XX IMEKO World Congress Metrology for Green Growth September 914, 2012, Busan, Republic of Korea UNCERTAINTY ANALYSIS OF CELL COUNTING BY METABOLIC ASSAYS C.Divieto1, G.Sassi1,2, and M.P.Sassi1 1 Istituto Nazionale di Ricerca Metrologica I.N.Ri.M., Torino, Italy, [email protected] 2 Department of Material Science and Chemical Engineering Politecnico di Torino, Torino, Italy, [email protected] Abstract: Cell counting is an important procedure in cell The possibility to perform sequential measurement in biology and its uncertainty evaluation is fundamental to the same cell population is extremely important in cell improve cell-based measurement comparability which is proliferation analysis. However, cell counting currently variable. In this study, for the first time, the methodologies are mostly destructive or invasive. In uncertainty of a metabolic assay for cell counting based on addition, in the last few years, three-dimentional (3D) fluorescence intensity measurement has been evaluated. cultures, mimicking the cells native microenvironment Results, in 2D cell culture, revealed that three are the main better than the traditional bi-dimensional (2D) culture [4], components: repeatability and reproducibility of the are being developed and used. However, often in 3D measurement system and a contribution due to the sample cultures the detachment of cells as well as the observation of loading step procedure. This study represents the basis of a cells are not possible, thus indirect assays are the only 3D cell culture in which metabolic methodologies are the applicable methods to assess the cell number quantification. most suitable method to monitor cell growth. In the present study a metabolic, indirect, non destructive method has been used to assess cell proliferation. Keywords: 2D and 3D cell culture, scaffold, cell Metabolic assays measure a metabolic activity proliferation rate, CellTiter Blue, resazurin, uncertainty accounting for vital cells only and quantify the number of analysis. individuals under the hypothesis that all the active individuals show a metabolic activity and that the mean of 1. INTRODUCTION these individual activities is stable. The metabolic activities of a cell vary with the cell Cell counting is a fundamental procedure in living cell phenotype (e.g. undifferentiated and differentiated culture-based experiments and protocols in which the cell phenotype) and the phenotype changes depend on several number determination is required. Cell number phenomena (influence parameters) occurring in cell culture quantification is one of the parameters to investigate several environments, such as glucose limitation or pH modification cell culture features such as cell viability, proliferation, of the medium, which influence the metabolic activity and growth, fitness and metabolism which require a monitoring introduce artefacts into the metabolic assays [5]. Hence, as function of time [1]. To quantify cells number is when the cell number estimation is based on a cell metabolic considered a measurement service requested by NMIs activity it is extremely important to consider that metabolic related to the quantification of cells in cells adhered and 3D assays are affected by various influence parameters and their cells measurement in tissues [2]. Cell counting is the entire characterization requires a deep and intense analysis quantification of the quantity of organism in a bioreactor. A of the expressed phenotype, aiming to evaluate the bioreactor is any limited volume in which a cell culture uncertainty of the methods. In a 3D system (also called evolves. scaffold) the metabolic activity measurement is obtained by Cell number can be determined by removing cells from the measuring the concentrations of a substrate or a specific bioreactor and counting them with either a hemocytometer metabolic product over time. or an electronic cell counter, or by cell image analysis, or by There are at present some metabolic reaction-based assaying a metabolic activity of the cells within the methods in the literature for monitoring animal cell number bioreactor (e.g. intracellular enzymes activity) [3], when the (giving information on cell proliferation) over time [6, 7]. activity is proportional to the number of cells explicating However, most of them requires the use of substances that that activity. are cytotoxic [6], or results in cell lysis to measure the The measurement of cell viability and proliferation in metabolic product [6,7,8]. The method chosen to be vitro provides important information about the bioreactor analyzed in this work is based on the reduction of resazurin biocompatibility and its influence on cells state, including into the end product resorufin. It is not cytotoxic and do not cell fitness, cell metabolism and cell differentiation. In require any cell damaging steps [9]. addition, by monitoring cell proliferation an differentiation This work aims to emphasize the procedures for the in vitro, many assumptions and predictions about cell uncertainty evaluation of a metabolic method for measuring behaviour in in vivo situations, can be made. the cell number. The innovative contribution of this work is because metabolic methods to determine the cell number, although widely used, have never been metrologically The number of cells in the bioreactor is considered characterized, neither in 2D nor in 3D cell cultures. proportional to the total metabolic activity of cells. The This study is applied to human mesenchymal stem cell metabolic activity is the reaction rate and can be calculated (hMSC) 2D cultures, using the CellTiter-blue® method by the substrate consumption rate or by the product (CTB), a metabolic assay based on the resazurin conversion formation rate. The substrate can have a limiting effect, thus to resorufin to evaluate the cell number and estimate the cell is usually supplied in excess. The formation of the product is proliferation. measured. The product concentration in the culture medium is detected by measuring the fluorescence intensity due to an 2. MATERIALS AND METHODS adequate excitation. The reaction rate can be calculated as the variation of product concentration over time. Usually, CellTiter-blue® proliferation assay: The CTB the measure of product concentration at a standard time proliferation assay is a fluorimetric and metabolic assay to point provides a measure of the mean reaction rate from the determine the number of living cells in a biological in vitro starting time point to the standard time point of system. It is based on a redox reaction: the reduction of measurement. resazurin (dark blue in color, redox dye with a slight Under the hypothesis that the metabolic activity of the intrinsic fluorescence) into the end product resorufin (pink, cells is stable, i.e., the measuring time is very lower than highly fluorescent molecule, 579Ex/584Em), made by redox duplication characteristic time, the number of cells N in the enzymes. Only living cells can perform this reaction because bioreactor is proportional to the product molar concentration nonviable cells rapidly lose metabolic capacity and do not in the liquid phase CProd,t available in the bioreactor at time t generate any fluorescent signal [9]. O’Brien et al. (2000) and to the volume of the liquid phase in the bioreactor VLF. proposed the resazurin dye, the original name of the Alamar Blue dye, to assess the mammalian 2D cell cultures N K1CProd,tVLF (1) cytotoxicity [10]. Resazurin solution is added to the bioreactor as 10-20% in volume. Fluorescence intensity has The product concentration at time t in the liquid phase been measured after a contact time (0.5 to 6 h). is proportional to the fluorescence intensity emitted by the molecules of product I . Fluorescence measurement system: The GloMax®- f,Prod,t Multi Microplate Multimode Reader (Promega) was used to measure fluorescence intensity. Excitation was performed at CProd,t K 2 I f,Prod,t (2) 525 nm wave length while emission was measured in the range 580-640 nm. 96 well plates were used. The cell culture medium and the substrate could emit in the same spectrum region and lead to modifications of the Cell seeding and cell culture medium: hMSCs were fluorescence intensity. It is, hence, necessary to measure the purchased from Lonza (Basel, Switzerland). They are bone blank (b) in absence of cells but with times and treatments marrow derived-hMSCs from donor. hMSCs were expanded analogous to those of the samples (s) with cells. and maintained in a complete non differentiating growth medium (aMEM, Listarfish) supplemented with 10% fetal I I I bovine serum (FBS, Listarfish), 1% L-glutamine, 1% f,Prod,t f,s,t f,b,t (3) antibiotics (kanamycin). Cells were grown in monolayers, until 80-85% of confluence, they were washed once with 1X Hence, the measurand equation can be written as: Phosphate Buffer Saline (PBS), detached with 0.05% trypsin, counted by means of a hemocytometer and N KI f,s,t I f,b,t VLF suspended at several concentrations (cells/ml) in 100 μl (4) complete non differentiating growth medium to be seeded The fluorescence intensity is determined by the instrument and cultured statically in 96 well plates. Then, cells are used for the measure. An influence quantity is the sample volume loaded in the well. Thus, an analysis of the posed in the incubator at 37°C with 5% CO2 over night to let the cells to adhere to the bottom of the wells. fluorescence intensity sensitivity with respect to the sample volume loaded in the well needs to be carried out to evaluate