by
Dr Norshuhaila Mohamed Sunar
CHAPTER 1CELL CULTURE TECHNOLOGY
Outline
1.1 Definition and Introduction1.2 Culture Environment1.3 Culture Media1.4 Fields Of Applications, Techniques And
Products of Culture Technology
1.1 Definition and Introduction
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
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
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
Making a Primary Culture
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)
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
Cell morphologies vary depending on cell type
Fibroblastic
Endothelial
Epithelial
Neuronal
1.2 Culture Environment
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
1.3 CULTURE MEDIA
PLANT AND ANIMAL CELL CULTURE MEDIUM
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
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%).
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.
RPMI Media
Eagles Minimum Essential Media
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.
Culture Media
Requires
Ions; Na+, K+, Ca++, Mg++, Cl-, PO4-, HCO3
-
Trace elements; iron, zinc, selenium
Sugars such as glucose
Amino Acids
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
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.
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.
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 .
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
Antibiotic & Antimycotic
Antibiotics and fungicides can be used as a supplement to aseptic technique to prevent microbial contamination.
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
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.
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)
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
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.
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
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
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
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
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
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
SOLUTIONS USED IN CELL CULTURE
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.
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
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
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.
1.3 FIELDS OF APPLICATION, TECHNIQUES AND PRODUCTS
CO2 incubator
maintains CO2 level (5-10%), humidity and temperature (37o C) to simulate in vivo conditions.
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
Vacuum pump
For permanent aspiration of liquids (media, PBS).
Use unplugged glass pasteur pipets, throw into sharps box when done.
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
Mechanics of phase microscopyMechanics of phase microscopy
Shifting of phase by ½ a wavelengthAdd and subtract amplitudes to createmore contrast
A comparison
Phase contrast microscopy Light microscopyCan be used on living cells requires stain, thus killing cells
Technique for cultivating animal cell
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
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
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
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?
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.
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.
Condition for animal cell culture
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.
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
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.
Thank you