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. 2015 Apr 18:7:9-16.
doi: 10.1016/j.btre.2015.04.004. eCollection 2015 Sep.

Validation of three viable-cell counting methods: Manual, semi-automated, and automated

Daniela Cadena-Herrera  1 , Joshua E Esparza-De Lara  2 , Nancy D Ramírez-Ibañez  2 , Carlos A López-Morales  2 , Néstor O Pérez  2 , Luis F Flores-Ortiz  2 , Emilio Medina-Rivero  2
Affiliations

Validation of three viable-cell counting methods: Manual, semi-automated, and automated

Daniela Cadena-Herrera et al. Biotechnol Rep (Amst). .

Abstract

A viable cell count is essential to evaluate the kinetics of cell growth. Since the hemocytometer was first used for counting blood cells, several variants of the methodology have been developed towards reducing the time of analysis and improving accuracy through automation of both sample preparation and counting. The successful implementation of automated techniques relies in the adjustment of cell staining, image display parameters and cell morphology to obtain equivalent precision, accuracy and linearity with respect to the hemocytometer. In this study we conducted the validation of three trypan blue exclusion-based methods: manual, semi-automated, and fully automated; which were used for the estimation of density and viability of cells employed for the biosynthesis and bioassays of recombinant proteins. Our results showed that the evaluated attributes remained within the same range for the automated methods with respect to the manual, providing an efficient alternative for analyzing a huge number of samples.

Keywords: Cell count; Cell viability; Trypan blue exclusion.

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Figures

None
Graphical abstract
Fig. 1
Fig. 1
Comparison of images acquired by the Countess® camera at different trypan blue concentrations: (a) Presents the images as were acquired by the instrument camera, while (b) Represents images as they were analyzed by the software. At the concentration of 1.0% the background noise interfered with the capability of the instrument to perform more accurate and precise counts.
Fig. 3
Fig. 3
Viability linearity obtained for: (A) CHO-K1 cells by hemocytometer, (B) U937 cells by Countess®, and (C) CHO-K1 cells by Vi-CELL® XR; all compared versus viability standards.
Fig. 2
Fig. 2
Concentration linearity obtained for: (A) CHO-K1 cells by hemocytometer, (B) U937 cells by Countess®, and (C) CHO-K1 cells by Vi-CELL® XR; all compared versus concentration beads.
See this image and copyright information in PMC

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