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 culturePrimary explantCell cultureSecondary cellsTransformed/immortalised/continuous

Monolayersuspension

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

ANIMAL CELL CULTURE

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 functionsinvolved 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

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(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

CHARACTERISTICS OF ANIMAL continue(in relation to cells)

HISTORY196

5 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 continue1885 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 techniqueCulturing connective tissue for extended periodsShow the contractility of heart muscle tissue over 2-3 mths period

1920 Established procedures for organ culture

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 -1954

Beginning of cell culture experimentationPerfected methods and defined mediaDescribed 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

HISTORY continue

Reninerythropoietin

Mouse kidney cells

Secondary Hamster kidney cellsPrimary cell cultures split several times:

disadvantage: fibroblasts overgrow the epithelial cells

Types of investigations/research involving Tissue Culture

Intracellular activityEg the replication and transcription of DNA, protein synthesis,energy metabolism, drug metabolism

Intracellular fluxRNA, hormone receptors, metabolites, calcium signal transduction and membrane trafficking,

Cell-cell interactionMorphogenesis, 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

Environmental interactionInfection, drug action, ligand receptor interactions, cytoxicity, mutagenesis, Carcinogenesis

Cell productsSecretion, biotechnology, bioreactor design, product harvesting, downstream processing

GeneticsGenetic analysis,transfection, infection, transformation,immortalization, senescence

Types of investigations involving Tissue Culture.. 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 proteinscompared 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 ofParkinson’s disease patient

Production of Vaccines: polio, measles, mumps, rubella chicken pox; viral vectored vaccines, gene deleted vaccines

TISSUE CULTURE APPLICATION..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

TISSUE CULTURE APPLICATIONS..continue

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

TYPE OF TISSUE CULTURE

kidney

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

organ

Selectionof tissue

dissection

Pieces of tissue

Grow in media•Explants •Explants with outgrowth

Whole organ ( limited by size)

ORGAN CULTURE

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,EmbryoEggs(best: for TC : embryo, young) because stage of differentiation)

organ

explantGrow in media•Explants •Explants with outgrowth

Primary explant

Finely cut

Finely cut tissue or explant

Derived from dispersed cells taken from primary explant outgrowth or the original tissue

Cells dispersed (mechanical or enzyme) in suspensionthen 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

3. Cell culture

•Mouse, mammals,•Embryo•Embryonated Eggs(best: for TC : embryo, young) because stage of differentiation)

organ

explant

Grow in media-monolayer-suspension cells

Cell culture

Finely cut

Finely cut tissue or explant

Enzymic digestion

4. Secondary cells

Explanted from a donor organismGiven the right culture conditions-divide and grow forsome time in vitro e.g. 50-1900 generations

Do not continue to divide indefinitely, physical characteristicsmay changeWill 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 productionUndergo 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 cultureEg.-aggregates in suspensionHigh 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 typesAfter 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 conditionsUnderstanding the complexity of cells-environment-media requirementAbility to detect microbial (and mycoplasma) contamination and cross contamination with other cell linesTo troubleshoot, diagnose and solve TC related- problems

QuantityLarge expenditure of efforts and materials – production of relatively little tissue

Small laboratories 1-10g Larger laboratory 10-100g Industrial pilot plant scale: >100g

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 linesUnstable aneuploid chromosomal constitutionHeterogeneity 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

LIMITATIONS OF TISSUE CULTURE..continue

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

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 cultureThis 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

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.