Cell and Tissue Culture Flashcards
Describe cell culture:
The process by which prokaryote or eukaryote cells are grown in vitro under controlled conditions
Tends to refer to eukaryote mammalian cells
Describe why we study cells in vitro:
Allows for the investigation of cellular functions in a controlled setting: Growth and metabolism Gene expression/intracellular biology Molecular/intracellular biology Test therapies
Describe what cells as a model system can study:
Normal physiology and biochemistry of cells
Effects of therapeutic or toxic compounds (including drug screening)
Effects of mutatgenesis and carcinogenesis
Manufacture of biologicals (e.g. vaccines, therapeutic proteins)
Describe the advantages of cell culture over other model systems:
Consistency and reproducibility (batches of clonal cells means cells should respond in exactly the same way)
Ease of genetic manipulation (transfection)
Ease of imaging (2D vs. 3D imaging)
Simple biology (no extracellular matrix interactions)
Describe the disadvantages of cell culture:
Artificial system (2D, simple biology, not an in vivo representation) Transformed super cells (not natural or normal, such as cancer cell lines, something has bee done to immortalise cells and allow them to replicate indefinitely) Overexpression
List some of the commercial/clinical applications of cell culture:
Production of hormones, growth factors etc.
Culture of pathogens
Production of gene therapy reagents
Culture of new tissue/embryos
Describe the assumptions and limitations of tissue culture as an experimental tool:
Assumption: Behaviour of cells in vitro reflects their in vivo characteristics
Limitations: Surface composition, cell-cell interactions, cellular environment, nutrient/pH environment are all different from in vivo
Describe the basic types of tissue culture:
Primary culture: cells cultured directly from subject - can be cultured as dissociated cells or tissue explants
Cell lines: established or immortalised cells with ability to proliferate indefinitely
Name the two types of cultures cells can grow in:
Monolayers (adherent)
Suspensions (non-adherent)
Describe the advantages of primary culture:
Retain phenotype characteristics e.g. ability to release and respond to neurotransmitters
Can culture heterogeneous cell populations
Describe the disadvantages of primary culture:
Time-consuming to isolate, difficult to purify and grow
Get overgrown fibroblasts
Can’t be propagated long-term (or sub-cultured)
Expensive
Difficult to obtain enough tissue for ongoing study
Primary cells may differe from one animal to another
Collection requires on ethical approval
Describe the advantages of cell lines:
Limitless availability (transformed to grow indefinitely) Readily available Standard and well-characterised Quick-growing and stable Inexpensive
Describe the disadvantages of cell lines:
May retain few in vitro characteristics of tissue of interest
Must be shipped on dry ice
Important to obtain from reputable sources
After many passages, cells may mutate into sub-clones with different properties
Transformation may change their nature/response
Describe the origins of primary tissue and cell lines:
Primary tissue:
- Organs/biopsies
- Includes explants, embryos, single cell suspensions, cultured separately
Cell lines:
- Transformed primary tissue
- Obtained from tumour tissue
- Other investigators
Describe the process of creating cell lines from resected tissue:
Resected tissue –> Cell or tissue culture in vitro –> Primary culture - sub-culture –> Cell line:
- Sucessive sub-culture –> senscence
- Immortalisation
a. immortalised cell line
b. lose control of growth –> transformed cell line
Describe the importance of knowing cell morphologies:
Cells can easily become contaminated
e.g. HeLa is an invasive cell line
Describe cell growth in culture:
Rates of growth depend on seeding density, growth factor requirements and media requirements
Growth exhibits lag, exponential (log) and stationary phases
Cells cannot grow and divide forever (become dormant after a certain number of divisions, lose particular characteristics - genetic drift)
Describe the requirements of mammalian cells in culture:
Sterile environment
Steady supply of nutrients (overgrowth if all the nutrients are used)
Suitable growth surface
Stable temperature and pH
Describe the maintenance of cells in culture:
Cells need:
Substrate (adherent) or liquid (suspension)
Nutrients
Environment
Basal media
Supplements
Describe cell culture supplements:
Serum (source of proteins)
Binds and neutralises toxins, contains GFs
Fetal calf serum (batch variability, risk of contamination, hgih protein content can complicate downstream assays)
Calf or human serum
GFs
Factors inducing differentiation
Antibiotics/antimycotics for infection control
Describe a chemically defined media:
Contains no serum (unknown proteins)
Additives need to contain protein, peptides, fatty acids and lipids
Required for human clinical use
Describe buffering and pH control in cell culture:
pH needs to be optimised for growth pH 7.2-7.4
Buffering system (bicarbonate or chemical)
pH indicator of phenol red
Describe growing surfaces for cells in culture
Plasticware
- Modified polystyrene, irradiated for sterility, differ depending on applications
ECM proteins
Feeder layer (secrete GFs)
Describe cell and tissue dissociation:
Tissue usually disintergrated into individual cells for subsequent culture using enzymatic or mechanism dispersion (collagenase, dispase, trypsin)
Dissociated cells can be further purified by
- Differentiatial centrifugation
- Immunoaffinity chromatography
- Selective substrate adherence
Describe the process of passaging and subculturing cells:
Aims to maintain cells in culture (prevent overgrowth) and to increase cell number for experiments/storage
Check confluency (70-80%) –> Remove media –> Wash with PBS –> Incubate with trypsin/EDTA –> Resuspend in serum-containing media –> Split into new flask
Describe plating cells for experiments:
Plate type:
Size, material, coating
Cell density:
Consider final density required
Consider density required during key treatments
Consider doubling time of cells and assay duration
Run a pilot assay with different cell densities
Describe cell quantification:
For reproducibility of experiments, start with a known number of cells
Decide optimal plating density for experiment
Scaling up of experiment for experiment (density, drug concentration, volume of media)
e.g. haemocytometer
Describe freezing and storing cells:
Prolonged culture of cell lines results in changes in phenotype, morphology and genetic drift
Set up cell bank to ensure constant supply fo fresh cells of early passge with original phenotype
Cell lines stored in liquid nitrogen (vapour phase and liquid phase)
Cells must be frozen in cryovials to withstrand low temperature
Describe freezing and thawing cells:
Freeze slow, defrost fast
Optimal freezing rate 1-3 per minute degrees
DMSO used as a cyroprotectant but is toxic to cells
Describe infection control:
Infection is a constant problem - must maintain sterility for optimal cell health
Sources of infection carried over from primary tissue or introduced by personnel
Infection may be visible or undetectable by the naked eye
Antibiotics could be added by affect function and not allowed in human clinical use
List common microbial contaminants:
Bacteria
Yeast
Mycoplasma
Viruses
Antibiotic/mycotics used to control low grade infection
Source/route of infection should be determined to prevent repeat contamination
Describe disinfection in cell culture:
UV light 70% ethanol Hypochlorite Virkon, trigene Should be varied to prevent resistant strains developing
List some common tissue culture assays:
Image-based assays ICC Immunofluorescence FRAP Western blot qPCR Colometric/fluorescent/luminescent assays Enzyme assays Cytoxicity and viability Gene expression reporters Transfections to study gene effects ICC to study changes in protein levels/localisation
List some cell death/survival or proliferation assays for use following drug treatment:
LDH release
MTT
alamarBlue
Describe the LDH release assay:
Principle:
- Lactate dehydrogenase
- Released into media by dead/damaged cells
- Colorimetric assay to quantify cell death (=more colour)
- Cell-free supernatant incubated with yellow tetrazolium salt (LDH release reduces salt to red formazan dye)
Include background, low and high controls, substance control
Describe the MTT assay:
Principle:
- Colorimetric assay to measure cell viability (more cells=more colour)
- Can measure effect of treatment on cell death or cell proliferation
- In viable cells mitochondrial nreductase reduces yellow tetrazolium salt to a purple formazan product
- Optimise incubation time for each cell line
- Measures increase or decrease in metabolic activity
- Can only measure once
- Background/substance control
Describe the alamarBlue assay:
Principle:
Blue coloured, non-fluorescent resazurin is reduced to red fluorescent compound resorufin by live cells
- Incubate treated cells with reagent
- Available commercially
- Highly sensitive and can be measured on fluorescence or absorbance readers
- Cell lysis is not required - can measure over time
List how cell number can be measured:
Trypan blue, propidium iodide - excluded by viable cells
DAPI, hoechst - apoptosis
BRDU incorporation
Describe transfection:
Introduction of foreign genetic material into a eukaryote cell Methods for getting DNA into cell - CaPO4 - Commerical transfection reagents - Electroporation - Viral transduction Not all cells transfect well
Describe lipofectamine transfection:
Cationic lipid transfection
Nucleic acid-containing liposomes fuse with the plasma membrane of living cells and are engulfed and contents released into the cell for replication or expression
High transfection expression performance with low cytotoxicity to many cell types
Describe how to optimise lipofectamine transfection:
Cell density is important
Need to optimise the amount/ratio of lipofectamine and DNA for particular cell type
Use PLUS reagent to enhance efficiency
Serum can interfere with transfection efficiency
Describe the process of generating stably transfected cell lines:
Overcome variability seen with transient transfections
Transfect cells with transgene and resistance gene –> treat cells with selection agent –> pick colonies –> test presence, function of transgene
BUT it is unknown where the gene will integrate and protein detected may not correlate to in vivo levels
Describe how inducible transfections overcome the limitations of stably transfected lines:
Negative effects of constitutive knockout or overexpression Control over expression Reversibility Dose-dependent expression Stable expression systems
Describe the tet-on/tet-off expression system basics:
Based on bacterial Tet operon System consists of: A regulatory plasmid An inducible response plasmid Systems available in a single plasmid
Describe the mechanisms of the Tet system:
Fusion of TetR to herpes simplex VP16 yields a strong transcriptional activator
OFF: Activity can be switched off in the presence of the Tet analogue dox
ON: Mutant form of TetR that does not bind to TetO in the absence of dox, becomes active in the presence of dox and leads to transgene expression
Describe the advantages of the Tet system:
Promoter is tightly regulated
Regulatory elements absent in eukaryotes (lack of pleiotropy)
No endogenous tet action
Well-characterised induction agent
Fast induction
High induction at low concentrations (sub-therapeutic doses of dox are sufficient)
Dox crosses BBB
Describe electric cell-substrate impedance sensing:
A real-time method to study the activities of cells grown in tissue culture
Cells are grown on plates that have gold electrodes etched onto them
A small AC current is applied across the electrode - as cells attach, they act as insultors and increase impedance relative to their morphology and nature of cell attachment
As cells are stimulated to change their function, the accompanying changes in morphology alter the impedance
Describe frequencies in ECIS:
Uses different frequencies to analyse different components of the impedance
At low frequency (40000Hz) more current flows directly through insulting membranes
By analysing at a range of frequencies different components of the cellular response can be modelled
List the behaviours ECIS can be used to study:
Attachment and spreading Proliferation Toxicity Cell invasion Differentiation and stem cell biology Wound healing and barrier formation
Describe the advantages and disadvantages of ECIS:
Advantages:
Allows for real time analysis of response, allowing to determine subtle differences or changes in response to treatment - can determine appropriate timepoint to carry out more in-depth analysis
Disadvantages:
Doesn’t provide a direct link to the molecular changes occurring
Expensive
