A first approach to evaluate the cell dose in highly porous scaffolds by using a nondestructive metabolic method

Abstract

Background: In cell-based therapies, in vitro studies on biomimetic cell-scaffold constructs can facilitate the determination of the cell dose, a key factor in guaranteeing the effectiveness of the treatment. However, highly porous scaffolds do not allow a nondestructive evaluation of the cell number. Our objective was to develop a nondestructive method for human mesenchymal stem cells dose evaluation in a highly porous scaffold for bone regeneration.

Materials & measurement method: Proliferation trend of human mesenchymal stem cells on Biocoral^® scaffolds was measured by a resazurin-based assay here optimized for 3D cultures. The method allows to noninvasively follow the cell proliferation on biocorals over 3 weeks with very high reproducibility.

Conclusion: This reliable method could be a powerful tool in cell-based therapies for cell dose determination.

Keywords: Biocoral®; cell dose; cell number; cell proliferation; hMSCs; nondestructive methods; porous scaffolds.

Figure 1.. Biocoral scaffolds images.

(A) Biocorals have been acquired with digital camera, and (B–E) scanning electron microscopy. (B & C) show a scaffold before seeding: in (B) the scaffold surface where cells are usually seeded (scale bar = 4 mm) and in (C) the scaffold internal pores, after scaffold mechanical fragmentation(scale bar = 1 mm). (D & E) show a scaffold after cell seeding andcell fixation: in (D) fixed cells (arrows) occupied some pores (bar =500 μm) and in (E) at higher magnification two cells in two different pores (arrows; scale bar = 300 μm).

Figure 2.. Calibration curve for 3D cell culture on Biocoral^®.

Fluorescence intensity of resazurin metabolized by cells on biocorals after 2 h incubation. The correlation between the fluorescence intensity and the nominal cell number, was found linear in the range between 5 × 10³ and 3.0 × 10⁵ cells. The correlation coefficient was 0.967. Error bars: horizontal bars are the uncertainty if the manual counting estimated as 5%; vertical bars are the uncertainty of the fluorescence measurements estimated as 5.9%.

Figure 3.. Calibration curve for 2D cell culture.

Cells were seeded in six-well plate in the range of 1.0 × 10³ and 2.5 × 10⁵ per well. Resazurin was incubated with cells for 2 h. The correlation coefficient was 0.995. Error bars: horizontal bars are the uncertainty if the manual counting estimated as 5%; vertical bars are the uncertainty of the fluorescence measurements (estimated as 5%), n = 3.

Figure 4.. Experimental reproducibility: fluorescence intensity on biocorals seeded with 0.5, 1.0 and 3.0 × 10⁵ cells.

Mean fluorescence intensity from human mesenchymal stem cells at P7 seeded at three nominal cell number (0.5 and 1.0 and 3.0 × 10⁵ cells) on biocorals (n = 3 for 0.5 and 1.0 × 10⁵ cells; n = 6 for 3.0 × 10⁵ cells) and measured over time at days 1, 4, 7, 10, 14, 18, 21. Error bars represent the uncertainty of the measurement calculated quadratically composing the experimental repeatability and the experimental reproducibility.

Figure 5.. Pore distribution of a representative portion of Biocoral^® scaffolds.

The pore size distribution ranges from 100 to 700 μm.

Figure 6.. Resazurin and resorufin emission spectra.

A low number and a high number of cells after 2 h incubation metabolize differently a resazurin 44 μl solution. The excitation was at 540 nm and the maximum emission (peaks) was found around 635 nm for resazurin metabolized by low number of cells (i.e., low concentrated resorufin) and 600 nm for resazurin metabolized by high number of cells (i.e., high concentrated resorufin).

Figure 7.. Incubation time is cell number dependent.

Cells seeded at higher density need a shorter incubation time to avoid off-scales. Points on curves at 4 and 6 h were off-scale after 5.0 × 10⁴ and 1.0 × 10⁵, respectively (missing points on curves). The optimal incubation time was found to be 2 h allowing to measure intensity on the entire range of cell number with no need for dilution.

Figure 8.. Effect of dilution.

Dilution 1:9 of the same metabolized resazurin analyzed and shown in Figure 4 are represented here.

Figure 9.. Incubation time and temperature effect.

The fluorescence measurement should be done as soon as the cells, or the metabolized resazurin, are taken from the incubator. The temperature decrease modifies the fluorescence intensity presumably due to the temperature-dependent pH decrease in absence of CO₂ or other pH buffers. Error bars represent the standard deviation of three samples (n = 3).

Figure 10.. Sensitivity of the method: fluorescence intensity on biocorals seeded with 2.5, 5.0 and 10.0 × 10³ cells.

Error bars (n = 3) represent the uncertainty of the measurement, calculated quadratically composing the fluorescence intensity measurement uncertainty and the experimental repeatability.

Figure 11.. Cell proliferation on Biocoral^®.

Proliferation curves on Biocorals expressed as number of cells (n) versus time (days in culture). Scaffolds seeded with 1.0 × 10⁴ nominal human mesenchymal stem cells and scaffolds seeded with 5.0 × 10⁴ nominal human mesenchymal stem cells. Error bars are the standard deviation among three scaffolds (n = 3).

Figure 12.. 2D and 3D cell cultures proliferation trend.

Fluorescence intensity emitted by cells as function of the days in culture. In 2D cell cultures at high density the proliferation trend showed the typical lag and log phases and a final plateau, the medium and low densities showed a lag and log phases both supposed being close to reach the plateau. In 3D cell cultures the trend is very similar in all three initial densities, however the lag phase is much longer and the log phase just starts at day 6. Error bars represent the uncertainty of the measurement as described in the Materials & measurement method section ‘Experimental repeatability and reproducibility on 3D cell cultures’.

Figure 13.. Cell viability assay: cell colonization of external and internal surfaces.

Fluorescence microscopy and confocal microscopy images of human mesenchymal stem cells on Biocorals^®. In green are shown human mesenchymal stem cells after 7 and 21 days of 3D cell culture at 5×, 10×, 20× and 40× magnification. In (A–E) cells were imaged after 7 days in 3D culture by fluorescence microscopy: in (A) and (B) cells within the Biocoral^® pores appear black with rounded shape in (A), bar = 150 μm; in (C) and (D) details of two single cells adherent to the scaffold structure, bar = 50 μm; in (E) a cell adherent to the wall of a single pore bar = 100 μm. In (F–J) cells were imaged after 21 days in 3D culture by confocal microscopy: in (F) cells on the external surface delineating the scaffold boundaries, bar = 150 μm; in (G) cells within the scaffold, bar = 50 μm; in (H) and (I) cells on the surface, bar = 100 μm; in (J) it is visible one pore containing three cells bar = 50 μm.