Current methods for assessing cell proliferation in 3D scaffolds rely on changes in metabolic activity or total DNA, however, direct quantification of cell number in 3D scaffolds remains a challenge. To address this issue, we developed an unbiased stereology approach that uses systematic-random sampling and thin focal-plane optical sectioning of the scaffolds followed by estimation of total cell number (StereoCount). This approach was validated against an indirect method for measuring the total DNA (DNA content); and the Bürker counting chamber, the current reference method for quantifying cell number. We assessed the total cell number for cell seeding density (cells per unit volume) across four values and compared the methods in terms of accuracy, ease-of-use and time demands. The accuracy of StereoCount markedly outperformed the DNA content for cases with ~ 10,000 and ~ 125,000 cells/scaffold. For cases with ~ 250,000 and ~ 375,000 cells/scaffold both StereoCount and DNA content showed lower accuracy than the Bürker but did not differ from each other. In terms of ease-of-use, there was a strong advantage for the StereoCount due to output in terms of absolute cell numbers along with the possibility for an overview of cell distribution and future use of automation for high throughput analysis. Taking together, the StereoCount method is an efficient approach for direct cell quantification in 3D collagen scaffolds. Its major benefit is that automated StereoCount could accelerate research using 3D scaffolds focused on drug discovery for a wide variety of human diseases.

Biomedical Center Faculty of Medicine in Pilsen Charles University Alej Svobody 76 Pilsen Czech Republic

Department of Computer Sciences and Engineering College of Engineering University of South Florida 4202 E Fowler Ave Tampa FL USA

Department of Histology and Embryology Faculty of Medicine in Pilsen Charles University Alej Svobody 76 Pilsen Czech Republic

Department of Pathological Physiology Faculty of Medicine in Pilsen Charles University Alej Svobody 76 Pilsen Czech Republic

Imaging and Optics Facility Institute of Science and Technology Austria Am Campus 1 Klosterneuburg Austria

SRC Biosciences 1810 W Kennedy Blvd Tampa FL USA

Sci Rep. 2023 Jun 21;13(1):10090. doi: 10.1038/s41598-023-37265-z. PubMed

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