ANIMAL CELL CULTURE

PRINCIPLES OF CELL AND TISSUE CULTURE

CHARACTERISTICS OF ANIMAL
(in relation to cells)

HISTORY

Types of investigations/research involving Tissue Culture

TYPES OF TISSUE CULTURE

Organ culture
Primary explant
Cell culture
Secondary cells
Transformed/immortalised/continuous

Monolayer
suspension
 ANIMAL CELL CULTURE

ADVANTAGES OF TISSUE CULTURE

•Ability to control the environment for maximising cell growth

•Characterization and homogeneity of samples
•Economy, Scale and mechanization
•Invivo modelling

LIMITATIONS

•Necessary Expertise
•Quantity and Cost
•Dedifferentiation and Selection
•Origin of cells
•Instability
PRINCIPLES OF CELL AND TISSUE CULTURE

The Cell
(basic unit of structure for living organisms
building block of life)

Hereditary information: DNA, regulates all cellular functions

Transmit information to the next generation of cells

Carry out specialized and vital functions

involved in the metabolism and anabolism of the cells

Extract and use chemical energy stored derived

from metabolic pathways

Take in nutrients and convert them into energy

Response to external and internal stimuli such as

changes in temperature, pH or nutrient levels
 CHARACTERISTICS OF ANIMAL
(in relation to cells)
 Multicellular-composed of many cells (>100 trillion cells)

 Vertebrate has more than 100 types of cells

 Animal consist of numbers of different types of cells which differ in

a) size
b) shape
c) structure
d) function

 Cell specialization: Cell associates in a very organised patterns to

perform specialised functions —each cell does not have to carry
out all the activity necessary for the life of the organisms
 CHARACTERISTICS OF ANIMAL continue
(in relation to cells)

 Each cell type has its own role eg. as secretory cells, to
contract, to store, to transmit electrical impulses etc.

 Many animal cells can with special care be induced to

grow outside of their organ or tissue of origin

 Cell tissue, organ can be isolated and grown in a plastic

ware (substrate) at different temperature, supplemented
with medium containing all nutrients and growth factors
 HISTORY
1965 Robert Hooke
Described cork cells as small rooms that monks lived in
Latin cellula-a small room
- Anthony van Leeuwenhoek
Designed the first simple microscope

1830 Mathias Schleiden and Theodore Schwann

Developed the `Cell Theory’ that states
(1) All organisms are composed of one or more cells
(2) Cell is a `basic unit’ of structure for all organisms

1855 Rudof Virchow

All cell can only arise by cell division from pre-existing cells

Eg. procaryotic cells: bacteria: binary fission

Eucaryotic cells: fungi, plants animals : mitosis and meiosis
 HISTORY continue
1885 Wilhelm Roux
Establish the principle of tissue culture
Removed a portion of the medullary plate of a chicken
embryo and maintained in warm saline for several days

1907 Ross Granville Harrison -zoologist

Established the methodology of tissue culture
demonstrated the growth of frog nerve cell processes in a
medium of clotted lymph

1912 Perfection of the aseptic technique

Culturing connective tissue for extended periods
Show the contractility of heart muscle tissue over 2-3 mths
period

1920 Established procedures for organ culture

 HISTORY continue
1943 Established – primary culture from chick embryo tissue
- continuous cell line from rodent tissue
- transplantable tumours
1952 Produced continuous human cell line from human tumour
tissues

1950 Beginning of cell culture experimentation

-1954 Perfected methods and defined media
Described attachment factor and feeder layers

1961 Human cells have finite lifespan

1975 Developed monoclonal antibodies

1988 Cultivate embryonic stem cells/hematopoeitic cell lines/stem

cells therapy
Renin
erythropoietin
 Mouse kidney cells

Secondary Hamster kidney cells

Primary cell cultures split several times:

disadvantage:
fibroblasts overgrow the epithelial cells
 Types of investigations/research involving Tissue Culture

 Intracellular activity
Eg the replication and transcription of DNA, protein synthesis,
energy metabolism, drug metabolism

 Intracellular flux
RNA, hormone receptors, metabolites, calcium signal transduction
and membrane trafficking,

 Cell-cell interaction
Morphogenesis, paracrine control, cell proliferation kinetics,
matrix (glycoproteins and proteoglycans) interaction,
metabolic cooperation, cell adhesion and motility

Paracrine Growth factors: growth factors act as morphogens eg. KGF produced
by dermal fibroblasts :
Regulates and influences epidermal differentiation
Types of investigations involving Tissue Culture.. continue

 Environmental interaction
Infection, drug action, ligand receptor interactions,
cytoxicity, mutagenesis, Carcinogenesis

 Cell products
Secretion, biotechnology, bioreactor design, product
harvesting, downstream processing

 Genetics
Genetic analysis,transfection, infection,
transformation,immortalization, senescence
 TISSUE CULTURE APPLICATION..continue

Production of Biological products using

recombinant DNA technology in animal cells: Expression of authentic
recombinant proteins
•animal cells are of a higher order than bacterial cells
•giving good post-translational modifications of proteins
compared to bacterial cells.
•More complex proteins can be made eg. lymphokines, interferons,
hormones like human growth factor, erythropoietin

Tissue engineering: Reconstituton and replacement of

damaged tissues and cells eg. Skin grafting,
neural graft – to replace chemical to the brain of
Parkinson’s disease patient

Production of Vaccines: polio, measles, mumps, rubella chicken pox;

viral vectored vaccines, gene deleted vaccines
 TISSUE CULTURE APPLICATIONS..continue
To study processes that take place in animal cells-
Understanding the metabolic and anabolic pathways, to
reengineer cells to differ its functions

Monoclonal antibodies production: Kohler and Milstein

produced the first hybridoma

Immunosuppression therapy

Gene targeting

Amniocentesis

Cytotoxicity testing

Diagnostics for viral diseases, isolation and identification of

viruses.

Cell surface receptors study

TYPE OF TISSUE CULTURE
 TYPE OF TISSUE CULTURE
1. Organ culture

•Retain 3D architecture characteristic in vivo

•retain differentiated properties
•Do not grow rapidly, only at the periphery
•Restricted to embryonic tissues, poor reproducibility
•Size limitation

kidney
•Mouse, mammals,
•Embryos ORGAN CULTURE
•Embryonated Eggs
(best: for TC : embryo, young)
because stage of differentiation)

dissection
Grow in media
Selection •Explants
of tissue •Explants with outgrowth

Pieces of tissue
organ Whole organ ( limited by size)
2. Primary explant culture

Fragment of tissues placed at glass/plastic-liquid interphase, cell

attachment and migration

explanted directly from a donor organism

Capable of one or two divisions, eventually senesce and die

e.g. Intestinal, tracheal explant (epithelial cells with beating cilia)

Best experimental models for invivo situations

Express characteristics not seen in cultured cells – eg cell surface

receptors
Mouse, mammals,
Embryo Primary explant
Eggs
(best: for TC : embryo, young)
because stage of differentiation)

Finely cut

Finely cut
tissue or explant

explant
Grow in media
•Explants
•Explants with outgrowth

organ
 3. Cell culture
Derived from dispersed cells taken from primary explant outgrowth
or the original tissue

Cells dispersed (mechanical or enzyme) in suspension

then cultured into adherent monolayer on solid substrate or
as cell suspension in medium

-able to propagate into continuous cell lines (subculture and passage)

- predominance of high growth cell - Increase total number of cells
- increase in uniformity
- phenotypic changes due to uptake of new genetic material
(transformation)

-These cultures have lost their histotypic architecture and often

some of the biochemical properties associated with it, However they
-can be propagated
-can be characterized
-can be stored by freezing
•Mouse, mammals,
•Embryo Cell culture
•Embryonated Eggs
(best: for TC : embryo, young)
because stage of differentiation)
Finely cut

Grow in media
-monolayer
Finely cut -suspension cells
tissue or explant

explant

organ
Enzymic digestion
 4. Secondary cells

Explanted from a donor organism

Given the right culture conditions-divide and grow for
some time in vitro e.g. 50-1900 generations

Do not continue to divide indefinitely, physical characteristics

may change
Will eventually senesce and die

Factors which control the replication of such cells in vitro :

related to the degree of differentiation of the cell

Eg. MRC5 cells : secondary human lung fibroblasts, for study of

viruses and vaccine production
Undergo between 60-70 doublings before senescence
5. Transformed or Immortalised or continuous cells

apparently capable of an unlimited number of population doublings;

Can grow and divide indefinitely in vitro – correct culture

conditions are maintained

Transformed- growth properties have been altered

Usually cancer or tumour cells

Transformation – complex processes, can occur by many

different routes

e.g. infection by transforming tumour viruses or

chromosomal changes
Monolayer culture

•Cell migrate/proliferate by attachment

•Anchorage dependent

•Commonly exhibited by most normal cell type

Suspension cell

•Can proliferate without attachment (anchorage independent)

•Restricted to hematopoietic cell, transformed cell or tumors

Histotypic culture or organotypic culture-attempt to mimic 3

dimensional structure of origin tissues in cell culture
Eg.-aggregates in suspension
High density perfusion in microcapillary bundles or membranes
ADVANTAGES OF TISSUE CULTURE
•Ability to control the environment for maximising cell growth
•Characterization and homogeneity of samples
•Economy, Scale and mechanization
•Invivo modelling

LIMITATIONS

•Necessary Expertise
•Quantity and Cost
•Dedifferentiation and Selection
•Origin of cells
•Instability
 ADVANTAGES

1. Control of the environment

a)Physiochemical parameters

i.e. pH, temperature, osmotic pressure, oxygen, carbon dioxide

b)Physiological conditions

•supplementation of medium with defined constituents i.e serum,

hormones and other regulatory substances
•Nutrient concentrations need to be regulated

c)Microenvironment

•Regulation of matrix (cell attachment and growth improved by pretreating

the subtrate : fibronectin, denatured collagen, cell-cell interaction, gaseous
diffusion)
Control of environment..continue

Treatment with specific biological compounds, can induce specific

alterations in the attachment and behavious of specific cell types.

Eg chodronectin enhances chondrocytes adherence ,

laminin promotes epithelial cells

Preservation of cell lines indefinitely - stored in liquid nitrogen

(-196oC)
 2. Characterization and homogeneity of cells

Tissue samples are invariably heterogenous

- consists of many types of cells

Replicates from one tissue – many cell types

After further subculturing (1-2 passgess)
– homogeneity attained
– uniform type of cells

-selective pressure of culture conditions tends to produce

a homogenous culture of the most vigorous cell types

Further replicates at each subculture – virtually identical

to each other
-reduced the need for statistical analysis of variance
 Characterization and homogeneity of cells..continue

Characterization: chromosomal analysis and DNA content,

cytology and immunostaining

Free of contamination (extraneous bacteria, viruses, fungi,

mycoplasma)

Free of contamination from other cell lines

Characteristic of line may be perpetuated over several generation

Validation and accreditation: Records of origin, history and purity

 3. Economy, scale and mechanization

Less reagent or media – cheaper

Lower and defined concentration – direct access to the cell

Compared to in vivo: 90% loss by excretion and distributon to

tissues not under study

Screening test: duplicates, triplicates , many variables

Reduction of animal use: legal, moral,ethical questions of

animal experimentaion is avoided

Microtitration, robotics- save time and economics of scale

4. Invitro Modeling of invivo condition

Development of histotypic (one-cell type) and

organotypic (more than cell types) models increased
accuaracy of the invitro modeling.

delivery of specific experimental compounds to be

regulated: C (concentration), T (duration of exposure)
and metabolic state
 LIMITATIONS OF TISSUE CULTURE

Expertise

Strict aseptic conditions

Understanding the complexity of cells-environment-media requirement
Ability to detect microbial (and mycoplasma) contamination
and cross contamination with other cell lines
To troubleshoot, diagnose and solve TC related- problems

Quantity
Large expenditure of efforts and materials
– production of relatively little tissue

Small laboratories 1-10g

Larger laboratory 10-100g
Industrial pilot plant scale: >100g
 LIMITATIONS OF TISSUE CULTURE.. continue
Origin of cells

If differentiation are lost – difficult to relate the cultured cells

to functional cells in the tissues where they are derived

Markers are not always expressed.

Media/culture condition may need to be modified, therefore markers

are expressed

Genetic Instability

Major problem with many continuous cell lines

Unstable aneuploid chromosomal constitution
Heterogeneity in growth rate and capacity to differentiate with
the population can produce variability from one passage to the next
 LIMITATIONS OF TISSUE CULTURE..continue

Dedifferentiation

Definition: `irreversible loss of the specialised properties that a cell would have
expressed in vivo’

Or `the loss of differentiated properties of tissue when it becomes malignant or

growth in culture (A mature cell returning to a less mature state).

Loss of the phenotypic characteristics typical of the tissue from which the cells
had been isolated (original)

Process reversal to differentiation: due to overgrowth of undifferentiated cells of

the same or a different lineage

Need to provide correct conditions so that many of the differentiated properties

of these cells may be restored

Serum-free selective media –allowed for the isolation of specific lineages

Major differences between animal cells in vivo and
tissue culture in vitro

Differences in cell behaviour between cultured cells

and their in vivo stem

Invivo 3D geometry and in vitro - In 2D monolayers

Lost heterotypic cell-cell interaction

Specific cell interactions characteristic of the histology

of the tissues are lost

Cells spreadout, become mobile-Proliferate – increased population

When cell line forms, it may represent only one or two cell
types-heterotypic
Major differences between animal cells in vivo and
tissue culture in vitro ..continue

The culture environment lacks the several systemic components

involved in homeostatic regulation in vivo eg. Hormones

Without this control, cellular metabolism maybe more constant in vitro

than in vivo but may not be truly representative of the tissue from which
the cells were derived
 Terminology

Senescence: The point at which a cell or cell culture terminally ceases to grow.

Serum free media: specialised medium that contains additional supplements

and growth factors so that cells can grow in the absence of animal sera.
It is still the case that only cells adapted to serum-free growth will
prosper in serum-free media.

Phenotype. The expressed characteristics of a cell or cell culture

This includes the morphology, markers, products secreted and all other
physical attributes.

A culture started from cells, tissues or organs taken directly from organisms.
A primary culture may be regarded as such until it is successfully subcultured
for the first time, when it becomes a 'cell line'.

Aneuploid: The situation which exists when the nucleus of a cell does not
contain an exact multiple of the haploid number of chromosomes; one or more
chromosomes being present in greater or lesser number than the rest.
The chromosomes may or may not show rearrangements.
 Terminology..continue
• Histotypic culture: a high density or tissue
like culture of one cell type.

• Organotypic culture: a high density or

tissue culture of more than one cell type.