A comparative study of mesenchymal stem cells cultured as cell-only aggregates and in encapsulated hydrogels

Abstract

There is increasing evidence that cells cultured in three-dimensional (3D) settings have superior performance compared to their traditional counterparts in monolayers. This has been attributed to cell-cell and cell-extracellular matrix interactions that more closely resemble the in vivo tissue architecture. The rapid adoption of 3D cell culture systems as experimental tools for diverse applications has not always been matched by an improved understanding of cell behavior in different 3D environments. Here, we studied human mesenchymal stem/stromal cells (hMSCs) as scaffold-free self-assembled aggregates of low and high cell number and compared them to cell-laden alginate hydrogels with and without arginine-glycine-aspartic acid peptides. We observed a significant decrease in the size of cell-only aggregates over 14 days in culture compared to the cells encapsulated in alginate hydrogels. Alginate hydrogels had persistently more living cells for a longer period (14 days) in culture as measured by total DNA content. Proliferation studies revealed that a weeklong culture of hMSCs in 3D culture, whether as aggregates or cell-laden alginate hydrogels, reduced their proliferation over time. Cell cycle analysis found no significant differences between days 1 and 7 for the different culture systems. The findings of this study improve our understanding of how aggregate cultures differ with or without a hydrogel carrier, and whether aggregation itself is important when it comes to the 3D culture of hMSCs.

Keywords: 3D cell culture; alginate hydrogels; regenerative medicine.

FIGURE 1

Human mesenchymal stem/stromal cells (hMSCs) in four different cell culture systems. Schematic illustration of the four 3D cell culture systems: (a) hMSCs seeded as low cell number aggregates; (b) hMSCs seeded as a high cell number aggregate; (c) hMSCs encapsulated in alginate hydrogels modified with arginine‐glycine‐aspartic acid (RGD); and (d) hMSCs encapsulated in alginate hydrogels without modification

FIGURE 2

Alginate hydrogels have higher DNA content over time compared to cells in aggregates. Human mesenchymal stem/stromal cells (hMSCs) were seeded in four different cell culture systems: low cell number aggregates, high cell number aggregate, alginate hydrogels without modification and alginate hydrogels modified with arginine‐glycine‐aspartic acid (RGD). The DNA content was analyzed using the PicoGreen assay at (a) day 1; (b) 7; and (c) 14. The same data were also used to compare the different culture systems over time: (d) hMSCs seeded as low cell number aggregates; (e) hMSCs seeded as a high cell number aggregate; (f) hMSCs encapsulated in alginate hydrogels modified with RGD; and (g) hMSCs encapsulated in alginate hydrogels without modification. Data are from three independent experiments. The dotted line indicates the approximate DNA content of 100,000 cells. Statistical significance was determined using one‐way ANOVA with Tukey's test for multiple comparisons. Except for a, all comparisons are statistically significant unless mentioned otherwise; *p < 0.02; n.s: not significant. Data are represented as median with range

FIGURE 3

The cell culture systems all maintain viable cells over 14 days in culture. Human mesenchymal stem/stromal cells (hMSCs) were seeded in four different cell culture systems and labeled with calcein‐AM (green; live) and ethidium homodimer‐1 (red; dead) at days 1, 7, and 14. (a–c) Fluorescence micrographs of hMSCs seeded as low cell number aggregates; (d–f) a high cell number aggregate; (g–i) encapsulated in alginate hydrogels modified with arginine‐glycine‐aspartic acid (RGD); and (j–l) encapsulated in alginate hydrogels without modification. Scale bars represent 100 μm. Data are representative of at least three independent experiments with similar results

FIGURE 4

Long‐term culture of human mesenchymal stem/stromal cells (hMSCs) as aggregates and in alginate hydrogels suppresses proliferation. hMSCs were seeded in four different cell culture systems and were subjected to 5‐ethynyl‐2'‐deoxyuridine (pink) for 48 h prior to analysis on days 2, 7 and 14. The samples were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue). (a–c) Fluorescence micrographs depict hMSCs seeded as low cell number aggregates; (d–f) high cell number aggregate; (g–i) encapsulated in alginate hydrogels modified with arginine‐glycine‐aspartic acid (RGD); and (j–l) encapsulated in alginate hydrogels without modification at day 2, 7, and 14. Scale bars represent 100 μm. Data are representative of at least three independent experiments with similar results

FIGURE 5

Low cell number aggregates have less cell cycle progression. Human mesenchymal stem/stromal cells (hMSCs) that were seeded in four different cell culture systems and were labeled for cellular DNA content followed by flow cytometry at days 1 and 7. Bar graph represents the quantitative measurement cell cycle phases (G0/G1, S, G2/M) at (a) day 1; (b) 7. The same data were also used to compare the different culture systems over time using stacked bars: hMSCs seeded as (c) low cell number aggregates; (d) a high cell number aggregate; (e) encapsulated in alginate hydrogels modified with arginine‐glycine‐aspartic acid (RGD); and (f) encapsulated in alginate hydrogels without modification. Error bars represent mean ± SD. Data are from three independent experiments. Statistical significance was determined using two‐way ANOVA with Tukey's test for multiple comparisons: *p < 0.04