Cell Culture Techniques Flashcards
History of Cell Culture
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
Define Cell/Tissue culture
Laboratory method (in vitro) by which cells are grown under controlled conditions outside their natural environment
List some advantage of cell culture
- 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
What are the types of cells in culture
- Primary tissue cell
- Immortalised cell lines
What are the differences between primary tissue cells and immortalised cell lines
Primary Tissue cells:
- Limited lifespan retains cell identity
- Taken from the tissue
- Pre-characterised and ready to use
- study cells with varied donor characteristics
Immortalised cell lines:
- Infinite lifespan, loses cell specificity
- from a vial with high mutations and clonal selections
- authentication required before use
- study single donor repeatedly
What are the characteristics of primary tissue cells
- 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
What are the methods of isolation for primary tissue cells
- Cells allowed to migrate out of an explant
- Mechanical and/or 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
What are the methods of isolation for hematopoietic cells
- Density centrifugation
- Immune-purification
- Fluorescence activated cell sorter (FACS)
Give some examples of Non-haematopoetic primary cells
- Liver
- Endothelial cells
- Muscle
- Skin
- Nerves
- Fibroblast
- Prostate
Give some examples of haematopoetic primary cells
- Stem, progenitor cells
- T and B cells
- Monocyte
- Dendritic cells
- Neutrophils
- Erythrocytes
- Megakaryocytes, Platelets
What are some disadvantages of primary 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 some characteristics of Cell lines
- 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
What are some methods of production of cell lines
- Isolated from cancerous tissues (e.g. HeLa cells)
- Immortalisation of healthy primary cultures (usually through genetic manipulation)
Describe the production of cell lines through genetic manipulation
- To generate cell lines we target processes that regulate cellular growth and ageing (p53, pRb and Telomerase)
- As cells divide over time, telomeres shorten, and eventually cell division stops (hayflick’s limit)→ Apoptosis (p53, pRb)
- Inactivation of p53 and RB can cause immortality
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?
- Take advantage of viral oncoproteins which are able to target p53 and RB
Simian Virus-40
Human Papiloma virus
How does the viral oncoprotein work
- 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
How does telomerase play a role in cell immortalisation
- The telomerase gene can also be introduced into a target primary cell.
- Some cells need both introductions 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
How is TERT transfected into cells
- Used plasmid which contains genes for neomycin resistance and TERT
- Plasmid is circularised and transfected into cells
- Cells grown in neomycin solution to only produce colonies which have been positively transfected
What is the difference between 2D and 3D cell cultures
2D is grown on a flat surface whereas 3D is grown in a matrix either in a spheroid or organoid culture
What are 3D cultures
An artificially created environment in which cells are permitted to grow or interact with their surroundings in all three dimensions.
Compare 2D and 3D cultures
2D Cultures:
- 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 Cultures:
- 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
Describe what is a Spheroid culture
Generated from cell lines
A 3d cellular aggregate is composed of one or more cell types that grow and proliferate.
May exhibit enhanced physiological responses but do not undergo differentiation or self-organisation
Describe what is a Organoid culture
Generated from primary tissue
3d structure derived from either PSCs, neonatal tissue stem cells or adult progenitors in which they can self-organisation and differentiate. Can resemble their in vivo counterparts and recapitulate some functions of organs
How are patient derived organdies good for cancer drug resistance studies
- biopsy cell straight from the patient
- can create organoids from the tumour biopsy cells
- test which drugs on the organoid model
- use the results to better treat the tumour in the patient
