TYPES OF CELL

CULTURE
Presented by:
Y. Bhavya sree
M. Pharmacy
Department of pharmacoloy
contents
 What is cell culture?
 Media composition.
 Types of cell culture.
 General procedure.
 Isolation of cells.
 Cryopreservation.
 Applications.

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Cell culture

Cell culture is the process by which cells are grown under controlled
conditions, generally outside of their natural environment.

OR

• Tissue or cell Culture is the general term for the removal of cells, tissues, or
organs from an animal or plant

• And their subsequent placement into an artificial environment for growth.

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Media composition
• Most animal cell culture media are generally having
following basic components and they are as follows:
• Energy sources: Glucose, Fructose, Amino acids
• Nitrogen sources: Amino acids
• Vitamins: Generally water soluble vitamins B & C.
• Inorganic salts: Na+, K+, Ca+2, Mg+2
• Fat and Fat soluble components: Fatty acids, cholesterols,
Antibiotics
• Growth factors and hormones
• Oxygen and CO2 concentration.
• The physical environment includes the optimum pH,
temperature, osmolality and gaseous environment, supporting
surface and protecting the cells from chemical, physical, and
mechanical stresses
• CO2 incubators are used and designed to mimic the
environmental conditions of the living cells.

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• An inverted microscope is used for visualizng cell cultures
invitro.
• For most animal cell cultures low speed centrifuges are needed
• Cryo preservation is storing of cells at very low temperature (-
1800C to -196 OC) using liquid nitrogen. DMSO is a
cryopreservative molecule which prevents damage to cells.
• Serum is essential for animal cell culture and contains growth
factors which promote cell proliferation

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 Types of cell culture
Cell cultures have been divided into the following three main
types:
1. Primary cell culture
2. Secondar cell culture
3. Cell lines

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Primary cell culture
• A primary culture is made by inoculating cells obtained from
animal or human tissue directly in a suitable growth medium.
• The tissue isolated from the animal or human body is excised
into very small fragments with the help of scissors and forceps.
• These fragments are placed in a sterile medium and the tissue
fragments are disaggregated into individual cells by treating
them with proteolytic enzyme, trypsin.
• One can easily select grow certain types of cells by carefully
choosing the composition of the growth medium.

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• For establishing a primary culture of blood cells, the suspension may
be subjected to density gradient centrifugation to separate the
different components of blood.
• The gradients necessary for the separation of different blood
components are established by using ‘Ficoll’ and 'PercolΓ media.
This method has been widely used for separation of lymphocytes

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Secondary cell culture
• A secondary cell culture is obtained by subculturing of the primary
cell culture.
• For sub-culturing cells of suspension culture, the primary culture is
diluted with fresh growth media to obtain the secondary cell culture,
for example with lymphocytes.
• For subculturing anchorage dependent cells, some of the cells of the
primary culture are removed and inoculated in a fresh culture
medium.
• In relation to sub-culturing, a term ‘passage number’ is used.

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• The passage number refers to the number of times sub-culturing has
been done, after original isolation of cells from the primary culture.
• Split ratio is the ratio which tells us about the number of new culture
produced after each sub-culturing.
• This split ratio forms a basis of relationship between the passage
number and generation number and is given in the following equation
• Generation number = Passage number × split ratio/2

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Cell lines
• 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.
• Cells with the highest growth capacity predominate, resulting in a degree of
genotypic and phenotypic uniformity in the population.

Cell lines are known by:

1. A code: Eg-NHB for normal human brain.
2. A cell line number: This is applicable when several cell lines are the Same cell
culture Soure. Eg- NHB1, NHB2.
3. Number of population doubling, The cell line has already undergone.
Eg- NHB2/2 means two doubling.
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Commonly used cell lines:
• HeLa cells which are used to proliferate the famous polio vaccine.
• HEK 293 or Human embryonic kidney- derived epithelial cells. Cell line
of choice in transient and stable transformation experiment for protein
expression and even in electrophysiological experiment.
• CHO Cells are mostly used in various experiments such as
recombinant protein production and studies of the epidermal growth
factor.
Types of cell lines:
On the basis of the life span of the cell culture Cell lines are categorized
into 2 types:
1. Finite cell line
2. Continuous cell line.
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 Types of culture in cell lines

1. Monolayer Cultures: When the bottom of the culture vessel is covered

with a continuous laver of cells, usually one cell in thickness, they are
referred to as monolayer cultures.

2. Suspension cultures: Majority of the continuous cells grow as mono

layers. Some of the cells with are non adhesive.

Eg- cells of leukemia or certain cells which can be mechanically kept in

suspension. Can be propagated in suspension.
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General procedure

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 Cryopreservation
• Cryopreservation is a process that preserves organelles, cells, tissues or any
other biological constructs by cooling the samples to very low temperature.
• At very low temperature all the biological activities of the cells stop and the
cells dies. (Enzyme activity, gene expression, metabolic activity).
Applications:
• Blood bank
• Seed bank
• Freezing of cell culture.
• Sperm bank
• Storage of rare germplasm
• Organ preservation.

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Cryodamage:
• At very low temperature: formation of crystals in the cells, undergo
damage of the cells and lead to death.
• At gradual temperature: solvent around the cell is frozen and solute
concentration is increased and water is lost from cell and cell get
dehydrated and lead to death.
How to prevent cryodamage:
• Using cryoprotectants- Used to protect biological tissue from freezing
damage. Such chemicals are some times called antifreeze agents.
• Eg- glycerol, ethylene glycol, propylene glycol and dimethyl sulfoxide
(DMSO).

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 Applications
• Excellent model system for studying
• Normal physiology, cell biology and biochemistry of cells
• The effects of drugs, radiation and toxic compounds on the cell
• The interaction between disease causing agents and cells (mutagenesis,
carcinogenesis)
• Nutritional studies
• The process and triggers of aging.
• Toxicity testing
• Genetic engineering and gene therapy-Studies dealing with genetics (eg: gene transfer,
genetic analysis, transformation, immortalization, senescence)

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• Cell-based manufacturing-Laboratory production of medical and
pharmaceutical compounds for wide range of applications.
• vaccines, insulin, interferon, hormones, other therapeutic protein
• Genetic counselling (amniocentesis)
• Virology- Cultivation of virus for vaccine production, also used to study the
infectious cycle.
• Cancer research Study the functions of various chemicals, virus and
radiation to convert normal cultured cells to cancerous cells.
• Drug screening and development

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