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