Construction of Microsphere Culture System for Human Mesenchymal Stem Cell Aggregates

Abstract

Stem cells cultured in cell aggregates exhibit higher cell survival rates and enhanced anti-inflammatory and angiogenic effects compared to single cells, constructing a stable and economical cell aggregate culture system that can accurately adjust the mass transfer distance of nutrients, which contributes to improving the therapeutic effects of stem cell aggregates. In this study, an alginate hydrogel microsphere culture system (Alg-HM) was prepared using electrostatic spraying technology and refined by optimizing the electrostatic spraying technology parameters, such as the sodium alginate concentration, voltage, electrospray injection speed, and nozzle inner diameter. Furthermore, by setting the Tip-dropped culture system (Tip-D culture system, created by dropping the resuspended hMSC aggregate-hydrogel solution with a tip to form the hydrogel microsphere) and Matrigel culture system (created by dropping the resuspended hMSC aggregates-Matrigel solution with a tip to form the Matrigel culture system) as the control group and Alg-HM as the experimental group, the culture effect of hMSC aggregates in the optimized Alg-HM culture system was tested; CCK-8 detection and Ki-67 immunofluorescence staining showed that the Alg-HM culture system significantly enhanced the cell proliferation activity of hMSC aggregates after 7 and 14 days of culture. The Calcein-AM/PI cell staining results showed that the Alg-HM culture system can significantly reduce the central necrosis of hMSC aggregates. The RNA sequencing results showed that the Alg-HM culture system can significantly activate the signaling pathways related to cell proliferation in hMSCs. This culture system is helpful for the culture of cell aggregates in vitro and efficient transplantation in vivo.

Keywords: alginate hydrogel; cell aggregate; electrostatic spraying technology; mesenchymal stem cells.

Figure 1

Effects of different parameters on particle size of Alg-HM. (a) Model of Alg-HM preparation process, (b) Alg-HM prepared by different concentrations of alginic acid solution and its particle size statistics, (c) Alg-HM prepared at different voltages and its particle size statistics, (d) Alg-HM prepared at different injection rates and its particle size statistics, (e) Alg-HM prepared at different nozzle inner diameters and its particle size statistics (scale bar: 200 µm).

Figure 2

The Alg-HM culture system significantly reduces the degree of central cell necrosis of hMSC aggregates. (a) Construction of different hMSC aggregate culture systems (scale bar: 200 µm). (b) hMSC aggregates in different culture systems were stained with Calcein-AM (green for live cells) and PI (red for dead cells) at day 7 and day 14 (scale bar: 50 µm). (c) Quantification of the fluorescence intensity of PI in each group (n = 5; *** p < 0.001 relative to Tip-D).

Figure 3

The Alg-HM culture system promotes the proliferation of the hMSC aggregates. (a) hMSC aggregates in different culture systems were stained with Ki-67 (green for proliferative cells) and DAPI (blue) at day 7 and day 14 (scale bar: 50 µm). (b) Quantification of the fluorescence intensity of Ki67 in each group. (c) Proliferation of hMSC aggregates in different groups detected via CCK-8 at days 1, 3, 5, 7, and 14 (n = 5; ** p < 0.01, *** p < 0.001 and **** p < 0.0001 relative to Tip-D).

Figure 4

The RNA sequencing results revealed that the Alg-HM culture system can significantly alter the transcriptome of hMSCs in 7-day culture. (a) PCA of the transcriptomes in different groups. (b) A volcano map showing differentially expressed genes (DEGs) of hMSCs cultured in the Alg-HM culture system compared to the Tip-D culture system. RNA-seq analysis of the transcriptomes of hMSCs cultured for 7 days, including 2340 up-regulated and 1722 down-regulated genes. (c) Hierarchical clustering of DEGs of hMSCs cultured in different culture systems for 7 days. (d) Hierarchical clustering of DEGs of hMSCs cultured in different culture systems for 7 days in the cell cycle. (e) Hierarchical clustering of DEGs of hMSCs cultured in different culture systems for 7 days in the PI3K-AKT signaling pathway.

Figure 5

The RNA sequencing results revealed that the Alg-HM culture system can significantly activate the cell signaling pathways related to the cell cycle process in hMSCs in 7-day culture. (a) The GO enrichment analysis of the DEGs of Alg-HM vs. Tip-D at 7 days. Red frame: Positive regulation of the cell cycle process. (b) The KEGG enrichment analysis of the DEGs of Alg-HM vs. Tip-D at 7 days. Red frame: PI3K-Akt signaling pathway, cell cycle. (c) The gene set enrichment analysis (GSEA) of the genes related to the cell cycle of Alg-HM vs. Tip-D at 7 days. (d) The gene set enrichment analysis (GSEA) of the genes related to the PI3K-Akt signaling pathway of Alg-HM vs. Tip-D at 7 days. (e) The percentage of cells in different phases of the cell cycle assessed via flow cytometry.