Cultured Cells: Characteristics, Growth Parameters and Synchronization

Read this article to learn about the characteristics, growth parameters and synchronization of cultured cells.

Characteristics of Cultured Cells:

Some of the important distinguishing properties of cultured cells are given below:

1. Cells which do not normally proliferate in vivo can be grown and proliferated in cultures.

2. Cell to cell interactions in the cultured cells are very much low.

3. The three dimensional architecture of the in vivo cells is not found in cultured cells.

4. The hormonal and nutritional influence on the cultured cells differs from that on the in vivo cells.

5. Cultured cells cannot perform differentiated and specialized functions.

6. The environment of the cultured cells favours proliferation and spreading of unspecialized cells.

Environmental influence on cultured cells:

The environmental factors strongly influence the cells in culture. The major routes through which environmental influence occurs are listed:

i. The nature of the substrate or phase in which cells grow. For monolayer cultures, the substrate is a solid (e.g. plastic) while for suspension cultures, it is a liquid.

ii. The composition of the medium used for culture nutrients and physicochemical properties.

iii. Addition of hormones and growth factors.

iv. The composition of the gas phase.

v. The temperature of culture incubation.

The biological and other aspects of cultured cells with special reference to the following parameters are briefly described:

1. Cell adhesion.

2. Cell proliferation.

3. Cell differentiation.

4. Metabolism of cultured cells.

5. Initiation of cell culture.

6. Evolution and development of cell lines.

Cell Adhesion:

Most of the cells obtained from solid tissues grow as adherent monolayers in cultures. The cells, derived from tissue aggregation or subculture, attach to the substrate and then start proliferating. In the early days of culture techniques, slightly negatively charged glasses were used as substrates. In recent years, plastics such as polystyrene, after treatment with electric ion discharge, are in use.

The cell adhesion occurs through cell surface receptors for the molecules in the extracellular matrix. It appears that the cells secrete matrix proteins which spread on the substrate. Then the cells bind to matrix through receptors. It is a common observation that the substrates (glass or plastic) with previous cell culture are conditioned to provide better surface area for adhesion.

Cell adhesion molecules:

Three groups of proteins collectively referred to as cell adhesion molecules (CAMs) are involved in the cell-cell adhesion and cell-substrate adhesion.

Cell-cell adhesion molecules:

These proteins are primarily involved in cell-to-cell interaction between the homologous cells. CAMs are of two types — calcium-dependent ones (cadherin’s) and calcium-independent CAMs.

Integrin’s:

These molecules mediate the cell substrate interactions. Integrin’s possess receptors for matrix molecules such as fibronectin and collagen.

Proteoglycans:

These are low affinity trans membrane receptors. Proteoglycans can bind to matrix collagen and growth factors. Cell adhesion molecules are attached to the cytoskeletons of the cultured cells.

Cell Proliferation:

Proliferation of cultured cells occurs through the cell cycle, which has four distinct phases (Fig. 35.1)

M phase:

In this phase (M = mitosis), the two chromatids, which constitute the chromosomes, segregate to daughter cells.

G₁ phase:

This gap 1 phase is highly susceptible to various control processes that determine whether cell should proceed towards DNA synthesis, re-enter the cycle or take the course towards differentiation.

S phase:

This phase is characterized by DNA synthesis wherein DNA replication occurs.

G₂ phase:

This is gap 2 phase that prepares the cell for reentry into mitosis. The integrity of the DNA, its repair or entry into apoptosis (programmed cell death) if repair is not possible is determined by two check points-at the beginning of DNA synthesis and in G₂ phase.

Control of cell proliferation:

For the cells in culture, the environmental signals regulate the cell cycle and thereby the cell proliferation. Low density of the cells in a medium coupled with the presence certain growth factors (e.g. epidermal growth factor, platelet-derived growth factor) allows the cells to enter the cell cycle.

On the other hand, high cell density and crowding of cells inhibits the cell cycle and thereby proliferation. Besides the influence of the environmental factors, certain intracellular factors also regulate the cell cycle. For instance, cyclins promote while p53 and Rb gene products inhibit cell cycle.

Cell Differentiation:

The various cell culture conditions favour maximum cell proliferation and propagation of cell lines.

Among the factors that promote cell proliferation, the following are important:

i. Low cell density

ii. Low Ca²⁺ concentration

iii. Presence of growth factors

For the process of cell differentiation to occur, the proliferation of cells has to be severely limited or completely abolished.

Cell differentiation can be promoted (or induced) by the following factors:

i. High cell density.

ii. High Ca²⁺ concentration.

iii. Presence of differentiation inducers (e.g. hydro­cortisone, nerve growth factor).

As is evident from the above, different and almost opposing conditions are required for cell proliferation, and for cell differentiation. Therefore if cell differentiation is required two distinct sets of conditions are necessary.

1. To optimize cell proliferation.

2. To optimize cell differentiation.

Maintenance of differentiation:

It is now recognized that the cells retain their native and original functions for long when their three dimensional structures are retained. This is possible with organ cultures. However, organ cultures cannot be propagated.

In recent years, some workers are trying to create three dimensional structures by per-fusing monolayer cultures. Further, in vitro culturing of cells on or in special matrices (e.g. cellulose, collagen gel, matrix of glycoproteins) also results in cells with three dimensional structures.

Dedifferentiation:

Dedifferentiation refers to the irreversible loss of specialized properties of cells when they are cultured in vitro. This happens when the differentiated in vitro cells lose their properties (Fig. 35.2). In the in vivo situation, a small group of stem cells give rise to progenitor cells that are capable of producing differentiated cell pool (Fig. 35.2A).

On the other hand, in the in vitro culture system, progenitor cells are predominantly produced which go on proliferating. Very few of the newly formed cells can form differentiated cells (Fig. 35.2B). The net result is a blocked differentiation. Dedifferentiation implies an irreversible loss of specialized properties of the cells. On the other hand, de-adaptation refers to the re-induction of specialized properties of the cells by creating appropriate conditions.

Metabolism of Cultured Cells:

The metabolism of mammalian cultured cells with special reference to energy aspects is depicted in Fig. 35.3. The cultured cells can use glucose or glutamine as the source of energy. These two compounds also generate important anabolic precursors.

As glucose gets degraded by glycolysis, lactate is mainly produced. This is because oxygen is in limited supply in the normal culture conditions (i.e. atmospheric oxygen and a submerged culture) creating an anaerobic situation. Lactate, secreted into the medium, accumulates.

Some amount of pyruvate produced in glycolysis gets oxidized through Krebs cycle. A small fraction of glucose (4-9%) enters pentose phosphate pathway to supply ribose 5-phosphate and reducing equivalents (NADPH) for biosynthetic pathways e.g. synthesis of nucleotides.

Glutamine is an important source of energy for the cultured cells. By the action of the enzyme glutaminase, glutamine undergoes deamination to produce glutamate and ammonium ions. Glutamate, on transamination (or oxidative deamination) forms a-ketoglutarate which enters the Krebs cycle.

Pyruvate predominantly participates in transamination reaction to produce alanine, which is easily excreted into the medium. In the rapidly growing cultured cells, transamination reaction is a dominant route of glutamine metabolism.

Deamination of glutamine releases free ammonium ions, which are toxic to the cultured cells, limiting their growth. In recent years, dipeptides glutamyl-alanine or glutamyl-glycine are being used to minimize the production of ammonia. Further, these dipeptides are more stable in the medium.

As already stated, α-ketoglutarate obtained from glutamine (via glutamate) enters the Krebs cycle and gets oxidized to carbon dioxide and water. For proper operation of Kerbs cycle, balancing of the intermediates of the cycle is required.

Two metabolites of Kerbs cycle namely malate and oxaloacetate leave the cycle and get converted respectively to pyruvate and phosphoenol pyruvate. The latter two compounds can reenter the Krebs cycle in the form of acetyl CoA. Thus, the continuity of Kerbs cycle is maintained. Glucose as well as glutamine gets metabolised by the cultured cells to supply energy in the form of ATP.

Initiation of Cell Culture:

The cell culture can be initiated by the cells derived from a tissue through enzymatic or mechanical treatments. Primary culture is a selective process that finally results in a relatively uniform cell line. The selection occurs by virtue of the capacity of the cells to survive as monolayer cultures (by adhering to substrates) or as suspension cultures.

Among the cultured cells, some cells can grow and proliferate while some are unable to survive under the culture environment. The cells continue to grow in monolayer cultures, till the availability of the substrate is occupied.

The term confluence is used when the cultured cells make close contact with one another by fully utilizing the available growth area. For certain cells, which are sensitive to growth limitation due to density, the cells stop growing once confluence is reached. However, the transformed cells are insensitive to confluence and continue to overgrow.

When the culture becomes confluent, the cells possess the following characters:

1. The closest morphological resemblance to the tissue of origin (i.e. parent tissue).

2. The expression of specialized functions of the cells comparable to that of the native cells.

Evolution and Development of Cell Lines:

The primary culture grown after the first subculture is referred to as cell line. A given cell line may be propagated by further sub culturing. As the subcultures are repeated, the most rapidly proliferating cells dominate while the non- proliferating or slowly proliferating cells will get diluted, and consequently disappear.

Senescence:

The genetically determined event of cell divisions for a limited number of times (i.e. population doublings), followed by their death in a normal tissue is referred to as senescence. However, germ cells and transformed cells are capable of continuously proliferating. In the in vitro culture, transformed cells can give rise to continuous cell lines.

The evolution of a continuous cell line is depicted in Fig. 35.4. The cumulative cell number in a culture is represented on Y-axis on a log scale, while the X-axis represents the time in weeks. The time for development of a continuous cell line is variable. For instance, for human diploid fibroblasts, the continuous cell line arises at about 14 weeks while the senescence may occur between 10 to 20 weeks; usually after 30 and 60 cell doublings.

Development of continuous cell lines:

Certain alterations in the culture collectively referred to as transformation, can give rise to continuous cell lines. Transformation may be spontaneously occurring, chemically or virally- induced. Transformation basically involves an alteration in growth characteristics such as loss of contact inhibition, density limitation of growth and anchorage independence. The term immortalization is frequently used for the acquisition of infinite life span to cultured cells.

Genetic variations:

The ability of the cells to grow continuously in cell lines represents genetic variation in the cells. Most often, the deletion or mutation of the p⁵³ gene is responsible for continuous proliferation of cells. In the normal cells, the normal p⁵³ gene is responsible for the arrest of cell cycle. Most of the continuous cell lines are aneuploid, possessing chromosome number between diploid and tetraploid value.

Normal cells and continuous cell lines:

A great majority of normal cells are not capable of giving rise to continuous cell lines. For instance, normal human fibroblasts go on proliferating for about 50 generations, and then stop dividing. However, they remain viable for about 18 months. And throughout their life span, fibroblasts remain euploid. Chick fibroblasts also behave in a similar fashion. Epidermal cells and lymphoblastic cells are capable of forming continuous cell lines.

Characterization of Cultured Cells:

Characterization of cultured cells or cell lines is important for dissemination of cell lines through cell banks, and to establish contacts between research laboratories and commercial companies.

Characterization of cell lines with special reference to the following aspects is generally done:

1. Morphology of cells

2. Species of origin.

3. Tissue of origin.

4. Whether cell line is transformed or not.

5. Identification of specific cell lines.

Morphology of Cells:

A simple and direct identification of the cultured cells can be done by observing their morphological characteristics. However, the morphology has to be viewed with caution since it is largely dependent on the culture environment. For instance, the epithelial cells growing at the center (of the culture) are regular polygonal with clearly defined edges, while those growing at the periphery are irregular and distended (swollen).

The composition of the culture medium and the alterations in the substrate also influence the cellular morphology. In a tissue culture laboratory, the terms fibroblastic and epithelial are commonly used to describe the appearance of the cells rather than their origin.

Fibroblastic cells:

For these cells, the length is usually more than twice of their width. Fibroblastic cells are bipolar or multipolar in nature.

Epithelial cells:

These cells are polygonal in nature with regular dimensions and usually grow in monolayers. The terms fibroblastoid (fibroblast-like) and epitheloid (epithelial-like) are in use for the cells that do not possess specific characters to identify as fibroblastic or epithelial cells.

Species of Origin of Cells:

The identification of the species of cell lines can be done by:

a. Chromosomal analysis.

b. Electrophoresis of isoenzymes.

c. A combination of both these methods.

In recent years, chromosomal identification is being done by employing molecular probes.

Identification of Tissue of Origin:

The identification of cell lines with regard to tissue of origin is carried out with reference to the following two characteristics:

1. The lineage to which the cells belong.

2. The status of the cells i.e. stems cells, precursor cells.

Tissue markers for cell line identification:

Some of the important tissue or lineage markers for cell line identification are briefly described.

Differentiated products as cell markers:

The cultured cells, on complete expression, are capable of producing differentiation markers, which serve as cell markers for identification.

Some examples are given below:

a. Albumin for hepatocytes.

b. Melanin for melanocytes

c. Hemoglobin for erythroid cells

d. Myosin (or tropomyosin) for muscle cells.

Enzymes as tissue markers:

The identification of enzymes in culture cells can be made with reference to the following characters:

a. Constitutive enzymes.

b. Inducible enzymes.

c. Isoenzymes.

The commonly used enzyme markers for cell line identification are given in Table 35.1.

Tyrosine aminotransferase is specific for hepatocytes, while tyrosinase is for melanocytes. Creatine kinase (MM) in serum serves as a marker for muscle cells, while creatine kinase (BB) is used for the detection of neurons and neuroendocrine cells.

Filament proteins as tissue markers:

The intermediate filament proteins are very widely used as tissue or lineage markers.

For example:

a. Astrocytes can be detected by glial fibrillary acidic protein (GFAP).

b. Muscle cells can be identified by desmin.

c. Epithelial and mesothelial cells by cytokeratin.

Cell surface antigens as tissue markers:

The antigens of the cultured cells are useful for the detection of tissue or cells of origin. In fact, many antibodies have been developed (commercial kits are available) for the identification cell lines (Table. 35.2). These antibodies are raised against cell surface antigens or other proteins.

The antibodies raised against secreted antigen a-fetoprotein serves as a marker for the identification of fetal hepatocytes. Antibodies of cell surface antigens namely integrin’s can be used for the general detection of cell lines.

Transformed Cells:

Transformation is the phenomenon of the change in phenotype due to the acquirement of new genetic material. Transformation is associated with promotion of genetic instability.

The transformed and cultured cells exhibit alterations in many characters with reference to:

a. Growth rate

b. Mode of growth

c. Longevity

d. Tumorigenicity

e. Specialized product formation.

While characterizing the cell lines, it is necessary to consider the above characters to determine whether the cell line has originated from tumor cells or has undergone transformation in culture.

Identification of Specific Cell Lines:

There are many approaches in a culture laboratory to identify specific cell lines:

a. Chromosome analysis

b. DNA detection

c. RNA and protein analysis

d. Enzyme activities

e. Antigenic markers.

Chromosome analysis:

The species and sex from which the cell line is derived can be identified by chromosome analysis. Further, it is also possible to distinguish normal and malignant cells by the analysis of chromosomes. It may be noted that the normal cells contain more stable chromosomes. The important techniques employed with regard to chromosome analysis are briefly described.

Chromosome banding:

By this technique, it is possible to identify individual chromosome pairs when there is little morphological difference between them. Chromosome banding can be done by using Giemsa staining.

Chromosome count:

A direct count of chromosomes can be done per spread between 50-100 spreads. A camera Lucida attachment or a closed circuit television may be useful.

Chromosome karyotyping:

In this technique, the chromosomes are cut, sorted into sequence, and then pasted on to a sheet. The image can be recorded or scanned from the slide. Chromosome karyotyping is time consuming when compared to chromosome counting.

DNA detection:

The total quantity of DNA per normal cell is quite constant, and is characteristic to the species of origin, e.g. normal cell lines from human, chick and hamster fibroblasts. However, the DNA content varies in the normal cell lines of mouse, and also the cell lines obtained from cancerous tissues. Most of the transformed cells are aneuploid and heteroploid. DNA analysis is particularly useful for characterization of such cells. Analysis of DNA can be carried out by DNA hybridization and DNA fingerprinting.

DNA hybridization:

The popular Southern blotting technique can be used to detect unique DNA sequences. Specific molecular probes with radioisotope, fluorescent or luminescent labels can be used for this purpose. The DNA from the desired cell lines is extracted, cut with restriction endonucleases, subjected to electrophoresis, blotted on to nitrocellulose, and then hybridized with a molecular (labeled) probe, or a set of probes. By this approach, specific sequences of DNA in the cell lines can be detected.

DNA fingerprinting:

There are certain regions in the DNA of a cell that are not transcribed. These regions, referred to as satellite DNA, have no known functions, and it is believed that they may provide reservoir for genetic evolution. Satellite DNA regions are considered as regions of hyper variability. These regions may be cut with specific restriction endonucleases, and detected by using cDNA probes.

By using electrophoresis and autoradiography, the patterns of satellite DNA variations can be detected. Such patterns referred to as DNA fingerprints are cell line specific. In recent years, the technique of DNA fingerprinting has become a very popular and a powerful tool to determine the origin of cell lines.

RNA and protein analysis:

The phenotype characteristics of a cell line can be detected by gene expression i.e. identification of RNAs and/or proteins. mRNAs can be identified by Northern blot technique while proteins can be detected by Western blot technique.

Enzyme activities:

Some of the in vivo enzyme activities are lost when the cells are cultured in vitro. For instance, arginase activity of the liver cells is lost within a few days of culturing. However, certain cell lines express specific enzymes that can be employed for their detection e.g. tyrosine aminotransferase for hepatocytes, glutamyl synthase activity for astroglia in brain. For more examples of enzymes useful in cell line detection, refer Table 35.1.

Isoenzymes:

The multiple forms of an enzyme catalysing the same reaction are referred to as isoenzymes or isozymes. Isoenzymes differ in many physical and chemical properties—structure, electrophoretic and immunological properties, K_m and V_max values.

The isoenzymes can be separated by analytical techniques such as electrophoresis and chromatography. Most frequently, electrophoresis by employing agarose, cellulose acetate, starch and polyacrylamide is used. The crude enzyme is applied at one point on the electrophoretic medium. As the isoenzymes migrate, they distribute in different bands, which can be detected by staining with suitable chromogenic substrates.

Isoenzymes are characteristic to the species or tissues. Isoenzymes of the following enzymes are commonly used for cell line detection:

a. Lactate dehydrogenase

b. Malate dehydrogenase

c. Glucose 6-phosphate dehydrogenase

d. Aspartate aminotransferase

e. Peptidase B.

Isoenzyme analysis is also useful for the detection of interspecies cross-contamination of cell lines. For instance, contamination of mouse cell line with hamster cell line can be identified by using peptidase B isoenzymes.

Antigenic markers:

Cell lines can be characterized by detection of antigenic markers through the use of antibodies. The antigenic markers may be located on the cell surface or secreted by the cells into the culture medium. Some of the antibodies in common use for the detection of different cell types are given in Table 35.2 (See p. 430).

Measurement of Growth Parameters of Cultured Cells:

Information on the growth state of a given culture is required to:

a. Design culture experiments.

b. Routine maintenance of culture.

c. Measurement of cell proliferation.

d. Know the time for subculture.

e. Determine the culture response to a particular stimulus or toxin.

Some of the commonly used terms in relation to the measurement of growth of cultured cells are explained.

Population doubling time (PDT):

The time interval for the cell population to double at the middle of the logarithmic (log) phase.

Cell cycle time or generation time:

The interval from one point in the cell division to the same point in the cycle, one division later. Thus cell cycle time is measured form one point in the cell cycle until the same point is reached again.

Confluence:

It denotes the culture stage where­in all the available substrate (growth area) is utilized, and the cells are in close contact with each other.

Contact inhibition:

Inhibition of cell motility and plasma membrane ruffling when the cells are in complete contact with other adjacent cells. This mostly occurs at confluence state, and results in the ceasation of the cell proliferation.

Cell density:

The number of cells per ml of the medium.

Saturation density:

The density of the cells (cells/ml², surface area) in the plateau phase.

Growth Cycle of Cultured Cells:

The growth cycle of cultured cells is conventionally represented by three phases — the lag phase, the log (exponential) phase and the plateau phase (Fig. 35.5). The properties of the cultured cells vary in the phases.

The lag phase:

The lag phase represents a period of adaptation during which the cell forms the cell surface and extracellular matrix (lost during trypsinization), attaches to the substrate and spreads out. There is an increased synthesis of certain enzymes (e.g. DNA polymerase) and structural proteins, preparing the cells for proliferation.

The production of specialized products disappears which may not reappear until the cell proliferation ceases. The lag phase represents preparative stage of the cells for proliferation following subculture and reseeding.

The log phase:

The log phase is characterized by an exponential growth of cells, following the lag phase.

The duration of log phase depends on the cells with reference to:

a. Seeding density.

b. Growth rate.

c. Density after proliferation.

During the log phase, the cultured cells are in the most uniform and reproducible state with high viability. This is an ideal time for sampling. The log phase terminates after confluence is reached with an addition of one or two population doublings.

The plateau phase:

As the cells reach confluence, the growth rate is much reduced, and the proliferation of cultured cells almost stops.

This stage represents plateau or stationary phase, and is characterized by:

a. Low motility of cells.

b. Reduced ruffling of plasma membrane.

c. Cells occupying minimum surface area.

d. Contact inhibition.

e. Saturation density.

f. Depletion of nutrients and growth factors.

g. Reduced synthesis of structural proteins.

h. Increased formation of specialized products.

The majority of normal cultured cells that form monolayers stop growing as they reach confluence. Some of the cells however, with replenishment of medium continue to grow (at a reduced rate) after confluence, forming multilayers of cells. The transformed cultured cells usually reach a higher cell density compared to the normal cells in the plateau phase (Fig. 35.6).

Plating Efficiency of Cultured Cells:

Plating efficiency, representing colony formation at low cell density, is a measure used for analyzing cell proliferation and survival.

When the cells, at low densities, are cultured in the form of single cell suspensions, they grow as discrete colonies. Plating efficiency is calculated as follows.

Plating efficiency = No. of colonies formed/No. of cells seeded × 100

The term cloning efficiency is used (instead of plating efficiency) when each colony grows from a single cell.

Seeding efficiency representing the survival of cells at higher densities is calculated as follows.

Seeding efficiency = No. of cells recovered/No. of cells seeded × 100

Cell Synchronization:

Synchronization literally means to make two or more things happen exactly simultaneously. For instance, two or more watches can be synchronized to show exactly the same time. The cells at different stages of the cell cycle in a culture can be synchronized so that the cells will be at the same phase. Cell synchrony is required to study the progression of cells through cell cycle. Several laboratory techniques have been developed to achieve cell synchronization.

They are broadly categorized into two groups:

1. Physical fractionation for cell separation.

2. Chemical blockade for cell separation.

Cell Separation by Physical Means:

Physical fractionation or cell separation techniques, based on the following characteristics are in use:

a. Cell density.

b. Cell size.

c. Affinity of antibodies on cell surface epitopes.

d. Light scatter or fluorescent emission by labeled cells.

The two commonly used techniques namely centrifugal elutriation and fluorescence-activated cell separation are briefly described hereunder.

Centrifugal elutriation:

The physical characteristics—cell size and sedimentation velocity are operative in the technique of centrifugal elutriation. Centrifugal elutriator (from Beckman) is an advanced device for increasing the sedimentation rate so that the yield and resolution of cells is better. The cell separation is carried out in a specially designed centrifuge and rotor (fig. 35.7). The cells in the medium are pumped into the separating chamber while the rotor is turning.

Due to centrifugal force, the cell will be pushed to the edges. As the medium is then pumped through the chamber in such a way that the centripetal flow is equal to the sedimentation rate of cells. Due to differences in the cells (size, density, cell surface configuration), the cells tend to sediment at different rates, and reach equilibrium at different positions in the chamber.

The entire operation in the elutriator can be viewed through the port, as the chamber is illuminated by stroboscopic light. At the equilibrium the flow rate can be increased and the cells can be pumped out, and separated in collecting vessels in different fractions. It is possible to carry out separation of cells in a complete medium, so that the cells can be directly cultured after separation.

Fluorescence-activated cell sorting:

Fluorescence-activated cell sorting is a technique for sorting out the cells based on the differences that can be detected by light scatter (e.g. cell size) or fluorescence emission (by pretreated DNA, RNA, proteins, antigens). The procedure involves passing of a single stream of cells through a laser beam so that the scattered light from the cells can be detected and recorded. When the cells are pretreated with a fluorescent stain (e.g. chromomycin A for DNA), the fluorescent emission excited by the laser can be detected.

There are two instruments in use based on the principle of fluorescent-activated cell sorting:

1. Flow cytometer:

This instrument is capable of sorting out cells (from a population) in different phases of the cell cycle based on the measurements of a combination of cell size and DNA fluorescence.

2. Fluorescent-activated cell sorter (FACS):

In this instrument, the emission signals from the cells are measured, and the cells sorted out into collection tubes.

Comparison between physical methods:

For separation of a large number of cells, centrifugal elutriator is preferred. On the other hand, fluorescent-activated cell sorting is mostly used to obtain high grade pure fractions of cells from small quantities of cells.

Cell Separation by Chemical Blockade:

The cells can be separated by blocking metabolic reactions. Two types of metabolic blockades are in use — inhibition of DNA synthesis and nutritional deprivation.

Inhibition of DNA synthesis:

During the S phase of cell cycle, DNA synthesis can be inhibited by using inhibitors such as thymidine, aminopterine, hydroxyurea and cytosine arabinoside. The effects of these inhibitors are variable. The cell cycle is predominantly blocked in S phase that results in viable cells.

Nutritional deprivation:

Elimination of serum or isoleucine from the culture medium for about 24 hours results in the accumulation of cells at G₁ phase. This effect of nutritional deprivation can be restored by their addition by which time the cell synchrony occurs.

Some Highlights of Cell Synchronization:

a. Cell separation by physical methods is more effective than chemical procedures.

b. Chemical blockade is often toxic to the cells.

c. Transformed cells cannot be synchronized by nutritional deprivation.

d. A high degree of cell synchrony (>80%) can be obtained in the first cycle, and in the second cycle it would be <60%. The cell distribution may occur randomly in the third cycle.

Cellular Senescence and Apoptosis:

As the cells grow in culture, they become old due to aging, and they cannot proliferate any more. The end of the proliferative life span of cells is referred to as senescence.

Cellular Senescence:

The growth of the cells is usually measured as population doublings (PDs). The PDs refer to the number of times the cell population doubles in number during the period of culture and is calculated by the following formula.

Log₁₀ (No. of cells harvested) – log₁₀ (No. of cells seeded)/ log10²

The phenomenon of senescence has been mostly studied with human fibroblast cultures. After 30-60 populations doublings, the culture is mainly composed of senescent fibroblasts. These senescent fibroblast are unable to divide in response to mitotic stimuli. It must be noted that the cells do not appear suddenly, but they gradually accumulate and increase in number during the life span of the culture.

The different parameters used for the measurement of cell growth in cultures are listed below:

a. Direct measure of cell number.

b. Determination of DNA/RNA content.

c. Estimation of protein/ATP concentration.

Measurement of Senescence:

The direct measurement of senescent cells is rather difficult.

Some of the indirect measures are:

a. Loss of metabolic activity

b. Lack of labeled precursor (³H-thymidine) incorporation into DNA.

c. Certain histochemical techniques.

Senescence-associated β-galactosidase activity assay

There occurs an overexpression of the lysosomal enzyme β-galactosidase at senescence. This enzyme elevation is also associated with an increase in the cell size as the cell enters a permanent non-dividing state. The number of senescent cells in a culture can be measured by senescence-associated β-galactosidase (SA-β) assay.

The assay consists of the following stages:

1. Wash the cells and fix them using a fixative (e.g. para formaldehyde), and wash again.

2. Add the staining solution (X-gal powder in dimethylformamide dissolved in buffer) to the fixed cells and incubate.

3. The senescent cells display a dense blue colour which can be counted.

Apoptosis:

The process of programmed cell death (PCD) is referred to as apoptosis. The cell death may be initiated by a specific stimulus or as a result of several signals received from the external environment. Apoptosis occurs as a result of inherent cellular mechanisms, which finally lead to self-destruction. The cell activates a series of molecular events that cause an orderly degradation of the cellular constituents with minimal impact on the neighbouring tissues.

Reasons for in situ apoptosis:

1. For proper development:

The formation of fingers and toes of the fetus requires the removal of the tissues between them. This is usually carried out by apoptosis.

2. Destruction of cells that pose threat to the integrity of the organism:

Programmed cell death is needed to destroy and remove the cells that may otherwise damage the organisms.

Some examples are listed:

a. Cells with damaged DNA during the course of embryonic development. If they are not destroyed, they may result in birth defects.

b. Cells of the immune system, after their appropriate immune function, undergo apoptosis. This is needed to prevent autoimmune diseases e.g. rheumatoid arthritis.

c. Cells infected with viruses are destroyed by apoptosis.

3. Cell destruction due to negative signals:

There are several negative signals within the cells that promote apoptosis. These include accumulation of free radicals, exposure to UV rays, X-rays and chemotherapeutic drugs.

Mechanism of apoptosis:

The programmed cell death may occur due to three different mechanisms:

1. Apoptosis due to internal signals.

2. Apoptosis triggered by external signals e.g. tumor necrosis factor-α (TNF-α), lymphotoxin.

3. Apoptosis triggered by reactive oxygen species.

Role of caspases in apoptosis:

A group of enzymes namely activated proteases play a crucial role in the programmed cell death. These proteases are actually cysteinyl aspartate specific proteinases or in short, commonly referred to as caspases. There are about ten different types of caspases acting on different substrates ultimately leading to cell death. For instance, capsase I cleaves interleukin 1β.

Inhibition of caspase activities:

Since the caspases are closely involved in apoptosis, it is possible to prevent cell death by inhibiting their activities. Certain specific peptides that can inhibit caspases, and thus apoptosis have been identified.

Measurement of Apoptosis:

A simple and easy way of detecting dead or dying cells is the direct microscopic observation. The dying cells are rounded with dense bodies which can be identified under phase contrast microscope. The cells that have undergone apoptosis contain fragmented chromatin which can be detected by conventional staining techniques. In recent years, more sensitive and reliable techniques have been developed for measuring apoptosis.

Some of them are briefly described:

Determination ADP/ATP ratio:

Both the growth and apoptosis of cells require ATP. But when there is growth arrest, an elevation of ADP occurs. Thus measuring ADP/ATP ratio will throw light on the dead cells. In fact, some assay systems for measuring ADP/ATP ratios are commercially available.

TUNEL assay:

A significant biochemical event for the apoptosis is the activation of endogenous nuclease activity. This enzyme cleaves DNA into fragments with free 3-hydroxyl groups. The newly formed small DNA fragments can be extended by employing the enzyme DNA polymerase. If labeled nucleotides are used for DNA fragment extension, they can be detected.

TUNEL is an abbreviation for TdT-mediated dUTP nick end-labeling assay. TUNEL is very fast and effective for the determination of DNA fragments formed by endogenous nuclease activity. The apoptotic nuclei can be identified by a fluorescent technique using fluorescein isothiocyanate (FITC) and 4, 6-diaminophenylindole.

DNA laddering test:

During the course of apoptosis, the genomic DNA is cleaved to mono — and oligonucleosomal DNA fragments. These fragments can be separated by agarose electrophoresis, and detected. The nucleosomal fragments of apoptotic cells give a characteristic ladder pattern on electrophoresis.

Limitations of the test:

DNA laddering test is not very specific since several cells that have undergone apoptosis may not show DNA laddering. Further, some cells not subjected to apoptosis may also show DNA ladders, for these reasons, DNA laddering test is coupled with some other test for measurement of apoptosis.