The combination of three-dimensional and rotary cell culture system promotes the proliferation and maintains the differentiation potential of rat BMSCs

Abstract

Bone marrow mesenchymal stem cells (BMSCs) are a good candidate for tissue engineering and clinical application. One of the challenges in its cell therapy is how to quickly obtain an adequate number of seed cells and meanwhile maintain suitable differentiation potential. In this study we combined three-dimensional (3D) collagen porous scaffolds with rotary cell culture system (RCCS) (RCCS-3D) to create a stereoscopic dynamic environment for the amplification of rat BMSCs in vitro. The results revealed that this RCCS-3D system could enhance BMSCs' proliferation and colony formation, as well as maintain the differentiation potential compared with conventional static two-dimensional (2D) and 3D cell culture conditions. In addition, high-throughput microarray analysis showed that gene expressions of RCCS-3D system displayed significant differences in cell proliferation and differentiation compared with static-2D conditions. Thus, RCCS-3D system could provide an effective means for BMSCs cell proliferation in vitro and meanwhile maintain differentiation potential in tissue engineering.

Figure 1

(a) Phase-contrast image of BMSCs colonies in the primary culture. Scale bar = 200 μm. (b) Phase-contrast image of BMSCs after three passages. Scale bar = 200 μm. (c) Fluorescent-activated cell sorting (FACS) analysis revealed that cells were positive for CD29 and CD90 expression and negative for CD11b/c and CD45.

Figure 2

The morphology of BMSCs on 2D culture plate and 3D scaffolds. (a) Macrophotograph of 3D collagen porous scaffolds. (b) SEM image of 3D porous scaffolds. Scale bar = 400 μm. (c) Macrophotograph of BMSCs cultured in RCCS-3D group. (d) Macrophotograph of BMSCs cultured in static-2D group and static-3D group.

Figure 3

Confocal images of BMSCs stained with FDA in static-2D group after 3 days (a), 7 days (d), static-3D group after 3 days (b), 7 days (e), and RCCS-3D group after 3 days (c), 7 days (f). Scale bar = 200 μm.

Figure 4

SEM images of BMSCs grown in static-2D group after 3 days (a), 7 days (d), static-3D group after 3 days (b), 7 days (e), and RCCS-3D group after 3 days (c), 7 days (f). Scale bar = 20 μm.

Figure 5

Cell number and CFU analysis of BMSCs cultured in static-2D, static-3D and RCCS-3D groups. (a) Proliferation curves of BMSCs in static-2D, static-3D and RCCS-3D groups. The fold increase of cell number was calculated at 0, 1, 3, 5 and 7 day, normalized with the fold increase of cells was set as 0 on the seeding day. The difference on the 7th day was significant by one-way ANOVA analysis (*p < 0.05). (b) Loading efficacy of cells in static-2D, static-3D and RCCS-3D groups. (c) Statistical analysis of colony numbers of BMSCs in each group (n = 3, *p < 0.05). (d–f) Morphology of colonies formed by the cells in static-2D group (d), static-3D group (e) and RCCS-3D group (f) and the colonies were stained with crystal violet.

Figure 6

The RCCS-3D vs static-2D group comparison that gene ontology analysis of differentially expressed genes in biological process. (a) Top 20 terms based on most significant p value of biological processes. (b) Top 12 terms based on the differential genes were most extensive transcriptional domain coverage.

Figure 7

The RCCS-3D vs static-2D group comparison that gene ontology analysis of differentially expressed genes in cellular component. (a) Top 20 terms based on most significant p value of biological processes. (b) Top 12 terms based on the differential genes were most extensive transcriptional domain coverage.

Figure 8

The RCCS-3D vs static-3D group comparison that gene ontology analysis of differentially expressed genes in biological process. (a) Top 20 terms based on most significant p value of biological processes. (b) Top 12 terms based on the differential genes were most extensive transcriptional domain coverage.

Figure 9

The RCCS-3D vs static-3D group comparison that gene ontology analysis of differentially expressed genes in cellular component. (a) Top 20 terms based on most significant p value of biological processes. (b) Top 12 terms based on the differential genes were most extensive transcriptional domain coverage.

Figure 10

Adipogenic and osteogenic differentiation of BMSCs in static-2D, static-3D and RCCS-3D groups. (a) Q-PCR analysis of adipogenic gene expression levels and osteogenic gene expression levels before BMSCs in each group transplanted to 2D plates for 21 days adipogenic and osteogenic differentiation (N = 3, *p < 0.05). (b) Q-PCR analysis of adipogenic gene expression levels and osteogenic gene expression levels after BMSCs transplanted to 2D plates for 21 days adipogenic and osteogenic differentiation of BMSCs in each group (N = 3, *p < 0.05).

Figure 11

Oil red O staining (a–f) and Von kossa staining (g–i) of BMSCs transplanted to 2D plates for 21 days adipogenic and osteogenic differentiation.