A multicompartment holder for spinner flasks improves expansion and osteogenic differentiation of mesenchymal stem cells in three-dimensional scaffolds

Abstract

In the tissue engineering field dynamic culture systems, such as spinner flasks, are widely used due to their ability to improve mass transfer in suspension cell cultures. However, this culture system is often unsuitable to culture cells in three-dimensional (3D) scaffolds. To address this drawback, we designed a multicompartment holder for 3D cell culture, easily adaptable to spinner flasks. Here, the device was tested with human mesenchymal stem cells (MSCs) seeded in 3D porous chitosan scaffolds that were maintained in spinner flasks under dynamic conditions (50 rpm). Standard static culture conditions were used as control. The dynamic conditions were shown to significantly increase MSCs proliferation over 1 week (approximately 6-fold) and to improve cell distribution within the scaffold. Moreover, they also promoted osteogenic differentiation of MSCs, inducing an earlier peak in alkaline phosphatase (ALP) activity, and a more homogenous ALP staining and matrix mineralization in the whole scaffolds, but particularly in the center. Overall, this study shows a new multicompartment holder to culture 3D scaffolds that can broaden the application of spinner flasks.

FIG. 1.

Details of the multicompartment holder system for three-dimensional (3D) dynamic cell culture in spinner flask. (A) Perforated container and lid. (B) Stirring bar made of stainless steel. (C) Assembly of the multicompartment holder with the lid; the impeller support was modified to accommodate the container. (D) Scheme of the impeller shaft (1) with a magnet support (2) and a magnet (3), holding two containers (4) and a lid (5). (E) Scheme of two multicompartment holders with a lid in the spinner flask (6). Color images available online at www.liebertpub.com/tec

FIG. 2.

Viability of mesenchymal stem cells (MSCs) cultured in chitosan (Ch) 3D scaffolds in static and dynamic conditions. Representative CSLM images (z-stacks) from LIVE/DEAD cytotoxicity/viability assay performed at days 7 and 14 of culture (Calcein AM stains live cells in green; EthD-1 stains dead cells in red; scale bar, 200 μm). Color images available online at www.liebertpub.com/tec

FIG. 3.

Proliferation and metabolic activity of MSCs cultured in Ch 3D scaffolds under static and dynamic conditions. (A) DNA content of cell-loaded scaffolds was quantified using PicoGreen assay. DNA content is significantly higher (**p<0.025) in dynamic versus static cultures. In static, DNA content significantly increases (**p<0.025) from day 7 to 14. Results are shown as mean±standard deviation (SD) (n=5). (B) Glucose and (C) lactic acid concentration (g/L) in culture supernatants collected during time in culture. Results are shown as mean±SD (n=3 replicates of one from three independent experiments).

FIG. 4.

Comparison of MSCs distribution in Ch 3D scaffolds in static and dynamic cultures at days 7 and 14. Hematoxylin&eosin (H&E) staining of scaffolds' cross sections (7 μm photomicrographs, scale bars: 500 and 200 μm) show cells (in purple) distributed along periphery and center of the scaffolds (in pink). Color images available online at www.liebertpub.com/tec

FIG. 5.

MSCs differentiation into osteogenic lineage when cultured in Ch 3D scaffolds in static and dynamic conditions. (A) Representative images of alkaline phosphatase (ALP) staining in whole scaffolds (scale bar, 1 mm). (B) Quantification of ALP activity (nmol/min/mg total protein). Results are shown as mean±SD (n=4); (*p<0.05; ALP activity is significantly higher in dynamic versus static conditions). (C) Representative images of von Kossa staining after 28 days of culture (scale bar, 1 mm). (D) Immunostaining of collagen type I (Col I) produced by MSCs after 28 days of culture (7-μm scaffolds' cross sections; scale bars: 200 and 50 μm). OM, osteoinductive medium. Color images available online at www.liebertpub.com/tec

FIG. 6.

Comparison of MSCs distribution and metabolic activity in Ch 3D scaffolds in static and dynamic cultures under osteogenic conditions. (A) H&E staining of scaffolds' cross-sections (7-μm photomicrographs; scale bar, 200 μm) show cells (in purple) distributed along periphery and center of the scaffolds (in pink). (B) Glucose and (C) lactic acid concentrations (g/L) were quantified in culture supernatants. Results are shown as mean±SD (n=3 replicates from n=1 representative experiment). Color images available online at www.liebertpub.com/tec