CCT Flashcards
What are advantages of cell cultures?
→Control of the physiochemical environment (pH, temperature, osmolarity) and physiological conditions (levels of hormones and nutrients)
→Control of the micro-environment of the cells
→Cells can be easily characterised by cytological or immune-staining techniques and visualised using imaging techniques
→Cells can be stored in liquid nitrogen for long periods (cryopreservation)
→Cells can be easily quantified
Reduces use of animals in scientific experiments
→Cheaper to maintain
What are the two types of cell cultures?
→primary tissue cells
→immortalised cell lines
What are the differences between primary tissue cells and cell line culture?
→primary tissue cells have limited lifespan retain cells
but CL has infinite lifespan and loses specificity
→pre-characterised and ready to use but CL requires authentication required before use
→study cells with varied donor characteristics but CL study single donor repeatedly
→PTC are less heterogenous
What are the characteristics of PTC?
→Cells derived directly from tissues/patients (unmodified)
→Finite lifespan (~6-7 divisions)
→Cells divide and/or differentiate
→Cells carry out normal functions
→Living conditions similar to body environment
What are the two methods for isolation of cells?
→explant culture-Cells allowed to migrate out of an explant
→Mechanical (mincing, sieving, pipetting) or/and enzymatic dissociation (trypsin, collagenase, hyaluronidase, protease, DNAase)
Why are haemotopoeic cells an exception when isolating cells?
→Do not need to be disaggregated
→already are as individual cells circulating in blood.
→Use density centrifugation instead
Why is centrifugation used for haematopoietic cells?
→Takes advantage of the different densities of diff blood cells
Which cells are densest?
→Granulocytes
What are other methods of isolation?
→Immuno-purification- using antibody-coated magnetic beads
→Fluorescence activated cell sorter
What are the non-haematopoietic cells?
→liver →muscle →endothelial →skin →nerves → fibroblasts
What are the disadvantages of primary tissue cells?
→Inter-patient variation →Limited number (small amount at high cost) →Finite lifespan and hard to maintain →Difficult molecular manipulation →Phenotypic instability →Variable contamination
What are the characteristics of cell lines?
→Immortalised cells from one type of primary tissue 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 if same cell lines are used
What are the two types cell lines?
- Isolated from cancerous tissues (e.g. HeLa cells)
2. Immortalisation of healthy primary cultures (usually through genetic manipulation)
What are the targets in cells for production of cell lines through genetic manipulation?
→processes that regulate cellular growth and ageing
What are process that regulate cellular growth and ageing?
→p53
→pRB
→telomerase
What happens as telomeres shorten?
→cell division stops
→ Apoptosis (p53, pRb)
How can we inhibit the function of tumour suppressor proteins, or introduce telomerase in order to alter a cell’s capability for its finite number of divisions?
→taking advantage of viral ‘oncoproteins
What are the viruses used to alter a cell’s capability for its finite number of divisions and their viral oncoproteins?
→Simian Virus-40
(SV40)- Large T antigen, Small t antigen
→Human Papilloma
Virus (HPV), E6 E7
What are the targets for viruses used in cell line division numbers?
→p53
→pRb
How does SV40 T-antigen work in cell line generation?
→interacts with p53 and pRb
What does use of SV40 T-antigen in cell line allow?
→increased growth without loss of function of these proteins
What does E6 and E7do?
→targets p53 for degradation, and E7 binds to pRb inactivating it
What is the phenotype like in cell lines made using E6 and E7?
→maintain a differentiated phenotype
What do some cells need in generating cell lines?
→need both introduction of the telomerase gene
→inactivation of the pRb/p53 for “immortalisation”
Are TERT enzymes active in somatic cells?
→no
How is TERT activated in somatic cells?
→need to be transfected
→Make a vector containing selection marker eg neomycin(antibiotic resistance) and telomerase gene.
→Plasmid has to be circularised
→Treat cells with antibiotic and only cells with it will survive
What are the advantages of 2D cell culture?
Simple, well established
- Affordable
What are the disadvantages of 2D cell cultures?
→Forced apical-basal polarity
→High stiffness
→Limited communication with other cells
→No diffusion of gradients
→Results not relevant to human physiology
→Cells lose their original phenotype and functional characteristics
What are the advantages of 3D cultures?
→Adhesion in all three dimensions
→No forced polarity
→ Variable stiffness
→ Diffusion gradients of nutrients and waste products
→More relevant to human physiology
→Allow cells to aggregate- balls of cells
What are the disadvantages of 3D cultures?
→More complex
→Added expense
What are the two types of shapes of 3D cultures?
→spheroids
→organoids
Describe spheroids
→3D
→cellular aggregate composed of 1(or more) cell types that grow and proliferation
→exhibit enhanced physiological responses
→do not differentiate or self-organise
Describe organoids
→3D culture →derived from PSCs, AdSC → cells spontaneously self organise into properly differentiated functional cells types → resemble in vivo counterpart →resemble some function of organ
What do patient derived organoids allow?
→the study cancer drug resistance
→Make treatment based on results of the treatment on culture
Why are patient-derived cells organoids?
→derived from primary tissue cells
What is the percentage mimicry of phenotype in organoids?
→85%
Define transefection
process by which foreign DNA is deliberately introduced into a eukaryotic cell through non-viral methods including both chemical and physical methods in the lab.
Give examples of non-viral transfection methods
→plasmid,
→ CRISPR/Cas9 complex
Give examples of chemical transfection
→Lipofection
→Calcium phosphate
→Cationic polymer
→Magnet-mediated transfection
Why is transfection performed in 2D?
→if 3D not all cells would be transfected
Give examples of physical transfection
→Electroporation →Nucleofection →Microinjection →Laserfection/ optoinjection →Biolistic Particle Delivery
What is lipofection?
→Using cationic lipid transfection systems
→uses liposomes
Why are liposomes used for lipofection?
→has unilamellar liposomal structure
→has phospholipid bilayer
→net positive charge and the DNA/plasmid will have a negative charge
Describe lipofection
- Interaction with the cell membrane
- Taken up by endocytosis
- Release from the endosome
→4. Transport to the nucleus
→5. Entry to the nucleus inefficient and may need mitosis
Do liposomes deliver hydrophobic or hydrophilic drugs?
→hydrophobic or hydrophilic
How do you ensure the drug is delivered into the tissue of interest?
→Attach tissue specific antigens to the liposomes
How is electroporation carried out?
→High electric field
→Forms pores- increase permeability
→DNA goes through cell which then reseal
What does the rate of pore sealing in electroporation dependent on?
→temperature
What are the characteristics of nucleofection?
→Combination of electroporation and lipofection
- Increased efficiency particularly of non-dividing cells
- Technology is protected under patent
- Different solution and protocols are used for each cell type
Describe viral infection
1. plate HEK293T cells →transfect plasmids →collect 1st supernatant →refrigerate →collect 2nd supernatant →collect viral pellet after centrifuging →use for transduction
What is HEK293T?
→used as vehicle for production of material in viral transfection
What types of cells are used in viral infection?
→Retrovirus,
→Adenovirus,
→Lentivirus commonly
are used.
What enables HEK293T cells to produce recombinant proteins within plasmid vectors?
→contains the SV40 large T antigen
