Transcript
Page 1: Chapter2 principles of cell culture

by

Dr Norshuhaila Mohamed Sunar

CHAPTER 1CELL CULTURE TECHNOLOGY

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Outline

1.1 Definition and Introduction1.2 Culture Environment1.3 Culture Media1.4 Fields Of Applications, Techniques And

Products of Culture Technology

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1.1 Definition and Introduction

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Cell Culture in vitro - A brief history

1885: Roux maintained embryonic chick cells alive in saline solution for short lengths of time

1912: Alexis Carrel cultured connective tissue and showed heart muscle tissue contractility over 2-3 months

1943: Earle et al. produced continuous rat cell line

1962: Buonassisi et al. Published methods for maintaining differentiated cells (of tumour origin)

1970s: Gordon Sato et al. published the specific growth factor and media requirements for many cell types

1979: Bottenstein and Sato defined a serum-free medium for neural cells

1980 to date: Tissue culture becomes less of an experimental research field, and more of a widely accepted research tool

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Isolation of cell lines for in vitro culture

Resected Tissue

Cell or tissue culture in vitro

Primary culture

Secondary culture

Sub-culture

Cell Line

Sub-culture

ImmortalizationSuccessive sub-cultureSingle cell isolation

Clonal cell line Senescence

Transformed cell line

Immortalised cell line

Loss of control of cell growth

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Primary cultures

• Derived directly from animal tissueembryo or adult? Normal or

neoplastic?• Cultured either as tissue explants or single

cells• Initially heterogeneous – become

overpopulated with fibroblasts(a cell in connective tissue)

• Finite life span in vitro

Types of cell cultured in vitro

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Making a Primary Culture

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Types of cell cultured in vitro

Secondary cultures

• Derived from a primary cell culture• Isolated by selection or cloning• Becoming a more homogeneous cell population• Finite life span in vitro• Cells taken from a primary culture and passed or

divided in vitro.• These cells have a limited number of divisions or

passages. After the limit, they will undergo apoptosis.

(Apoptosis is programmed cell death)

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Types of cell cultured in vitro

Continuous cultures

• Derived from a primary or secondary culture• Immortalised:

• Spontaneously (e.g.: spontaneous genetic mutation)• By transformation vectors (e.g.: viruses &/or plasmids)

• Serially propagated in culture showing an increased growth rate

• Homogeneous cell population• Infinite life span in vitro• Genetically unstable

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Cell morphologies vary depending on cell type

Fibroblastic

Endothelial

Epithelial

Neuronal

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1.2 Culture Environment

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Cell culture environment (in vitro)

What do cells need to grow?

• Substrate or liquid (cell culture flask or scaffold material)chemically modified plastic or coated with proteinssuspension culture

• Nutrients (culture media)

• Environment (CO2, temperature 37oC, humidity)Oxygen tension maintained at atmospheric but can be

varied

• Sterility (aseptic technique, antibiotics and antimycotics)Mycoplasma tested

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1.3 CULTURE MEDIA

PLANT AND ANIMAL CELL CULTURE MEDIUM

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Animal Culture Medium Basics

Medium components Buffer(s) to maintain pH Salts for osmosis and cell needs Amino acids – essential and other Growth stimulants (hormones and agonists) Serum 血清 (fetal calf/bovine) Lipids including cholesterol Vitamins Food (typically glucose) Trace Minerals for metabolism/enzyme function Nucleic Acids

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Animal Culture Medium Basics

“Base” medium Just components without additives. no serum or

antibiotics“Complete” medium

5 or 10% serum Basic medium Antibiotics or antimycotics

“low serum or starving” medium Same as complete but with no or low levels of

serum (0.5%).

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Selecting Media (animal cells)

The choice of cell culture medium is extremely important, and significantly affects the success of cell culture experiments.

Different cell types have highly specific growth requirements, and the most suitable medium for each cell type must be determined experimentally.

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RPMI Media

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Eagles Minimum Essential Media

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Culture Media

These contain a mixture of amino acids, glucose, salts, vitamins, and other nutrients, and are available either as a powder or as a liquid from various commercial suppliers.

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Culture Media

Requires

Ions; Na+, K+, Ca++, Mg++, Cl-, PO4-, HCO3

-

Trace elements; iron, zinc, selenium

Sugars such as glucose

Amino Acids

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Media Selection

Cell Line Cell Type Species Tissue Medium

293 Fibroblast Human Embryonic Kidney

MEM10% HI Horse Serum

3T6 Fibroblast Mouse Embryo DMEM10% FBS

A549 Epithelial Human Lung Carcinoma

F-12K10% FBS

H9 Lyphoblast Human T-Cell Lymphoma

RPMI-164010% FBS

HeLa Epithelial Human Cervix Carcinoma

MEM10% FBS

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Serum

Serum is a partially undefined material that contains growth and attachment factors, and may show considerable variation in the ability to support growth of particular cells.

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Serum

Fetal calf serum (FCS) is the most frequently used serum, but for some applications less expensive sera such as horse or calf serum can be used.

Different serum batches should be tested to find the best one for each cell type.

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FCS vs FBS

Fetal Calve / Bovine Serum – typically used interchangeably but are different. Fetal Calf Serum – taken from newborn Calves Fetal Bovine Serum – from fetus

Some variation in content of growth factorsVariability in lot to lot and location to location

– often times tested for several viruses that my impact cells.

Either can be different if mother or calve has been nursing or grazing 放牧 .

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L-glutamine

L-glutamine is an unstable amino acid that, with time, converts to a form that cannot be used by cells, and should be added to medium just before use. Provides nitrogen for nucleotides Serves (like pyruvate) as secondary energy source for

metabolism Breaks down (40%) within 3 to 4 weeks, faster if in

cultured cells. In culture, glutamine breakdown generates

ammonium Some supplements (glutamax) are more stable and

can replace glutamine for long term culturing of slow cells

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Antibiotic & Antimycotic

Antibiotics and fungicides can be used as a supplement to aseptic technique to prevent microbial contamination.

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Antibiotic & Antimycotic

Antibiotic / Antimycotic Working Concentration

Stability at 37°C

Amphotericin B 2.5 µg / ml 3 days

Ampicillin 100 µg / ml 3 days

Penicillin 100 U / ml 3 days

Streptomycin 100 µg / ml 3days

Puromycin 20 µg / ml Unknown

Kanamycin 100 µg / ml 5 days

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Media & Components

Media, serum, and supplements should be tested for sterility before use by incubation of a small aliquot at 37°C for 48 hours.

If microbial growth has occurred after this incubation, the medium or supplement should be discarded.

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Plant Culture Medium

Plant Culture Medium Requirements Varies: Plant cell type (woody, fern, orchid…) Maintenance of callus or shoot formation (stage II) Stimulation of Root and de-differentiation Protoplast, suspension or batch cultures

General Components: Macronutrients, micronutrients, vitamins, amino

acids, nitrogen, phosphorous, sugar, organic supplements and solidifying agents/support systems

AND growth regulators (hormones)

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Plant Culture Medium

Macronutrients (macroelements) - Needed in media in large amounts and make up ~0.1% of dry weight of plant: Nitrogen – supplied in form of ammonium ion

(H4NO3+) and nitrate (KNO3) – best if both are present

and together act to buffer pH. Some amino acids can supplement N requirements or take

place as the N is removed via TCA and transglutaminases High ammonium causes a pale, glassy culture (vitrification 玻

璃化 )

Potassium – come as counter ion with NO3-and PO4

-2

Phosphorus – K2HPO4, H4NO3(HPO4)2,

High concentrations of phosphate will lead to ppt with Ca+2 and other cations

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Plant Culture Medium

Micronutrients (Microelements): Trace amount elements and salts necessary for growth:Fe (FeSO4) –The complex allows for a slow

continuous release and avoids free metal generation of radical oxides after reaction with water.

Others include: Zn, Cu, B, and Mo.

Carbon and Energy Source – cultures do little if any photosynthesis (heterotrophs). Must supply carbon to metabolize ATP and other energy molecules. Sucrose is usually used Galactose, sorbitol and maltose also are used.

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Plant Culture Medium

Organic Supplements – Wide range of various needs Amino Acids – can provide nitrogen and support for metabolism

as well as biosynthesis for new proteins, lipids and nucleotides Casine (milk protein) hydrolysates typically are the source of amino

acids Vitamins: Vitamin B1 (thiamin) and Vitamin B6 (nicotinic acid

pyridoxine), and myo-inositol. The latter is not a vitamin but used as one for plant culture media.

Activated Charcoal (AC) –Used for it’s ability to bind hydrophobic compounds which inhibit growth. The actual role isn’t always clear nor is it always included in medium.

Gelling Agents (support systems) – Solidified surface typically from the complex carbohydrates (non-digestible) extracted from seaweed (agar). Lots of variation between batches and suppliers Gums from plants, agarose can also be used

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Plant Culture Medium

Growth Regulators- Five main classes; auxin, cytokinin, gibberellins, abscisic acid and ethylene.

Auxins- Promote cell division and growth – most auxins are synthetic and not found in plants. Naturally produced 1H-indol-3-acetic acid, is unstable to both heat and light.

• Naturally produced in apical and root meristems seeds and developing fruit

• Alters proton pump and ATP production in target cells• Induces cell elongation • Suppresses lateral bud growth and stimulates adventitious roots• Synthetic form(s) include 2-4 dichorophenoxyacetic acid (2-4D)• Acts as a herbicide by inducing unsustainable growth in broad leaf

(dicot) weeds – corn, rice and wheat all have one leaf (monocot).• Can be used for trees to hold fruit for development

2-4-D

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Plant Culture Medium

Growth Regulators- Five main classes; auxin, cytokinin, gibberellins, abscisic acid and ethylene.

Cytokinins – Promote cell division and are produce in young leaves fruits and seeds.

• Used to stimulate cell division, induce shoot formation and auxiliary shoot proliferation while inhibiting root formation. Not good for stage III.

• Delays cell aging and increases as some fruits bloom and grow

• Used to induce bud growth in orchids and daylilies

• Prevents browning in salads• When mixed with gibberellins – can increase

the size of a fruit (30-50% in pears and mangos)

Zeatin – first isolated from corn

Kinetin– first isolated herring spirm

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Plant Culture Medium

Growth Regulators- Five main classes; auxin, cytokinin, gibberellins, abscisic acid and ethylene.

Ratio of Auxin and cytokinin control root formation

Root initiation occurs when more auxin than cytokinin is in media and adventitious and shoot growth takes place when more cytokinin than auxin ratio

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Plant Culture Medium

Growth Regulators- Five main classes; auxin, cytokinin, gibberellins, abscisic acid and ethylene.

Gibberellins & Abciscic acid- Regulate cell elongation and determine plant height.

Gibberellins increase growth of low-density cultures, enhance callus growth and elongate dwarf plants

Abscisic acid alters callus growth, enhance bud and shoot formation, and inhibit cell division. Commonly used in somatic embryogenesis

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Plant Culture Medium

Growth Regulators- Five main classes; auxin, cytokinin, gibberellins, abscisic acid and ethylene.

Ethylene- volatile gas produced during ripening, stress, mechanical damage or infection.

Produced from methyl group of methionine Nearly all plant tissues can produce Natural role is to encourage fruit ripening and flower

blooming Used commercially to initiate flowering and ripen

tomatoes, citrus and bananas – why brown bags? Specific protein receptors for ethylene have been found

which act as transcription factors Can be a problem in culture without proper air circulation

H2C=CH2

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SOLUTIONS USED IN CELL CULTURE

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Phosphate Buffered Saline - Ca2+ Mg2+ Free (PBS)

Used to wash/remove excess serum that inhibits the function of Trypsin-EDTA. trypsin is inhibited by serum 

Must be warmed in the water bath before use so cells are not shocked by cold liquid.

PBS without Ca2+/Mg2+ wash adherent cell cultures before detaching them from the growing surface with trypsin.

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Trypsin EDTA

An enzyme used to detach the cells from a culture dish. Trypsin cleaves peptide bonds (LYS or ARG) of the

extracellular matrix. EDTA chelates calcium ions in the media that would

normally inhibit the function of trypsin. Trypsin will self digest and become ineffective if left in

water bath more than 20 minutes. Trypsinizing cells too long will reduce cell viability

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Trypan Blue

An exclusion dye(dye exclusion method. )Living cells cannot take up the dye and will

appear bright and refractile.Live cells or tissues with intact 完整 cell

membranes are not coloured.Dead cells with broken membranes will

absorb the dye and appear blue.Usually add 200 l of trypan blue to 200 l of

cell suspension in eppendorf tube

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Bleach

Used to destroy any remaining cells in dishes and tubes before they are tossed in the trash can.

Add enough to change media to clear, wait 5 minutes, rinse solution down sink throw away the dish/flask/plate in the trash can.

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1.3 FIELDS OF APPLICATION, TECHNIQUES AND PRODUCTS

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CO2 incubator

maintains CO2 level (5-10%), humidity and temperature (37o C) to simulate in vivo conditions.

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Water bath

To warm media PBS before placing on cells

Can harbor 包庇 fungi and bacteria, spray all items with 70% ethanol before placing in the hood.

Usually takes 10 -15 minutes for media to warm, 5-10 min to thaw 解冻

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Vacuum pump

For permanent aspiration of liquids (media, PBS).

Use unplugged glass pasteur pipets, throw into sharps box when done.

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Inverted Phase Microscope

A phase contrast microscope with objectives below the specimen.

A phase plate with an annulus 环 will aid in exploiting differences in refractive indices in different areas of the cells and surrounding areas, creating contrast

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Mechanics of phase microscopyMechanics of phase microscopy

Shifting of phase by ½ a wavelengthAdd and subtract amplitudes to createmore contrast

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A comparison

Phase contrast microscopy Light microscopyCan be used on living cells requires stain, thus killing cells

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Technique for cultivating animal cell

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How do people culture cells in the laboratory?

Revive复活 frozen cell populationIsolate from tissue

Maintain in culture (aseptic technique)

Sub-culture (passaging)

Cryopreservation

Count cells

Containment level 2 cell culture laboratory

Typical cell culture flask

Used to freeze cells

冷冻保存

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Passaging Cells

Why passage cells? To maintain cells in culture (i.e. don’t overgrow) To increase cell number for experiments/storage

How? 70-80% confluency Wash in PBS to remove dead cells and serum Trypsin digests protein-surface interaction to release

cells (collagenase also useful) EDTA enhances trypsin activity Resuspend in serum (inactivates trypsin) Transfer dilute cell suspension to new flask (fresh

media) Most cell lines will adhere in approx. 3-4 hours

Check confluency of cells

Remove spent medium

Wash with PBS

Resuspend in serum containing media

Incubate with trypsin/EDTA

Transfer to culture flask70-80% confluence 100% confluence

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Passage cells

Resuspend cells in serumcontaining media

Centrifuge & Aspirate supernatant

Transfer to cryovialFreeze at -80oC

Resuspend cells in 10% DMSO in FCS

Why cryopreserve cells?• Reduced risk of microbial contamination.• Reduced risk of cross contamination with other cell

lines.• Reduced risk of genetic drift and morphological

changes.• Research conducted using cells at consistent low

passage.

How?• Log phase of growth and >90% viability• Passage cells & pellet 丸 for media exchange• Cryopreservant (DMSO) – precise mechanism

unknown but prevents ice crystal formation• Freeze at -80oC – rapid yet ‘slow’ freezing• Liquid nitrogen -196oC

Transfer to liquid nitrogen storage tank

Cryopreservation of Cells

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ContaminationA cell culture contaminant can be defined as some element in the culture system that is undesirable because of its possible adverse effects on either the system or its use.

1-Chemical ContaminationMediaIncubatorSerumwater

2-Biological ContaminationBacteria and yeastVirusesMycoplasmasCross-contamination by other cell culture

How Can Cell Culture Contamination Be Controlled?

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Technique of cultivating animal cells

Excise 摘除 tissues from specific organ of animals (lung, kidney) under aseptic conditions.

Transfer tissues into a growth medium containing serum and antibiotics in small T-flasks.

These cells form a primary culture that usually attach onto the glass surface of flask in monolayer form.

The cells growing on support surfaces are known as anchorage-dependent cells.

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Some cells grown in suspension culture and are known to be nonanchorage-dependent cells.

Then a cell line appear from the primary culture and known as secondary culture.

Remove cell from the surface of flasks using trypsin and add serum to the culture bottle.

The serum containing suspension is then use to inoculate secondary cultures.

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Condition for animal cell culture

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Culture condition

It is critical to find a ‘HAPPY’ environment for cell cultures.

Happy environment = allows cells to increase in number by undergoing cell division (mitosis).

Provide the cells with appropriate temp, good substrate for attachment and proper culture medium.

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Temperature

Usually set at the same point as the body temp of the host from which the cell obtained

Cold-blooded vertebrates – 18-25°CMammalian cells – 36-37°CTemp maintained by use of carefully calibrated

and frequently checked incubators

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pH

Most cells in culture grow best at pH 7.4 Common used buffer bicarbonate-CO2 or

HEPESKeep the pH medium in a range 7-7.4When using bicarbonate-CO2 buffer, need to

regulate the amount of CO2 dissolved in the medium

Done by using an incubator with CO2 control set to provide an atmosphere with between 2% and 10% CO2.

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Thank you


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