Introduction to Tissue culture

Objectives
After the session, students should be able to explain
the meaning of tissue culture and various types of tissue culture the application of tissue culture the advantages and disadvantages of each type of tissue culture the significant of culture environment on tissue culture the basic procedure of tissue culture the safety consideration for tissue culture work

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What is tissue culture?

In vitro culture (maintain and/or proliferate) of cells, tissues or organs Types of tissue culture
Organ culture Tissue culture Cell culture

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Organ culture
The entire embryos or organs are excised from the body and culture Advantages
Normal physiological functions are maintained. Cells remain fully differentiated.

Disadvantages
Scale-up is not recommended. Growth is slow. Fresh explantation is required for every experiment.
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Tissue Culture
Fragments of excised tissue are grown in culture media Advantages
Some normal functions may be maintained. Better than organ culture for scale-up but not ideal.

Disadvantages
Original organization of tissue is lost.

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Cell Culture
Tissue from an explant is dispersed, mostly enzymatically, into a cell suspension which may then be cultured as a monolayer or suspension culture. Advantages
Development of a cell line over several generations Scale-up is possible

Disadvantages
Cells may lose some differentiated characteristics.

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Why do we need Cell culture?

Research
To overcome problems in studying cellular behavior such as:
confounding effects of the surrounding tissues variations that might arise in animals under experimental stress

Reduce animal use

Commercial or large-scale production

Production of cell material: vaccine, antibody, hormone

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Cell culture application

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Advantages of Cell culture

Advantages:
Absolute control of physical environment Homogeneity of sample Less compound needed than in animal models

Disadvantages:
Hard to maintain Only grow small amount of tissue at high cost Dedifferentiation Instability, aneuploidy
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Types of Cell culture

1. Primary Cultures
Derived directly from excised tissue and cultured either as
Outgrowth of excised tissue in culture Dissociation into single cells (by enzymatic digestion or mechanical dispersion) usually retain many of the differentiated characteristics of the cell in vivo initially heterogeneous but later become dominated by fibroblasts. the preparation of primary cultures is labor intensive can be maintained in vitro only for a limited period of time.
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Advantages:
Disadvantages:

Types of Cell culture

2. Continuous Cultures
derived from subculture (or passage, or transfer) of primary culture
Subculture = the process of dispersion and re-culture the cells after they have increased to occupy all of the available substrate in the culture

usually comprised of a single cell type can be serially propagated in culture for several passages There are two types of continuous cultures
Cell lines Continuous cell lines
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Types of continuous culture

1) Cell lines

finite life, senesce after approximately thirty cycles of division usually diploid and maintain some degree of differentiation. it is essential to establish a system of Master and Working banks in order to maintain such lines for long periods

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Types of continuous culture

2) Continuous cell lines
can be propagated indefinitely generally have this ability because they have been transformed
tumor cells. viral oncogenes chemical treatments.

the disadvantage of having retained very little of the original in vivo characteristics

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Transformation VS Transfection
Transformation
Spontaneous or induced permanent phenotypic changes resulting from change in DNA and gene expression growth rate mode of growth (loss of contact inhibition) specialized product formation longevity loss of need for adhesion

Transfection
Introduction of DNA into a cell (like viral DNA)

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Initiation of culture
Tissue

dispersion
Primary cell culture Subculture

Stored

Cell line Finite numbers

Continuous cell line Indefinite numbers

Stored

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Cell Culture Morphology

Morphologically cell cultures take one of two forms:
growing in suspension (as single cells or small free-floating clumps)
cell lines derived from blood (leukaemia, lymphoma)

growing as a monolayer that is attached to the tissue culture flask.

cells derived from solid tissue (lungs, kidney), endothelial, epithelial, neuronal, fibroblasts

Hela-Epithelial

BAE1-Endothelial

MRC5-Fibroblast

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SHSY5Y-Neuronal

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Special types of Cell culture

Cells in the culture can be grown to adopt in vivo characteristic Histotypic culture
Single cell lineage

Organotypic culture
Multiple cell lineages

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Biology of Culture cells

Cell growth and differentiation in the culture depends on:
The nature of cells The culture environment
the nature of the substrate on which cell grow the physicochemical and physiological constitution of culture medium the constitution of gas phase the incubation temperature the cell-cell and cell-matrix interaction
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Cell cycle
G2 check point DNA replicated cell big
environment suitable

M Mitosis

Metaphase check point chromosome align on spindle

G2 Gap2

G1 Gap1

G0 S Synthesis
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G1 check point cell big

environment suitable
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Cell cycle
Interphase:
generally lasts at least 12 to 24 hours in mammalian tissue the cell is constantly synthesizing RNA, producing protein and growing in size
Gap 0 (G0): cell will leave the cycle and quit dividing temporary or more permanent Gap 1 (G1): Cells increase in size, RNA and protein synthesis, there is a G1 Checkpoint S Phase: The DNA replication occurs Gap 2 (G2): The cell will continue to grow and produce new proteins. There is a G2 Checkpoint

Mitosis or M Phase:
Cell growth and protein production stop the cell cycle divides into two similar daughter cell Mitosis last perhaps only one to two hours there is a Checkpoint in the middle of mitosis (Metaphase Checkpoint) that ensures the cell is ready to complete cell division.
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Factors affecting cell proliferation

Promotion of cell proliferation
low cell density (leaves the cell with free edge) signals from environment: Growth factors

Inhibition of cell proliferation

Density limitation: high cell density Contact inhibition: cell contact signals from environment: p53 gene product
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Factors affecting cell diferentiation

Cell differentiation is important for normal cell functions Factors promoting cell differentiations
high cell density cell-cell and cell-matrix interaction inducers: hydrocortisone, retinoid, matrix

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Factors affecting cell adhesion

Cell adhesion is important for cell proliferation and differentiation (signaling
through cytoskeleton)

Cell adhesion molecule

Cell-cell interaction: CAMs, cadherins Cell-matrix interaction: integrin, transmembrame proteoglycan

Tight junctional complex in epithelial cells for cell-cell interaction

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Factors affecting cell adhesion

Enzymatic disaggregation digests the adhesion molecule and extracellular matrix Most cells from solid tissues grow as adherent monolayer Matrix-coated surface promotes cell proliferation and differentiation

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Factors affect cell culture success

Appropriate cells Suitable environment
Solid phase
substrate or phase on which the cell grow eg. glass, plastic, collagen, agar

Liquid phase
physicochemical and physiological constitution of the medium

Gaseous phase Temperature Aseptic environment

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Solid phase
Anchorage dependent cells require a nontoxic, biologically inert to attach and allow movement for growth The most convenient vessels are polystyrene plastic other growth surface such as glass, filter wells The surface can be treated by
coated with matrix substrate eg. Collagen, poly-l-lysine, matrigel Feeder layers: monolayer of supporting cells, perhaps promote cell growth and differentiation by cell contact and substance secreted
Neurons on glial cell feeder layers

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Liquid phase
Components of culture media
Inorganic Salts retain the osmotic balance of the cells regulate membrane potential by provision of sodium, potassium and calcium ions. are required in the cell matrix for cell attachment and as enzyme cofactors. Carbohydrates Most media contain 4-20 mM glucose main source of energy from glycolysis
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Liquid phase
Proteins and Peptides are used to replace those normally present in serum eg. transferrin, fibronectin Amino acids important for cell proliferation and differentiation glutamine can enter Krebs cycle Fatty Acids and Lipids important in serum free media e.g. cholesterol and steroids essential for specialized cells.

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Liquid phase
Vitamins vitamins B are necessary for cell growth and proliferation precursors for numerous co-factors The vitamins commonly used in media include thiamine, riboflavin and biotin Trace Elements zinc, copper, selenium and tricarboxylic acid intermediates. Selenium is a detoxifier and helps remove oxygen free radicals.

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Liquid phase
Buffering Systems

most cells need optimal pH conditions in the range 7.2 - 7.4 close control of pH is essential for optimum culture conditions
bicarbonate/CO2 buffering systems Chemical buffering: HEPES

Most commercial culture media include phenol red as a pH indicator

yellow (acid) or purple (alkali) Osmolarity

similar to plasma osmolarity 290 mOsm

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Liquid phase
Serum Undefined factors: complex mix of albumins, growth factors and growth inhibitors increase the buffering capacity of cultures able to bind and neutralize toxins can be important for slow growing cells or where the seeding density is low Subject to batch to batch variation Heat inactivation of serum (incubation at 56C for 30 minutes) can help to reduce the risk of contamination

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Gaseous phase
Carbondioxide
important for buffering system
5-10% CO2 Endogenous production: pyruvate
Pyruvate Dehydrogenase
O H3 C C O C O

HSCo A H3C

O C S Co A

+ CO2

pyruvate

NAD+ NADH

acetyl-CoA

Oxygen
most cells in culture require low oxygen tension anaerobic glycolysis high oxygen can produce toxic free radical
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Temperature
The optimum temperature depends on
the body temperature of animals from which the cells were obtained anatomical variation of temperature (skin temperature may be lower than the rest of the body)

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Aseptic techniques
Microorganism remains a major problem in cell culture prevention of contamination
Antibiotics improvement of laboratory condition Aseptic techniques Clean and tidy work surface Personal hygiene
hand washing caps, gowns, face mask

Reagents and media Culture vessels

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Cryopreservation of Cell Lines

The aim of cryopreservation is to enable stocks of cells to be stored to prevent the need to have all cell lines in culture at all times Reduced risk of microbial contamination Reduced risk of cross contamination with other cell lines Reduced risk of genetic drift and morphological changes Work conducted using cells at a consistent passage number Reduced costs (consumables and staff time)

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Cryopreservation of Cell Lines

Method Advantages Disadvantages
Ease of maintenance Steady temperature Low running costs Requires liquid nitrogen back-up Mechanically complex Storage temperatures high relative to liquid nitrogen

Electric (-135C) Freezer

Liquid Phase Nitrogen

Steady ultra-low (-196C) temperature Simplicity and mechanical reliability

Requires regular supply of liquid nitrogen High running costs Risk of cross-contamination via the liquid nitrogen - 196C

Vapor Phase Nitrogen

No risk of crosscontamination from liquid nitrogen Low temperatures achieved Simplicity and reliability

Requires regular supply of liquid nitrogen High running costs Temperature fluctuations between - 135C and - 190C

Risk Assessment
Risks depend on: Source of material the nature of operation being carried out Assesment: Pathogenicity Route of transmission Agent stability Infectious dose Concentration Availability of data from animal studies Availability of an effective prophylaxis Medical surveillance Experience and skill level of at-risk personnel
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Risk groups for animal cell culture

The level of risk depends on the cell line to be used and is based on whether the cell line is likely to cause harm to humans. Low risk
Non human/non primate continuous cell lines and some well characterized human diploid lines of finite lifespan

Medium risk
Poorly characterized mammalian cell lines.

High risk
Cell lines derived from human/primate tissue or blood. Cell lines with endogenous pathogens (the precise categorization is dependent upon the pathogen) Cell lines used following experimental infection where the categorization is dependent upon the infecting agent
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Safety aspects of cell culture

SAFETY CONSIDERATIONS Assume all cultures are hazardous since they may harbor latent viruses or other organisms The following safety precautions should also be observed:
pipetting: use pipette aids to prevent ingestion keep aerosols down to a minimum no eating, drinking, or smoking wash hands after handling cultures and before leaving the lab decontaminate work surfaces with disinfectant (before and after) autoclave all waste use biological safety cabinet (laminar flow hood) use aseptic technique dispose of all liquid waste after each experiment and treat with bleach

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Risk Group (RG)

Classification is based on the potential effect of biological agent on healthy human adult RG1-agents are not associated with disease RG2-agents are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available RG3-agents are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available RG4-agents are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available
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Biosafety cabinets
The Class I BSC provides personnel and environmental protection, but no product protection. The Class I BSC is hard-ducted to the building exhaust system, thimble-connected, or recirculated back into the room depending on use. The Class II (Types A, B1, B2, and B3)24 biological safety cabinets provide personnel, environmental and product protection.
Laminar flow
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Recommended Biosafety Levels for Infectious Agents

BSL
1

Agents
Not known to consistently cause disease in healthy adults

Practices
Standard Microbiological Practices

Safety Equipment (Primary Barriers)

None required

Facilities (Secondary Barriers)

Open bench top sink required

Associated with human disease, hazard = percutaneous injury, ingestion, mucous membrane exposure

BSL-1 practice plus: Limited access Biohazard warning signs "Sharps" precautions Biosafety manual defining any needed waste decontamination or medical surveillance policies BSL-2 practice plus: Controlled access Decontamination of all waste Decontamination of lab clothing before laundering Baseline serum

Primary barriers = Class I or II BSCs or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials; PPEs: laboratory coats; gloves; face protection as needed

BSL-1 plus: Autoclave available

Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences

Primary barriers = Class I or II BCSs or other physical containment devices used for all open manipulations of agents; PPEs: protective lab clothing; gloves; respiratory protection as needed

BSL-2 plus: Physical separation from access corridors Self-closing, doubledoor access Exhausted air not recirculated Negative airflow into laboratory

Dangerous/exotic agents which pose high risk of life-threatening disease, aerosol-transmitted lab infections; or related agents with unknown risk of transmission

BSL-3 practices plus: Clothing change before entering Shower on exit All material decontaminated on exit from facility

Primary barriers = All procedures conducted in Class III BSCs or Class I or II BSCs in combination with full-body, airsupplied, positive pressure personnel suit

BSL-3 plus: Separate building or isolated zone Dedicated supply and exhaust, vacuum, and decon systems Other requirements outlined in the text

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References
R. Ian Freshney. Culture of Animal cells a manual of basic technique. 4th edition. Wiley-Liss, New York. 2000.

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