**6. Continuous assay**

Continuously tracking the cell growth is important for accurate assessment of drug effects and/or growth condition changes. Even though various endpoint assays [75, 82, 83] can be terminated at various time point to obtain the time-dependent cell proliferation curve, the procedures are labor-intensive and have high experimental error. Therefore, a nondestructive continuous assay is highly desirable and critical for the precise evaluation of drug potency and efficacy. Besides the traditional turbidimetric assay by spectrophotometry that has been used for years in microbial assays, four new technologies have been developed that can be used for continuous assays.

### **6.1 High-content analysis**

High-content analysis is a cell imaging and analysis system which includes automatic high-speed microscopic imaging, fully automatic image analysis, and data management. Through snapshot cellular microscopic imaging of a 96-well plate followed by synchronous analysis, the high-content analysis, such as the CloneSelectTM imaging system, accurately measures the cell number without any cell damage. Compared with the MTT method, the experimental deviation of dose pharmacodynamic curve obtained by CloneSelectTM imaging system is smaller and more reproducible. Therefore, high-content analysis has become a reliable choice for big pharmaceutical companies to evaluate drug antiproliferation effects in high-throughput screening, although mostly for attached mammalian cells.

#### **6.2 Electrical impedance technology**

Electrical impedance technology-based real-time cellular analysis (RTCA) [84–86] and Epic BenchTop optical biosensors methods [87, 88] have been used in measuring bacterial growth. However, the methods require cells to be cultured on the working electrode or a sensor array of complex structures. The methods [89] cannot detect changes in the cells themselves, so the cell damage caused by drugs and the understanding of the mechanism of drug action are difficult to assess accurately; this inevitably brings detection error and interference. In addition, the requirement of complex laboratory infrastructure further limits their utility.

#### **6.3 Raman spectroscopy analysis**

Raman spectroscopy provides a spectroscopic fingerprint of a substance. Based on the difference in monochromatic light with vibrational modes, Raman spectroscopy can be used for qualitative and quantitative measures of the changes in biochemical composition. For example, Raman spectroscopy was used as a noninvasive method to distinguish cells at different stages in the cell cycle [90]; to identify living cells from dead cells [91–95]; to image cellular organelles [96]; to track drug distribution [97] and metabolism [91]; to monitor cell apoptosis [94], death, and cytotoxicity [92, 95]; and to study cell responses to external stimuli [97–101]. However, the analysis of Raman results requires expertise in identifying the spectroscopic fingerprint of a substance.

#### **6.4 EZMTT dye-based cell proliferation analysis**

The EZMTT dye [81] was initially designed to overcome the stability issue of WST-8 (CCK-8) reagent which can cause false positives in the presence of antioxidants such as BME (**Figure 5A**) or EGCG. Later, the EZMTT dye was found to be essentially nontoxic and stable in various media [102]. After a single dye addition (**Figure 5B**), the EZMTT method showed linear dose–response to cell numbers and higher signal to background ratio than other relevant methods (**Figure 5C**). The IC50 values measured by the EZMTT method are precise and essentially the same as the other methods (**Figure 5D**). Interestingly, when both EZMTT and WST-8 (CCK-8) methods were applied to track the growth of

#### **Figure 5.**

*EZMTT assays: (A) EZMTT dye is stable in the presence of up to 6 mM BME. (B) One step addition of the EZMTT dye allows sensitive measurement of cell growth. (C) Comparison of the signal to background ratio of various tetrazo-based assay (MTS, MTT, CCK-8, EZMTT) in the presence of the same amount of A549 cancer cells. (D) Essentially the same IC50 values were obtained from the MTS, MTT, CCK-8, or EZMTT-based assays. (E) Cell growth followed by the EZMTT method. (F) Cell growth followed by the WST-8 method. (G) For a good inhibitor, essentially the same IC50 values were obtained from the CTG or EZMTT-based assays. (H) CB839 could only achieve up to 80% inhibtion; (I) the DIP rate of CB839.*

**51**

treating the disease.

*Cell Growth Measurement*

exponential phase.

was observed [105].

**7. Conclusions**

*DOI: http://dx.doi.org/10.5772/intechopen.86835*

A549 lung cancer cells, WST-8 (CCK-8)-treated cells stopped growth in 1 day, whereas the EZMTT-treated cells could grow till saturation (**Figure 5E** and **F**) and allowed easy determination of the cell density and the doubling time in the

In addition, when the EZMTT method was used to track the drug-induced proliferation (DIP) rate changes, the EZMTT method demonstrated high sensitivity and reliability in detecting drug resistance. **Figure 5H** and **I** compares the dose–response curve obtained by the CellTiter-Glo (CTG) method and the EZMTT method. For a sensitive inhibitor, both methods showed essentially the same IC50 and % inhibition (**Figure 5H**). When a partial inhibitor is tested, both methods showed essentially the same EC50 values, but % inhibition was lower in the EZMTT method, because the partially inhibited cells are still growing (**Figure 5I**). Recently, several KGA allosteric inhibitors were rediscovered as partial inhibitors. For example, CB839 had shown 100% inhibition in CTG assay [103], whereas the EZMTT assay showed approximately 80% inhibition [104], and this is further confirmed by the (DIP) rate measurement. At the steady state, 10 μM CB839 did not completely inhibit cancer cell growth. Interestingly, when 64 nM CB839 and 24 μM ebselen were used in combination, synergistic effects were observed; even though individual compounds only showed partial inhibition, when used in combination, nearly complete inhibition of cancer cell growth

As a new assay format, EZMTT showed powerful applications in cell growth analysis, clinical diagnosis of trivial drug resistance, precise medicine for drug combination, and cost-effective drug discovery by building better correlation between

Cell proliferation assays are widely used in molecular biology, tumor biology, pharmacology, and pharmacokinetics. It is important in studying not only the basic biological characteristics of cells but also a basic method for analyzing cell states and studying genetic traits. The increase of cell numbers can be simply measured by manual counting of cells under microscopy, using the dye exclusion method (i.e., trypan blue) to count only viable cells. Less fastidious, scalable methods include the use of cytometers; especially the flow cytometry allows combining cell counts ("events") with other specific parameters such as fluorescent probes for membranes, cytoplasm, or nuclei which allows distinguishing dead/viable cells, cell types, cell differentiation, and expression of a biomarker such as Ki67. Beside counting the increasing number of cells, cells can also be assessed based on the metabolic activity, such as the CFDA-SE or calcein-AM method measures not only the membrane functionality (dye retention) but also the functionality of cytoplasmic enzymes (esterases). Also, the MTT-type assays or the resazurin assay (fluorimetric) measures the mitochondrial redox potential. Most of these assays are endpoint assays and may or may not correlate well with the cell proliferation, depending on cell growth conditions, populations of different cells, drug interferences, or toxicity. For precise evaluation of druginduced proliferation rate changes, a continuous assay is highly desirable. Among various assay formats, the EZMTT dye showed initial promise in precise and sensitive detection of partial inhibition which is the cause of worldwide crisis in drug resistance, and the EZMTT method is expected to provide valuable information for exploring the pathogenesis of the disease, diagnosing the disease, and

the in vitro cell-based assay and in vivo animal models.

*Cell Growth*

**6.3 Raman spectroscopy analysis**

the spectroscopic fingerprint of a substance.

**6.4 EZMTT dye-based cell proliferation analysis**

Raman spectroscopy provides a spectroscopic fingerprint of a substance. Based on the difference in monochromatic light with vibrational modes, Raman spectroscopy can be used for qualitative and quantitative measures of the changes in biochemical composition. For example, Raman spectroscopy was used as a noninvasive method to distinguish cells at different stages in the cell cycle [90]; to identify living cells from dead cells [91–95]; to image cellular organelles [96]; to track drug distribution [97] and metabolism [91]; to monitor cell apoptosis [94], death, and cytotoxicity [92, 95]; and to study cell responses to external stimuli [97–101]. However, the analysis of Raman results requires expertise in identifying

The EZMTT dye [81] was initially designed to overcome the stability issue of WST-8 (CCK-8) reagent which can cause false positives in the presence of antioxidants such as BME (**Figure 5A**) or EGCG. Later, the EZMTT dye was found to be essentially nontoxic and stable in various media [102]. After a single dye addition (**Figure 5B**), the EZMTT method showed linear dose–response to cell numbers and higher signal to background ratio than other relevant methods (**Figure 5C**). The IC50 values measured by the EZMTT method are precise and essentially the same as the other methods (**Figure 5D**). Interestingly, when both EZMTT and WST-8 (CCK-8) methods were applied to track the growth of

*EZMTT assays: (A) EZMTT dye is stable in the presence of up to 6 mM BME. (B) One step addition of the EZMTT dye allows sensitive measurement of cell growth. (C) Comparison of the signal to background ratio of various tetrazo-based assay (MTS, MTT, CCK-8, EZMTT) in the presence of the same amount of A549 cancer cells. (D) Essentially the same IC50 values were obtained from the MTS, MTT, CCK-8, or EZMTT-based assays. (E) Cell growth followed by the EZMTT method. (F) Cell growth followed by the WST-8 method. (G) For a good inhibitor, essentially the same IC50 values were obtained from the CTG or EZMTT-based assays.* 

*(H) CB839 could only achieve up to 80% inhibtion; (I) the DIP rate of CB839.*

**50**

**Figure 5.**

A549 lung cancer cells, WST-8 (CCK-8)-treated cells stopped growth in 1 day, whereas the EZMTT-treated cells could grow till saturation (**Figure 5E** and **F**) and allowed easy determination of the cell density and the doubling time in the exponential phase.

In addition, when the EZMTT method was used to track the drug-induced proliferation (DIP) rate changes, the EZMTT method demonstrated high sensitivity and reliability in detecting drug resistance. **Figure 5H** and **I** compares the dose–response curve obtained by the CellTiter-Glo (CTG) method and the EZMTT method. For a sensitive inhibitor, both methods showed essentially the same IC50 and % inhibition (**Figure 5H**). When a partial inhibitor is tested, both methods showed essentially the same EC50 values, but % inhibition was lower in the EZMTT method, because the partially inhibited cells are still growing (**Figure 5I**). Recently, several KGA allosteric inhibitors were rediscovered as partial inhibitors. For example, CB839 had shown 100% inhibition in CTG assay [103], whereas the EZMTT assay showed approximately 80% inhibition [104], and this is further confirmed by the (DIP) rate measurement. At the steady state, 10 μM CB839 did not completely inhibit cancer cell growth. Interestingly, when 64 nM CB839 and 24 μM ebselen were used in combination, synergistic effects were observed; even though individual compounds only showed partial inhibition, when used in combination, nearly complete inhibition of cancer cell growth was observed [105].

As a new assay format, EZMTT showed powerful applications in cell growth analysis, clinical diagnosis of trivial drug resistance, precise medicine for drug combination, and cost-effective drug discovery by building better correlation between the in vitro cell-based assay and in vivo animal models.
