**Chapter 4** Cell Growth Measurement

*Ning Xu, Xingrou Chen, Jingjing Rui, Yan Yu, Dongshi Gu, Jennifer Jin Ruan and Benfang Helen Ruan*

## **Abstract**

*Cell Growth*

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The cell is the basic structural and functional unit of all living organisms. As the smallest unit and building blocks of life, cells differ in size, shape, metabolism, reproduction, and growth requirements. Cells reproduce through cell division involving a four-phase (G1, S, G2, M) cell cycle, which is tightly regulated at multiple checkpoints. The resulting growth curve demonstrates that cell population increases in three sequential steps: incubation, exponential hyperplasia, and stagnation/death phases. Cell growth is subject to changes in disease state and/or environmental conditions. This chapter will focus on methods for cell growth measurement, which are grouped into five sections: cell cycle, apoptosis, growth curve, druginduced proliferation (DIP), and continuous assays. Among the continuous assays, the EZMTT dye allows for long-term tracking of cell growth under various conditions and shows promise in precision medicine by early detection of drug resistance.

**Keywords:** cell cycle, apoptosis, growth curve, drug-induced proliferation, continuous assays, drug resistance

#### **1. Introduction**

The cell is the smallest unit of living organisms [1] and grows both in population and size. Cellular growth [2] is tightly regulated and usually shows three sequential steps, including incubation, exponential hyperplasia, and stagnation/ death phases [3]. Unrestricted cell growth causes cancer, and drugs cure the disease by regulating the cell growth back to normal. Therefore, precise measurement of cell growth is very important in biomedicine, including cancer, aging, drug resistance, drug discovery, environment contamination, material biocompatibility [4, 5], fermentation, immunology, etc.

Cells grow by cell division which includes four major components: the G1, S, G2, and M phases in sequence [6], as shown in **Figure 1**. The signature of the G1 phase is the synthesis of enzymes that are required for DNA replication. During the S phase, DNA is replicated to produce two identical sets of chromosomes. The G2 phase is mainly involved in the production of microtubules that are required during the process of division, the mitotic phase. Increases in cell volume are observed during the interphase (G1, S, G2 phase). The M phase consists of prophase, metaphase, anaphase, and telophase in sequence, and the parent cell is divided into two daughter cells through nuclear division (karyokinesis), cytoplasmic division (cytokinesis), and formation of a new cell membrane [7].

Cell division is more complex in eukaryotes whose cell division involves either mitosis or a more complex process called meiosis. Mitosis and meiosis are two

#### **Figure 1.** *Cell cycle.*

different "nuclear division" processes. Through binary fission, mitosis [8] produces two daughter cells with the same number of chromosomes as the parental cell. Meiosis, also called reductive division, is the division of a germ cell involving two fissions of the nucleus to form four gametes that have half the normal cellular amount of DNA. A male and a female gamete can then combine to produce a zygote, a cell which again has the normal number of chromosomes [9]. Therefore, the enlargement of cell volume and changes in DNA content are two parameters commonly used in cell cycle measurement.

The cell cycle is tightly regulated at multiple checkpoints [10]. Various growth conditions such as the temperature, nutrients, cell density, and drug treatment can block the cell cycle at various stages. Instead of unrestricted growth, the cell growth curve shows that the cell population increases through three phases: incubation period → exponential hyperplasia → stagnation period. During exponential growth, cells demonstrate great variation in required cell density and doubling times that are highly dependent on cell type and growth conditions.

Cell death occurs in each generation. Acute cellular injury causes traumatic cell death (necrosis) [11], whereas apoptosis is a highly regulated and programmed cell death that occurs each day in multicellular organisms. The average adult human loses between 50 and 70 billion cells each day due to apoptosis [12] which is critical, because uncontrolled cell proliferation is closely related to the occurrence of human diseases such as tumors. Commonly used analyses for apoptosis are morphological analysis, detection of apoptotic biomarkers, and flow cytometric analysis of cellular DNA content.

Inhibiting cancer cell or infectious microbial growth is the purpose of drug treatment. However, drug resistance is the notorious worldwide crisis that prolongs hospital stays and considerably increases mortality. Identification of specific genetic mutations has been the major effort in understanding drug resistance, but the

**39**

*Cell Growth Measurement*

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

in early discovery of drug resistance.

**2.1 Parameters used for DNA content analysis**

rates than the diploid ones [18].

range is 0.9 to 1.1) [19].

**2. Cell cycle**

and apoptosis.

fore, very important in early detection of drug resistance.

enough to detect the survival of a minor population of cells.

continuous assays that can be used easily with various cell types.

results have had little diagnostic value [13]. Recently, many research groups [14–17] demonstrated that drug resistance develops owing to a small population of cells resistant to the drug, and drug treatment results in the selection for the growth of the small drug-resistant cell population. Recognition of partial efficacy is, there-

Traditionally, the dose–response curve is used to evaluate the potency of an inhibitor (IC50). Commonly used cell proliferation assays are metabolic activitybased methods such as the tetrazo-based cellular NAD(P)H detection system (MTT, CCK8, EZMTT) and the cellular ATP detection system (CellTiter-Glo Assay). Unfortunately, due to experimental error, the endpoint assays are not sensitive

For precise measurement of drug efficacy, the drug-induced proliferation (DIP)

Therefore, this chapter will mainly discuss the methods used for measuring cell cycle, apoptosis, growth curve, and drug-induced proliferation, continuous assays that can track the growth condition-induced cell proliferation, and its applications

Each cell cycle involves the G1, S, G2, and M phases in sequence, and each phase is associated with its signature protein biomarkers, DNA content, and cell size, as shown in **Figure 1**. DNA ploidy and protein biomarker analyses are commonly used in cell cycle studies and have important applications in clinical cancer diagnosis, drug efficacy evaluation, prognosis prediction, cell dynamics,

DNA ploidy, DNA index (DI), S phase fraction (SPF), and potential doubling

DNA ploidy refers to the number of chromosomes or the total DNA content in cells. DNA ploidy analysis in combination with clinical pathological diagnosis has a great value in early diagnosis and in prognosis prediction for malignant tumors, for both solid tumors and cancer cell extracts from body fluids, glandular secretions, and exfoliated tissue cells. Aneuploid tumors showed significantly higher recurrent

The DNA index (DI) refers to the ratio between the cells in G0/G1 peak of the tumor samples and that of the normal diploid samples. The calculation equation is DI (DNA index, number of judgment ploidy) = (average number of G0/G1 phase cell peaks in sample)/(average number of G0/G1 phase cell peaks in normal diploid cells). A DI of 1 means a normal diploid sample (generally the normal

The S phase fraction (SPF) shows the percentage of cells in S phase and indicates the proliferative activity of the cells. The calculation equation is SPF (S phase fraction %) = S cells / (G0/G1 + S + G2M) cells × 100%. Another cell proliferation parameter is proliferation index (PI), and the calculation equation is PI (prolifera-

The potential doubling time (Tpot) refers to the time required to double the cell

time (Tpot) are commonly used parameters for cell cycle analyses.

tion index %) = (S + G2M) cells/ (G0/G1 + S + G2M) cells × 100%.

number, which occurs during exponential growth.

rate has been proposed as a better parameter than the IC50 or MIC (80% inhibition) measurement [15]. However, the measurement of a precise DIP rate calls for results have had little diagnostic value [13]. Recently, many research groups [14–17] demonstrated that drug resistance develops owing to a small population of cells resistant to the drug, and drug treatment results in the selection for the growth of the small drug-resistant cell population. Recognition of partial efficacy is, therefore, very important in early detection of drug resistance.

Traditionally, the dose–response curve is used to evaluate the potency of an inhibitor (IC50). Commonly used cell proliferation assays are metabolic activitybased methods such as the tetrazo-based cellular NAD(P)H detection system (MTT, CCK8, EZMTT) and the cellular ATP detection system (CellTiter-Glo Assay). Unfortunately, due to experimental error, the endpoint assays are not sensitive enough to detect the survival of a minor population of cells.

For precise measurement of drug efficacy, the drug-induced proliferation (DIP) rate has been proposed as a better parameter than the IC50 or MIC (80% inhibition) measurement [15]. However, the measurement of a precise DIP rate calls for continuous assays that can be used easily with various cell types.

Therefore, this chapter will mainly discuss the methods used for measuring cell cycle, apoptosis, growth curve, and drug-induced proliferation, continuous assays that can track the growth condition-induced cell proliferation, and its applications in early discovery of drug resistance.
