Lecture

Animal Biotechnology
Haji Akbar

Establishment of cell
culture:
Many types of animal cells can grow in vitro
such as tumour cells, neuroblastoma cells etc.
Based on experiment continuous (immortal)

cell line can be developed from cultured

tissue.
It is not necessary each cell that place on

medium start growth.

Basis on growth responses culture cells

are divided into three types:

Precursor

or master cell or stem cells,

undifferentiated but committed cells and
mature differentiated cells

Cell cultures are at a stage of equilibrium of

these types of cells which may be shifted by

manipulating growth conditions.

e.g. in the presence of low serum, suitable

hormones, cell matrix interaction and high
cell density, cell differentiation is promoted
where as in the presence of high serum

suitable growth factors and low cell density

cell proliferation is promoted.

In addition the type cells growing in culture

determined by their representative sources

from where these have been derived.

e.g. high Stem cell will be found in

culture driven from embryos

b.c they are capable of frequent cell

division
culture.

as

compared

to

adult

cell

And cells from stress conditions such as

muscles fibroblast etc will be committed

precursor . (C.P cells have limited life)

i. Evolution of cell
line:
Primary culture sub culture in

special
growth medium under control condition.

Some cells attached and proliferate to

yield single cell line.

The suspension should be diluted with

fresh medium.

ii. Primary Cell culture:

After disaggregation culturing ------- primary

cell culture.
Refers to cultures prepared directly from the

tissue directly taken from animals.

Proteolytic enzymes (trypsin and

Collagenase) are commonly used to break

the protentious material between cells.

Primary culture becomes cell line only after

first subculture.
A cell line is a permanently established cell

culture that will proliferate indefinitely

given appropriate fresh medium and space.
Subculture is needed when nutrient present

in medium for cell growth diminish.

Secondary cell culture

Primary
cell culture
and
cell
line:can be passaged to form
secondary cell culture.

Primary Cell culture cannot viable for a long

time because the cell utilize all nutrients of

the medium. Therefore sub culturing is
needed.
During repeated sub culturing and selection

the cell line gets evolved and properly

established consisting of proliferating cells.

Thus the unaltered form of cells line is called

continues cell line which propagated in logarithmic

ways.
Any change in continues cell line may discontinue

the increase in cell number. Brought about by

chemical, spontaneous mutation or viruses (EBV).
The phenomenon of alteration in continuous cell

line is called in vitro transformation.

Cell line consists of several similar and dissimilar

lineages. A defined cell lineage

properties is called Cell strain.

having

specific

Nomenclature of cell
line:
Represented in abbreviated form either
derived from the source of Cells, name of
institute or association with virus
e.g. EB, Epstein Barr; WI, Wistar Institute)
Numbered If more than one cell line (e.g.

EB1, EB2)
Furthermore, a cell line is given the number

of population doubling e.g. EB1/1.

Types of cell line:

Different types of cell line produced from

different tissues or organs.

Broadly they are grouped into: Finite cell line

and continuous cell line.

Finite cell line:
Grow through a limited number of cell
generations and have limited life.
Grow slowly and form monolayer.
Doubling time ranges from 24 to 96 hours.
Anchorage dependent, contact inhibition and
density limitation.

The transformed cells in vitro bears

the following differences.
Enhanced growth and proliferation due to

rapid growth rate.

Existence of alter ploidy i.e. aneuploidy or

heteroploidy due to alter chromosomes

number.

Different cell shape and organization of

microfilaments.

Ability to translocate,

Factors affecting subculture in

vitro:

Several factors that influence differentiation

and proliferation of cells when subculture in

vitro as below;

Mammalian cells need attachment to a

solid surface so their maximum numbers

are limited by the surface area available.

Serum is added in medium (mixture of

several kinds of growth promoting and

growth inhibitory factors).

Somatic cell fusion:

Fusion of two different cells to produce a hybrid

cell.
Application in biotechnology:
Study control gene expression and

differentiation.
Gene mapping
Malignancy
Viral replication
Antibodies production via hybridoma technology

Continue!!!
Macrophages

fuse around
bacterial cell in the tissues.

the

foreign

body

or

Bones cells also known to undergo somatic fusion.

In culture cells are induce to fuse by some viruses

e.g. Sendai virus.

This virus induces first to form heterokaryon and

during mitosis chromosomes of heterokeryon are

brought two poles and which latter on to form hybrid.
Some chemicals such as polyethylene glycol also

induce somatic cell fusion.

Organ culture:
Means the maintenance of a part of tissue or part

of organ or whole organ in vitro.

or

The culture of complete living organs (explants) of

animals and plants outside the body in a suitable

culture medium.
Animal organs must be small enough to allow the

nutrients in the culture medium to penetrate all

the cells.
It is easier to culture embryonic organ than the

adult animals.(O2 95% required).

Different

methods are required for

culturing of embryonic and adult organs.

Embryonic organ can be culture by any of the

following methods:
Organ culture on plasma clots:
Prepared by mixing 5 drops of embryo extract

with 15 drops of plasma in a watch glass on a

cotton wool pad (put in Petri dish).
Moistened

evaporation.

the

cotton

to

decrease

Organ culture on agar medium:

Solidify culture medium with agar is also

used for organ culture.

Adults fail to survive whereas embryonic

organ grow well.

The media consist of ingredients:
Agar (1%basal salt solution) chick embryo

extracts and horse serum in the ratio of 7:3:3

Organ culture in liquid medium:

Consist of all the ingredients except agar.

Perforated metal gauze or cellulose acetate or

a raft of lens paper is used to provide support.
Whole embryo culture:
Chick embryo: eggs incubation at 38C for 40-42

hrs. sterilization (70% ethanol)

Blastoderm placed on watch glass containing

medium
incubate
developments

at

37.5C

for

further

Growth phase of Cells in culture

* Lag phase
: adapt to new environment; repair cell membrane
damage

* Log phase

: exponential growth: 90-100% of cells are dividing

* Plateau or Stationary phase

: cell growth ~ 0-10%
: Contact inhibition of movement
: Density limitation of growth

Advantage of using cell culture

Can be observed microscopically

Genetic homogeneity
Environment

(pH, Temp., osmotic pressure, O2 and CO2 tension

Rapid
Requires less amount of material

Primary cell culture and Establishment cell line

Preparation of cell suspension from intact tissue

1. Single cell preparation

: use mechanical, Chemical, and/or enzymatic method

2. Disaggregate or dissociate cell

: cutting, homogenizing, rotary shaker, vortex,
pipette, teasing

 Preparation of cell suspension from intact tissue

(cont.)
** Enzymes **

Trypsin (crude)
: from cattle and pigs pancrease

: contain Chymotrypsin, elastase, ribonucleas

deoxyribonuclease and amylase

** Enzymes **

Collagenase
: for connective tissue
Pronase
: for fibroblast
Elastase
: for fibroblast protein
Deoxyribonuclease
: for DNA

 Preparation of cell suspension from intact tissue

(cont.)
Source of tissue
: Young animals
e.g. kidney (Monkey, Dog, Rabbit),
Chick embryo
: Old animal tissue contains a large
amount
of connective tissue

 Preparation of cell suspension from intact tissue

(cont.)
1. Removal of tissue and place in Isotonic
(or growth medium with antibiotic)
2. Trim off unwanted parts and cut into smaller pieces
3. Dissociate cells using enzyme

4. Remove large debris and wash cells

 Preparation of cell suspension from intact tissue

(cont.)
5. Suspend cells in growth medium accordingly

6. Pipette into culture vessel and incubate

Primary cell culture

subculture
Cell line

cloning

Physical separation

Subline or cell strain

 Establishment of Suspension culture

: Heteroploid cell line ; suspension culture

1. Resuspend trypsinized cells to 5 x 105 cells/ml

2. Continous stirring

3. Cellular product be removed

and replenished with fresh medium

Contamination

fungal contamination
Bacterial contamination
Mycoplasma contamination
Viral contamination
Other cell line

Sources of contamination

Original tissue : primate virus, mycoplasma

Biological: Serum
Laboratory personnel : from body, aerosols
Laboratory environment
: culture vessel cap
: humidified Incubator
: Water bath
: Insect

Other Parameters

Incubator : humidified
Incubation temperature
pH and Buffer
Gas phase
Osmolarity and water grade

Cell Storage

*** Prevent genetic drift ***

: Freezing Medium
* Serum (~ 20-90%)
* Culture medium
* Cryoprotective agent : ~ 5-10%

(DMSO, Glycerol)

Cell Storage

: cell concentration ~ 5 x 106-2 x 107 cells/ml

: Temp. decline rate 1-10oC/min
* (-20oC) Freezer
* (-70oC) Freeze :

6M-2 yr

* liquid nitrogen: Years

: % cell viability is decrease 2-3%/yr

Cell Thawing and culture

** Slow Freeze : Quick Thaw **

1. Quick thaw in 37oC water bath

2. Pipette to culture vessel
3. Slowly growth medium adding
4. Incubate for overnight
5. Refresh with new growth medium

Fibroblast-liked cell

Epithelium-liked cell

Monolayer cell culture