Animal tissue culture

• Animal tissue culture is a technique used in biological and medical

research to grow and maintain animal cells and tissues outside the
organism's body.
• To successfully perform animal tissue culture, you need to meet
certain requirements and follow specific protocols.
Animal tissue culture

• 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

• Antibiotics and Antimycotics: To prevent bacterial and fungal

contamination, antibiotics like penicillin and streptomycin, as well as
antimycotics like amphotericin B, can be added. However, some
experiments may require antibiotic-free media.
 Preparation of Animal Cell Culture Medium

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.

3.BSS in Ophthalmology: BSS is used in ophthalmology, particularly during eye surgery

and procedures like cataract surgery. It helps maintain proper intraocular pressure and
provides a balanced environment for the eye's structures.
 NATURAL MEDDIA USED IN ANIMAL CELL CULTURE

• Natural media can be more complex and may contain extracts or

components derived from natural sources.
• Serum: Fetal bovine serum (FBS) is a common component in cell
culture media, and it is derived from the blood of fetal cows.
• While it is an animal-derived product, it is a natural component that
provides growth factors, hormones, and nutrients necessary for cell
growth. However, serum-free media are available for researchers who
want to reduce or eliminate the use of serum in their cultures.
 NATURAL MEDDIA USED IN ANIMAL CELL CULTURE

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

tissues to provide cells with a natural microenvironment that includes tissue-specific

growth factors and extracellular matrix components. For example, brain-derived extracts

can be used for neural cell culture.

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

fluid-derived stem cells.

 NATURAL MEDIA USED IN ANIMAL CELL CULTURE

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

factors that support cell growth and differentiation.

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

can affect cell responses.

 Role of CO2 in animal tissue culture

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.

2.Electrolytes: Serum contains a range of electrolytes, including sodium (Na+), potassium

(K+), calcium (Ca2+), magnesium (Mg2+), chloride (Cl-), and bicarbonate (HCO3-).

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

1.Hormones: Various hormones, such as insulin, thyroid hormones, and

cortisol, can be found in serum. Hormones play a crucial role in regulating
various physiological processes and are often measured in clinical
diagnostics to assess endocrine function.
2.Metabolites: Serum contains a wide range of metabolic byproducts and
intermediates that reflect the body's ongoing metabolic activities.
Examples include lactate, bilirubin, and various enzymes like alanine
aminotransferase (ALT) and aspartate aminotransferase (AST).
3.Lipids: Serum contains lipids such as cholesterol and triglycerides, which
are important for energy storage and the structure of cell membranes.
4.Gases: Serum contains dissolved gases, such as oxygen and carbon
dioxide, which are involved in gas exchange and transportation throughout
the body.
 Role of serum in animal cell culture
1. Growth Factors and Cytokines: Serum contains a variety of growth factors and cytokines that promote
cell growth, proliferation, and differentiation. These factors include epidermal growth factor (EGF),
fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), and
more. They play a crucial role in stimulating cell division and maintaining cell health.

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

1.Attachment Factors: Serum contains adhesive proteins, such as fibronectin,

vitronectin, and laminin, which facilitate cell attachment to the culture vessel or
substrate. Proper cell attachment is crucial for cell survival and growth in culture.

2.Proteins and Albumin: Serum contains a variety of proteins, including albumin,

which serves as a carrier for hormones, nutrients, and other small molecules,
helping to transport them to cells. Albumin also contributes to maintaining the
osmotic balance of the culture medium.

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.

3.Immune Factors: Serum contains immunoglobulins (antibodies) and complement proteins

that can help protect cells from contamination by microorganisms.
 Role of serum free animal cell culture &applications

• Research and Experimentation: a. Basic Cell Biology Studies: Serum-

free media are often used for studying fundamental cellular
processes, signaling pathways, and gene expression without the
interference or variability introduced by serum constituents.

• Drug Discovery: Serum-free media can be employed in high-

throughput drug screening assays to test the effects of potential drug
compounds on cells in a more controlled and consistent environment.
 Role of serum free animal cell culture &applications
• Biotechnology and Bioprocessing: a. Bioproduction of Recombinant Proteins: Serum-free media

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

lines used in biomanufacturing.

• 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

cells and immune cells.

 Role of serum free animal cell culture &applications

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.

2.Compliance with Regulatory Requirements: In some applications, such as clinical

research and cell therapy production, regulatory authorities may require the use of serum-
free media to ensure the safety and consistency of the cell-based products.

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:

• Drug screening and development.

• Mutagenesis and carcinogenesis.
• Normal physiology and biochemistry of cells.
• Potential effects of drugs and toxic compounds on the cells.

• 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.

➢Primary cultures may contain specific receptors

➢Under proper culture conditions may become organotypic (for organoids).
➢Since primary organ or slice cultures do not require cell dissociation, they
maintain the three-dimensional structure of the tissue and cell-to-cell contact

Disadvantages of primary cell culture.

➢Difficult to obtain
➢Relatively short life span in culture
➢Very susceptible to contamination
Secondary cell culture. This simply refers to the first passaging of cells, a switch to a different
kind of culture system, or the first culture obtained from a primary culture.
Techniques for the development of primary cell cultures

• Mechanical disaggregation.
• Enzymatic disaggregation.

• Mechanical disaggregation: The mechanical approach involves slicing or harvesting

tissue and subsequent harvesting of spill out cells. This can be achieved by sieving, syringing
and pipetting.

This procedure is inexpensive, rapid and simple, however,

all these approaches involve the risk of cell damage, thus
mechanical disaggregation is only used when the viability of
the cells in the final yield is not very important
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 normal physiology and biochemistry of cells.

➢Used for studying the effects of drugs and toxic compounds on the cells and
mutagenesis and carcinogenesis.

➢ It is also used in drug screening and development.

➢It is used in large scale manufacturing of biological compounds (e.g., vaccines,

therapeutic proteins).

➢Used in tissue engineering-artificial organs.

 Cell lines
A cell line can be defined as a permanently established cell culture which will propagate
forever, provided the continuous supply of suitable fresh medium and the availability of space
for the cells to propagate
Types of cell lines
➢ Finite cell lines: cell lines that lose their ability to divide after a limited period of time. i.e.
these cell lines have a limited life span. Usually, finite cell lines contain cells which can
divide 20–100 times before losing their capability to divide
➢ Continuous cell lines/ Infinite cell lines:
Continuous subculturing of cells or treatment of cells with carcinogens (chemicals),
oncogenic viruses, etc, results in changes in phenotypical characteristics, in particular
morphology, which can alter cells and lead to the development of cells that grow faster than
normal cells.
Cell lines obtained from these altered cells have infinite life spans. Such types of cell lines are
referred to as continuous cell lines.
Commonly used cell lines
Nomenclature of cell line:
• Represented in abbreviated form either derived from the source of Cells,
name of institute or association with virus
• e.g. EB, Epstein Barr; WI, Wistar Institute)

• Numbered If more than one cell line (e.g. EB1, EB2)

 HeLa cell lines
HeLa is an immortal cell line used in scientific research. It is the oldest and most commonly used
human cell line

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

A typical growth curve for cultured cells

displays a sigmoid pattern of proliferation. The
growth phases associated with normal cells are
divided in to 4 phases
 Growth kinetics of cells in culture/Phases of Cell Growth

1.Lag Phase – at this stage the cells do not divide.

During this period the cells adapt to the culture
conditions
2.Logarithmic (Log) Growth Phase – cells
actively proliferate and an exponential increase in
cell density arises. The cell population is considered
to be the most viable at this phase; therefore, it is
recommended to assess cellular function at this
stage.
3.Plateau (or Stationary) Phase – cellular
proliferation slows down due to the cell population
becoming confluent.

4.Decline Phase – cell death predominates in this phase and

there is a reduction in the number of viable 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.
 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

nutrients and microorganisms uniformly.

Stirrer: Mixes the gas bubbles throughout the liquid culture medium.
Baffle: Prevents the counterflow or vortex formation by breaking
down the gas bubbles to improve aeration efficiency.
Sparger: It supplies oxygen into the culture medium through the
perforated tubes.
Drain point: Withdraws cells or medium for the continuous
fermentation.
Cooling jacket: It is fitted externally to the fermentation vessel which
allows the passage of steam or cold water to balance the heat
generated during the process.
• Controlling elements monitor the parameters like (temperature, pH,
acid, bases, oxygen supply, pressure etc.) that are necessary for the
product formation and it includes:

• Pt-100: Monitors the temperature in the culture vessel.

Foam probe: It senses foam formation.
•
pH electrode: Monitors the pH in the culture vessel.
Oxygen sensor: Maintains the dissolved oxygen content level.

Heating pad: Provides heat to the medium.

Cold finger: It is a pipe that passes cold water inside a vessel to cool
the contents.

Rotameter: Provides variable airflow into the culture vessel.

Pressure valve: Maintains the pressure.

Air pump: Supplies air throughout the medium.

 PROPERTIES OF A FERMENTER
▪ It should be reliable for long-term operation.

▪ 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.

▪ It should consume less power.

▪ A fermentor must be equipped with controlling probes that can maintain the temperature, pH, oxygen level etc.

▪ It facilitates the passage of inoculum and media into the vessel.

▪ A bioreactor does not allow excessive evaporation loss.

▪ It minimizes the labour input for the operation, harvesting, cleaning and maintenance.
BIOREACTORS USED FOR CELL CULTURE

The reactors used for large-scale cell cultures are

–Continuous Stirred bioreactors

–Packed bed bioreactor
–Airlift bioreactor
– Fluidized Bed Bioreactor
 CONTINUOUS FLOW 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.

It is operated in the plug-flow mode (velocity of the fluid is assumed

to be constant), with a minimum of back mixing

By selecting an appropriate flow rate, it is possible to achieve a very high

reaction rate or even complete conversion of the substrate in a single pass
of the column

Packed bed bioreactors have the advantages of simplicity of

operation and low cost. Shear forces are low in packed bed
reactors

Packed bed bioreactors have been widely used for the production of ethanol, acetone-
butanol-ethanol, lactic acid and enzymes
Fluidized Bed Bioreactor

In the fluidized bed bioreactor, the immobilized cells are maintained in

motion by a continuous flow of the feed solution

The fluidized bed bioreactor provides conditions that are intermediate

to those of the stirred tank and packed bed bioreactor.

Mixing in the fluidized bed is better than packed bed and the shear rate
is lower compared to the stirred tank bioreactor.

However, fluidized bed bioreactors are subject to unstable bed expansion

because of the changes in bead density with time due to cell growth and gas
formation, resulting in washout.
Air-lift bioreactor
The air-lift bioreactor can be regarded as a special variation of the fluidized
bed 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

Compared to the common fluidized bed bioreactor, the main

advantage of the air-lift bioreactor is the improved fluidization
characteristics.
 AIR-LIFT FERMENTER CULTURE

• The major limitation of scale-up in suspension culture is inadequate mixing and

gas exchange.
• For small cultures, stirring of the medium is easy, but the problem is with large
cultures.
• The design of fermenter should be such that maximum movement of liquid is
achieved with minimum shear to damage the cells.
• A 5% CO2 in air is pumped through the bottom of the fermenter.
• The bubbles formed move up to agitate and aerate the culture.
• These bubbles carry a flow of liquid along with them and release at the top which
goes to the bottom for recycling. It is possible to continuously supply O2 to the
culture in this technique
• . Air-lift fermenter culture technique is suitable for fragile animal cells. This
fermenter is extensively used in the biotechnology industry for culture capacities
up to 20,000 litres.
ANIMAL CELL CULTURE: SCALE-UP PROCESS

• 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

• It is usually necessary to maintain cell strains in stirred

suspension cultures, by agitation (or stirring) of the medium.
• The stirring of the culture medium is achieved by a magnet
encased in a glass pendulum or by a large surface area paddle.
• The stirring is usually done at a speed of 30-100 rpm.
• This is sufficient to prevent sedimentation of cells without creating
shear forces that would damage cells.
• Static suspension cultures: Some cells can grow in suspension
cultures, without stirring or agitation of the medium, and form
monolayer cells. However, static suspension cultures are
unsuitable for scale-up.
Factors in Scaling-Up:

• For appropriate scale-up, the physical

and chemical requirements of cells have
to be satisfied.
• Physical parameters:
• i. Configuration of the bioreactor.
• ii. Supply of power.
• iii. Stirring of the medium.
• Chemical parameters: i. Medium and
nutrients. ii. Oxygen. iii. pH and buffer
systems. iv. Removal of waste products.
 COMMONLY USED TECHNIQUES FOR STIRRED SUSPENSION CULTURE
• The size of the stirrer flask is in the range of 2-10 litres.
• It is fitted with a magnetized rotating pendulum, and two
side arms — one for the addition of cells and medium, and
the other for the supply of CO2.
• The stirrer culture vessel is autoclaved and is then set up
• The flask is seeded with the culture.
• Then medium along with an antifoam agent is added.
• The flask is connected to CO2 and stirred at a speed of 60
rpm. The flask is incubated for about 2 hours. The contents
of the small stirrer flask are transferred to a large flask and
the entire set up is restarted.
• Incubation at 37°C is carried out for 4-7 days. The growth
of the cells is monitored daily, and the cells are counted.
There is a tendency of the cells to enter apoptosis, if the
concentration exceeds 1 × 106 cells/ml.
 CONTINUOUS FLOW 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.
OTHER SYSTEMS FOR SUSPENSION CULTURE
• Rotating chambers:
• The mixing and aeration of the culture medium can be achieved by 2 or 3 rotating
chambers.
• The chambers are so designed that the cell suspension and mixing are high in one
chamber while the product and spent medium remain in the other chamber.
• These chambers are separated by semipermeable membrane. Perfused suspension
culture:
• This also has two compartments. The cells are kept in a low-volume compartment at
high concentration, while the medium is perfused in adjacent compartment. The product
can be collected in a third compartment
 SCALE-UP IN MONOLAYER:

• The monolayer culture are anchorage- dependent. Therefore, for the scale-up
of monolayer cultures, it is necessary to increase the surface area of the
substrate in proportion to the number of cells and volume of the medium.
• Suspension cultures are preferred as they are simple. The advantages and
disadvantages of monolayer cultures are listed.

• Advantages: i. Change of medium and washing of cells easy. ii. It is easy to

perfuse immobilized monolayer cells. iii. The cell product formation
(pharmaceutically important compounds e.g. interferon, antibodies) is much
higher.

• iv. The same set up and apparatus can be repeatedly used with different media
and cells.
• Tedious and costly.
• ii. Require more space.
• iii. Growth of cells cannot be monitored effectively.
• iv. Difficult to measure control parameters (O2 pH, CO2 etc.)
• For scale-up of monolayer cultures, a wide range of tissue cultures and
system have been developed. A selected few of them are briefly described.
 MICRO-CARRIER CULTURE:

• Monolayers can be grown on small spherical carriers or micro-beads (500

μm in diameter diameter) referred to as micro-carriers.

• The micro-carriers are made up of any one the following materials

• i. Plastic (acrobeads, bioplas).
• ii. Glass (bioglass, ventreglas).
• iii. Gelatin (ventregel, cytodex-3).
• iv. Collagen (biospex, biospheres)
• v. Cellulose (DE-52/53).
• vi. DEAE Dextran (cytodex I, dormacell).
 MICRO-CARRIER CULTURE:

• The micro-beads provide maximum surface area for monolayer cultures.

• This actually depends on the size and density of the beads.
• The cells can grow well on the smooth surface at the solid-liquid interface.
• However, micro-carriers need efficient stirring without grinding the beads.
The main advantage with micro-carrier culture is that it can be treated as a
suspension culture for all practical purposes.
• Micro-carriers can be cultured in stirrer flask or in continuous suspension
• In fact, the suppliers of micro-carriers provide the technical literature and
other relevant information for setting up a micro-carrier culture
FACTORS AFFECTING MICRO-CARRIER CULTURE:

i. Composition and coating of beads (gelatin and collagen beads

are preferred as they can be solubilized by proteases).
ii. Higher stirring speed is usually required.
iii. Glass beads are used when the micro-carriers need to be
recycled.
Analysis of micro-carrier culture: The cell counting techniques
are difficult to be used for micro-carrier cultures. The growth rate
can be detected by analyzing DNA or protein.
MICRO-CARRIER CULTURE:
 ROLLER BOTTLE 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

• The main limitation of cell factory is that it is very difficult

to monitor the growth of cells.
• The major advantage however, is its simple operation to
produce large number of cells.
REQUIREMENTS FOR A BIOREACTOR FOR ANIMAL CELL CULTURE

1) Well-controlled environment (Temperature, pH,)

2) Supply of nutrients

3) Gentle mixing (avoid shear damage to cells)

4) Gentle aeration (add oxygen slowly to the culture medium, but avoid the formation of
large bubbles which can damage cells on contact).

5) Removal of wastes