cell Tissue culture

1. Basics of Cell Culture and
Establishment of Cell Line

2. Introduction
•Cell culture is the process by which prokaryotic,
eukaryotic or plant cells are grown under controlled
conditions. But in practice it refers to the culturing
of cells derived from animal cells.
•Cell culture was first successfully undertaken by
Ross Harrison in 1907
•Roux in 1885 for the first time maintained
embryonic chick cells in a cell culture

3. Major development’s in cell
culture technology
• 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.

4. Why is cell culture used for?
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

5. Contd….
• Virology
Cultivation of virus for vaccine production, also
used to study there 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

6. Tissue culture
• In vitro cultivation of organs, tissues & cells at defined
temperature using an incubator & supplemented with a
medium containing cell nutrients & growth factors is
collectively known as tissue culture
• Different types of cell grown in culture includes connective
tissue elements such as fibroblasts, skeletal tissue, cardiac,
epithelial tissue (liver, breast, skin, kidney) and many
different types of tumor cells.

7. Primary culture
• Cells when surgically or enzymatically removed from an
organism and placed in suitable culture environment will
attach and grow are called as primary culture
• Primary cells have a finite life span
• Primary culture contains a very heterogeneous population of
cells
• Sub culturing of primary cells leads to the generation of cell
lines
• Cell lines have limited life span, they passage several times
before they become senescent
• Cells such as macrophages and neurons do not divide in vitro
so can be used as primary cultures
• Lineage of cells originating from the primary culture is called
a cell strain

8. Continous cell lines
• Most cell lines grow for a limited number of generations
after which they ceases
• Cell lines which either occur spontaneously or induced
virally or chemically transformed into Continous cell lines
• Characteristics of continous cell lines
-smaller, more rounded, less adherent with a higher
nucleus /cytoplasm ratio
-Fast growth and have aneuploid chromosome number
-reduced serum and anchorage dependence and grow
more in suspension conditions
-ability to grow upto higher cell density
-different in phenotypes from donar tissue
-stop expressing tissue specific genes

9. Types of cells
On the basis of morphology (shape & appearance) or on
their functional characteristics. They are divided into three.
• Epithelial like-attached to a substrate and appears flattened
and polygonal in shape
• Lymphoblast like- cells do not attach remain in suspension
with a spherical shape
• Fibroblast like- cells attached to an substrate appears
elongated and bipolar

10. Establishment of Cell Line

11. Introduction to Cell lines
 A cell line is a permanently established cell culture that will proliferate
indefinitely given appropriate fresh medium and space.
 A cell culture developed from a single cell and therefore consisting of cells
with a uniform genetic make-up.

12.  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 established.
Cell Culture

13.  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).
 At this stage, the cells have to be subcultured (i.e., passaged) by
transferring them to a new vessel with fresh growth medium to provide
more room for continued growth.
Primary culture

14.  After the first subculture, the primary culture becomes known as a cell
line or subclone.
 The term cell line refers to the propagation of culture after the first
subculture
 Cell lines derived from primary cultures have a limited life span (i.e., they
are finite), 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 line or subclone

15.  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 line.
Cell strain

16. •Mouse, mammals,
•Embryo
•Embryonated Eggs
because stage of differentiation)
organ
explant
Grow in media
-monolayer
-suspension cells
Cell culture
Finely cut
Finely cut
tissue or explant
Enzymic digestion

17. STAGES OF CULTURE
Isolated tissue
(disaggregation)
Primary cell culture(limited lifespan after certain proliferations undergo
senescence)
Finite cell cultures
continuous cell lines(immortalized cell line acquires ability to proliferate indefinitely
by transformation)

18. Culture Conditions
Culture conditions vary widely for each cell type, but the artificial
environment in which the cells are cultured invariably consists of a suitable
vessel containing the following:
a substrate or medium that supplies the essential nutrients (amino acids,
carbohydrates, vitamins, minerals)
growth factors
hormones

19.  gases (O2, CO2)
 a regulated physico-chemical environment (pH, osmotic pressure,
temperature)
 Most cells are anchorage-dependent and must be cultured while
attached to a solid or semi-solid substrate (adherent or monolayer
culture), while others can be grown floating in the culture medium
(suspension culture).

20. Cryopreservation
If a surplus of cells are available from subculturing, they should be treated
with the appropriate protective agent (e.g., DMSO or glycerol) and stored at
temperatures below –130°C (cryopreservation) until they are needed.

21. Morphology of Cells in Culture
Cells in culture can be divided in to three basic categories based on their
shape and appearance (i.e., morphology).
Fibroblastic (or fibroblast-like) cells are
bipolar or multipolar, have elongated
shapes, and grow attached to a
substrate.

22. Epithelial-like cells are polygonal
in shape with more regular
dimensions, and grow attached
to a substrate in discrete
patches.
Lymphoblast-like cells are
spherical in shape and usually
grown in suspension without
attaching to a surface.

23. Cell line Types
Norm
al
Transfor
med
Stem
cell
Taken from a
tumor tissue
and cultured
as a
single cell
type
Normal cells
underwent a
genetic change to
be tumor cells
They are Master
Cells that
generate
Other
differentiated
cell types

24. Types of Cell Lines:
Finite cell lines
Continuous cell lines

25. Finite Cell Lines :
Normal cells usually divide only a limited number of times before losing their
ability to proliferate, which is a genetically determined event known
as senescence; these cell lines are known as finite.
The cells normally divide 20 to 80 times (i.e. is 20-80 population doublings)
before extinction. The actual number of doublings depends on the species, cell
lineage differences, culture conditions etc.
The human cells generally divide 50-80 times, while murine cells divide 30-50
times before dying.

26. Continuous Cell Lines or Immortal cell lines
When a finite cell line undergoes transformation and acquires the ability to
divide indefinitely, it becomes a continuous cell line.
The continuous cell lines are transformed, immortal and tumorigenic. The
transformed cells for continuous cell lines may be obtained from normal
primary cell cultures (or cells strains) by treating them with chemical
carcinogens or by infecting with oncogenic viruses.

28. Property Finite cell line Continuous cell line
 Growth rate
 Mode of growth
 Yield
 Transformation
 Ploidy
 Anchorage dependence
 Contact inhibition
 Cloning efficiency
 Serum requirement
 Markers
Slow
Monolayer
Low
Normal
Euploidy
Yes
Yes
Low
High
Tissue specific
Fast
Suspension or Monolayer
High
Immortal, tumorigenic
Aneuploid
No
No
High
Low
Chromosomal, antigenic or
enzymatic
Comparison of properties of finite and continuous cell lines

29. Nomenclature of Cell Lines:
It is a common practice to give codes or designations to cell lines for their
identification. For instance, the code NHB 2-1 represents the cell line from
normal human brain, followed by cell strain (or cell line number) 2 and clone
number 1.
While naming the cell lines, it is absolutely necessary to ensure that each
cell line designation is unique so that there occurs no confusion when
reports are given in literature.
Further, at the time of publication, the-cell line should be prefixed with a
code designating the laboratory from which it was obtained e.g. NCI for
National Cancer Institute, Wl for Wistar Institute.

30. Selecting the Appropriate Cell Line
Consider the following criteria for selecting the appropriate cell line for your
experiments:
Species:
In general, non-human cell lines have less risk of biohazards, hence preferred.
However, species differences need to be taken into account while
extrapolating the data to humans.
Functional characteristics: What is the purpose of your experiments? For
example, liver- and kidney-derived cell lines may be more suitable for
toxicity testing.

31. Finite or continuous cell lines:
Cultures with continuous cell lines are preferred as they grow faster, easy to
clone and maintain, and produce higher yield. But it is doubtful whether
the continuous cell lines express the right and appropriate functions of the
cells. Therefore, some workers suggest the use of finite cell lines, although it
is difficult.

32. Normal or transformed:
Transformed cell lines usually have an increased growth rate and higher
plating efficiency, are continuous, and require less serum in media, but they
have undergone a permanent change in their phenotype through a genetic
transformation.

33. Growth characteristics:
The following growth parameters need to be considered:
i. Population doubling time
ii. Ability to grow in suspension
iii. Saturation density (yield per flask)
iv. Cloning efficiency

34. Stability:
The stability of cell line with particular reference to cloning, generation of
adequate stock and storage are important.
Phenotypic expression:
It is important that the cell lines possess cells with the right phenotypic
expression.

35. EXAMPLES OF ESTABLISHED CELL LINES
 May be derived from Normal or Tumor cells.
Cell line Organism Origin Tissue
HeLa Human Cervical cancer
293-T Human
Kidney (embryonic)
A-549
Human Lung carcinoma
ALC
Murine Bone marrow
CHO
Hamster Ovary
HB54
Hybridoma Hybridoma
FM3 Human
Metastatic lymph node

36. Animal cell lines and products
Cell line Product
Human tumour Angiogenic factor
Human leucocytes Interferon
Mouse fibroblasts Interferon
Human Kidney Urokinase
Transformed human kidney cell line, TCL-598
Single chain urokinase-type plasminogen activator (scu-
PA)
Human kidney cell (293) Human protein (HPC)
Dog kidney Canine distemper vaccine
Cow kidney Foot and Mouth disease (FMD) vaccine
Chick embryo fluid Vaccines for influenza, measles and mumps
Duck embryo fluid Vaccines for rabies and rubella
Chinese hamster ovary (CHO) cells
Tissue-type plasminogen activator (t-PA)
B-and gamma interferons
Factor VIII

37. Applications
 Screening of the anti cancer drugs
 Cell based bioassay
 To determine the cytotoxicity
 In vitro screening of several drugs
 Production of antiviral vaccines
 Cancer research, which required the study of uncontrolled cell division
in cultures
 Cell fusion techniques
 Genetic manipulation
 Study of the effects of toxins & pollutants using cell lines
 Study of the function of nerve cells
 Chromosome analysis of cells derived from womb