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

as compared to adult cell culture.

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 and cell line:
Primary cell culture 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 having specific properties is called Cell
strain.
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 the foreign body or bacterial
cell in the tissues.

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 the cotton to decrease evaporation.

 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 at 37.5C for further developments
 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