Chapter 5: Animal cell culture [B. Pharma SEM 3]

1. PREPARED BY
Bhargavi Mistry
[Assistant Professor, School of
Pharmacy Rk University]
ANIMAL CELL
CULTURE

2. LIST OF CONTENT
01
Introduction
02
Types of cell culture
03
Types of animal cell
culture
04
Requirements for
animal cell culture
05
Advantages of
animal cell culture
06
Applications
Prepared by: Bhargavi Mistry 2

3. Ø Cell culture refers to the process by which cells are grown under controlled
conditions, generally outside their natural environment. (In vitro)
Ø Animal cell culture defines the cells taken from the organ of an experimental animal.
The cells may be removed directly or by mechanical or enzymatic action.
Ø Examples of cells used to culture are fibroblasts, lymphocytes, cells from cardiac
and skeletal tissues, cells from the liver, breast, skin, kidney, and different types of
tumor cells.
Ø Roux in 1885 for the first time maintained embryonic chick cells in cell culture.
Ø Cell culture was first successfully undertaken by Ross Harrison in 1907.
INTRODUCTION
Prepared by: Bhargavi Mistry 3

4. CELL CULTURE
Precursor cell
culture
Differentiate cell
culture
Stem cell culture
An undifferentiated
cell committed to
differentiate
Completely differentiated
cells that have lost the
c a p a c i t y t o f u r t h e r
differentiate.
Undifferentiated cells go
on to develop into any
type of cell.
TYPES OF CELL CULTURE
Prepared by: Bhargavi Mistry 4

5. CELL CULTURE
Primary cell culture Secondary cell culture & cell line
TYPES OF ANIMAL CELL CULTURE
a) Adherent cells
b) Suspension cells
a) Finite cell lines
b) Indefinite cell lines
Prepared by: Bhargavi Mistry 5

6. Ø This is the cell culture obtained straight from the cells of the host tissue.
Ø The cells dissociated from the parental tissue are grown on a suitable container
and the culture thus obtained is called primary cell culture.
Ø Such culture comprises mostly heterogeneous cells and most of the cells divide only
for a limited time. However, these cells are much similar to their parents.
Primary cell culture
01
Prepared by: Bhargavi Mistry 6

7. These cells are anchorage-dependent and propagate as a monolayer. These cells need to
be attached to a solid or semi-solid substrate for proliferation.
E.g. Fibroblasts and Epithelial cells
Suspension cells do not attach to the surface of the culture vessels. These cells are also
called anchorage-independent or non-adherent cells which can be grown floating in the
culture medium.
E.g. Hematopoietic stem cells (derived from blood, spleen, and bone marrow) and
tumor cells
a) Adherent cells:
b) Suspension cells:
Prepared by: Bhargavi Mistry 7

8. Ø The culture established from primary culture is known as secondary culture or
subculture or sub clone.
Ø The process involves removing the growth media and disassociating the adhered
cells(usually enzymatically).
Ø Sub-culturing of primary cells to different divisions leads to the generation of cell
lines.
Secondary cell culture & cell line
02
Prepared by: Bhargavi Mistry 8

9. Cell line
The primary culture, when subculture becomes a cell line or cell strain that can be finite
or continuous, depending on its lifespan in culture.
a) Finite cell lines:
Cell lines with a limited number of cell generations and growth. These cells are finite cell
lines.
a) Indefinite cell lines:
Cell lines obtained from in vitro transformed cell lined or cancerous cells are indefinite
cell lines and can be grown in monolayer or suspension form.
Prepared by: Bhargavi Mistry 9

10. REQUIREMENTS FOR ANIMAL CELL CULTURE
Substrates for cell growth
Glass, Plastic, & Palladium as
a substrate
Prepared by: Bhargavi Mistry 10
01
02
03
Culture media
Natural media: Eg, Clots,
Artificial media: Eg. Serum
containing media
Sterilization of Glassware,
Equipments and culture media
04
Equipment required
Laminar air flow, Centrifuged, Microscope
05
06
Aseptic room, Culture room
Data collection/observation room

11. GROWTH REQUIREMENTS
Ø The culture media used for cell cultures are generally complex, and culture condition
widely varies for each cell type.
Ø However, media generally include amino acids, vitamins, salts (maintain osmotic
pressure), glucose, a bicarbonate buffer system (maintain a pH between 7.2 and
7.4), growth factors, hormones, O2, and CO2.
Ø To obtain the best growth, the addition of a small amount of blood serum is usually
necessary, and several antibiotics, like penicillin and streptomycin, are added to
prevent bacterial contamination.
Prepared by: Bhargavi Mistry 11

12. Ø Temperature varies on the type of host cell. Most mammalian cells are maintained
at 370C for optimal growth, while cells derived from cold-tolerate a wider
temperature range (i.e. 150C to 26°C).
Ø Actively growing cells of the log phase should be used which divide rapidly during
culture.
Prepared by: Bhargavi Mistry 12

13. PROCESS TO OBTAIN PRIMARY CELL CULTURE
Ø Primary cell cultures are prepared from fresh tissues.
Ø Pieces of tissues from the organ are removed aseptically; which are usually minced
with a sharp sterile razor and dissociated by proteolytic enzymes (such as
trypsin)apart the intercellular cement.
Ø The obtained cell suspension is then washed with a physiological buffer (to remove
the proteolytic enzymes used).
Ø The cell suspension is spread out on the bottom of a flat surface, such as glass or petri
dish.
Ø This thin layer of cells adhering to the glass or plastic dish is overlaid with a Suitable
culture medium and is incubated at a suitable temperature.
Prepared by: Bhargavi Mistry 13

14. Prepared by: Bhargavi Mistry 14
Figure: Animal cell culture for normal cells

15. Prepared by: Bhargavi Mistry 15
Figure: Animal cell culture for continuous cells

16. ASEPTIC TECHNIQUES
Ø Microbial infections (bacterial or fungal), can occur in cell culture creating a problem
to identify and eliminate the cell culture.
Ø Therefore, all cell culture methods should be carried out in a sterile environment with
proper aseptic techniques.
Ø Work should be done in laminar airflow with the constant unidirectional flow of
HEPA-filtered air.
Ø All materials, solutions, and the whole atmosphere should be contamination free.
Prepared by: Bhargavi Mistry 16

17. CRYOPRESERVATION
Ø If a surplus of cells is available from sub-culturing, they should be treated with the
appropriate protective agent ( DMSO or glycerol) and stored at temperatures below
-130°C until they are needed.
Ø ADVANTAGES:
1. Reduced risk of microbial contamination.
2. Reduced risk of cross-contamination with other cells.
3. Reduced risk of genetic drift and morphological changes.
4. Work conducted using cells at a consistent passage number.
5. Reduced costs.
Prepared by: Bhargavi Mistry 17

18. Prepared by: Bhargavi Mistry 18
ADVANTAGES OF ANIMAL TISSUE CULTURE
01
02
03
04
Cell cultures are easy to store
for a longer period in liquid
nitrogen.
Cell categorization is easy for
immunological cytological
studies.
Biological studies can be
easily done using cell
cultures.
Cell culture techniques can
reduce the use of animals
in various studies.

19. APPLICATIONS
Prepared by: Bhargavi Mistry 19
01 02 03 04 05 06 07
Vaccine
production
Cancer
research
Virology
Cell-based
manufacturing
Genetic
engineering
Drug screening
& development
Gene therapy

20. Ø The most important uses of cell culture is in research and production of vaccines.
Ø The ability to grow large amounts of virus in cell culture eventually led to the
creation of the polio vaccine, and cells are still used today on a large scale to
produce vaccines for many other diseases, like rabies, chicken pox, hepatitis B,
and measles.
Ø In early times, researchers had to use live animals to grow poliovirus, but due to
the development of cell culture technique they were able to achieve much greater
control over virus production and on a much larger scale which eventually develop
vaccines and various treatments.
Vaccine production
01
Prepared by: Bhargavi Mistry 20

21. Ø Since both normal cells and cancer cells can be grown in culture, the basic
differences between them can be closely studied.
Ø In addition, it is possible, by the use of chemicals, viruses and radiation, to convert
normal cultured cells to cancer causing cells. Thus, the mechanisms that cause
the change can be studied.
Ø Cultured cancer cells also serve as a-test system to determine suitable drugs and
methods for selectively destroying types of cancer.
Cancer research
02
Prepared by: Bhargavi Mistry 21

22. Ø Cell cultures are widely used in the clinical detection and isolation of viruses, as
well as basic research into how they grow and infect organisms.
Ø The large-scale production of viruses for use in vaccine production.
E.g. polio, rabies, chicken pox, hepatitis B and measles.
Ø The large-scale production of cells that have been used to produce genetically
engineered proteins that have medicinal or commercial value.
E.g: monoclonal antibodies, insulin, hormones, etc.
Ø Production of artificial skin cells.
Virology
03
Cell-based manufacturing
04
Prepared by: Bhargavi Mistry 22

23. Ø The ability to transfect or reprogram cultured cells with new genetic material
(DNA and genes) has provided a major tool to molecular biologists
Ø Cell-based assays have become increasingly important for the pharmaceutical
industry, not just for cytotoxicity testing but also for high throughput screening of
compounds that may have potential use as drugs.
Genetic engineering
05
Drug screening & development
06
Prepared by: Bhargavi Mistry 23

24. Ø The ability to genetically engineer cells has also led to their use for gene therapy.
Ø Cells can be removed from a patient lacking a functional gene and the missing or
damaged gene can then be replaced.
Ø The cells can be grown for a while in culture and then replaced in the patient.
Ø An alternative approach is to place the missing gene into a viral vector and then
“infect” the patient with the virus in the hope that the missing gene will then be
expressed in the patient's cells.
Gene therapy
07
Prepared by: Bhargavi Mistry 24

25. REFERENCES
Prepared by: Bhargavi Mistry 25
1) Dr. Imtiyaz Wani, Pharmaceutical Microbiology, PV books, First color edition,
Page no. 221 to 230
2) Dr. Vimal Kumar, Pharmaceutical Microbiology, Technical Publications, Page
no. 15-1 to 15-10
3) Animal cell culture: https://biologyreader.com/animal-cell-culture.html

26. QUESTIONS
Prepared by: Bhargavi Mistry 26
1) Write a short note on the establishment of Primary cell culture.
2) Describe animal cell culture in brief.
3) Write applications of animal cell culture.
4) Define the following terms:
Cell culture, Cell line, and Primary cell culture, Secondary cell culture.

27. THANK YOU
Prepared by: Bhargavi Mistry 27