The Influence of Cell Culture Density on the Cytotoxicity of Adipose-Derived Stem Cells Induced by L-Ascorbic Acid-2-Phosphate

Yuan-Kun Wu,Yuan-Kun Tu,Jiashing Yu,Nai-Chen Cheng

doi:10.1038/s41598-019-56875-0

Abstract

Ascorbic acid-2-phosphate (A2-P) is an oxidation-resistant derivative of ascorbic acid that has been widely employed in culturing adipose-derived stem cells (ASCs) for faster expansion and cell sheet formation. While high dose ascorbic acid is known to induce cellular apoptosis via metabolic stress and genotoxic effects, potential cytotoxic effects of A2-P at high concentrations has not been explored. In this study, the relationship between ASC seeding density and A2-P-induced cytotoxicity was investigated. Spheroid-derived ASCs with smaller cellular dimensions were generated to investigate the effect of cell-cell contact on the resistance to A2-P-induced cytotoxicity. Decreased viability of ASC, fibroblast, and spheroid-derived ASC was noted at higher A2-P concentration, and it could be reverted with high seeding density. Compared to control ASCs, spheroid-derived ASCs seeded at the same density exhibited decreased viability in the A2-P-supplemented medium. The expression of antioxidant enzymes (catalase, SOD1, and SOD2) was enhanced in ASCs at higher seeding densities. However, their enhanced expression in spheroid-derived ASCs was less evident. Furthermore, we found that co-administration of catalase or N-acetylcysteine nullified the observed cytotoxicity. Collectively, A2-P can induce ASC cytotoxicity at higher concentrations, which can be prevented by seeding ASCs at high density or co-administration of another antioxidant.

Highlights

Fluorescent microscopic images showed calcein AM staining of adipose-derived stem cells (ASCs) cultured for 1 day at different seeding densities (1250, 2500, 5000, 10000 cells/cm2) under different A2-P concentrations (0, 32.5, 62.5, 125, 250 μM)
Since A2-P is a vital supplement for ASC culture to increase cell yield and fabricate cell sheets, it is crucial to determine the potential cytotoxicity associated with A2-P concentration and cell culture density
We observed a dose-dependent relationship between A2-P concentration and viable ASC numbers per high power field

Summary

Introduction

Fluorescent microscopic images showed calcein AM staining of ASCs cultured for 1 day at different seeding densities (1250, 2500, 5000, 10000 cells/cm2) under different A2-P concentrations (0, 32.5, 62.5, 125, 250 μM). At the same seeding density, increasing A2-P concentration resulted in significantly fewer stained live cells. At a lower seeding density, fibroblasts exposed to increasing levels (100 to 1000 μM) of AA displayed reduced viability[18] This phenomenon can be readily explained by the insufficient recruitment of anti-oxidative capabilities when the total cell number is low in each culture well. When we subjected low-density ASCs to the A2-P-supplemented medium, a drastic decrease in cell viability with marked cell death was observed. This phenomenon could not merely be attributed to the low cell number in each culture well. We further investigated the underlying mechanism contributing to the survival of ASCs under various seeding densities and A2-P concentrations

Methods

Results

Discussion

Conclusion