Professional Documents
Culture Documents
• Aseptic Technique: Maintain strict aseptic (sterile) conditions in the laboratory to prevent
contamination of the cell cultures.
• This includes working in a laminar flow hood or biosafety cabinet, sterilizing equipment and
media, and wearing appropriate protective gear, such as gloves and lab coats.
• Cell Culture Equipment:
• Tissue culture incubator: Maintain a controlled environment with a temperature of 37°C (or
other suitable temperature), 5% CO2 (for most mammalian cells), and high humidity.
• CO2 supply: To regulate the CO2 concentration in the incubator.
• Microscope: For monitoring cell growth and assessing cell morphology.
• Centrifuge: Used for cell harvesting and other procedures.
• Pipettes and pipette tips: To handle cells, media, and reagents with precision.
• Autoclave: For sterilizing glassware, media, and equipment.
Animal tissue culture
1.Cell Culture Dishes and Flasks: These come in various sizes and are
used for growing and passaging cells. They may be made of plastic or
glass and should be sterile.
2.Culture Media:
1. Growth medium: Nutrient-rich liquid or solid (agar) medium containing
essential nutrients, including salts, amino acids, vitamins, and serum (usually
fetal bovine serum) for cell growth.
2. Serum-free media: Some experiments require specialized, serum-free media.
3. Antibiotics and antimycotics: Added to prevent contamination.
4. Phenol red (for some media): Used as a pH indicator.
ANIMAL TISSUE CULTURE
1.Sterile Technique and Reagents: Ensure that all solutions, reagents, and equipment that
come into contact with the cells are sterile.
2.Cell Lines: Use established cell lines or primary cells derived from animals. These cells
should be characterized and free from contamination. Cells can be obtained from
reputable cell banks.
3.Growth Factors and Supplements: Depending on the specific cell type, growth factors and
supplements may be required to support cell growth and proliferation.
4.Passaging Tools: Tools for subculturing or passaging cells, such as trypsin-EDTA solution,
cell scrapers, and cell dissociation reagents.
5.Cryopreservation Equipment: For long-term storage of cell stocks, including freezing
medium and liquid nitrogen storage tanks.
6.Monitoring and Record-keeping: Maintain detailed records of cell culture conditions,
passages, and experimental data. Regularly inspect cells for signs of contamination or
deterioration.
ANIMAL TISSUE CULTURE
1.Quality Control: Regularly test cells for mycoplasma contamination, perform karyotype
analysis, and check cell identity.
2.Ethical Considerations: Ensure that you have the necessary ethical approvals for
working with animal tissues and cells, and follow all relevant ethical guidelines and
regulations.
Animal cell culture media
• Animal cell culture media are specifically designed to support the growth and maintenance
of animal cells, which can include a wide range of cell types, from mammalian cells to avian
or amphibian cells.
• The composition and preparation of animal cell culture media can vary depending on the
specific cell type, but I can provide you with a general guideline for the composition and
preparation of a basic animal cell culture medium, suitable for mammalian cell lines.
• Adjustments may be needed based on your specific cell type and research goals.
• Basal Medium: The basal medium provides essential nutrients and a buffer system to maintain pH.
Common basal media for mammalian cell culture include Dulbecco's Modified Eagle Medium (DMEM)
and RPMI-1640 for adherent cells, and Minimum Essential Medium (MEM) for suspension cells.
These basal media can be modified by adding other components to meet the specific needs of the
cells.
Animal cell culture media
1.Serum: Fetal bovine serum (FBS) is often included as a source of growth factors,
hormones, and proteins that support cell growth. The concentration of FBS
typically ranges from 5% to 10%, although this may vary depending on the cell
type and experimental requirements. Serum-free media are also available for
some applications.
2.Amino Acids: A mixture of non-essential and essential amino acids is added to
support protein synthesis. Amino acids can be obtained as a 100x concentrated
solution and added to the medium as needed.
3.Vitamins: Vitamins, such as ascorbic acid and various B vitamins, are included
to support various metabolic processes in the cells.
4.Minerals and Salts: Various inorganic salts, such as sodium chloride, potassium
chloride, calcium chloride, and magnesium sulfate, are added to provide essential
ions for cell function and osmotic balance.
ANIMAL CELL CULTURE MEDIA
1.Glucose: Dextrose or glucose is used as the primary energy source for the cells and is
typically added at a concentration of 1,000 mg/L (approximately 5.5 mM).
2.Buffer System: A bicarbonate buffer system is used to maintain the pH of the medium
around 7.4. This is essential for cell viability and function. CO2 is typically supplied in the
incubator to help maintain pH.
3.pH Indicator: Phenol red is commonly used as a pH indicator. It turns the medium pink
when it is alkaline and yellow when it is acidic.
ANIMAL CELL CULTURE MEDIA
1.Start with a sterile environment. Use a laminar flow hood or biosafety cabinet and practice
sterile technique.
2.Weigh and measure all the components accurately, and filter-sterilize them using a 0.22-
micron filter to remove any contaminants.
3.Combine the sterile basal medium, serum, amino acids, vitamins, minerals, and other
components in a sterile container, following the specific recipe for your medium.
4.Adjust the pH of the medium to around 7.4 using 1 M HCl or 1 M NaOH as necessary.
5.Sterile-filter the medium through a 0.22-micron filter to ensure sterility.
6.Dispense the medium into sterile containers, such as bottles or flasks, and store them at the
appropriate temperature (usually 4°C for short-term storage or -20°C for long-term storage for
some components).
7.Prior to using the medium, warm it to the desired incubation temperature (usually 37°C) and
equilibrate it in a CO2 incubator to reach the desired pH and gas conditions.
Balanced Salt Solution (BSS)
• A Balanced Salt Solution (BSS) is a type of isotonic, buffered salt solution that is
commonly used in various biological and medical applications, including cell culture,
tissue preservation, and ophthalmology.
• BSS solutions are designed to closely mimic the physiological salt concentrations and pH
of body fluids, making them suitable for maintaining cells and tissues under conditions
that resemble their natural environment.
• There are different formulations of BSS depending on the intended application. Here are a
few common types:
Balanced Salt Solution (BSS)
1.Phosphate-Buffered Saline (PBS): This is one of the most widely used types of BSS. It
contains a balanced mixture of sodium chloride (NaCl), potassium chloride (KCl), sodium
phosphate, and potassium phosphate, along with a pH buffer to maintain a near-neutral pH.
PBS is often used in cell culture, immunohistochemistry, and as a wash buffer in various
laboratory procedures.
2.Hank's Balanced Salt Solution (HBSS): HBSS is a buffered salt solution containing
various salts, including calcium chloride (CaCl2), magnesium chloride (MgCl2), and sodium
bicarbonate (NaHCO3), in addition to sodium and potassium salts. It is commonly used for
maintaining cells and tissues in vitro, such as during cell washing, trypsinization, and tissue
dissociation.
Balanced Salt Solution (BSS)
1.Ringer's Solution: Ringer's solution is used in medical applications and consists of sodium
chloride, potassium chloride, and calcium chloride in water. It is used for intravenous
administration to replace fluids and electrolytes in patients.
2.Lactated Ringer's Solution: Similar to Ringer's solution, lactated Ringer's contains sodium
chloride, potassium chloride, and calcium chloride but also includes sodium lactate (sodium
salt of lactic acid). It is used for fluid resuscitation and to correct electrolyte imbalances in
medical settings.
1.Coconut Water: Coconut water has been explored as a natural and low-cost alternative
to commercial cell culture media. It contains various nutrients, including sugars, vitamins,
and minerals, which can support the growth of certain cell types.
2.Plasma: For specific applications, plasma, the liquid component of blood, has been used
in cell culture media to maintain cell viability and function. This is especially relevant in
studies that require the presence of blood-related factors.
NATURAL MEDDIA USED IN ANIMAL CELL CULTURE
1.Tissue Extracts: Some researchers use tissue extracts or homogenates from animal
growth factors and extracellular matrix components. For example, brain-derived extracts
2.Amniotic Fluid: Amniotic fluid contains nutrients, proteins, and growth factors and has
been used in specialized cell culture applications, particularly in the study of amniotic
1.Urine-Derived Media: Urine contains various components that can be used in cell culture
media, especially for certain types of stem cells. Urine-derived media may contain specific
2.Plant Extracts: Some natural plant extracts, such as phytochemicals or herbal extracts,
have been explored for their potential to influence cell behavior or to be used as
supplements in cell culture media. These extracts may contain bioactive compounds that
pH Regulation: CO2 is used to regulate the pH of the culture medium. When CO2 is dissolved in
water, it forms carbonic acid (H2CO3), which can act as a buffer to maintain the pH of the
medium within the optimal range (usually around 7.2 to 7.4 for most cell lines).
This is essential for cell viability and proper cell function. Cells are sensitive to changes in pH, so
maintaining a stable pH is crucial for successful tissue culture.
Carbon Source: CO2 serves as a source of carbon for cells. In tissue culture, cells require a
source of carbon for their metabolic processes, and CO2 provides this carbon in the form of
bicarbonate ions (HCO3-).
Cells can use bicarbonate ions as a carbon source to produce energy and build essential
biomolecules.
Role of CO2 in animal tissue culture
1.Osmotic Balance: CO2 helps in maintaining the osmotic balance of the culture medium. It
contributes to the overall solute concentration and osmolarity of the medium, which is
important for cell volume regulation and preventing cell damage due to osmotic stress.
2.Gas Exchange: CO2 is involved in gas exchange with oxygen (O2) in the culture
environment. Cells consume oxygen during respiration and produce CO2 as a metabolic
byproduct.
The balance between O2 and CO2 levels is essential for cell growth and viability. CO2
incubators are often used in tissue culture to control and maintain the appropriate gas
concentrations.
Role of CO2 in animal tissue culture
• Buffering Capacity: CO2 helps maintain the culture medium's buffering capacity, which is
important for stabilizing the pH in the presence of acidic or basic compounds that may be
produced during cell metabolism.
Constituents of serum &its role
• Serum is the liquid portion of blood that remains after blood coagulation, and it contains a
variety of constituents that serve important roles in the body. Some of the key constituents
of serum and their roles include:
1.Water: Water makes up the largest proportion of serum and is essential for maintaining
the overall volume and viscosity of blood.
3.These electrolytes are crucial for maintaining the body's acid-base balance, osmotic
pressure, and proper functioning of nerves and muscles.
Constituents of serum &its role
1.Proteins:
1. Albumin: Albumin is the most abundant protein in serum and plays a key role in
maintaining colloidal osmotic pressure, which helps regulate the distribution of fluid
between blood and tissues.
2. Globulins: These include various proteins, such as immunoglobulins (antibodies) that
play a vital role in the immune system and clotting factors (e.g., fibrinogen) involved
in blood coagulation.
3. Fibrinogen: This soluble protein is essential for blood clot formation when an injury
occurs. It converts into insoluble fibrin during the clotting process.
2.Nutrients: Serum contains a range of nutrients, including glucose, amino acids, fatty
acids, vitamins, and minerals. These are important for energy production, tissue repair,
and various metabolic processes.
3.Waste Products: Urea, creatinine, and other metabolic waste products are present in
serum. These waste products are filtered out of the blood by the kidneys and excreted in
urine.
Constituents of serum &its role
2. Hormones: Serum contains hormones such as insulin, transferrin, and various steroid hormones. These
hormones can influence cell metabolism and function, helping cells to maintain their physiological
processes.
3. Nutrients: Serum provides essential nutrients for cells, including amino acids, lipids, vitamins, minerals,
and glucose. These nutrients are required for cellular energy production, biosynthesis of cellular
components, and overall cell metabolism.
Role of serum in animal cell culture
3.Anti-Apoptotic Factors: Serum can contain factors that protect cells from apoptosis
(programmed cell death), promoting cell survival and longevity in culture.
Role of serum in animal cell culture
1.Antioxidants: Serum contains antioxidants that help protect cells from oxidative stress, a
common challenge in cell culture. These antioxidants can reduce the damage caused by
reactive oxygen species (ROS) and help maintain cell viability.
2.Buffering Capacity: Serum can help stabilize the pH of the culture medium by acting as a
buffer, which is important for maintaining an optimal pH environment for cells.
are commonly used in bioprocessing applications, such as the production of recombinant proteins
and monoclonal antibodies, as they offer more defined and consistent growth conditions for cell
• Vaccine Production: Serum-free media can be used in vaccine production processes to grow
cells or viruses used in vaccine manufacturing. This helps ensure the purity and safety of the
vaccine product.
• Cell Therapy and Regenerative Medicine: Serum-free media are crucial for the ex vivo expansion
and maintenance of cells used in cell-based therapies and regenerative medicine, including stem
1.Clinical and Diagnostic Research: a. In Vitro Diagnostics: Serum-free media are used in
various in vitro diagnostic tests and assays that require the cultivation of specific cell
types under defined conditions. b. Cancer Research: Researchers studying cancer cells
may use serum-free media to better control and standardize the conditions in which they
grow and test cancer cells.
2.Transgenic and Genetically Modified Cells: Serum-free media can be ideal for the
cultivation of genetically modified or transgenic cell lines, as they allow for consistent
growth and characterization of these cells.
Role of serum free animal cell culture &applications
1.Reduced Contamination Risk: Serum-free media can reduce the risk of contamination
with adventitious agents, such as viruses and prions, that may be present in animal-
derived sera.
3.Reduction of Ethical and Economic Concerns: Serum-free media can be more ethical, as
they eliminate the need for the use of animal-derived sera. They can also reduce the cost
associated with purchasing and testing large quantities of serum.
Animal cell culture
Tissue culture involves the in vitro maintenance and propagation of cells in optimal conditions.
Culturing animal cells, tissue or organs in a controlled artificial environment is called animal
tissue culture.
Animal cell culture involves isolation of cells from a tissue before establishing a culture in a
suitable artificial environment. Initial isolation of the cells from the tissues can be achieved by
disaggregation using enzymatic or mechanical methods.
Why do we need animal cell culture?
Animal cell culture offers suitable model systems for investigating the following factors:
• It also permits reliable and reproducible results, and is thus considered as a significant
model system in cellular and molecular biology.
Terms frequently used in animal tissue culture
Cell culture : The process of removing cells from an animal and their subsequent growth in
an artificially controlled environment
Primary cell culture :This is the first culture (a freshly isolated cell culture) or a culture
which is directly obtained from animal or human tissue by enzymatic or mechanical
methods.
The primary objective of this culture is to maintain the growth of cells on an appropriate
substrate, available in the form of glass or plastic containers, under controlled environmental
conditions.
These cells are typically slow growing, heterogeneous and carry all the features of the tissue of
their origin.
Since they are directly obtained from original tissue they have the same karyotype (number and
appearance of chromosomes in the nucleus of a eukaryotic cell) as the original tissue.
Once subculture, primary cell cultures can gives rise to cell lines, which may either die
after several subcultures (such cell lines are known as finite cell lines) or may continue to
grow indefinitely (these are called continuous cell lines).
An increase in cell numbers in a primary culture results in exhaustion of the substrate and nutrients,
which can influence cellular activity and lead to the accumulation of high levels of toxic
metabolites in the culture.
Summary of primary cell culture
It is the first culture that is established from the in vivo environment
Cells derived from a primary cell line mimics the exact in vivo conditions, however, it is
challenging to maintain these cells.
Primary cell cultures require a nutrient medium containing a high amount of different amino
acids, micronutrients and, occasionally, some types of hormones or growth factors
The biological response received from a primary culture will be closer to that in an in vivo
environment
Primary cell cultures can be efficiently utilized up to a few passages, about two to four,
afterwards their risk of contamination is higher.
Advantages of primary cell culture.
• Mechanical disaggregation.
• Enzymatic disaggregation.
➢ This approach involves efficient disaggregation of cells with high yield by using enzymes
such as trypsin, collagenase and others
➢ Enzyme based disaggregation allows hydrolysis of fibrous connective tissue and the
extracellular matrix.
➢ Currently, the enzymatic method is extensively used as it offers high recovery of cells
without affecting the viability of cells.
APPLICATION OF ANIMAL CELL CULTURE:
➢Used for studying the effects of drugs and toxic compounds on the cells and
mutagenesis and carcinogenesis.
The line was derived from cervical cancer cells taken on February 8, 1951
from Henrietta Lacks. a patient who died of cancer on October 4, 1951.
The cell line was found to be remarkably durable and prolific, which gives rise to its extensive use
in scientific research.
The cells were taken from Henrietta Lacks's cancerous cervical tumor.
Previously, cells cultured from other human cells would only survive for a few days. Scientists
would spend more time trying to keep the cells alive than performing actual research on them
But, Cells from Henrietta Lacks' tumor behaved differently. These cells were labled
'HeLa’, the first two letters of the patient's first and last name; this became the name of the
cell line
HeLa cell lines
These were the first human cells grown in a lab that were naturally "immortal", meaning that
they do not die after a set number of cell divisions.
HeLa cells, like other cell lines, are termed "immortal" in that they can divide an unlimited
number of times in a laboratory cell culture plate as long as fundamental cell survival
conditions are met (i.e., being maintained and sustained in a suitable environment)
The stable growth of HeLa enabled a researcher at the University of Minnesota hospital to
successfully grow polio virus, enabling the development of a vaccine, and by 1952, Jonas
Salk developed a vaccine for polio using these cells.
Growth kinetics of cells in culture/Phases of Cell Growth
It is important to know and record the growth characteristics of the cell line in use before
starting any experiments
An alteration in cellular growth can indicate a significant problem within the cell line and if
undetected, can have detrimental effects on experimental results
As they are relatively large and slow growing, they are easily damaged when cultivated in an
artificial environment
Animal cell culture using bioreactors plays an important role in the production of biological
products .
The biological products produced by animal cell culture include monoclonal antibodies, protein
pharmaceuticals, and viral vaccines.
Bioreactors have also been used to produce quantities of cells in short supply naturally, such as
stem cells.
Bioreactors/ Fermenter for large scale culture of cells
Animal cells, unlike microbial, plant, or other eukaryotic cells, have no tough cell wall, and
their membranes are fragile.
As they are relatively large and slow growing, they are easily damaged when cultivated in an
artificial environment
Animal cell culture using bioreactors plays an important role in the production of biological
products .
The biological products produced by animal cell culture include monoclonal antibodies, protein
pharmaceuticals, and viral vaccines.
Bioreactors have also been used to produce quantities of cells in short supply naturally, such as
stem cells.
Design of Fermenter
✓Basic elements
✓Controlling elements
• Top-plate: It is the cover that is generally made of stainless steel.
Inoculation pipe: It helps to port the inoculum inside the fermentor.
Drive motor: It drives the impeller shaft.
Impeller shaft: Holds the agitator centrally.
Impeller: Acts as an agitating device for mixing up the
▪ A fermentor should be capable of being operated aseptically or should provide sterile conditions.
▪ The bioreactor provides adequate aeration and agitation for uniform mixing of the contents in the vessel.
▪ A fermentor must be equipped with controlling probes that can maintain the temperature, pH, oxygen level etc.
▪ It minimizes the labour input for the operation, harvesting, cleaning and maintenance.
BIOREACTORS USED FOR CELL CULTURE
• In a continuous flow culture, it is possible to keep the cells at a desired and set
concentration, and maintain. This is carried out by a bio-stat or chemo-stat
• Continuous flow culture consists of growing the cells at the mid-log phase, removal of
a measured volume of cells, and replacement by an equal volume of medium.
• The equipment, specially designed for this purpose has the facility for removal of the
cells and addition of medium.
• The flow rate of the medium addition can be determined from the growth rate of the
culture.
• The medium flow can be regulated by a peristaltic pump.
• By this technique, it is possible to keep the culture conditions constant rather than to
produce large number of cells.
• The continuous flow cultures are useful for monitoring metabolic changes in relation to
cell density.
• However, these cultures are more susceptible to contamination.
CONTINUOUS FLOW CULTURE:
Packed bed bioreactor It consists of a column packed with immobilized cells through which
the substrate solution flows.
Packed bed bioreactors have been widely used for the production of ethanol, acetone-
butanol-ethanol, lactic acid and enzymes
Fluidized Bed Bioreactor
Mixing in the fluidized bed is better than packed bed and the shear rate
is lower compared to the stirred tank bioreactor.
It usually contains an internal loop gas draft tube inside the column.
Gas sparging induces the liquid up flow with the suspended particles
in the inner draft tube.
Subsequently, gas escapes from the top of the bioreactor and the liquid
with the suspended particles is led through the gas-free down comer
• The scale-up process is divided into two categories. The two categories
are:
• (1) Scale-up in Suspension and
• (2) Scale-up in Monolayer
• Scale-up in suspension is the preferred method as it is simpler. Scale-up
of suspension culture primarily involves an increase in the volume of the
culture. Small scale generally means the culture capacity less than 2 litres
volume (or sometimes 5 litres).
STIRRED SUSPENSION CULTURES AND STATIC SUSPENSION CULTURES
• In a continuous flow culture, it is possible to keep the cells at a desired and set
concentration, and maintain. This is carried out by a bio-stat or chemo-stat
• Continuous flow culture consists of growing the cells at the mid-log phase, removal of
a measured volume of cells, and replacement by an equal volume of medium.
• The equipment, specially designed for this purpose has the facility for removal of the
cells and addition of medium.
• The flow rate of the medium addition can be determined from the growth rate of the
culture.
• The medium flow can be regulated by a peristaltic pump.
• By this technique, it is possible to keep the culture conditions constant rather than to
produce large number of cells.
• The continuous flow cultures are useful for monitoring metabolic changes in relation to
cell density.
• However, these cultures are more susceptible to contamination.
MICRO-CARRIER CULTURE:
• In roller bottles, the cells adhere to the total curved surface area of the micro carrier
beads, thereby markedly increasing the available space for growth.
• These tissue culture bottles can be used in specialized CO2 incubators with
attachments that rotate the bottles along the long axis.
• After each complete rotation of the bottle, the entire cell monolayer has transiently
been exposed to the medium.
• The volume of medium need only be sufficient to provide a shallow covering over
the monolayer
• Roller bottle culture has certain advantages. i. The medium is gently and constantly
agitated. ii. The surface area is high for cell growth. iii. Collection of the supernatant
medium is easy.
• There are limitations in roller culture. i. Monitoring of cells is very difficult. ii.
Investment is rather high.
MULTI-SURFACE CULTURE:
• The most commonly used multi-surface propagator of
monolayer is Slunclon cell factory (in short Nunc cell
factory).
• It is composed of rectangular petri dish-like units with
huge surface area (1,000-25,000 cm2).
• The units are inter-connected at two adjacent corners by
vertical tubes.The medium can flow between the
compartments from one end.
• The cell factory is almost like a conventional petridish or Slunclon cell
a flask with multiplayer units. factory
2) Supply of nutrients
5) Removal of wastes
CELL VIABILITY
• The number of viable cells in the culture provides an accurate indication of
the health of the cell culture
• Trypan blue and erythrosin B determine cell viability through the loss of
cellular membrane integrity.
• Both these dyes are unable to penetrate the cell membrane when the
membrane is intact, but are taken up and retained by dead cells (which lack
an intact membrane).
The toxic chemicals in the culture medium affect the basic functions of cells.
The cytotoxicity effect can lead to the death of the cells or alterations in their
metabolism.
Methods to access viable cell number and cell proliferation rapidly and accurately
is the important requirement in many experimental situations that involve in vitro
and in vivo studies.
The cell number determination can be useful for determining the growth factor
activity, concentration of toxic compound, drug screening, duration of exposure,
change in colony size, carcinogenic effects of chemical compounds, and effects
of solvents (such as ethanol, propylene, etc.).
CYTOTOXICITY
3.ATP assay.
MORPHOLOGIC CHANGES-CYTOTOXICITY
β-Galactosidase staining at pH 6.0 on MCF-7 cells untreated (left) and senescent MCF-7 cells treated with etoposide (12.5 μM,
24 hr) and allowed to recover for 4 days (right).
MTT ASSAY- THE ASSAYS TO MEASURE VIABLE CELLS (VIABILITY ASSAYS)
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay
• The MTT assay allows simple, accurate, and reliable counting of metabolically
active cells based on the conversion of pale yellow tetrazolium MTT.
• Nicotinamide adenine dinucleotide in metabolically active viable cells reduces
tetrazolium compounds into brightly colored formazan products or reduces
resazurin into fluorescent resorufin
• MTT and resazurin assays are widely used, as they are inexpensive and can
be used with all cell types
• The protease marker assay utilizes the cell-permeant protease substrate
glycylphenylalanyl-aminofluorocoumarin (GF-AFC). The substrate, which
lacks an aminoterminal blocking moiety, is processed by aminopeptidases
within the cytoplasm to release AFC.
• Tetrazolium as an electron acceptor reduces to formazan by the
enzyme succinate dehydrogenase .
• In MTT assay, yellow tetrazolium salt in the live cells reduces to purple
insoluble crystals of formazan
• Thus the higher the live cells, the more formazan are produced
• Formazan crystals are dissolved by a detergent and measured in a 96-
well plate by Elisa Reader
• This method was first introduced by Mosmann in 1982
In order to produce MTT solution,5 mg of MTT powder is dissolved in PBS solution (5mg/ml)
For this purpose, first 104cells and 100µl of culture medium are added to each well of 96-well plate
Three replicates were consider for each concentration (3 wells as control (containing culture medium and cell)
and 3 wells as blank (containing only the medium)
Then it is incubated for 18-24 h in order for the cells to adhere to the plate bottom (CO25% and 37o C) .
After this period, various concentrations of the desired compound is added to the wells and incubated for 24,48
and 72 h.
After the desired intervals for the test, 10µl of MTT solution is added to each of the wells for each 100µl of the
culture medium and the plates are incubated for 3-5 h
Then the supernatant is removed and to dissolve the yielded formazan, the suitable detergent (Isopropanol or
DMSO is added to the wells.
• Protease release.
• DNA staining.
PRINCIPLE OF THE LDH RELEASE ASSAY .
• The viable cells in culture have intact outer membranes.
. The GF-AFC substrate can penetrate live cells where cytoplasmic aminopeptidase activity
removes the gly and phe amino acids to release aminofluorocoumarin (AFC) and generate
a fluorescent signal proportional to the number of viable cells
• The released proteases cleave the substrate to liberate aminoluciferin, which serves as a
substrate for luciferase and leads to the production of a “glow type” signal
INTRODUCTION
• Growth Factor is a protein molecule made by the body; it functions to regulate cell division & cell
survival.
• Growth factors bind to receptors on the cell surface, with the result of activating cellular
proliferation and/or differentiation, promote cell growth.
• Growth factors are proteins that function as growth stimulators (mitogens) and/or growth inhibitors,
stimulate cell migration, act as chemotactic agents, inhibit cell migration, inhibit invasion of tumor
cells, modulate differentiated functions of cells, involved in apoptosis, involved in angiogenesis and
promote survival of cells without influencing growth and differentiation.
• Examples for Growth Factors are EGF, FGF, NGF, PDGF, VEGF, IGF, GMCSF, GCSF, TGF,
Erythropoietin.
GROWTH FACTORS
• Growth Factors are biologically active poly-peptides which function as hormone like
regulatory signals, controlling the growth and differentiation of responsive cells. The distinction
between growth factors and hormones is frequently arbitrary.
• Growth Factors are involved in cell differentiation and are essential to normal cell cycle, and
are thus vital elements in the life of animals from conception to death.
• Growth Factors mediate fetal development, play a role in maintenance and repair of tissues,
stimulate production of blood cells & participate in cancerous processes.
• Growth factors, which generally considered as a subset of cytokines, refer to the diffusible
signaling proteins that stimulate cell growth, differentiation, survival, inflammation, and tissue
repair.
EGF(Epidermal Growth Factor)
• EGF stands for Epidermal Growth Factor which is a protein found in both humans and other
animals such as mice.
• When present in humans, it’s often located in various tissues of the body.
• Two of the most common areas are the parotid gland and submandibular gland.
• Upon discovering human EGF for the first time, it was called urogastrone.
• Epidermal growth factor is often found in various liquids throughout the body, particularly;
urine, milk, blood plasma, and saliva.
• It is thought that epidermal growth factor is produced largely -testosterone stimulation. As
such, it’s often more commonly present in male bodies than female.
• Epidermal growth factor (EGF) and EGF receptor (EGFR) play an essential role in wound
healing through stimulating epidermal and dermal regeneration.
• In addition, EGFR inhibitors (EGFRis) have become a therapeutic option for the treatment of
cancer. EPIDERMAL GROWTH FACTOR (EGF)
FUNCTION & INTERACTIONS
• The main function of EGF is to help encourage cell growth. It does this by binding with a receptor
known as EGFR.
• When this binding occurs, it leads to a rapid increase in the cell count, differentiation within cells, and
cell survival. • This is particularly prevalent in salivary EGF proteins.
• When EGF is produced in the salivary glands, it is linked to maintaining and repairing gastric tissue,
along with oro-oesophagal maintenance.
• As a consequence, many problems within this part of the body can be healed thanks to EGF.
• The best example of this is ulcers in the mouth and throat, which can be healed when this protein is
secreted
• Nerve growth factors (NGFs) are a family of proteins that play a
crucial role in the development, maintenance, and survival of nerve
cells (neurons). These proteins promote the growth and
differentiation of neurons, as well as the maintenance of their overall
health.
• The prototypical nerve growth factor is called NGF, and it was the first
discovered member of this family. NGF is essential for the
development and survival of sensory and sympathetic neurons. It is
produced by various cells, including those in the skin, connective
tissues, and certain immune cells.
1. NGF Function: NGF promotes the growth, maintenance, and survival of neurons. It plays a critical role
in the development of the nervous system during embryonic and early postnatal stages.
2. Neurotrophins: NGF belongs to a family of proteins known as neurotrophins. Other members of this
family include brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5
(NT-4/5). Each neurotrophin has specific functions and affinities for different types of neurons.
3. Receptors: Neurotrophins exert their effects by binding to specific receptors on the surface of neurons.
The Trk (tropomyosin receptor kinase) family of receptors and the p75 neurotrophin receptor are
involved in mediating the actions of NGF and other neurotrophins.
4. Neuronal Survival and Plasticity: NGF is crucial for the survival of certain neurons, and it also plays a
role in synaptic plasticity, which is the ability of synapses to strengthen or weaken over time in response
to activity.
5. Implications in Neurological Disorders: Dysregulation of nerve growth factors has been implicated in
various neurological disorders. For example, reduced levels of NGF and BDNF have been associated
with neurodegenerative diseases like Alzheimer's disease.
6. Therapeutic Potential: Because of their role in promoting neuronal health and survival, nerve growth
factors have been investigated for their therapeutic potential in various conditions, including
neurodegenerative diseases and nerve injuries. However, the development of effective therapies has
proven to be challenging.
Nerve growth factors (NGFs)
• Nerve growth factors (NGFs) are a family of proteins that play a crucial role in the
• NGF is essential for the development and survival of sensory and sympathetic neurons.
• It is produced by various cells, including those in the skin, connective tissues, and certain
immune cells.
Nerve growth factors (NGFs)
1.NGF Function: NGF promotes the growth, maintenance, and survival of neurons. It plays a
critical role in the development of the nervous system during embryonic and early postnatal
stages.
3.Receptors: Neurotrophins exert their effects by binding to specific receptors on the surface
of neurons.
Nerve growth factors (NGFs)
1.Neuronal Survival and Plasticity: NGF is crucial for the survival of certain neurons, and it
also plays a role in synaptic plasticity, which is the ability of synapses to strengthen or weaken
over time in response to activity.
3.Therapeutic Potential: Because of their role in promoting neuronal health and survival, nerve
growth factors have been investigated for their therapeutic potential in various conditions,
including neurodegenerative diseases and nerve injuries. However, the development of
effective therapies has proven to be challenging.
Fibroblast Growth Factors (FGFs)
• FGFs exert their effects by binding to and activating cell surface receptors,
known as FGF receptors (FGFRs).
Fibroblast Growth Factors (FGFs)
1. Tissue Repair and Regeneration: FGFs contribute to tissue repair and regeneration in adults. They
play a role in wound healing, bone regeneration, and the repair of other tissues after injury.
2. Angiogenesis: FGFs are potent stimulators of angiogenesis, the formation of new blood vessels. This
is particularly important in processes such as embryonic development, wound healing, and the growth
of tumors.
3. Implications in Disease: Dysregulation of FGF signaling has been implicated in various diseases,
including cancer, developmental disorders, and metabolic diseases. Abnormal FGF signaling can
contribute to uncontrolled cell growth and angiogenesis, hallmarks of cancer progression.
2. Receptors: The biological effects of PDGF are mediated by binding to specific cell surface receptors
known as Platelet-Derived Growth Factor Receptors (PDGFRs). There are two types of PDGFRs:
PDGFR-α and PDGFR-β. These receptors are receptor tyrosine kinases, meaning they activate
intracellular signaling pathways by adding phosphate groups to tyrosine residues.
3. Cellular Effects: PDGF stimulates cell proliferation, migration, and survival. It plays a crucial role in
various physiological processes, including embryonic development, tissue repair, and angiogenesis
(formation of new blood vessels).
4. Therapeutic Implications: Given its role in tissue repair and various diseases, PDGF and its
receptors have been explored as potential therapeutic targets. Researchers are investigating ways to
modulate PDGF signaling for the treatment of conditions such as chronic wounds, fibrosis, and certain
cancers.
Platelet-Derived Growth Factor (PDGF)
1.Wound Healing: PDGF is a key player in the wound healing process. After injury,
platelets release PDGF, which attracts and stimulates the proliferation of fibroblasts and
smooth muscle cells. This helps in the formation of granulation tissue and contributes to
the repair of damaged tissue.
2.Fibrosis and Tissue Repair: PDGF is involved in the regulation of tissue repair and
remodeling. In some cases, persistent or excessive PDGF signaling may contribute to
pathological conditions, such as fibrosis.
3.Cancer: Abnormal PDGF signaling has been implicated in certain types of cancer. In
some cases, tumors may overproduce PDGF or express abnormal PDGFRs, leading to
uncontrolled cell growth and migration.
Platelet-Derived Growth Factor (PDGF)
primarily produced in the kidneys and to a lesser extent in the liver. EPO stimulates the formation of red blood cells
(erythropoiesis) from precursor cells in the bone marrow, known as erythroid progenitor cells.
1. Erythropoiesis Regulation: The primary function of EPO is to regulate and stimulate the production of red blood cells. Red
blood cells are crucial for transporting oxygen from the lungs to various tissues and organs throughout the body.
2. Production Site: EPO is mainly produced in the kidneys in response to low oxygen levels in the blood. Hypoxia, or low
oxygen, stimulates the release of EPO, which then acts on the bone marrow to increase the production of red blood cells.
3. EPO Receptors: The effects of EPO are mediated through its binding to specific receptors, called erythropoietin receptors
(EPOR), on the surface of erythroid progenitor cells in the bone marrow. This binding triggers intracellular signaling pathways
that promote the survival, proliferation, and differentiation of these cells into mature red blood cells.
Erythropoietin (EPO)
1. Medical Applications: Recombinant human erythropoietin (rhEPO) has been developed and is used medically to treat
conditions associated with low red blood cell counts, such as anemia. It is often prescribed for patients with chronic kidney
disease, cancer patients undergoing chemotherapy, and individuals with certain types of anemia.
2. Athletic Doping: EPO has gained notoriety in the context of sports doping. Athletes have used synthetic EPO to artificially
boost their red blood cell counts, leading to increased oxygen-carrying capacity and improved endurance. However, the
use of EPO for performance enhancement is considered unethical and is banned in competitive sports.
3. Neuroprotective Effects: Beyond its role in erythropoiesis, research has suggested that EPO may have neuroprotective
effects. Some studies have explored its potential in treating neurological conditions and injuries, although more research is
needed in this area.
• Understanding the role of erythropoietin in regulating red blood cell production has significant implications for the treatment
of anemia and related conditions. Ongoing research continues to explore the therapeutic potential of EPO in various
medical contexts, as well as its broader effects on different physiological systems.