cell culture techniques Flashcards
what are cell cultures
lab method by which cells are grown under controlled conditions outside their natural environment
advantages of cell culture
control of physiochemical environment and physiological conditions
control of micro environment of cells
cells easily characterised by cytological or immune staining techniques and visualised using imaging techniques
cells can be stored in liquid nitrogen for long periods
cells easily quantified
reduces use of animals in scientific experiments
cheaper to maintain
describe primary tissue cells
derived from tissues/patients
good for personalised medicines
finite lifespan
cells divide and or differentiate
cells carry out normal functions
primary tissue cells: methods of isolation
- cells allowed to migrate out of an explant
- mechanical and enzymatic dissociation
exception = haemopoietic cells, don’t need to be disaggregated, already are as individual cells circulating in blood
named methods of isolation for primary tissue cells
density centrifugation
fluorescence activated cell sorter
immune-purification
examples of primary tissue cells
non haematopoietic: liver endothelial cells muscle skin nerves fibroblasts prostate
haematopoietic stem, progenitor cells T and B cells monocyte osteoblasts dendritic cells neutrophils erthrocytes megakaryocytic, platelets
disadvantages of tissues cells
inter-patient variation limited number finite lifespan and hard to maintain difficult molecular manipulation phenotypic instability variable contamination
cell lines characteristics
immortalised cells
less limited number of cell divisions or unlimited
phenotypically stable, defined population
limitless availability
easy to grow
good reproducibility
good model for basic science
cell lines: methods for production
- isolated from cancerous tissues
2. immortalisation of healthy primary cultures
cell lines: production through genetic manipulation
to generate cell lines we target processes that regulate cellular growth and ageing
- as cells divide over time, telomeres shorten and eventually cell division stops = apoptosis
how can we inhibit the function of tumournsuppresor proteins or introduce telomerase in order to alter a cells capability for its finite number of divisions
taking advantage of viral oncoprotein
e.g SV40,HPV use large t antigen or small t antigen, E6, E7 to target p53, pRb
cell lines 2
SV40 = interact with p53 and pRb. can cause increased growth without loss of functions of these proteins
telomerase gene can be introduced into target primary cells
some cells need both intro of telomerase and inactivation of p53/prb for immortalisation
2D vs 3D cell cultures
2D = forced apical-basal polarity, high stiffness, limited communication with other cells, no diffusion of gradients, results not relevant to human physiology, simple and affordable
3D =
adhesion in all three dimensions, no forced polarity, variable stiffness, diffusion gradients of nutrients and waste products, more relevant to human physiology, more complex and added expense
spheroids
3D cellular aggregate composed of 1 cell type that grow and proliferate, may exhibit enhanced physiological responses but don’t undergo differentiation
organoids
3D structure derived from either PSCs, neonatal tissue stem cells or AdSCs/adult progenitors, cells spontaneously organise into diff functional cell types and progenitors, and resemble their invivo counterpart and recapitulate at least some function of the organ
