Cell Culture Techniques Flashcards
What is a cell culture?
Laboratory method (IN VITRO) by which cells are grown under controlled conditions outside of they’re natural environment.
What are the advantages of cell cultures?
- control of the physiochemical environment (pH, temperature) and physiological environment (levels of hormone and nutrients)
- control of micro-environment of cells (matrix, cell-cell interactions)
- 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 of time
- cells can be easily quantified
- reduces use of animals in scientific experiments
- cheaper to maintain
What is the difference between primary tissue cells and immortalised cell lines?
Primary living cells:
- taking from living tissues
- limited lifespan retains cell identitiy
- pre-characterised and ready to use
- study cells with varied donor characteristics
Immortalised cell lines:
- derived from one cell
- infinite, lifespan, loses cell specifity
- from a vial with high mutations and colonal selections
- authentication required before use
- study single donor repeatedly
What are primary tissue cells?
Cells derived directly from tissue/patients, good for personalised medicine
Finite lifespan (6-7 divisions)
Cells divide and/or differentiate
Cells carry out normal functions
What methods of isolations are used for primary tissue cells?
- Cells allowed to migrate out of explant
- Mechanical (mincing, sieving, pipetting) or enzymatically (trypsin, protease, DNAase) isolate the tissue
ONLY EXCEPTION - haematopoetic cells do not need to be disaggregated - they already are as individual cells circulating blood
- Haematopoetic cells are separated using:
- density centrifugation
- immuno-purification (mixed with antibody coated magnetic beads and antigen expressing cells are isolated)
- fluorescent activated cell sorter (FACS)
Give some examples of non-haematopoetic primary cells?
- liver
- skin
- endothelial cells
- muscle
- nerves
- fibroblasts
- prostate
Give some examples of haematopoetix primary cells?
- T cells and B cells
- stem progenitor cells
- dendritic cells
- monocyte
- neutrophils
- erythrocytes
- megakaryotyes
- osteoblasts
What are the disadvantages of primary tissue cells?
- inner patient variation
- limited number (small amount at high cost)
- finite lifespan and hard to maintain
- difficult molecular manipulation
- phenotypic instability (have different characteristics)
- variable contamination
What are cell lines?
Immortalised cells
Less limited number of cell divisions (~30) or unlimited
Phenotypically stable, define population
Limitless availability
Easy to grow
Good reproducibility
Good model for basic science
What are methods for producing cell lines?
- Isolated from cancerous tissues
- Immortalisation of healthy primary cultures (genetic manipulation)
How can cell lines be produced from genetic manipulation?
Targeting processes that regulate cellular growth and ageing
- p53 inactivation
- telomerase activated
- pRb inactivation
As cells divide over time, telomeres shorten , and eventually cell division stops (APOPTOSIS)
How can we inhibit the function of tumour suppressor proteins, or introduce telomerase in order to alter a cells capability for its finite number of divisions?
Taking advantage of viral “Oncoproteins”
SV40:
- SV40 T antigen interacts with p53 and pRb
- This can cause increased growth without loss of function of these proteins
E6+E7:
- targets p53 for degradation, and E7 binds to pRb inactivating it
USUAL METHOD
Telomerase gene can also be introduced into target primary cell - using plasmid cultures with antibiotics
SOME CELLS NEED BOTH INTRODUCTION OF TELOMERASE GENE AND INACTIVATION OF THE PRB/p53 for “immortalisation”
What is a 3D cultures?
Artificially created environment in which cells are permitted to grow or interact with their surroundings in all three dimensions
What are the advantages of using 3D cultures?
- adhesion in all three dimensions
- no forced polarity
- variable stiffness
- more relevant to human physiology
- diffusion gradients of nutrients and waste products
What are the negatives of 3D cell cultures?
- more complex
- added expenses
