A brief description of animal cell culture and its applications

1. By:
Dr. Fahad Khan
Assistant Professor
Department of Biotechnology
Noida Institute of Engineering & Technology, Greater
Noida
Basic of Animal Cell Culture

2. Introduction to Cell Culture
History
Major Advancements
Applications
Terminologies
Types of cell culture
Cell culture media, types and Components
Factors affecting media
Basic requirements
Cell culture techniques, Disaggregation methods
Passaging
Cell counting and cell viability
Contamination
Cryopreservation

3. *Cell culture has become one of the major tools used in the life
sciences today.
*Cell culture involves the distribution of cells in an artificial
environment (in vitro) which is composed of the necessary
nutrients, ideal temperature, gases, pH and humidity to allow
the cells to grow and proliferate.
* In this procedure cells are directly isolated from body or
disaggregated by enzymatic or mechanical procedure or they
may be derived from cell lines or cell strains.
*This environment usually consists of a suitable glass or plastic
culture vessel containing a liquid or semisolid medium that
supplies the nutrients essential for survival & growth.

4. *1878: Claude Bernard proposed that physiological systems of an
organism can be maintained in a living system after the death of an
organism.
*1885: Roux maintained embryonic chick cells in a saline culture.
*1897: Loeb demonstrated the survival of cells isolated from blood and
connective tissue in serum and plasma.
*1907: Harrison cultivated frog nerve cells in a lymph clot and observed
the growth of nerve fibers in vitro for several weeks. He was considered
by some as the father of cell culture.
*1910: Burrows succeeded in long term cultivation of chicken embryo
cell in plasma clots. He made detailed observation of mitosis.

5. *
*1911: Lewis and Lewis made the first liquid media consisted of sea
water, serum, embryo extract, salts and peptones.
*1913: Carrel introduced strict aseptic techniques so that cells could be
cultured for long periods.
*1916: Rous and Jones introduced proteolytic enzyme trypsin for the
subculture of adherent cells.
*1940s: The use of the antibiotics penicillin & streptomycin in culture
medium decreased problem of contamination in cell culture.
*1948: Earle isolated mouse L fibroblasts which formed clones from
single cells.
*1952: G. O. Gey established a continuous cell line HeLa from a human
cervical carcinoma patient Henrietta Lacks.
*Dulbecco developed plaque assay for animal viruses using confluent
monolayers of cultured cells.

6. *
*1955: Eagle studied the nutrient requirements of selected cells in
culture & established first widely used chemically defined medium.
*1965: Harris and Watkins were able to fuse human and mouse cells
by the use of a virus.
*1975: Kohler and Milstein produced the first hybridoma capable of
secreting a monoclonal antibody.
*1978: Sato established the basis for the development of serum-free
media from cocktails of hormones and growth factors.
*1982: Human insulin became the first recombinant protein to be
licensed as a therapeutic agent.
*1985: Human growth hormone produced from recombinant
bacteria was accepted for therapeutic use.

7. *First development was the use of antibiotics which inhibits
the growth of contaminants.
*Second was the use of trypsin to remove adherent cells to
subculture further from the culture vessel.
*Third was the use of chemically defined culture medium.

8. Areas where cell culture technology is currently playing a
major role:
Model systems for:
*Studying basic cell biology, interactions between disease causing
agents and cells, effects of drugs on cells, process and triggering of
aging & nutritional studies.
Toxicity testing:
*Study the effects of new drugs.
Cancer research
*Study the function of various chemicals, virus & radiation to
convert normal cultured cells to cancerous cells.

9. *
Virology
*Cultivation of virus for vaccine production.
*Also used to study their infectious cycle.
Genetic Engineering
*Production of commercial proteins, large scale production of
viruses for use in vaccine production e.g. polio, rabies, chicken
pox, hepatitis B & measles.
Gene therapy
*Cells having a functional gene can be replaced to cells which are
having non-functional gene

10. *Controlled and defined physiological conditions.
*Cell homogeneity can be done by serial passages.
*Requirement of reagents and chemicals in smaller quantities than
in vivo experiment.
*Ethical, social and legal issues are less in comparison to
animal/human experiment.
Disadvantages:
• Expertise is required for doing animal cell culture.
• More expensive technique as compared to animal tissue.
• Unstable chromosome may be achieved.

11. *Primary Cell Culture: When cells are surgically removed from an
organism and placed into a suitable culture environment they will
attach, divide and grow.
*Cell Line: When the primary culture is subcultured and they
show an ability to continuously propagate.
*Anchorage dependency: Cells grow as monolayers adhering
to the substrate (glass/ plastic)
*Passaging/subculturing: The process of splitting the cells.
*Finite cells: When the cells has finite life span.
*Continuous cell lines: When the cells can grow upto infinite
lifespan.
*Confluent: Situation wherein cells completely cover the
substrate

12. Cell
Culture
Primary cell
culture
Secondary cell
culture/Cell
Line
Adherent
cell culture
Suspension
cell culture
Finite cell
culture
Continuous
cell culture

13. *Once live cells are isolated and separated from an individual
and placed into a suitable culture environment, these cells will
attach, proliferate and grow.
*Primary cell culture is the maintenance and growth of cells
dissociated from the primary or original tissue like liver, blood
or kidney.
*Cell are isolated by using enzymatic and /or mechanical
methods and cultures in specialized nutrient rich culture
medium in suitable glass or plastic containers.
*Cells grown in primary cell culture usually have the same
morphology and karyotype as the tissue they have been
originated from.
*Examples: Mouse embryos, Chick embryos, Human biopsy
materials

14. Depending on the type of cells growing in culture, primary cell culture can
be of two types- Adherent and Suspension:
Adherent cell culture:
*In this type of culture, adherent cells are generally obtained from tissues
of immobile organs such as liver or kidney where these were immobile
and are fixed in connective tissue.
*These cells shows the requirement of attachment for growth as these cells
need solid surface for attachment and only then can grow. These cells are
called as anchorage dependent cells.
Suspension cell culture:
*Suspension cultures are obtained from cells of the free floating organ as
blood system since these cells are suspended in plasma for e.g.
lymphocytes.
*These cell types do not require any attachment or surface for growth and
are known as anchorage independent cells or suspension cells.

15. *When primary culture is passaged or sub-cultured, it becomes
secondary culture or cell line.
*Passaging or Subculture means removal of medium and transfer
of cells from previous culture vessel to another fresh culture
vessel with fresh growth medium.
*It is regularly required for the supply of fresh nutrients and
increasing space for continuously dividing cells.
*Subculturing of cells from one culture vessel to another involves
disassociating the adhered cells by using enzyme trypsin and
adding fresh growth media.
*It depends on the characteristics and growth rates of particular
cell types to decide the subculture or splitting ratio.

16. *On the basis of the life span of culture, the cell lines are categorized
into two types - finite or continuous depending upon whether it has
limited culture life span or it is immortal in culture.
Finite Cell Lines:
*Cell lines which grows for a limited life span and have limited
number of cell divisions (around 20 - 80 population doublings or
subcultures) are known as finite cell lines.
*The factors which manage the division of these cells in vitro are
related to the type and differentiation level of the starting cell type.
Continuous Cell Lines:
*Those cell line which get transformed or immortalized either
automatically or manually during subcultures under in vitro culture
conditions changes into continuous cell lines.
*These types of cell lines gains potential for sub culturing
indefinitely, becomes anchorage independent, lack contact
inhibition, and accumulate aneuploidy or heteroploidy.

17. Cell line Organism Origin Tissue
HeLa Human Cervical cancer
293-T Human Kidney (embryonic)
A-549 Human Lung carcinoma
CHO Hamster Ovary
DU145 Human Prostate Cancer
Cervical
Cancer HeLa
cell line
Normal
HEK293 cells