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
What are the advantages of using 2D cell cultures?
- Simple, well established
- affordable
What are the disadvantages of the 2D cell culture?
- forced apical-based polarity
- high stiffness
- limited communication with other cells
- no diffusion of gradients
- results not relevant to human physiology
What are the two main types of 3D cultures?
Spheroids
- generated from cell lines
- no cell differentiation or organisation
- usually one cell type or two max
Organoids
- derived from primary tissues
- derived from stem cells in the adult tissue
- can self organise into properly differentiated cell types and progenitors
- recapitulate come function of the organ “mini organs”
How can organoiods allow the study of cancer drug treatment?
Patient derived organoids from tumour biopsy
We can treat the mini organ with different drugs to see which drugs are more effective with treating cancer cells
Can extrapolate results and make treatment decisions
What is cell transfection?
The process by which foreign DNA is deliberately introduced into a eukaryotic cell through NON VIRAL METHODS including both chemical and physical methods
Always performed in 2D because in 3D the transfection might not meet all the cells
What is lipofection?
A transfection technique that introduces DNA into the cell using liposomes
Liposomes are vesicles that can easily merge with cell membrane
Liposomes have a net posative charge
Liposomes can be used as potential drug carriers for drug delivery
Plasmid membrane of cells is negatively charged
Describe what happens during lipofection?
- Interaction with the cell membrane
- Taken up by ENDO cytosol
- Release from the ENDO some
- Transport to the nucleus
- Entry to the nucleus inefficient and may need mitosis
What is electroporation?
A type of transfection where a electric field is applied to the cell to increase they’re permiability, allowing drugs,DNA, to enter the cell (in plasmid)
What is nucleofection?
Combination of electroporation and lipofection
Increased efficiency particularly if non-dividing cells
Technology is protected under patient
Different solution and protocols are used for each cell type.
What is viral infection/transduction ?
A type of transfection that exploits the mechanism of viral infection
High transfection efficiency
Retrovirus, Adenovirus, but most commonly, lentivirus
Target cells need to express viral receptor to work
Saftey aspects to consider
