ANIMAL CELL SCIENCE & TECHNOLOGY UNIT - III

V. MAGENDIRA MANI

ASSISTANT PROFESSOR

PG & RESEARCH DEPARTMENT OF BIOCHEMISTRY

ISLAMIAH COLLEGE (AUTONOMOUS)

VANIYAMBADI

Mail: magendiramani@rediffmail.com

Download at : magendiramanivinayagam@academia.edu

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CULTURE MEDIA – SERUM & SERUM FREE MEDIUM

Introduction

Animal cell culture can be described as in vitro maintenance and propagation of animal cells

using a suitable nutrient media. Culturing is a process of growing animal cells artificially. The

most important and essential step in animal cell culture is selecting appropriate growth medium

for in-vitro cultivation. The selection of the medium depends on the type of cells to be cultured

and also the purpose of the culture. Purpose of animal cell culture can be growth, differentiation,

or even production of desired products like pharmaceutical compounds. Animal cells are

cultured using a completely natural media, or an artificial media along with some of the natural

products.

Natural Media:

In the early years of this in vitro cultivation of animal cell culture technique natural media are

obtained from biological sources were used. For example

1. Body fluid such as plasma, serum, lymph, amniotic fluid and much more are used. These

fluids used as animal cell culture media after testing for toxicity and sterility.

2. Tissue extract such as extract of liver, spleen, bone marrow and leucocyes also used as animal

cell culture media. But most commonly used tissue extract is from chick embryo.

3. Plasma clots are also used as media for animal cell culture and now they are commercially

produced as culture media.

4. Bovine embryo extract are also prepared using bovine embryos of up to 10 days age, and are

used as animal cell culture media.

Artificial Media:

1. The artificial media contains partly or fully defined components.

2. The basic criteria for choosing an artificial media for animal cell culture are

The culture media should provide all the required nutrients to the cell.

Media should maintain the physiological pH at around 7 with the help of buffering

system.

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The animal cell culture media should be sterile, and isotonic to the culturing cells. The

basis for the animal cell culture media is the balanced salt solution, which are used to

create a physiological pH and osmolarity required to maintain the animal cells in vitro or

in laboratory conditions.

For promoting cell growth and proliferation, many types of animal cell culture media are

designed by adding or varying different constituents for example serum containing media

and serum-free media.

SERUM MEDIA

Serum is commonly used as a supplement to cell culture

media. It provides a broad spectrum of macromolecules,

carrier proteins for lipoid substances and trace elements,

attachment and spreading factors, low molecular weight nutrients, and hormones and growth

factors. The most widely used animal serum supplement is fetal bovine serum (FBS). Since

serum in general is an ill-defined component in cell culture media, a number of chemically

defined serum-free media formulations have been developed in the last two decades. Besides

modern cell biological advances in cell and tissue culture and efforts towards a standardization of

cell culture protocols in Good Cell Culture Practice, in addition, considerable ethical concerns

were raised recently about the harvest and collection of fetal bovine serum. Thus, in order to

decrease the annual need for bovine fetuses in terms of the 3Rs through any reduction in the use

or partial replacement of serum, as well as in terms of an improvement of cell and tissue culture

methodology, serum-free cell culture represents a modern, valuable and scientifically well

accepted alternative to the use of FBS in cell and tissue culture.

1. Serum media is an example for natural media. Natural media are very useful and convenient

for a wide range of animal cell culture. But they also have got some disadvantages such as poor

reproductability due to lack of knowledge of exact composition of these natural media.

2. Major reasons for using synthetic media are for immediate survival of cells, for prolonged

survival, for indefinite growth and also for specialized functions.

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a. Balanced diet solution with specific osmotic pressure and pH are used for the immediate

survival

b. Serum or balanced salt solution along with amino acids, oxygen, vitamins and serum proteins

are used for long survival.

c. Minimum Essential Medium also known as Eagles media are used for mammalian cell culture

d. Role of serum in animal cell culture is very complex and also it contains mixture of many

biomolecules such as growth factors and also growth inhibitory factors.

Function of serum:

1. Serum is bound to proteins and basic nutrient in solution.

2. Serum also contains hormones and growth factors, which play a major role in stimulating cell

growth and function

3. Serum also helps in attachment of the cells

4. Serum also acts as spreading factor.

5. Serum also function as binding protein such as albumin carrying factor

6. Serum also helps in carrying hormones, vitamins, minerals, lipids and much more biological

substances.

7. Serum also minimizes mechanical damage and also damage caused by viscosity

8. Serum also acts as natural buffering agent and helps in maintaining the pH of the culture

media.

Disadvantage of Serum in Media:

1. Serum may contain inadequate amount of cell specific growth factors and may need to

supplement the media or it may also contain in abundance of cytotoxic compounds.

2. Serum media has got high risk of contamination with virus, fungi and mycoplasma.

3. There is no uniformity in the composition of serum, as still we do not know the exact

composition of the serum

4. Special tests are done to maintain the quality of the each batch of serum before it is used in

cell culture media.

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5. Serum may also contain some of the growth inhibiting factors; these in turn will inhibit the

cultured cell growth and proliferation.

6. Serum availability is restricted as they are extracted from cattle

7. Presence of serum in the culture media may interfere with the purification and isolation of cell

culture products, such as pharmaceutical compounds. Due to this additional steps are added for

the isolation of cell culture products.

Serum Free Media:

As using serum in animal cell culture media has got some disadvantages, to overcome this serum

free media are designed and developed.

Advantages of Serum Free Media:

1. Main and important advantage of serum free media is scientists or researchers can control the

growth of cultured cells as required by changing the composition of the media.

2. Serum free media can be designed using specific factors, which will help in differentiation of

cultured cells with specific desired functions.

Disadvantages of Serum Free Media:

1. Cell proliferation is very slow in serum free media.

2. Cultured cells may need more than one type of media to obtain desired cell culture products.

3. Purity of reagents used in the serum free media

4. Availability

5. The physiological variability

6. The shelf life and consistency,

7. The quality control,

8. The specificity,

9. Downstream processing,

10. The possibility of contamination,

11. The growth inhibitors,

12. The standardization and the costs.

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MEASUREMENT OF VIABILITY

The measurement of cell viability plays a fundamental role in all forms of cell culture.

Sometimes it is the main purpose of the experiment, such as in toxicity assays. Cell viability is a

determination of living or dead cells, based on a total cell sample. Viability measurements may

be used to evaluate the death or life of cancerous cells and the rejection of implanted organs. In

other applications, these tests might calculate the effectiveness of a pesticide or insecticide, or

evaluate environmental damage due to toxins.

Cell viability may also be examined in a population or certain risk group to further understand

the growth of cells. This is particularly the case with cancerous cells in human and animal

populations. A viability analysis can give researchers information about the ways in which

cancers grow, act, and react to treatment. These statistics can better inform treatment or help

doctors give patients more accurate statistics on outcomes of particular types of cancers.

Another example of viability testing in medicine is the analysis of cells in populations where

cells are routinely destroyed. For example, autoimmune conditions can attack normal and healthy

cells, causing a cell viability test to yield very few living cells. Evaluation of cell viability in

people with autoimmune disease may help determine progress of a disease or change treatment

goals and options.

Measuring cell viability is probably the most common procedure, besides assessing cell number

in the cell biology laboratory. A cell viability assay or test should determine whether the cells are

alive or dead. If too many dead cells are present, the cell suspension should be "cleaned up"

using a method that removes the dead cells.

There are two types of viability assay. These are:

Dye exclusion viability assays

Metabolic viability assays

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Dye Exclusion Viability Assays

As its name implies, a dye exclusion assay uses a dye or stain that can enter the cell and usually

intercalates with the DNA in the nucleus. The mere entry of the dye into the cell assumes that the

cell membrane has lost its integrity and that the cell is dead. In other words, live cells exclude the

dye, while dead cells allow the dye to enter. Dye or stains that are used in dye exclusion viability

assays include, but are not limited to:

Typan blue, often used to detect both viability and cell number using a hemocytometer or

similar in an automated instrument.

Propidium iodide (PI), which can be detected using manual and automated techniques,

including a flow cytometer. This stain is also used for cell cycle analysis and other assays.

7-Aminoactinomycin D (7-AAD), usually detected by flow cytometry and often used in

cellular therapeutic applications.

Acridine Orange, often used in hemocytometer procedures, but can also detected by flow

cytometry. This is a toxic compound.

Metabolic Viability Assays

Metabolic viability assays do not rely on the assumption

that the cell membrane must lose its integrity in order to determine whether a cell is alive or

dead. Metabolic viability assays usually rely on the cell's ability to perform a specific

biochemical reaction that can be measured usually by absorbance, fluorescence or luminescence

methods. These might include:

Reduction of a tetrazolium compound, e.g. MTT/XTT and measured by absorbance.

Conversion of non-fluorescent Calcein AM to a fluorescence-emitter after acetoxymethyl

ester hydrolysis by intracellular esterases.

Function of ion pumps and channels detected by fluorescence.

Change in pH indicators detected by fluorescence.

Cellular and mitochondrial metabolism detected by measuring ATP using luminescence

methodology.

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MEASUREMENT OF CYTOTOXICITY

Cytotoxicity is the quality of being toxic to cells. Examples of toxic agents are an immune cell

or some types of venom, e.g. from the puff adder (Bitis arietans) or brown recluse spider

(Loxosceles reclusa). Treating cells with the cytotoxic compound can result in a variety of cell

fates. The cells may undergo necrosis, in which they lose membrane integrity and die rapidly as a

result of cell lysis. The cells can stop actively growing and dividing (a decrease in cell viability),

or the cells can activate a genetic program of controlled cell death (apoptosis).

Cells undergoing necrosis typically exhibit rapid swelling, lose membrane integrity, shut down

metabolism and release their contents into the environment. Cells that undergo rapid necrosis in

vitro do not have sufficient time or energy to activate apoptotic machinery and will not express

apoptotic markers

Measuring cytotoxicity

Cytotoxicity assays are widely used by the pharmaceutical industry to screen for cytotoxicity in

compound libraries. Researchers can either look for cytotoxic compounds, if they are interested

in developing a therapeutic that targets rapidly dividing cancer cells, for instance; or they can

screen "hits" from initial high-throughput drug screens for unwanted cytotoxic effects before

investing in their development as a pharmaceutical.

Assessing cell membrane integrity is one of the most common ways to measure cell viability and

cytotoxic effects. Compounds that have cytotoxic effects often compromise cell membrane

integrity. Vital dyes, such as trypan blue or propidium iodide are normally excluded from the

inside of healthy cells; however, if the cell membrane has been compromised, they freely cross

the membrane and stain intracellular components. Alternatively, membrane integrity can be

assessed by monitoring the passage of substances that are normally sequestered inside cells to the

outside. One molecule, lactate dehydrogenase (LDH), is commonly measured using LDH

assay. Protease biomarkers have been identified that allow researchers to measure relative

numbers of live and dead cells within the same cell population. The live-cell protease is only

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active in cells that have a healthy cell membrane, and loses activity once the cell is compromised

and the protease is exposed to the external environment. The dead-cell protease cannot cross the

cell membrane, and can only be measured in culture media after cells have lost their membrane

integrity.

Cytotoxicity can also be monitored using the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-

tetrazolium bromide (MTT) or MTS assay. This assay measures the reducing potential of the

cell using a colorimetric reaction. Viable cells will reduce the MTS reagent to a colored

formazan product. A similar redox-based assay has also been developed using the fluorescent

dye, resazurin. In addition to using dyes to indicate the redox potential of cells in order to

monitor their viability, researchers have developed assays that use ATP content as a marker of

viability. Such ATP-based assays include bioluminescent assays in which ATP is the limiting

reagent for the luciferase reaction.

Cytotoxicity can also be measured by the sulforhodamine B (SRB) assay, WST assay and

clonogenic assay.

A label-free approach to follow the cytotoxic response of adherent animal cells in real-time is

based on electric impedance measurements when the cells are grown on gold-film electrodes.

This technology is referred to as electric cell-substrate impedance sensing (ECIS). Label-free

real-time techniques provide the kinetics of the cytotoxic response rather than just a snapshot like

many colorimetric endpoint assays.

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MEASUREMENT PARAMETERS FOR GROWTH IN ANIMAL CELL CULTURE

Cell culture is the process by which cells are grown under controlled conditions, generally

outside of their natural environment. In practice, the term "cell culture" now refers to the

culturing of cells derived from multi-cellular eukaryotes, especially animal cells. The laboratory

technique of maintaining live cell lines (a population of cells derived from a single cell and

containing the same genetic makeup) separated from their original tissue source became healthier

in the middle 20th century.

There are four main types of cell proliferation assays, and they differ according to what is

actually measured: DNA synthesis, metabolic activity, antigens associated with cell proliferation

and ATP concentration.

DNA synthesis cell proliferation assays

One of the most reliable and accurate assay types is measurement of DNA synthesized in the

presence of a label. Traditional cell proliferation assays involve incubating cells for a few hours

to overnight with 3H-thymidine. Proliferating cells incorporate the radioactive label into their

nascent DNA, which can be washed, adhered to filters and then measured using a scintillation

counter. Besides the length of the experiment, the obvious downsides to this method are the

hazards and hassle of using and disposing of radioactive materials. If that’s not your thing, you

can perform a similar protocol using 5-bromo-2'-deoxyuridine (BrdU), which also becomes

incorporated into newly made DNA. This adds a few steps because you must incubate with a

BrdU-specific monoclonal antibody, sometimes followed by a secondary antibody as a reporter,

before you can measure a colorimetric, chemiluminescent or fluorescent reporter signal. On the

other hand, you don’t need to work with radioactivity. This is suitable for

immunohistochemistry, immunocytochemistry, in-cell ELISAs, flow cytometry analysis and

high-throughput screening.

Metabolic cell proliferation assays

Another measure of cell proliferation is the metabolic activity of a population of cells.

Tetrazolium salts or Alamar Blue are compounds that become reduced in the environment of

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metabolically active cells, forming a formazan dye that subsequently changes the color of the

media. This is caused by increased activity of the enzyme lactate dehydrogenase during

proliferation. The absorption of the media-containing dye solution can be read using a

spectrophotometer or microplate reader in low- or high-throughput configurations. Four types of

tetrazolium salts are most common: MTT,XTT, MTS and WST1.

Detecting proliferation markers

A third way to measure cell proliferation is to detect an antigen present in proliferating cells, but

not nonproliferating cells, using a monoclonal antibody to the antigen. For example, in human

cells, the antibody Ki-67 recognizes the protein of the same name, expressed during the S, G2

and M phases of the cell cycle but not during the G0 and G1 (nonproliferative) phases. Using this

assay with tissue slices precludes high-throughput methods. On the other hand, this method

enjoys an advantage for cancer researchers because it’s suitable for assaying tumor cell

proliferation in vivo. Other common markers for cell proliferation and/or cell cycle regulation,

targeted by antibodies, include PCNA (proliferating cell nuclear antigen), topoisomerase IIB and

phosphohistone H3.

Measuring [ATP]

A final type of cell proliferation assay takes advantage of the tight regulation of intracellular

ATP within cells. Dying or dead cells contain little to no ATP, so there is a tight linear

relationship between cell number and the concentration of ATP measured in a cell lysate or

extract. The bioluminescence-based detection of ATP, using the enzyme luciferase and its

substrate luciferin, provides a very sensitive readout. In the presence of ATP, luciferase produces

light (proportional to the ATP concentration) that can be detected by a luminometer or any

microplate reader capable of reading luminescent signals. This approach is also well suited to

high-throughput cell proliferation assays and screening.