Flow Cytometry Applications in Cell Culture

Mohamed Al-Rubeai, A Nichol Emery, Mohamed Al-Rubeai, A Nichol Emery

• Publisher: CRC Press
• Year: 2020
• ISBN:
  9781000148503
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Cell Biology
• Index: Biological Sciences

This work present practical, biotechnological applications of flow cytometry techniques for the study of animal, plant and microbial cells, explaining methodologies for sample preparation, staining and analysis. It discusses cell variability in cell culture processes and shows how the quantitative analysis of heterogeneous populations aids in the biotechnological exploitation of cells.

1
Monitoring the Proliferative Capacity of Cultured Animal Cells by Cell Cycle Analysis

V. LEELAVATCHARAMAS, A. NICHOLAS EMERY, AND MOHAMED AL-RUBEAI
The University of Birmingham, Birmingham, England

I. AN INTRODUCTION TO CELL CYCLE DYNAMICS IN CULTURED CELLS

The advancement of the technology for large-scale cultivation of animal cells has relied on the implementation of strategies and techniques that can be grouped into four categories: maximization of viable cell concentration, improvement of medium formulation, implementation of high-performance reactor configurations, and maximization of specific production rate. However, the development of optimal-processing schemes has always been hindered by a lack of the equipment necessary for adequate state identification in the bioreaction processes. Most of the monitoring systems available are biochemically based, slow, off-line, and yield only global values for the reaction system. Even on-line analytical systems common in bacterial and fungal fermentations, such as the measurement of oxygen uptake rates and carbon dioxide production rates by off-gas analysis, are problematic in animal cell culture owing to the very low rates to be measured and consequent poor sensitivity.

Efficient and effective process monitoring of the identified major parameters that characterize growth is the first step toward achieving adequate and sensitive process design and control. The major constituent of growth is the cell cycle, the analysis of which may provide a reliable index for the prediction of growth potential and changes in cell number with time during the cultivation period. As the cell population at anytime during cultivation is a mixture of dividing cells and dying cells, for one to predict the cell number long before it can be assessed by cell counting it is necessary to separate the component of cell growth (sometimes referred to as the growth fraction) from that of cell death. In conditions of little or no cell loss, such as are encountered during the exponential growth phase of batch culture, the analysis of the cell cycle, specifically the fraction of S cells, can furnish reliable information on the proliferative dynamics of cell culture [1]. To indicate the ability of a feeding control strategy based on cell cycle analysis to meet the demands of cells for optimal growth in perfusion culture, Miltenburger's group successfully used analysis of the percentage of S-phase cells as an indicator for regulation of the medium flow rate to hybridoma cells [2],

Such analyses of the cell cycle and, indeed, of many other structural and physiological characteristics of individual cells in a population can be efficiently, rapidly, and accurately achieved only by flow cytometry (FC). Flow cytometry can provide the detailed and thorough analyses needed for the employment of strict control of the heterogeneous cell populations normally seen in cell culture.

There is now a widespread and rapidly growing interest in the use of FC for the monitoring of large-scale animal cell bioprocesses. In this chapter we present a number of examples of its use, even when the measurement is of only a single parameter (DNA), to obtain useful insights into the proliferation kinetics of Chinese hamster ovary (CHO) cells. This parameter, which can be easily and rapidly analyzed, can singularly indicate the state of the culture and characterize its population dynamics; hence, it is likely to make a significant contribution to the development of sophisticated population balance models suitable for control with a high degree of accuracy.

The measurement of DNA was one of the first, and still is, one of the most widespread applications of FC, examples of which include the characterization of aneuploid malignant cells, cytotoxic drug effects on cell growth, the detection of polyploid species, and the separation of sperm containing the X chromosome from those containing the Y chromosome. The DNA measurements are achieved by staining with one of a variety of fluorochromes, such as propidium iodide (PI), ethidium bromide (EB), Hoechst 33258, plicamycin (mithramycin), and at least another 15 other dyes. The interaction between these dyes and the DNA molecules depends on not only the concentration of the free dye, but also on the concentrations of electrolytes and on temperature [3].

The cell cycle is defined as the interval between completion of mitosis in the parent cell and completion of the next mitosis in one or both daughter cells [4], It can be divided into four phases. The G₁ phase is the period of a young cell, lasting from the completion of mitotic division until the start of the replication of its DNA. The S phase is the period of DNA synthesis, during which the genome is duplicated. The G₂ phase is the mature stage of the cell, lasting from the end of the genome duplication until the onset of the mitotic prophase. The M phase is the short period of mitosis, during which extensive structural changes appear, and at the end of which the division of the cytoplasm is initiated. For most cells, the durations of the S, G₂, and M phases are relatively constant lasting for 6-9, 2-5, and less than 1.0 h, respectively. G₁ is the most variable phase of the cell cycle: it can last 30 h or more in some cell lines, but can be lacking entirely in others (e.g., in one line of Chinese hamster lung cells [5]). Most cell populations consist of a mixture of dividing and nondividing fractions. The cell typically enters the nondividing state (or the G₀; [6]) from a point immediately after mitosis, but reenters the dividing state (or the cell cycle) in mid-G₁ [7]. Pardee et al. [8] divided the interval between G₀ and S phases into four subsections (Fig. 1a): a competent state (C), which can last for about 6 h; a point V, which is similar to that of cycling cells that have just completed mitosis; a control point R (the restriction point or start point [9]) beyond which growth factors are not required for progression through the cell cycle; and finally, a period of organization of the machinery for DNA synthesis (start of the S phase). The cell, after completing one cycle and reaching the next G₁, can then either proceed through another cycle or can enter the G₀ and stay there until it is stimulated. Not all cells can enter the G₀ state. Some transformed cells (and hybridomas) are examples of cycling cells for which their cell cycles lack the control point R, at which the entry into the S phase is determined (Fig. 1b). This situation is clear in many continuous cell lines in culture when depletion of nutrients results in reducing DNA and protein synthesis, thereby inducing cell death. Attempts to separate cell growth from cell death for the purpose of maintaining nondividing cells for high product productivity have been unsuccessful. These cells cannot be kept quiescent and viable over an extended period, and the increase in the G₁ cells seen in conditions of depleted nutrient is more likely to be due to a lack of precursors and energy sources necessary for protein and DNA synthesis. Linardos et al. [10] have studied the relationship of cell death to the cell cycle position and developed a model describing the steady-state growth and death rates of hybridoma cells. Their model was based on the transition probability model proposed by Smith and Martin [11] to explain the observed variability in the cell cycle. Their main observation, that the death rate in hybridoma cultures is proportional to the fraction of cells "arrested" in the G₁ phase, is in agreement with our own repeated observations in batch and continuous cultures [12]. Their use of the term arrest was understood to be a description of the fact that a high percentage of G₁ phase cells were obtained at suboptimal culture conditions. The lack of nutrients results in decreased biosynthetic activity, thereby reducing cell progression. In such cultures, the only evidence of arrest is when cells are observed entirely in G₁ after nutrient depletion, or death is restricted to G₁ cells, but neither situation has ever been observed in hybridoma culture.

FIGURE 1 Progression through the ceil cycle of normal cells (a) as proposed by Pardee et al. [8] and (b) of transformed or cancer cells for which growth factors are not critical for cell growth.

A typical pattern for the cell cycle distribution during simple batch culture of animal cells shows a substantial decrease in the S and G₂ fractions during the stationary and decline phases of culture. However, the increasing presence of dead cells and cell debris during these late culture phases complicates the analysis of the cell cycle distribution. To be able to use FC effectively for monitoring growth and productivity, it is first important to discriminate between viable and nonviable cells. Treatment of nonfixed cells with DNase to eliminate DNA from nonviable cells is an efficient method for the analysis of the proliferative capacity of such a heterogeneous culture [13,14].

II. SOME PROTOCOLS FOR CELL CYCLE ANALYSIS EXEMPLIFIED USING A CULTURE OF RECOMBINANT CHINESE HAMSTER OVARY CELLS

A. Cell Culture Protocol

The recombinant CHO 320 cell line used in the work described in the following, obtained from the Wellcome Foundation Ltd. (Beckenham, UK), expressed human interferon gamma, which was coamplified with dihydrofolate reductase by methotrexate selection.

The inoculum, which was taken from the late exponential phase of a 5% fetal calf serum (FCS)-supplemented culture, was spun down and resuspended in 255 ml RPMI 1640 with 1 μΜ methotrexate medium. In a typical experiment, in which the specific growth rates of a set of cultures were investigated by using a range of FCS concentrations, 50 ml of this suspension culture was inoculated into each batch of 1.0, 2.5, 5.0, 7.5, or 10.0% FCS culture to give the initial cell number of 2 × 10⁵/ml in 200 ml working volume. All the cultures, in stirred bottles, were incubated at 37°C in an incubator and magnetically stirred at an agitation rate of 150 rpm. Samples were taken every 6 h in the first 2 days and every 12 h for a further 2 days after incubation. After this, samples were taken every 24 h until the end of the batch. Samples were cell-counted and centrifuged at 1000 rpm for 5 min. The cells were fixed at 10⁶/ml with cold 70% ethanol and kep...