Automatic image analysis for quantification of apoptosis in animal cell culture by annexin-V affinity assay

Abstract

Apoptosis is a form of cell death in which the dying cell plays an active part in its demise. At the morphological level, it is characterised by cell shrinkage rather than the swelling seen in necrotic cell death. In cell culture, apoptosis limits the yield of economically and medically important products, and can result in synthesis of imperfect molecules. Therefore, this process must be identified, monitored and fully understood, so that a means to regulate it can be developed. We have developed a new automatic image analysis assay for detecting apoptosis in animal cell culture on the basis of the annexin-V affinity assay. The results of this assay were compared with data generated by flow cytometry and manual scoring. All three methods were found to correspond well but image analysis like flow cytometry offers operator-independent results, and can be used as a tool for rapid monitoring of viable cell number, apoptosis and necrosis in animal cell culture. Furthermore, reduction in cell size was measured and was found to precede the appearance of phosphatidylserine on the cell surface.

Introduction

Animal cell culture technology has become a key part of the bioprocess industry for producing pharmaceuticals, diagnostics and other biologically active products. Apoptosis has been observed in animal cell cultures (Cotter and Al-Rubeai, 1993) but during bioprocesses, cells may also die by necrosis. Necrosis is a passive process in which the cell typically exhibits distinctive morphological and physiological characteristics including changes in mitochondrial morphology and function (Zamzami et al., 1995) and the ability of the plasma membrane to regulate osmotic pressure (reviewed by Darzynkiewicz et al., 1997). Cell swelling is usually seen in a cell undergoing necrosis (Al-Rubeai, 1998). In contrast apoptosis is an active, regulated and controlled process with cells undergoing different and distinctive physiological and ultrastructural changes (Cohen, 1993; Schwarzmann and Cidlowski, 1993; Allen et al., 1997; Van Engeland et al., 1997). Apoptosis, which occurs as a normal regulatory mechanism in both fetal and adult tissues also plays a key role in certain diseases (Arends et al., 1994; Staunton and Gaffney, 1995; Solary et al., 1996) and treatments (Staunton and Gaffney, 1998). Various factors can trigger or repress apoptosis although the details of signal transduction pathways and the regulation of apoptosis by oncogenes and tumour suppresser gene products are not fully understood. However, the induction of apoptosis in bioreactors reduces product yield and can seriously affect product fidelity (Singh et al., 1994). Thus, in seeking to implement high performance bioprocess strategies, it is necessary to identify and control apoptotic cell death in bioreactors.

The clearest way to demonstrate the morphological or ultrastructural changes typical of apoptosis remains electron microscopy. An alternative method, dual staining of unfixed cells with acridine orange (AO) and propidium iodide (PI) can be used to differentiate apoptotic, necrotic and viable cells by fluorescence microscopy (Al-Rubeai, 1998). Two other detection methods are based on a characteristic biochemical feature of apoptosis, i.e., damage to nuclear chromatin. Electrophoresis of extracted DNA produces a characteristic ladder of oligonucleosomal fragments (180–200 bp) (Compton, 1992). In contrast, the DNA fragments produced in necrosis are irregular in size. Some flow cytometric assays are based on specific DNA fragmentation and membrane integrity to distinguish between apoptosis and necrosis (Darzynkiewicz et al., 1992; Li et al., 1996). The Terminal transferase Utilising Nick End Labelling (TUNEL) assay detects DNA strand breaks by labelling 3′OH termini with biotin or fluorochrome conjugated nucleotides in a reaction catalysed by exogenous TdT or DNA polymerase (nick translation) (O'Brian et al., 1997). However, it has been shown that the TUNEL assay can give false positive results in cells that have undergone non-apoptotic DNA strand-breaks (Cohen et al., 1992; Hendzel et al., 1998). Another alternative method for detecting apoptosis is based on the translocation of the membrane phospholipid phosphatidylserine from the inner to the outer leaflet of the plasma membrane during the early stages of apoptosis (Bratton et al., 1997; Emoto et al., 1997; Van den Eijnde et al., 1997a). In vivo, this translocation plays a role in allowing phagocytes to recognise apoptotic bodies and apoptotic cells (Fadok et al., 1992) so that the latter can be cleared from the body without invoking an inflammatory response. In vitro this phenomenon can be exploited as a method for detecting apoptotic cells (Kuypers et al., 1996; Lee and Pollard, 1997; Van den Eijnde et al., 1997b). Annexin-V is a protein which binds specifically to phosphatidylserine yet is unable to cross an intact plasma membrane. Thus, it can bind to phosphatidylserine on the outer leaflet of the membrane of apoptotic cells and when conjugated with fluorescein isothiocyanate (FITC) or other fluorochromes can be detected by fluorescence microscopy or flow cytometry (Van Heerde et al., 1995). Unfortunately, annexin-V will also bind to phosphatidylserine on the inner leaflet of the plasma membrane of necrotic cells because their loss of membrane integrity allows annexin-V into the cell. However, using PI (which intercalates into double stranded nucleic acid and is also membrane impermeable) as a counterstain to distinguish necrotic cells permits the annexin-V assay to be used to differentiate viable, apoptotic and necrotic cells by fluorescence microscopy and flow cytometry (Van Engeland et al., 1998).

Image analysis is a complement to manual microscopy, allowing routine quantification of microscopic observations. Typically, an image analysis system consists of a personal computer or workstation connected to a microscope via a video camera. The image is digitised in the computer (in space and tone) to produce an array of picture elements or “pixels”. Each pixel is assigned a “brightness” value, the intensity of which is used to distinguish features on monochrome images. For further information, see recent reviews by Thomas and Paul (1996)and Paul and Thomas (1998). Image analysis is now an established method for quantifying and characterising micro-organisms in biotechnological processes (Thomas and Paul, 1996). However, very few applications have been reported in the field of animal cell culture. Tucker et al. (1994)presented an automated image analysis system for quantifying viability and cell concentration on the basis of trypan blue exclusion. An on-line method for viability assessment was published by Maruhashi et al. (1994), where viability was calculated by a size distribution of cells and debris assuming that dead cells are smaller than viable cells. However, the latter method can give false results because necrotic cells can be larger than “normal” (Al-Rubeai et al., 1994). The TUNEL assay has been automated (Kong and Ringer, 1995) for detecting apoptosis in cell culture although it has been reported that DNA strand breaks and chromatin condensation are not necessarily related to apoptosis (Hendzel et al., 1998).

The annexin-V assay was chosen as the most appropriate method for monitoring apoptosis in animal cell cultures because it is a simple and robust method (good for future automation). In particular, it was readily portable to all three platforms to be compared in this study (manual microscopy, image analysis and flow cytometry) and there is a substantial literature on its use. Electron microscopy and DNA electrophoresis were discounted because they are clearly unsuitable for process monitoring. Simple AO/PI staining was discounted because there is no colour differentiation between viable and apoptotic cells; they are distinguished on the basis of nuclear morphology. Although this technique could be transferred to the image analysis platform, it was likely that difficulties would be encountered in adapting it to flow cytometry, since viable and apoptotic cells would have to be distinguished by light scatter properties. The TUNEL assay was discounted for two reasons: because of the reported false positives (Hendzel et al., 1998) and (ii) because the method is complex and likely to be difficult to fully automate; the ultimate goal being integrated online process monitoring and control.

In this work we describe and validate an automatic image analysis method based on the annexin-V affinity assay for detecting viable, necrotic and apoptotic cells in mammalian cell culture by comparison with results from established flow cytometry and manual fluorescence microscopy methods. Thus, the advantages of this third technology can be exploited in applications where its use is more appropriate than the existing methods.