the yield of MSCs is still limited due to the traditional cultural methods. Herein, we proposed a three-dimensional (3D) scaffold prepared using poly lactic-co-glycolic acid (PLGA) nanofiber with polylysine (PLL) grafting, to promote the growth and proliferation of MSCs derived from the human umbilical cord (hUC-MSCs). We found that the inoculated hUC-MSCs adhered efficiently to the PLGA scaffold with good affinity, fast growth rate, and good multipotency. The harvested cells were ideally distributed on the scaffold and we were able to gain a larger yield than the traditional culturing methods under the same condition. Thus, our cell seeding with a 3D scaffold could serve as a promising strategy for cell proliferation in the large-scale production of MSCs. Moreover, the simplicity and low preparation cost allow this 3D scaffold to extend its potential application beyond cell culture.

Supplementary information: The online version contains supplementary material available at 10.1186/s40643-023-00635-6.

Keywords: Biomedicine; Large-scale expansion culture; MSCs; PLGA@PLL scaffold.

Fig. 1

Cell seeding on the PLGA@PLL scaffold. A Wharton’s jelly was harvested in pieces from the human umbilical cord. B MSCs were cultured from Wharton’s jelly tissues and displayed a spindle-like shape. C SEM image of PLGA@PLL scaffold prepared using electrospinning technique. D A magnified SEM image of PLGA@PLL scaffold with a fibrous structure. E hUC-MSCs were incubated on the unmodified PLGA scaffold. F hUC-MSCs were incubated on the PLGA@PLL scaffold

Fig. 2

Cell proliferation was evaluated by cell counting and CCK-8 assay. A Cell growth curves of three groups, including culture dish, PLGA scaffold, and PLGA@PLL scaffold, were made according to cell counting. B Statistical analysis of cell proliferation according to CCK-8 assay (n = 6). C Cell growth curves of hUC-MSCs seeded on the culture dish, PLGA scaffold, and PLGA@PLL scaffold were made based on cell counting. D Statistical analysis of cell proliferation based on CCK-8 assay (n = 6). E Expression levels of the Ki67 and PCNA genes among the control and the scaffold groups were compared (n = 5). F Expression levels of the VCAM-1 and ICAM-1 genes among the control and the scaffold groups were compared (n = 5). The data were normalized to the internal control β-actin and are plotted. *indicates p < 0.05, **indicates p < 0.01, ***indicates p < 0.001, two-tailed t-test

Fig. 3

Cell senescence was analyzed by β-galactosidase staining and RT-qPCR. A native hUC-MSCs from Wharton’s jelly and B hUC-MSCs released from PLGA@PLL scaffold. After β-galactosidase staining, all samples were observed under a microscope and displayed in three random regions. C Expression levels of the key cellular senescence genes P16 and P21 among the control and the scaffold groups were compared. The real-time PCR data were normalized to the internal control β-actin and plotted (n = 5). **indicates p < 0.01, two-tailed t-test Scale bar: 100 μm

Fig. 4

Determination of the multipotency for hUC-MSCs. A Native MSCs from Wharton’s jelly, B hUC-MSCs, and C BM-MSCs released from the PLGA@PLL scaffold were incubated with a multipotency detection kit and then were induced to differentiate into chondroblasts stained with toluidine blue, osteoblasts stained with Oil red O, and adipoblast stained with alizarin red. Scale bar: 100 μm

Fig. 5

Specific hUC-MSCs markers were identified by flow cytometry. A Markers (CD90, CD44, CD105, and CD73) of the released hUC-MSCs from the PLGA@PLL scaffold. B The native MSCs from Wharton’s jelly served as controls