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