ROLAND INSTITUTE OF PHARMACEUTICAL

SCIENCES, BERHAMPUR

CELL CULTURE

SANJIV KUMAR PANDA

Dr. NIHAR RANJAN DAS [Link] 1st SEM
PROFESSOR PHARMACOLOGY
PHARMACOLOGY DATE-JUNE 12 2024
 Cell Culture
What is Cell Culture?
Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial
environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical
means before cultivation, or they may be derived from a cell line or cell strain that has already been already
established.
Primary Culture
Primary culture refers to the stage of the culture after the cells are isolated from the tissue and proliferated under the
appropriate conditions until they occupy all of the available substrate (i.e., reach confluence).
Sub-Culture (passaged)
Here, the cells have to be transferred to a new vessel with fresh growth medium to provide more room for continued
growth.
Cell Line
After the first subculture, the primary culture becomes known as a cell line or subclone. Cell lines derived from
primary cultures have a limited life span (i.e., they are finite; see below), and as they are passaged, cells with the
highest growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the population.
Cell Strain
If a subpopulation of a cell line is positively selected from the culture by cloning or some other method, this cell line
becomes a cell strain. A cell strain often acquires additional genetic changes subsequent to the initiation of the parent
Biosafety Levels
The regulations and recommendations for biosafety in the US are contained in the document Biosafety in
Microbiological and Biomedical Laboratories, prepared by the Centers for Disease Control (CDC) and the
National Institutes of Health (NIH), and published by the U.S. Department of Health and Human Services.
The document describes the microbiological practices, safety equipment, and facility safeguards for the
corresponding level of risk associated with handling a particular agent.
Biosafety Level 1 (BSL-1) - The basic level of protection common to most research and clinical
laboratories, and is appropriate for agents that are not known to cause disease in normal, healthy humans.
Biosafety Level 2 (BSL-2) - appropriate for moderate-risk agents known to cause human disease of
varying severity by ingestion or through percutaneous or mucous membrane exposure. Most cell culture
labs should be at least BSL-2, but the exact requirements depend upon the cell line used and the type of
work conducted.
Biosafety Level 3 (BSL-3) - appropriate for indigenous or exotic agents with a known potential for aerosol
transmission, and for agents that may cause serious and potentially lethal infections.
Biosafety Level 4 (BSL-4) - appropriate for exotic agents that pose a high individual risk of life-
threatening disease by infectious aerosols and for which no treatment is available. These agents are
restricted to high containment laboratories.
CULTURE ENVIRONMENT
• The following factors are needed for cell 3. Environmental factors:
survival: Temperature: 37°C.
1. Substrate (Solid and liquid suspension) – O2/CO2
Agar, glucose broths, Broths Buffers - pH.

2. Nutrients (energy source)

• So, the following physiochemical and
 'C' source - glucose, galactose
physiological conditions are to be maintained
 'N' source – Essential and Non-essential in a culture.
amino acids
• (a) Physiochemical: Temperature, pH, osmotic
 Vitamins pressure, O2 & CO₂ tension.
 Inorganic Salts - Na+, K+, CI
• (b) Physiological: Hormone and nutrient
 Growth factors concentrations.
 Hormones
• The physiochemical and physiological
 Antibiotics conditions are provided/controlled by the
 CELL CULTURE MEDIA
• Media provides necessary nutrients, hormones and growth factors for
cell growth as well as regulating the pH and osmotic pressure of the
culture. Serum is a source for hormones, minerals, growth factors,
lipids, enzymes, micro- nutrients, trace elements, etc.
• Media are of three types based on the requirement for
supplementation with serum.
1. Basal media
EXAMPLES OF CELL CULTURE
2. Reduced serum media
SERUM BASED SERUM-FREE
3. Serum free medium MEM MCDB 110
• Basal Media - Majority of media is basal media. Contain amino acid, DMEM MCDB 131
vitamins, inorganic salt, glucose ('C' source).Must be supplemented RPMI 1640 MCDB 170
with serum (animal). Disadvantage: Contamination due to added
Ham’s F12 MCDB 202
serum.
DMEM/F12 MCDB 302
• Reduced Serum Media - Basal media enriched with nutrients and
animal derived factors. Less amount of serum needed. McCoy’s 5A MCDB 402

• Serum Free Media - Animal sera is replaced with appropriate

nutritional and hormonal preparations/formulation.
 VARIOUS TYPES OF CELL CULTURE
• Primary cell culture • Secondary cell culture
• 2 types (a) Adherent culture • 2 types (a) Finite cell line
(b) Suspension culture (b) Continuous cell line
• These are the first cells taken from an • Primary cell culture → Subculture →
organism. The cells are dissociated from parent Secondary Culture
tissue (e.g. liver, kidney) using mechanical • Subculture refers to transfer of cell from one
method/enzymatic method. culture vessel to another culture vessel. This is
• Then they are grown in-vitro in culture periodically required to provide fresh nutrients,
medium (artificial) using suitable glass/plastic provide space for continuously growing cell
containers. lines. When a primary culture is subcultured, it
becomes secondary subculture.
• After at least one sub culture, cell lines are
produced.
 PRIMARY CELL CULTURE SECONDARY CELL CULTURE
A) Adherent Cell culture or anchorage Dependent A) Finite cell line is characterized by:
cells: • Limited time span
• These cells require an attachment for growth. The • Limited number of cells generation (20-80
cells are derived from immobile organs like population doublings)
kidney, liver, etc. • Slow growth rate
• The cells grow on the surface of a solid media • Doubling time 24-96 hrs
also known as Monolayer. It is easy to harvest on
• Anchorage dependence
any surface. So, growth is easy. But we get a
small number of cells as growth is halted after a • Continuous Cell lines is characterized by
single layer. • Transformed cell line
B) Suspension cell culture or anchorage • Immortal
Independent cells • Fast growth rate
• They do not require attachment for growth or • Doubling time 12-24hrs
don’t attach to the surface of the culture vessel. • Absence of anchorage dependence
These cells are derived from blood system
(lymphocytes). The cells freely float in the • Grow either as monolayer or suspension
culture medium (liquid). Here a greater number
of cells can be produced. But they grow in
haphazard.
 EQUIPMENTS
 Biological Safety Cabinet (laminar air flow cabinet):
 Provides aseptic environment of cell culture.
 Prevents contamination from bacteria, fungi, etc.
 Protects the operator from potential infection from the cultured cells.
They are of 3 classes based on the nature of cells and organisms being handled:
 Class I: Protects the operator but does not prevent contamination. So, it is useful for media preparation (without
antibiotics), for handling of nontoxic reagents and non-primate cell lines.
 Class II: Most commonly used. Both operator and cell culture are protected.
 Class III: It is also most commonly used. It prevents potentially hazardous infection from the cultured cells.
 The CO2 Incubator:

CO2-bicarbonate based buffer is used to buffer the cells in culture against changes in pH. Changes in atmospheric
CO₂ can alter the pH of the medium. So, exogenous CO₂ is used when media buffered with a CO2-bicarbonate
based buffer is used. 4-10% CO₂ is common for most cell culture experiment. The CO2 incubator ensures a
controlled environment for the optimum growth of cell lines. A water pan inside the incubator functions for
humidification while a CO2 monitoring device controls the CO2 tension. Natural convection functions for air
 Refrigerator (2-8°C) and Freezer (-20°C):
• Refrigerators are used for the local storage of media and reagents. On the other hand, unstable
media, serum and reagents are to be stored in a freezer.
 Soaking Bath or Sink:
• This is essential for the collection of wash up as well as for sanitizing with soap and water prior to
laboratory work.
 Pasteur Pipette:
• It is a device used for transfer of small quantities of liquid in the laboratory. It is essentially a glass
or plastic tube that is tapered to a narrow opening point at the lower end and fitted with a plastic or
rubber bulb at the upper end.
 Pipette Cylinder:
• These are needed for the collection of used pipettor tips. Gatorade glass bottles could be used for this
purpose.
 Liquid Nitrogen Freezer:
• A liquid nitrogen freezer or cryo-storage container is meant for the preservation of cell line stocks.
They use liquid nitrogen to freeze biological material in extremely cold temperatures (closer to -
 Inverted Microscope:
• This type of microscope is used for visualizing cell cultures in situ. The cells in culture vessel remain at the
bottom of the vessel and the medium floats above the growing cells. To observe these cells the inverted
microscope is used. The inverted microscope has the optical system at the bottom and the light source at the
top. This arrangement helps to observe the cultured cells in the plates.
 Centrifuges:
• Centrifuges separate or concentrate substances suspended in a liquid medium by density. A centrifuge is
needed for the removal of trypsin, preservatives, experimental additives and other substances.
 Water Bath:
• It is a container of water heated to a given temperature. It is used for heating substances placed in smaller
containers.
 Cell Counter:
• A cell counter is an instrument designed to measure cell count and viability (live, dead and total cells)
accurately and precisely in less than a minute per sample. It is essential for quantitative growth kinetics. It
is of great advantage when more than two or three cell lines are cultured in the laboratory.
 Sterilizer (Autoclave):
• An autoclave is a pressure chamber used to carry out industrial processes requiring elevated temperature
and pressure different from ambient air pressure. Autoclaves are used to perform sterilization.
REFERENCES
• Dr. Pratap Kumar Sahu, Dr. Prasant Tiwari; Cellular and Molecular
Pharmacology, 2021, Nirali Prakashan, pg no. 1.46-1.51, 4.1-4.2
• CELL CULTURE BASICS, invitogen, gibco
• [Link]
THANK
YOU