Cell Culture Techniques Flashcards
1
Q
What is a cell/tissue culture?
A
- A laboratory method (in vitro) by which cells are grown under controlled conditions outside their natural environment
- in vitro = lab method
- in vivo= natural organsim
2
Q
State the advantages of using cell cultures (8)?
A
- Control of environment: pH, temperature, osmolarity, levels of hormones, nutrients
- Control of the micro-environment of the cells
- Techniques: Cells can be stored in liquid nitrogen for long periods
- Cells can easily be quantified
- Cells can be easily characterised by cytological or immune-staining techniques and visualised using imaging techniques
- Reduces use of animals in scientific experiments
- Cheaper to maintain
3
Q
State the two types of cells found in cell cultures? VD
A
- Primary tissue cells
- Immortalised cell lines
4
Q
State characteristics of primary tissue cells? (4)
A
- Cells derived directly from tissues/patients (unmodified), good for personalised medicine
- Finite lifespan (~6-7 divisions)
- Cells can divide and/or differentiate
- Cells carry out normal functions - that they would normally in their tissue
5
Q
State the 2 main methods for isolation of primary tissue cells?
A
- Cells are allowed to migrate out of an explant - natural method of isolation
a. Explant = cells/tissue removed from body and placed in culture medium - Mechanical (mincing, sieving, pipetting) or/and enzymatic dissociation (trypsin, collagenase, hyaluronidase, protease, DNase) of tissue + method of detection of specific cell via immuno-purification
6
Q
State the exception to the standard methods of isolation of primary tissue
A
- Haemopoietic cells - Do not need to be disaggregated - They already are as individual cells circulating in blood
- HCs are SCs which give rise to other blood cells - any type of cell found in blood
7
Q
State the methods of isolation used here instead?
A
- Density centrifugation - separation of components of blood into layers dependent on density using density gradient medium
- Immuno-purification
- Fluorescence activated cell sorter (FACS)
8
Q
List examples of primary cells
Non haematopoietic with Haematopoietic
A
- Liver - Stem, progenitor cells
- Endothelial cells - T and B cells
- Muscle - Monocytes
- Skin - Osteoblasts
- Nerves - Dendritic cells
- Fibroblasts - Neutrophils
- Prostate - Erythrocytes / Platelets
9
Q
State disadvantages of primary cells (6)?
A
- Inter-patient variation: Different effects due to cells specific to each person
- Limited number (small amount at high cost)
- Finite lifespan and hard to maintain
- Difficult molecular manipulation
- Phenotypic instability
- Variable contamination
10
Q
What are immortal cell lines?
A
- Cell lines are cultures of animal cells that can be propagated repeatedly and sometimes indefinitely
- They arise from primary cell cultures
11
Q
State characteristics of immortalised cell lines? (7)
A
- Immortalised cells
- Less limited number of cell divisions (~30) or unlimited
- Phenotypically stable, defined population
- Limitless availability
- Easy to grow
- Good reproducibility
- Good model for basic science
12
Q
State the 2 methods of production of cell lines?
A
- Isolated from cancerous tissues (e.g. HeLa cells)
- Cancerous tissues used as these can grow very quickly or forever
- If this isn’t possible then - Immortalisation of healthy primary cultures (usually through genetic manipulation)
13
Q
How are cells lines produced via genetic manipulation? (Part 1)
A
- To generate cell lines we target processes that regulate cellular growth and ageing
- Telomerase, P53 and pRb
- As cells divide over time, telomeres shorten, and eventually cell division stops -> Apoptosis (p53, pRb)
- Telomeres shorten after each cell divison due to DNA end replication problem
14
Q
How are cells lines produced via genetic manipulation? (PART 2)
A
- p53 and pRB are the mediators of apoptosis
- Therefore p53 and pRB are inhibited so telomere stabilisation occurs and ‘immortality’ of cell lines
15
Q
State the two methods of genetic manipulation for cell line
production?
A
- Inhibition of tumour suppressor proteins
- Introduction of telomerase via transfection
16
Q
Describe the Inhibition of tumour suppressor proteins method of genetic manipulation for cell line production
A
- Take advantage of viral ‘oncoproteins’
- SV40’s T-antigen interacts with p53 and pRb. This can cause increased growth without loss of function of these proteins
- E6 targets p53 for degradation, and E7 binds to pRb inactivating it
17
Q
Describe the Introduction of telomerase via transfection method of genetic manipulation for cell line production
A
- The telomerase gene can also be introduced into a target primary cell
- Some cells need both introduction of the telomerase gene and inactivation of the pRb/p53 for “immortalisation”
- E6/E7 and telomerase transformations are believed to result in cell lines with a differentiated phenotype
18
Q
Describe the process of introduction of telomerase to form cell lines?
Comparison of primary tissue cell and cell lines
A
VD
19
Q
State the Key difference between 2D and 3D cell cultures?
A
- 2D = cells are grown on a flat dish
- 3D = artificially created environment in which cells are permitted to grow or interact with their surroundings in all three dimensions.
- Two types = spheroid and organoid
20
Q
State advantages and disadvantages of 2D cultures?
A
- Simple, well established
- Affordable |
- Forced apical basal polarity
- High stiffness
- Limited communication with other cells
- No diffusion of gradients
- Results not relevant to human physiology
21
Q
State advantages and disadvantages of 3D cultures?
A
- Adhesion in all three dimensions
- Variable stiffness
- No forced polarity
- Diffusion gradients of nutrients and waste products
- More relevant to human physiology |
- More complex
- Added expense
22
Q
State the difference between spheroid and organoid 3D cultures and what they are generated from?
A
- Spheroid
- Generated from immortalised cell lines
- Composed of 1 cell type which grows and proliferate
- But doesn’t undergo differentiation or self-organisation (non-stem cells)
- Can exhibit enhanced physiological response
23
Q
State the difference between spheroid and organoid 3D cultures and what they are generated from?
A
- Organoids
- Generated from primary tissue cells
- Composed of stem cells which can differentiate
24
Q
State a usage of patient-derived organoids?
A
Allows for the study of cancer drug resistance
25
Methods of cell transfection
What is transfection?
- Transfection is the process by which foreign DNA is deliberately introduced into a eukaryotic cell through non-viral methods including both chemical and physical methods in the labs e.g. a plasmid, a CRISPR/Cas9 complex.
- Always occurs in 2D
26
State the chemical and physical methods of transfection in the lab?
- Chemical: Lipofection, Calcium Phosphate, Cationic polymer, DEAE-Dextran, Magnetic mediated transfection, Activated dendrimers
- Physical: Electroporation, Nucleofection, Microinjection, Biolistic Particle Delivery, Laserfection/ optoinjection
- Viral: infection
27
State key methods of cell transfection?
- Chemical: Lipofection
- Physical: Electroporation, Nucleofaction
- Viral: Infection
28
State the components of lipofection?
- Liposomes: positive cationic head, linker and hydrophobic tail
- DNA: negatively charged
- When liposomes introduced to plasmid DNA via cationic lipid transfection systems, forms a lipoplexes with a net positive charge
29
State the process of lipofection?
- 1. Interaction with the cell memorane
- 2. Taken up by endocytosis
- 3. Release from the endosome
- 4. Transport to the nucleus
- 5. Entry to the nucleus is inefficient and may lead to mitosis
- Express protein of interest
30
What is the use of liposomes in drug delivery?
Used as potential drug carriers
31
Describe the method of electroporation?
- Plates of capacitor charged
- High electrical field temporarily forms pores on cell membrane of cell and increases permeability of cell. After electroporation the pores will reseal.
- Allows DNA to go through
- Rate of pore resealing is dependent on temperature
- decrease temp, decrease rate of pore resealing
32
What is nucleofaction and what is unique about it?
- Combination of electroporation and lipofection
- Used when the individual methods have been unsuccessful
- Increased efficiency particularly of non-dividing cells
- Technology is protected under patent
- Different solution and protocols are used for each cell type
33
What is viral infection/transduction?
- Exploits the mechanism of viral infection.
- High transfection efficiency.
- Usage: Retrovirus, Adenovirus, but most commonly Lentivirus are used.
- Target cells need to express the viral receptor to work.
- There are safety aspects to consider - as dealing with live virus
