Dr Siti Suri Lecture 1: Animal Cell Culture

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, contract, to store, to transmit electrical impulses etc.

to

 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

1885

HISTORY continue

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 -1954

Beginning of cell culture experimentation 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 cellsUnderstanding 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 •Embryonated Eggs (best: for TC : embryo, young) because stage of differentiation)

ORGAN CULTURE

dissection Selection of tissue organ

Grow in media •Explants •Explants with outgrowth

Pieces of tissue 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, Primary Embryo Eggs (best: for TC : embryo, young) because stage of differentiation)

explant

Finely cut

Finely cut tissue or explant explant

organ

Grow in media •Explants •Explants with outgrowth

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, Cell •Embryo •Embryonated Eggs (best: for TC : embryo, young) because stage of differentiation) Finely cut

Finely cut tissue or explant

explant

organ Enzymic digestion

culture

Grow in media -monolayer -suspension cells

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.