Cell and Tissue Culture

Question 1

Describe cell culture:

Answer 1

The process by which prokaryote or eukaryote cells are grown in vitro under controlled conditions
Tends to refer to eukaryote mammalian cells

Question 2

Describe why we study cells in vitro:

Answer 2

Allows for the investigation of cellular functions in a controlled setting:
Growth and metabolism
Gene expression/intracellular biology
Molecular/intracellular biology
Test therapies

Question 3

Describe what cells as a model system can study:

Answer 3

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)

Question 4

Describe the advantages of cell culture over other model systems:

Answer 4

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)

Question 5

Describe the disadvantages of cell culture:

Answer 5

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

Question 6

List some of the commercial/clinical applications of cell culture:

Answer 6

Production of hormones, growth factors etc.
Culture of pathogens
Production of gene therapy reagents
Culture of new tissue/embryos

Question 7

Describe the assumptions and limitations of tissue culture as an experimental tool:

Answer 7

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

Question 8

Describe the basic types of tissue culture:

Answer 8

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

Question 9

Name the two types of cultures cells can grow in:

Answer 9

Monolayers (adherent)

Suspensions (non-adherent)

Question 10

Describe the advantages of primary culture:

Answer 10

Retain phenotype characteristics e.g. ability to release and respond to neurotransmitters
Can culture heterogeneous cell populations

Question 11

Describe the disadvantages of primary culture:

Answer 11

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

Question 12

Describe the advantages of cell lines:

Answer 12

Limitless availability (transformed to grow indefinitely)
Readily available 
Standard and well-characterised
Quick-growing and stable
Inexpensive

Question 13

Describe the disadvantages of cell lines:

Answer 13

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

Question 14

Describe the origins of primary tissue and cell lines:

Answer 14

Primary tissue:

• Organs/biopsies
• Includes explants, embryos, single cell suspensions, cultured separately

Cell lines:

• Transformed primary tissue
• Obtained from tumour tissue
• Other investigators

Question 15

Describe the process of creating cell lines from resected tissue:

Answer 15

Resected tissue –> Cell or tissue culture in vitro –> Primary culture - sub-culture –> Cell line:

1. Sucessive sub-culture –> senscence
2. Immortalisation
   a. immortalised cell line
   b. lose control of growth –> transformed cell line

Question 16

Describe the importance of knowing cell morphologies:

Answer 16

Cells can easily become contaminated

e.g. HeLa is an invasive cell line

Question 17

Describe cell growth in culture:

Answer 17

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)

Question 18

Describe the requirements of mammalian cells in culture:

Answer 18

Sterile environment
Steady supply of nutrients (overgrowth if all the nutrients are used)
Suitable growth surface
Stable temperature and pH

Question 19

Describe the maintenance of cells in culture:

Answer 19

Cells need:
Substrate (adherent) or liquid (suspension)
Nutrients
Environment

Basal media
Supplements

Question 20

Describe cell culture supplements:

Answer 20

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

Question 21

Describe a chemically defined media:

Answer 21

Contains no serum (unknown proteins)
Additives need to contain protein, peptides, fatty acids and lipids
Required for human clinical use

Question 22

Describe buffering and pH control in cell culture:

Answer 22

pH needs to be optimised for growth pH 7.2-7.4
Buffering system (bicarbonate or chemical)
pH indicator of phenol red

Question 23

Describe growing surfaces for cells in culture

Answer 23

Plasticware
- Modified polystyrene, irradiated for sterility, differ depending on applications
ECM proteins
Feeder layer (secrete GFs)

Question 24

Describe cell and tissue dissociation:

Answer 24

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

Question 25

Describe the process of passaging and subculturing cells:

Answer 25

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

Question 26

Describe plating cells for experiments:

Answer 26

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

Question 27

Describe cell quantification:

Answer 27

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

Question 28

Describe freezing and storing cells:

Answer 28

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

Question 29

Describe freezing and thawing cells:

Answer 29

Freeze slow, defrost fast Optimal freezing rate 1-3 per minute degrees DMSO used as a cyroprotectant but is toxic to cells

Question 30

Describe infection control:

Answer 30

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

Question 31

List common microbial contaminants:

Answer 31

Bacteria Yeast Mycoplasma Viruses Antibiotic/mycotics used to control low grade infection Source/route of infection should be determined to prevent repeat contamination

Question 32

Describe disinfection in cell culture:

Answer 32

``` UV light 70% ethanol Hypochlorite Virkon, trigene Should be varied to prevent resistant strains developing ```

Question 33

List some common tissue culture assays:

Answer 33

``` 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 ```

Question 34

List some cell death/survival or proliferation assays for use following drug treatment:

Answer 34

LDH release MTT alamarBlue

Question 35

Describe the LDH release assay:

Answer 35

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

Question 36

Describe the MTT assay:

Answer 36

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

Question 37

Describe the alamarBlue assay:

Answer 37

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

Question 38

List how cell number can be measured:

Answer 38

Trypan blue, propidium iodide - excluded by viable cells DAPI, hoechst - apoptosis BRDU incorporation

Question 39

Describe transfection:

Answer 39

``` 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 ```

Question 40

Describe lipofectamine transfection:

Answer 40

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

Question 41

Describe how to optimise lipofectamine transfection:

Answer 41

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

Question 42

Describe the process of generating stably transfected cell lines:

Answer 42

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

Question 43

Describe how inducible transfections overcome the limitations of stably transfected lines:

Answer 43

``` Negative effects of constitutive knockout or overexpression Control over expression Reversibility Dose-dependent expression Stable expression systems ```

Question 44

Describe the tet-on/tet-off expression system basics:

Answer 44

``` Based on bacterial Tet operon System consists of: A regulatory plasmid An inducible response plasmid Systems available in a single plasmid ```

Question 45

Describe the mechanisms of the Tet system:

Answer 45

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

Question 46

Describe the advantages of the Tet system:

Answer 46

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

Question 47

Describe electric cell-substrate impedance sensing:

Answer 47

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

Question 48

Describe frequencies in ECIS:

Answer 48

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

Question 49

List the behaviours ECIS can be used to study:

Answer 49

``` Attachment and spreading Proliferation Toxicity Cell invasion Differentiation and stem cell biology Wound healing and barrier formation ```

Question 50

Describe the advantages and disadvantages of ECIS:

Answer 50

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