Methods for determining cell viability

Abstract

Method for determining cell viability in a cell culture of biological cells for the biotechnological production of proteins, in particular antibodies, characterized in that a fluorescent dye selected from 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate (Bis-ANS) or 8-anilinonaphthalene-1-sulfonate (ANS) is used as a detection agent, and wherein the fluorescent dye is present in the cell culture at a concentration of 0.1 µM to 10 µM, the fluorescent dye selectively penetrates cells with damaged cell membranes and binds to intracellular proteins, the labeled cells are detected by excitation in the UV range and detection of the fluorescence in the green wavelength range, and the determination of cell viability is carried out using an automated imaging fluorescence microscope.

Description

Field of invention The present invention relates to a method for determining cell viability. In particular, the invention relates to a method for determining cell viability in cell cultures of biological cells used for the biotechnological production of proteins, for example, antibodies. Background of the invention Substances, particularly proteins such as monoclonal antibodies (mAbs) for the diagnosis and treatment of diseases, are increasingly being produced biotechnologically in high-throughput bioreactors. This is primarily achieved using hybrid mammalian cells, with cell performance largely dependent on cell viability. The total cell count is determined by the number of dead cells and the number of living cells, with cell viability representing the proportion of living cells within the cell population. Continuous monitoring of cell viability is essential for the production of substances using cells to ensure the highest possible quality and yield of active pharmaceutical ingredients. Trypan blue, an anionic diazo dye, is used to determine cell viability.Living, intact cells are impermeable to the dye, while dead cells and cells with perforated cell membranes absorb the dye, which then binds to proteins within the cell. These damaged and dead cells are stained dark blue. In this way, the viability of cells within a cell population can be determined. State of the art Determining cell viability is a key parameter in the cultivation of biological cells, especially in cell cultures used for the biotechnological production of proteins. Monitoring cell viability is of great importance, for example, in the production of recombinant proteins or therapeutic antibodies. Various methods are known for determining cell viability. Fluorescent dyes, which can distinguish between living and dead cells, are frequently used. The publication by Emese Mozes et al., “A novel application of the fluorescent dye Bis-ANS for labelling neurons in acute brain slices,” Brain Research Bulletin, Volume 86, Issues 3-4, October 10, 2011, pp. 217-221, describes the use of the fluorescent dye 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate (Bis-ANS) for labeling neuronal structures in acute brain slices. It demonstrates that Bis-ANS can penetrate cells with damaged cell membranes and subsequently be detected by fluorescence microscopy. Patent application US 2014 / 0024074 A1 describes automated methods for determining cell parameters, in particular cell concentration and cell viability, using imaging analysis techniques. Fluorescence-based methods are used to analyze cell populations. Summary of the invention Starting from the known prior art, the object of the present invention is to provide an improved method for determining cell viability, which is particularly suitable for cell cultures used for the biotechnological production of proteins. In particular, a method is to be provided that enables a reliable and automated determination of cell viability. This object is achieved by a method with the features of claim 1. Advantageous embodiments of the invention with additional process steps are described below. Experiments have shown that cell viability can be measured at a Bis-ANS concentration of 0.1 µM to 10 µM. Another advantage is that the cells to be measured can be cultured in the presence of Bis-ANS, because at the concentration used, it exhibits no detectable cell toxicity. Preferably, the Bis-ANS concentration is approximately 1 µM. This eliminates the need to take a sample, stain it, and measure it; instead, cell culture viability can be measured directly and continuously using the cultured cells without an additional staining step. This allows for simpler, faster, and more accurate measurements. Due to Bis-ANS's ability to stain proteins, this method is particularly suitable for high-producer cell lines capable of high-throughput protein production. If the cell culture medium has a serum content higher than 0.5%, an additional washing step, e.g., in PBS buffer (phosphate-buffered saline), is required to remove serum proteins or reduce their concentration. Protein aggregates of varying sizes can form during the production of mAbs. To determine optimal production conditions, these protein aggregates must be measured during the production process. It is already known that the size of antibody aggregates in cell culture can be measured using Bis-ANS (Paul et al., 2015, Analytical and Bioanalytical Chemistry). Since Bis-ANS is used to determine cell viability according to the invention, the presence of the dye also allows for the determination of protein aggregates. The invention further comprises a reagent kit for labeling cells with a fluorescent dye to determine cell viability and an imaging fluorescence microscope that excites the dye in the UV range and measures its fluorescence in the corresponding wavelength range, wherein the fluorescent dye is Bis-ANS or ANS. Bis-ANS fluoresces in the green to blue range, so this would be the measurement range. The reagent kit may contain further reagents for carrying out the measurement. The following figures are intended to explain the invention in more detail, without limiting it to that. Brief description of the drawings The figures show in detail: Fig. 1 the staining of dead cells by Bis-ANS; Fig. 2 the number of viable cells as a function of the cultivation time; Fig. 3 a comparison of the dyes Bis-ANS and trypan blue; Fig. 4 the cytotoxicity of Bis-ANS; Fig. 5a a flow cytometry of CHO cells cultured without Bis-ANS as a control; and Fig. 5b a flow cytometry of CHO cells cultured with Bis-ANS. Detailed description of the drawings Figure 1 shows the staining of three different cell types with two different dyes for comparison. Three common cell lines were used: a CHO cell line (Chinese Hamster Ovary, Rentschler Biotechnology, Laupheim, Germany), a CAP amniocyte cell line (CEVEC Pharmaceuticals, Germany), and a murine hybridoma cell line. Cultivation was performed at 37°C, 5% CO2, and 85% relative humidity. The CHO and CAP cells were cultured in a shaking incubator (Kühner, Switzerland) at 140 rpm. The CHO cells were grown in chemically defined, serum-free SFM4CHO with 10 g / L glucose (Sigma-Aldrich) and 4 mM L-glutamine (Life Technologies). CAP cells were cultured in chemically defined, serum-free CAP-DM medium (CEVEC Pharmaceuticals, Germany). Hybridoma cells were cultured in DMEM medium with 10% FCS in static T175 flasks.The total number and the number of viable cells were determined using a Cedex XS Analyzer (Roche) with trypan blue. Figure 1 comprises nine different images acquired with a cell imager for fully automated fluorescence microscopy (NyONE, SynenTec, Germany). The top row, labeled CHO, shows three images of CHO cells. The middle row, labeled CAP, shows three images of CAP cells. The bottom row, labeled hybridoma, shows three images of hybridoma cells. All cells were stained with 10 µM Bis-ANS and imaged at 10x magnification. Column A shows the cells in brightfield. Column C shows the fluorescence images of the cells, which were excited to fluoresce with UV light and emit green light due to the Bis-ANS labeling. As a control, all cells were additionally stained with TO-PRO®-3, a fluorescent dye that binds to nucleic acids and is commonly used to detect dead cells. The TO-PRO®-3 dye concentration was 0.5 µM in each case.TO-PRO®-3 was excited with red light, and its red emission was measured. The corresponding fluorescence images are shown in column B. Figure 1 shows that in the CHO, CAP, and hybridoma cell lines, those cells that were specifically stained by TO-PRO®-3 were also stained by Bis-ANS. This demonstrates that Bis-ANS specifically stains dead cells and is suitable for detecting dead cells from various cell lines. The binding mechanism within the dead cell differs for the two dyes. Confocal microscopy has shown that TO-PRO®-3 binds to and stains nucleic acids, while Bis-ANS stains the entire cell interior (not shown). Furthermore, it was investigated whether the dye Bis-ANS is also suitable for determining cell viability. To determine the appropriate concentration, Bis-ANS was added to a suspension containing 0.04 × 10⁶ viable CHO cells / ml, with Bis-ANS concentrations ranging from 0.2 µM to 5 µM. The concentration series was performed using fluorescence microscopy at 20x magnification and showed that a Bis-ANS concentration of 0.2 µM is sufficient to stain all dead CHO cells (not shown). Figure 2 shows the absolute viable cell count as a function of the culture time, which is given in hours. 4 × 10⁵ viable CHO cells / mL were added to an SFM4CHO medium containing 10 g / L glucose and 4 mM glutamine. The CHO cells were then cultured in a shaking incubator at 140 rpm and a temperature of 37°C with 5% CO₂ and 85% relative humidity for 126 hours, and the viable cell count was determined. Figure 3 shows a comparison of the dyes Bis-ANS and trypan blue, which were used to determine cell viability as a function of cultivation time. Cultivation time is given in hours. Cultivation was carried out under the conditions described in Figure 2. Cell viability is shown as a percentage of the absolute viable cell count shown in Figure 2. During the period from 0 to 126 hours, cell viability was measured using fluorescence microscopy (white bar) with 0.2 µM Bis-ANS and using bright-field microscopy (black bar) with trypan blue. As Figure 3 shows, there were no significant differences within the first 54 hours of cultivation. The cell viability measured with both dyes was approximately 95%. After this period, the measurement with Bis-ANS showed a decrease in cell viability, while the value measured with trypan blue remained constant. This result is consistent with other studies that have shown that trypan blue tests overestimate cell viability in the range of high viable cell counts (Altman SA, Randers L, Rao G., 1993, Biotechnol Prog 9(6):671-4). Figure 4 shows the results of an experiment to determine the cytotoxicity of Bis-ANS. The concentration of CHO cells in cells / ml is plotted against the concentration of Bis-ANS in µM. The addition of various Bis-ANS concentrations to CHO cells shows an EC50 value of 5 ± 0.5 µM Bis-ANS. Since a Bis-ANS concentration of up to 1 µM is tolerated by CHO cells without cell damage and, on the other hand, allows for an accurate measurement of cell viability, this dye concentration is particularly well suited for addition to cell culture. Figure 4 thus shows that Bis-ANS can be added directly to cell suspensions to monitor the viability of the producing cells with high accuracy. It can therefore be concluded that cell viability can be determined directly during cultivation using Bis-ANS. Furthermore, it has been shown that the measurements are more accurate than with trypan blue. Figures 5a and 5b show fluorescence flow cytometry (“FACS”) of CHO cells cultured once without (Fig. 5a) and once with (Fig. 5b) Bis-ANS. Figure 5a shows a control measurement in which CHO cells were stained with TO-PRO®-3 after 120 hours of culture. TO-PRO®-3 was excited to fluorescence with red light, and the red emission was measured. The middle histogram in the top row of Figure 5a shows that approximately 19% of the cells were stained by TO-PRO®-3, or were TO-PRO®-3 positive, corresponding to a cell viability of approximately 80%. The middle histogram in the bottom row of Figure 5a shows that approximately 95% of the dead cells were stained by TO-PRO®-3. As a control, the CHO cells were excited with UV light, and the emission was measured in the green light range, where a signal from Bis-ANS is detected. However, due to the absence of Bis-ANS, no significant signal was measured, as expected. This is shown by the right-hand fluorescence histograms in the top and bottom rows of Figure 5a. Figure 5b shows a measurement using CHO cells as in Figure 5a, where the CHO cells were measured after 120 hours of culture. However, unlike Figure 5a, the CHO cells in Figure 5b were cultured in the presence of 1 µM Bis-ANS. The middle fluorescence histogram in the top row of Figure 5b shows that, after co-staining with TO-PRO®-3, 21.68% of the cells cultured in the presence of Bis-ANS were stained by TO-PRO®-3, i.e., TO-PRO®-3 positive. The right-hand fluorescence histogram in the top row of Figure 5b shows that 22.48% of the cells were Bis-ANS positive. Thus, the two measurements show very similar values. The lower center fluorescence histogram shows that 95% of the dead cells were stained by TO-PRO®-3 or were TO-PRO®-3 positive. The lower right fluorescence histogram shows that 91% of the dead cells were stained by Bis-ANS or were Bis-ANS positive. These flow cytometry data show that cells can be cultured in the presence of Bis-ANS without affecting cell viability, as cell viability is in the same range in Figures 5a and 5b: approximately 80% without Bis-ANS and approximately 79% with Bis-ANS. Furthermore, the data show that cell viability can be directly measured in cells cultured in the presence of Bis-ANS. This allows the measurement of cell viability to be performed more easily, quickly and accurately than with known methods.

Claims

Method for determining cell viability in a cell culture of biological cells for the biotechnological production of proteins, in particular antibodies, characterized in that a fluorescent dye selected from 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate (Bis-ANS) or 8-anilinonaphthalene-1-sulfonate (ANS) is used as a detection agent, and wherein the fluorescent dye is present in the cell culture at a concentration of 0.1 µM to 10 µM, the fluorescent dye selectively penetrates cells with damaged cell membranes and binds to intracellular proteins, the labeled cells are detected by excitation in the UV range and detection of fluorescence in the green wavelength range, and the determination of cell viability is carried out using an automated imaging fluorescence microscope. Method for determining cell viability according to claim 1, characterized in that the cells to be measured are cultured in the presence of Bis-ANS, preferably in a Bis-ANS concentration of about 1 µM. Method for determining cell viability according to one of the preceding claims, characterized in that the measurement is carried out directly and / or continuously with the cultured cells. Method for determining cell viability according to one of the preceding claims, characterized in that protein aggregates are additionally determined using Bis-ANS. Reagent kit for carrying out the method according to one of the preceding claims, comprising a fluorescent dye selected from Bis-ANS or ANS.

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