A resazurin-based, nondestructive assay for monitoring cell proliferation during a scaffold-based 3D culture process

Abstract

<jats:title>Abstract</jats:title> <jats:p>Development of viable cell estimation method without sacrificing proliferation and functions of cells cultured on regenerative biomaterials is essential for regenerative engineering. Cytotoxicity and depletion of resazurin are critical but often overlooked limitations that hindered applications of resazurin in viable cell estimation. The present work found that cytotoxicity and depletion of resazurin depended on cell concentration, resazurin concentration and resazurin incubation time. A simple strategy which only allowed cells to incubate with resazurin during each measurement was developed to eliminate negative effects of resazurin. This strategy was verified by monitoring proliferation of MC3T3-E1 preosteoblasts on poly(d,l-lactic acid) scaffold during a continuous 3D culture process for up to 21 days, comparing the accuracy with MTT assay which is a destructive assay with high sensitivity and accuracy and commonly used in regenerative engineering and comparing viability, proliferation and differentiation functions of MC3T3-E1, which were treated with/without this strategy for nondestructive evaluation. This method showed comparable linearity of standard curve and characteristics of growth curve to MTT assay. No major negative effects of this method on MC3T3-E1 viability and functions were found. Our work highlighted the importance of the concentration and incubation time of resazurin in designing application-specific nondestructive viability assay and would be helpful in improving the implanted medical devices as well as in regenerative engineering.</jats:p>

Development of Lab-on-a-Disc Integrated with Resazurin-based Assay for Total Bacterial Counting in Raw Milk

“…For a longer period of incubation time (120 min), the decrease of viable cells was significantly different (p < 0.05) with 20 %(v/v) and 30 %(v/v) when compared to 10 %(v/v) of resazurin. This result is consistent with a previous study which reported that high concentration of resazurin and longer incubation time could have an effect on a decrease of viable cells of bacteria [4]. The resazurin concentration of 44 μM was recommended due to the absence of cytotoxicity effect on various viable cells of bacterial microflora in raw milk including P. aeruginosa 1244, E. coli, and S. aureus [7].…”

“…A resazurin dye, a phenoxazine dye that is redox reaction sensitive is considered appropriate for testing viable cells. This dye has been regularly used to estimate the active growth of live organisms and measure metabolic activity of microorganisms [4]. The highly pink compound of resorufin in the presence of high viable cells will turn to the dihydroresorufin (white) during dye reduction [5].…”

Investigation of the Role of Neurokinin‐1 Receptor Inhibition Using Aprepitant in the Apoptotic Cell Death through PI3K/Akt/NF‐κB Signal Transduction Pathways in Colon Cancer Cells

“…The cell metabolic activity of AP was assessed by resazurin-based cytotoxicity assay. Resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) is a nonfluorescent dye that can reduce to resorufin and dihydro-resorufin (highly fluorescent), which is directly dependent on the number of viable cells [ 31 ]. Briefly, 2.5 × 10 4 cells were seeded in triplicates into 96-well plates (SPL Lifesciences, Pocheon, Korea).…”

Coordination Insertion Mechanism of Ring‐Opening Polymerization of Lactide Catalyzed by Stannous Octoate†

“…Biodegradable polymers are promising in many fields. [ 1‐6 ] In particular, aliphatic polyesters are very useful both as eco‐friendly thermoplastics for daily use and biomaterials for medical applications, [ 7‐19 ] since they are biocompatible and biodegradable, and exhibit excellent mechanical and processing properties. The polyester family includes mainly polylactide (PLA), polyglycolide (PGA), poly(ε‐caprolactone) (PCL), other small‐ring cyclic esters, and their copolymers.…”