cell culture techniques Flashcards
(35 cards)
outline the timeline of cell culture 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: George Otto Gey propagates HeLa cells from Henrietta Lacks.
1951: Jonas Salk and his team grow polio virus in monkey kidney cells.
1954: Fnders Weller and Robbins receive Nobel prize.
what is cell culture and why do scientists use it?
- lab method (in vitro) by which cells are grown under controlled conditions outside their natural environment.
- study how cells function.
- study how diseases develop
- test new treatments without endangering patients
What are advantages of cell culture?
- control the physiochemical environment (pH, temp, osmolarity) and physiological conditions (levels of hormones and nutrients)
- control micro-environment of the cells
- cells can easily be 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
- Cheaper to maintain
Why is it important cells can duplicate themselves?
- large quantity of cell culture needed so scientists can repeat the experiments over and compare results with other scientists
- so important to have population that duplicates themselves.
What is so special about HeLa cells?
- normal human cells have built in mechanisms that only lets them divide a certain number of times before apoptosis.
- Cancer cells ignore these signals and divide rapidly so we can generate large cell culture to study diseases BUT they can not survive Invitro (outside the human body , in labs)
- HeLa cells were the only cells that survived in labs (immortal)
- HeLa cells took up the polio virus and replicated, enabling Jonas Salk test a vaccine for polio.
What are two types of cells in culture?
- primary tissue cells
2. Immortalised cell lines.
What are characteristics of primary tissue cells?
- cells derived directly from tissue/patient, good for personalised medicine
- finite lifespan (~6/7 divisions)
- cells divide/differentiate
- cells carry out normal functions.
Outline method of isolation for primary tissue cells.
- cells allowed to migrate out of an explant.
2. Mechanical( mincing, sieving, pipetting) or enzymatic dissociation ( trypsin p, collagenases, proteases)
Define explant
-tissue which has been transferred from an animal/plant to a nutrient medium.
what are methods of mechanical dissociation?
- minicing
- sieving
- pipetting
What are
enzymatic agents used for dissociation?
- trypsin
- collagenase
- hyaluronidase
- protease
- DNAase.
Give an example where mechanical and enzymatic dissociation occurs.
- Magnetic immuno-purification of CD31+ Placental endothelial cells:
1. mincing = cutting into small uniform pieces.
2. dispase, trypsin, collagenase
What is an exception for dissociation of cells?
-Haemopoietic cells (blood cells) do not need to be disaggregated because they are already found as individual cells circulating in blood.
What are methods of isolation for hematopoietic cells?
- density centrifugation (sediment in test tube according to molecular weight)
- immunopurification
- Fluorescence activated cell sorter(FACS)
=> all filtering methods
What are the layers of in density centrifugation?
- plasma
- PMBCs
3.Density gradient medium - Granulocytes, Erythrocytes
=> 1-4 , smallest to largest molecular weight.
What are examples of non - hematopoietic primary cells?
- liver, endothelial cells, muscle, skin, nerves, fibroblast, prostate.
What are examples of haematopoietic cells?
- Stem, progenitor cells , T and B cells, monocyte, osteoblasts , dendritic cells, neutrophils, erythrocytes, megakaryocytes and platelets.
What are 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 characteristics of immortalised cell lines?
- immortalised cell lines are prolonged so scientists can study cells.
- less limited number of cell divisions
- phenotypically stable, defined population
- limitless availability(
- easy to grow
- good reproducibility
- good model for basic science
What is the method to produce cell lines?
- isolated from cancerous tissue (eg. HeLa)
2. immortalisation of healthy primary cultures (usually through genetic manipulation)
How do you produce immortal cells through genetic manipulation?
- target processes that regulate cellular growth and ageing
- p53, pRb involved in apoptosis so you genetically modify these/make them inactive, to prevent apoptosis = immortal cells line.
- telomerase mantains telomeres and prevents them from shortening so it needs to be activated = immortal cell line(tumour cells basically)
What genes and protein are involved in genetic manipulation?
- p53 = supports DNA repair by arresting cell cycle.
- pRb = tumour surpressor product of retinoblastoma susceptibility gene.
- Telomerase = reverse transcriptase which helps, maintains telomeres and prevents them from shortening, elongates telomere repeat sequences.
Why do we inhibit the function of tumour suppressor proteins, or introduce telomerase in order to turn primary cell line (finite) to immortalised cell line (infinite) ?
- tumour suppressor genes suppress the tumour gene preventing uncontrolled division leading to finite number of cells but to make a cell line (infinite cells) we need to inhibit tumour suppressor.
E6 (viral oncoprotein) targets tP53 for degradation and E7 binds to pRb inactivating it. - telomeres shorten over time with age and this leads to senescence and apoptosis (finite life span) telomerase sythesises telomere cells repeats reversing the loss of DNA in each round of replication ( infinite in cell lines)
=> the telomerase gene introduced into target primary cell
SV40’s T - antigen is responsible for viral DNA replication and it can cause increased growth of viral DNA by interacting with p53 and pRb (function of these proteins is intact)
- some cells need both introduction of the telomerase gene and inactivation of the pRb/p53 for immortalisation.
What is 3D culture?
- artificially created environment in which cells are permitted to grow or interact with their surroundings in all 3D.
