Background

Cell culture-based production is gaining increasing attention owing to the need for new manufacturing strategies. The advantages of cell-based production processes include the use of defined and serum-free cell cultures that allow greater consistency. Moreover, cell-based processes can be adapted to manufacturing processes involving bioreactors that are scalable and need less space (Aubrit et al., 2015). The mounting use of cell culture-based vaccine production, together with a quest for a more efficient and scalable process, have stimulated the development and implementation of carrier technology. Micro and macrocarriers are small compact surface support matrices for growing adherent cells. Those carriers (from the micrometer to the millimeter size range for micro and macrocarriers, respectively) and their density allows their maintenance in suspension with gentle stirring (Huang et al., 2020).

To better follow and understand a production process, it is necessary to quantify the number of cells on these carriers, as well as to track their viability and proliferation. The gold standard method for quantification of cells on micro and macrocarriers is nuclear staining (Sanford et al., 1951). In this method, cells are separated from the carriers, stained with crystal violet, and counted visually under a microscope. The method is tedious (Berry et al., 1996) and counts both live and dead cells, making it unsuitable for tracking cell condition. As a result, a method that can better quantify cells on micro and macrocarriers is desirable.

Counting cells on carriers poses a challenge to traditional assays due to the carriers’ shape. The main feature of the widely used methods to measure cell quantity (Riss et al., 2013) is dyeing the cells with a fluorescent or colorimetric dye and quantifying the results based on fluorescence or absorbance. Examples of such dyes are glycyl-phenylalanyl-aminofluorocoumarin (GF-AFC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1), and resazurin (Alamar blue). These assays are relatively inexpensive and have a homogeneous format, but their use is limited to single layer cultures (Mosmann, 1983; Berridge and Tan, 1993; Marshall et al., 1995). A limited number of assays for cell quantification on carriers have been developed, but they all require specialized equipment that is not commercially available, and are “matched” with specific micro or macrocarriers (Farrell et al., 2016). Thus, to date, no assays for quantification of cells on carriers that are simple, rapid, reliable, and low-priced are commercially available.

Here, a method for quantification of cells on carriers is described. The assay utilizes Alamar blue, a well-known and common marker for cell activity (Rampersad, 2012), and includes a calibration curve with known cell numbers. When added to cell cultures, the oxidized form of Alamar blue enters the cytosol and is converted to the reduced form by mitochondrial enzyme activity. This redox reaction is accompanied by a shift in the color of the culture medium from indigo blue to fluorescent pink, which can be easily measured by fluorometry (Al-Nasiry et al., 2007). The method can quantify cells in situ on the carriers without requiring separation of cells from the carriers. The method is simple, rapid, elegant, does not require specialized reagents or equipment, and results are strongly correlated with the gold standard assay. The method can be easily adapted for any cell-based process or other unique platforms for cell growth, including additional carriers and other scaffolds for culturing cells such as hydrogels.