Cell Culture Techniques Flashcards
History
1882: Sidney Ringer develops solutions of salt to maintain frog heart
1885: Wilhelm Roux cultures embryonic chick tissue
1940-50: Development of cell culture techniques for growing viruses
1951: Jonas Salk and his team grow polio virus in monkey kidney cells
1951: George Otto Gey propagates HeLa cells from Henrietta Lacks – ‘The Immortal Life of Henrietta Lacks’ – they have been used in ~75,000 studies
1954: Enders, Weller and Robbins receive Nobel prize
Why cell cultures?
Cell/tissue culture: laboratory method (in vitro) by which cells are grown under controlled conditions outside their natural environment.
Advantages
- Control of the physiochemical environment (pH, temperature, osmolarity..) and physiological conditions (levels of hormones and nutrients)
- Control of the micro-environment of the cells (matrix, cell-cell interactions and
cell substrates attachment)
- 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
Types of cells in culture
a) primary tissue cells
b) Cell lines
Primary tissue cells characteristics
- Cells derived directly from tissues/patients (unmodified), good for personalised medicine
- Finite lifespan (~6-7 divisions)
- 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 (mincing, sieving, pipetting) or/and enzymatic dissociation (trypsin, collagenase, hyaluronidase, protease,
DNAase)
Exception – Haemopoietic cells – Do not need to be disaggregated – They already are as individual cells circulating in blood
diagrams and examples
Primary tissue cells disadvantages
- Inter-patient variation
- Limited number (small amount at high cost)
- Finite lifespan and hard to maintain
- Difficult molecular manipulation
- Phenotypic instability
- Variable contamination
Cell line characteristics
- 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
Cell lines - Methods of production
- Isolated from cancerous tissues (e.g. HeLa cells)
- Immortalisation of healthy primary cultures (usually through
genetic manipulation)
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 (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?
Answer: taking 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
Cell lines made using E6/ E7 oncoproteins are believed to maintain a differentiated phenotype - 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
diagrams and tables
2D cultures vs 3D cultures
3D cell culture -artificially created environment in which cells are permitted to grow or interact with their surroundings in all three dimensions.
2D
- Forced apical-basal polarity
- High stiffness
- Limited communication with other cells
- No diffusion of gradients
- Results not relevant to human physiology
- Simple, well established
- 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
- Added expense
Patient-derived organoids allow the study cancer drug resistance
Spheroids
Generated from cell lines
A 3D cellular aggregate composed of 1 or more cell types that grow and proliferate, and may exhibit enhanced physiological responses but do not undergo differentiation or self-organisation (i.e. non-stem cells)
Organoids
Generated from primary tissue
A 3D structure derived from either PSCs, neonatal tissue stem cells or AdSC/adult progenitors, in which cells spontaneously self-organise into properly differentiated functional cell types and progenitors, and which resemble their in vivo counterpart and recapitulate at least some function of the organ.
Methods of cell 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 lab.
Chemical, physical, viral e.g.’s
e.g. a plasmid, a CRISPR/Cas9 complex
Lipofection
diagrams
- Interaction with the cell membrane
- Taken up by endocytosis
- Release from the endosome
- Transport to the nucleus
- Entry to the nucleus inefficient and may need mitosis
Electroporation
diagrams
