Cell Culture
Cell culture
Definition of tissue/cell culture
The artificial growth under controlled conditions of tissues and/or cells outside a living organism
Cell culture - isolated primary cells or established cell lines
Primary Culture
A culture that is started from cells taken directly from a tissue or organ
At this stage the cells are heterogeneous, they closely represent the parent cell types and express tissue-specific properties
A culture is regarded as primary until it is subcultured/passaged for the first time when it becomes a cell line
Limited lifespan
After a certain number of population doublings the cells undergo senescence and stop dividing while generally retaining viability
Monolayer
Anchorage dependent that grow attached to a substratum
When all substratum is covered by cells, confluence is reached
Suspension
Anchorage independent and grow without attachment
History of Cell Culture
1885
Wilheilm Roux removed part of an embryonic chicken medullary plate and maintained it in warm saline for several days
1907
Harrison established the first successful animal-tissue culture. He grew frog embryonic nerve tissue in lymphatic fluid
1913
Carrel showed that cells can grow for long periods in culture provided they are fed under aseptic conditions
1951
George Gey established a continuous line of cells(HeLa) from Henrietta Lacks who died of cervical cancer
1955
Eagle began to establish the essential nutritional requirements of cell in culture
1961
Hayflick and Moorhead show that human fibroblasts die after a finite number of divisions in culture#
UK Legal and Ethical Requirements
There are ethical and legal requirements for obtaining tissue for cell lines
Human tissue act 2004
The Human Tissue Authority regulates activities concerning the removal, storage, use and disposal of human tissue samples
Patient consent is required for the use of human tissue samples and ownership must be defined
Transfer of cell lines from one laboratory to another may require a material transfer agreement(MTA)
Cell culture advantages
Control of physiochemical environment
pH
Temperature
Osmotic Pressure
O2/CO2
Control of physiological conditions
Medium
Serum
Homogenous cultures
Good reproducibility
Unlimited supply of continuous cell lines
Infinite viable storage in liquid nitrogen
Economy
Low concentration of drugs etc compared to in vivo where 90% excreted
Avoids undue use of animals
Cell culture disadvantages
Expertise
Aseptic technique
Quantity
1-10g of cells
Outgrowth of undifferentiated cells
Stress of culture may create evolutionary pressure
Phenotypic drift and genotypic changes
Instability
Due to aneuploid chromosome constitution
Cells may adapt to the stress of culture environments by varying the activities of their enzymes
Loss of heterotypic interactions between different cell types
Loss of homeostatic regulation
Loss of 3D geometry
Producing primary culture
Explant method
Suitable for small amounts of material
Eliminates need for enzymes etc to disperse cells
Small pieces of tissue(1-2mm3) are placed in growth medium and held down with eg: cover-slip
Cells migrate out
Particularly useful epithelial cell cultures
Dissociation
For some tissue, mechanical dissociation is sufficient
For most tissues, these enzymes are useful
Trypsin
Collagenase
Deoxyribonuclease
The goal being a high yield of cells with high viability
Suspension Cultures
Seed cells directly into media
Cell line
Normally, a cell line isolated from normal tissue has a finite number of divisions called the Hayflick number before they stop dividing and die
If a cell's Hayflick limit is 50 for example
It will divide 50 times and then become senescent and subsequently die
Cell lines from tumours often undergo a 'crisis' whereby a population of smaller, faster growing, anchorage-independent cells takes over. These cells appear to be immortal and are termed transformed and become continuous cell lines.
Continuous cell lines show many differences to the primary cultures including
Morphology
Genetic changes
Continuous cell line
Defined as a culture that is apparently capable of an unlimited number of population doublings (i.e immortal)
A cell line should not be considered to be continuous until it has been grown in culture for at least 6 months
An immortal cell line is not necessarily neoplastically or malignantly transformed
Malignantly transformed cells will form tumours
A cell line established from a patient with a tumour is not necessarily a tumour cell line
Cancer cell lines are usually derived from the more aggressive and advanced cancers
Immortalisation
Immortalised cell lines have acquired the ability to proliferate indefinitely either through random mutation or deliberate modification (eg: artificial expression of telomerase. They are not limited by the Hayflick
Have the ability to produce continuous cell lines
Unlike transformed cells, immortalised cells are not necessarily cancerous. They show dependence on growth factors and are sensitive to growth inhibitors as well.
Normal cells in culture can be immortalised by transfection with oncogenic or viral DNA such as the SV40 large T antigen
Some cells can become immortalised spontaneously
Often done by inactivating tumour suppressor genes
Immortal cells will generally only grow as a single layer of cells in a dish
Due to contact inhibition
Transformation
Transformed cells will cause tumours in immunocompromised mice
Essentially loss of contact inhibition
Transformed cells may have these characteristics
Aneuploidy
Loss of contact inhibited growth
Density limitation of proliferation
Anchorage independence
Protease production
Promote angiogenesis
Invasiveness
Tumourigenesis
Growth in the absence of growth factors
Human embyronic stem cell lines
Harvested from the inner cell mass of the early embryo(4-5 days old) - ethical consent in US(2009)
Usually grown on a feeder layer of mouse embyronic skin cells
Provide environment for growth
Feeder cells do not divide
Can proliferate indefinitely while retaining the ability to give rise to any part of the body
Embryonic stem cells that have proliferated in culture for 6 months without differentiating are pluripotent and appear genetically normal are referred to as an embryonic stem cell line
Potential source of cells capable of replacing/repairing tissues that have been damaged by injury or disease
Parkinson's disease
Leukaemia
Arthritis
Diabetes
Infertility
Cell culture conditions
Gas phase
21% O2
Normal atmospheric
5% CO2
Good buffering capacity
Helps maintain culture pH (7.0-7.6) when used with bicarbonate buffers
Need loose or gas permeable lids on culture flasks
Temperature
37 C - incubator set to this temperature
High humidity
Reduces evaporation from unsealed culture vessels in order to prevent the cells from becoming hypertonic
Bottom of the tray filled with water
Substrate
Treated tissue culture plastic
Polystyrene
Glass
Microcarrier beads
Polystyrene
Polyacrylamide
DEAE cellulose
Collagen coated surfaces
Matrigel
Gelatin
Feeder-layer
Artificial capillaries
Co-culture
Feeder cells
Nutrients
Provide extracellular matrix requirements
Stroma cells - helps in leukemia
Mimics the BM microenvironment
Growth media
Many different media, largely based on standard salt mixes
Earle's Salts
Eagles minimum essential medium(EMEM)
RPMI
Most common - EMEM, RPMI
May contain any of
Amino acids
Glucose
Vitamins
Electrolytes
Growth Factors
Serum
Antioxidants
Cell stabilisers
Usually contain
Bicarbonate
Buffered with 5% CO2 in the gas phase
Maintained at 7.4
In event of CO2 incubator being unavailable or the experiment is carried out in the open
Medium is usually supplemented with HEPES buffer to maintain the pH
Another common - FCS
Added to between 5 and 20%
Most leukemia cells are grown here
Need to be tested by batch
Not used by defined media- replaced by EGF and hydrocortisone
Antibiotics can be used -
Give bacterial blue
Penicillin
Streptomycin
Antimycotics
Amphotericin B
Mycostatin
Media needs to be regularly changed
Replenish nutrients
Avoid build up of harmful metabolic by-products and dead cells
Carried out -
Suspension cells
Pelleted cells and resuspended in fresh media to be fed
Adherent cells
Remove old media and wash in PBS to remove residual FCS
Remove cells with the enzyme - usually trypsin
Seed into fresh culture media
Culturing cells growth
Fill up the available space or volume
Nutrient depletion
Accumulation of apoptotic/necrotic cells
Contact inhibition
Cell-cell contact
Splitting/Passaging Cells
Involves transferring a small number of cells into a new vessel
Avoids senescence associated with high cell density
Suspension cells are generally diluted into fresh media
Adherent cells are detached from the substrate with enzymes, usually trypsin or trypsin/EDTA and seeded into fresh media
Different cell lines require different splitting ratios
Often in 1:3-1:10
Some cultures will not grow well unless a minimum concentration of cells is initially added
Cells should not be passaged/sub-cultured continuously
The number of recommended passages depend on the cell line
Passaging should be minimised to reduce the possibility of phenotypic/genetic drift and contamination
Cell lines at high passage number may experience alterations in
Cell morphology
Response to stimuli
Growth rates
Protein expression
Transfection efficiencies
Signalling
Spheroid 3D cell cultures
Sphere shaped cell colonies in viscous scaffold
Hydrogel
Matrix
Collagen
They can grow within, on top or as suspension in the scaffold
Mimic growth of naturally occurring human tumours
Can be explained in a 3D model
Cells in contact with nutrients grow quickly
Kinetics of cells further inward depend upon diffusion
Important culture systems for research into
Tumour physiology
Radiation research
Drug screening
Toxicity testing
Metabolism studies
Generation of early stocks of a new cell line
New cell line from primary cells or bought from suppliers(ECACC, DSMZ, ATCC)
Continuous growth
Freeze aliquots
Stock cells
Grow
Freeze aliquots
When a cell line has been produced with the desired characteristics and free of contamination then seed stock should be stored frozen -
Freeze aliquots
Clean stocks of cells frozen
Actively growing cells harvested gently as possible - otherwise they will not survive the additional damage
Placed in a high density (1-10 million) in presence of a cryo-protective agent like glycerol or DMSO(both at 5-10%)
Increased serum concentration in the cryo-protective medium is often used to increase the survival rate of cells
Stored in liquid nitrogen (-196 C)
Cells frozen slowly at -1C/min. When reached -50C, transferred to LN2
When needed,
Thawed rapidly (37 C) and then re-seeded at high density to optimise recovery
Diluted in 1: 10 media to decrease concentration of the cryo-protective agent
Proper storage decreases
Chance of alterations
Loss of cell culture characteristics
Cell line verification
20% of human cell lines are not the cells were originally assumed because
Cross contamination
Most common containment - HeLa cells
Verification by
Morphology
Growth curve analysis
DNA barcoding(interspecies) and STR (intraspecies) identification
DNA fingerprinting
Multiplex PCR to amplify informative polymorphic markers
Antigen expression
Immunochemistry
FACS
Western blotting
Neoplasticity verification
Functional assays
Clonogenic cytogenetic abnormalities
Mycoplasma detection
Characterised by extracellular particles
Fluorescent dye Hoechst stain
Plasmo test - BDI
Disinfect the lab usually
Karyotype Chromosome spreading with banding
DNA protein/profiling
DNA microarrays
Proteomics
Reason to characterise cell line
Microbiological Contamination
Genetic/Epigenetic Change Acquisition
Contamination from other cell lines or misidentification/classification
Replicative Senescence
Cells usually stop dividing after finite divisions (Eg: human fibroblasts 25-40x)
Can be induced by intrinsic or extrinsic factors
Intrinsic
Telomeric shortening
Extrinsic
Irradiation
Oxidative Stress
Hostile Environment
Marked by changes in cell morphology, gene expression and metabolism
Senescence can be triggered by the activation of tumour suppressor proteins
Eg : p53 and Rb
Telomeres
Repetitive non-coding DNA sequences located at termini of chromosomes
Capping role along with associated proteins mean for the chromosome ends are not seen as double stranded breaks
Become shorter with each division because cells cannot copy all the way to the end of the chromosomes and that is why we age
When they are short enough, cell division stops and the cell dies
Ultimately they lead to induction of replicative senescence which prevents genetic instability
Re-entering proliferation can lead to crisis - gross chromosomal rearrangements and genomic instability; most cells die in this stage
However, cells that survive become immortalised through activation of telomerase
Telomerase
Enzyme that builds telomeres by addition of non-coding DNA sequence repeats
Human somatic cells
Telomerase is turned off, therefore, telomeres shorten with each cell division
Cancer cells produce plentiful telomerase
>90% cancers can maintain their telomeres
Transfection
Genetically modify cells working on for beneficial use
Process of introducing nucleic acids into eukaryotic cells (in vitro or in vivo) by non viral means
Reasons
Study of gene regulation and function
Promoter/Enhancer activity
Transcription Factor activity
Protein/Protein interactions
Generation of transgenic organisms
Transfection techniques for transgenic organisms
Expression and function of proteins
Protocols
Chemical agents
Artificial liposomes
Electroporation
Calcium phosphate and DEAE-dextran
Microinjection
Biolistic particle delivery/gene gun
It is a method that neutralises the issue of introducing negatively charged molecules into cells with a negatively charged membrane
Eg: phosphate backbones of DNA and RNA
Mammalian Cell Transfection Techniques
Determination of Optimal Transfection Method -
Safety Issue
Transgene Activity
Length of Expression
Cellular Context
Cell Type
Desired Efficiency
Time
Desired Viability
Cost
Expertise
Type of Delivered Molecule
Safety Issue
Transfection frequency (proportion of cells that have taken up the nucleic acid and are expressing protein) is affected by
Toxicity of procedure
Concentration and purity of nucleic acid
Cell number and degree of confluency
Transfection procedure itself
Concentration of reagents
Duration of exposure
And more…..
Expression period
Sensitivity of detection method
Transfection Techniques are required to be optimised for every individual cell type. Two main classes
Transient expression
DNA not inserted into nuclear genome
Foreign DNA lost when cells undergo mitosis
Stable transfection
Selects cells incorporated the foreign DNA into their genome by co-transfecting with the gene that confers a selection advantage
Resistance to a lethal agent such as neomycin resistance - G418
Types of Gene transfection
Non-viral methods
Chemical methods
Cationic polymers -
First reported nucleic acid transfection
DEAE dextran and polybrene are cationic polymers that tightly associate with negatively charged DNA
Positively charged polymer:DNA complex closely associate with the negatively charged membrane
Uptake is by endocytosis
Used for transient transfection
Calcium Phosphate
Reported by Graham and van der Eb
Mixture of calcium chloride, DNA and phosphate buffer results in the formation of a calcium phosphate co-precipitate of small insoluble particles that contain DNA
Particles adhere to cell membrane and enter into the cytoplasm of the target cell by endocytosis or phagocytosis
Technique prone to variability
Small pH changes can compromise transfection efficiency
Polyplus jetPrime
Can transfect DNA, siRNA or both
Artificial liposomes
Liposomes - lipid bilayers that form colloidal particles in solution
First commercially available liposome was Lipofectin
Cationic head of the lipid group associated with negatively charged DNA
Compacts the nucleic acid in a liposome/nucleic acid complex
Overall positive charge allows closer association between complex and cell membrane
Entry to the cell is by endocytosis or fusion with the cell membrane
Highly efficient technique and low concentrations required but difficult to know what lipid will work best for your cell line
Different lipids have widely variable transfection efficiencies in different cell lines
CPX-351-AML therapy
Liposomal delivery
CPX-351 is a dual-drug liposomal encapsulation of cytarabine and daunorubicin at a fixed 5:1 synergistic molar ratio
Superior antileukaemia activity vs free drugs when administered at the same drug ratios
Higher remission rates observed with CPX-351 than with 3+7 standard therapy with improved overall survival
Physical methods
Microinjection
Fine needle used to directly deliver nucleic acids into cultured cells or nuclei
Laborious
Costly
Not suitable for large amounts of cells
Electroporation
Brief electrical pulse of high strength induces a potential difference across the membrane that induces temporary pores to form
Optimisation (of pulse duration, voltage, capacitance, ionic strength of solution) is essential to reduce cytotoxicity
Can be adapted with a double pulse or using cuvettes that allow adherent cells to be electroporated
Biolistic particle delivery(gene gun)
DNA or RNA coated gold particles are located
Low pressure helium pulse delivers coated gold particles into target cell or tissue
Need large amount of starting cells due to high cell mortality
NanoParticles
Magnetofection
Based on the association of DNA with magnetic nanoparticles coated with cationic molecules
Resulting molecular complexes are then transported into cells through magnetic fields
Important aspects of careful optimisation to ensure successful transfection
Optimal amount of transfection reagent
DNA concentration
Cell density
Trying to ensure log growth
Incubation/contact time of reagent/DNA complexes
Transfection experiments yield best results when plasmid DNA is of highest quality - qiagen columns
Adequate expression period should be allowed
Usually monitored 24,48 and 72 hours post transfection
Selection for stable transfection occurs following the expression period
Selection methods
A number of agents are available for selection of stable transfections
Geneticin(G418) commonly used for stable selection of mammalian cells
Zeocin is rapidly becoming one of the most popular antibiotics to use for selection
Shows high toxicity in mammalian cells, fungi (including yeast), plants and bacteria
Detection of transfected cells - Reporters
Reporter gene concept initially put forth by Gorman uses bacterial CAT gene.
Now a variety of reporter genes are available.
Chloramphenicol acetyltransferase (CAT)
CAT enzyme activity
Traditionally measured using radioisotopic assays of CAT reaction products
Liquid scintillation counting(rapid and sensitive)
Cell extracts incubated in reaction mix containing 14C or 3H-labelled chloramphenicol
Thin layer chromatography separation of 14C chloramphenicol substrate and products
Less sensitive
Time consuming
But visual
Sandwich ELISA can be performed
Quicker
Safer
More accurate as it measures amount of protein synthesised, not enzyme activity
Green Fluorescent Protein(GFP)
From the jellyfish Aequorea victoria to monitor gene expression in vivo, in situ and in real time
Emits bright green light upon exposure to UV unlike other bioluminiscent reporters that require additional proteins, substrates or cofactors to emit light
Fluorescence is stable, species independent and can be monitored noninvasively in living cells
Also GFP variants including EGFP(GFPmut1) and EBFP
Can be used in combination for dual labelling
Useful for microscopy, monitoring gene expression from two different promoters in the same cell, monitoring localisation of two proteins in the same cells
Luciferase - bioluminiscence (luciferases and photon-emitting substrates luciferins)
pGL2 and pGL3 vector series
Modified firefly luciferase designed luc+
pRL contains cDNA encoding renilla luciferase(Rluc) from sea pansy
Firefly luciferase is measured by detecting light after adding luciferin and ATP
Light is produced by converting chemical energy of luciferin oxidation through an electron transition forming oxyluciferin
100 fold more sensitive than CAT assay
Detection by luminometers, plate readers or optical microscopes
b-galactosidase(b-Gal)
pSV b-GAL control vector to measure transfection efficiencies
SV40 early promoter and enhancer drive transcription of bacterial lacZ gene which is translated into the b-galactosidase enzyme
Assayed quickly in cell extracts using
Spectrophotometric assay
ONPG : O-nitrophenol-b-D-galactoside
Fluorometric assay
MUG : 4-methylumbellifery-b-D-galactoside
Histochemical assay
Using X-Gal
FACs assay
Using FDG
Luminescent assay
From Invitrogen - used chemiluminescent substrate Galacton-Star
Dual receptors
Commonly used to improve accuracy
Simultaneous expression and measurement of two reporters in a single system
Experimental reporter is associated with specific experimental conditions
Co-transfected control reporter provides internal control
Normalising experimental to control reporter minimises experimental variability
Secreted reporter protein
Requires no cell lysis(like GFP)
Versatile and suitable for a wide variety of cell types
Secrets human placental alkaline phosphatase(SEAP)
SEAP secreted from cells and activity in culture medium are proportional to changes in SEAP mRNA and protein
Advantages
Don’t need to lyse cells
Kinetics of gene expression can be studied by repeatedly sampling the medium
After samples taken cells can still be used for other methods
Sample collection can be automated
Background signals due to endogenous alkaline phosphatases are nearly absent
Tet-Off and Tet-On Gene Expression Systems
Quantitative regulation of gene expression upto 1000 fold induction in many cell types, inducible expression of toxic genes
Allow regulated gene expression in response to varying concentration of tetracycline(Tc) or Tc derivatives such as doxycycline(Dox)
In the Tet-off system
Gene expression is turned on in the absence of Tc or Dox
In the Tet-on system
Gene expression is activated in the presence of Dox
Serum can be contaminated with Tc or Dox
Molecular Imaging/Bioluminescent Imaging
Temporal analysis of tumour growth
C57BL/6 derived B16F10 malignant melanoma cell line was transfected with a plasmid encoding firefly luciferase and stable cell lines selected
SCID mice were given 2 x 104 labelled cells at a subcutaneous site and signals from animals imaged at the time points indicated using a cooled CCD camera
Tumour growth as well as central tumour necrosis after 2-3 weeks could be visualised and quantified
