W1 Mammalian cell culture Flashcards
3 main morphologies of cells
- fibroblast-like:
- attached to substrate; elongated and bipolar/multipolar; do not make tight cell-cell contacts - epithelial-like:
- attached to substrate; appear polygonal; grow to high densities; ,ale tight cell-cell contacts - lymphoblast-like:
- do not attach to substrate > grow in suspension; spherical shape
properties of primary cultures
surgically/enzymatically removed from organism > grown in suitable environment
finite life span
contain heterogenous population of cells
sub-culturing leads to generation of cell lines > immortalised primary cells
*macrophages and neurons do not divide in vitro > can only be used as primary cultures
common primary cultures
mouse embryonic fibroblasts (MEFs)
primary cortical or hipoocampal neurons (from embryonic or newborn mouse brains)
primary glial cells (astrocytes, from embryonic or newborn mouse brains)
blood cells
characteristics of continuous cell lines
divide infinitely and grow fast > continuous culturing
easy to culture and manipulate
often aneuploid chromosome number from duplication or deletion
loss of expression of tissue-specific genes > different in phenotype to the donor tissue/cell
characteristics of adult stem cells
multipotent, oligopotent or unipotent
rare population derived from adult tissues
differentiate into limited types of progenitors and terminally differentiated cells
mainly for repair and maintenance
characteristics of embryonic stem cells
pluripotent
derived from inner cell mass of blastocyst
differentiate into cells of the three germ layers
mainly for development
methods of iPSCs derivation
viral transduction
dna-based induction
mrna transfection
recombinant proteins
definition of ectopic expression
expression of a gene product in a cell culture system that does not express this gene product naturally
(Eg: expression of a neutron-specific protein in an epithelial cell)
types of transfection methods
calcium phosphate precipitation
poly-ethylene-imine (PEI)
cationic lipid-mediated
electroporation
viral transduction
microinjection
how does lipofection, calcium phosphate and PEI work
neutralise the negative charge of dna/rna backbone to allow them to pass through negatively charged lipid layer of cell membrane
difference between transfection and transduction
transfection: introduce dna/rna into eukaryotic cells using non-viral methods
transduction: same process but using viral-methods
pros and cons of transduction over transfection
pros: higher efficiency and less variable; easier screening for stable cell lines
cons: more time consuming
transient vs stable transfection
transient: temporary introduction of foreign dna into cells > short term expression
stable: permanent introduction into cells > integrate into chromosomal dna > continuous and long term expression
characteristics of transient transfection
short term expression
no integration into genome; plasmid gets diluted in culture as cells divide
expression typically very high
transfection efficiency varies
acute manipulation of specific gene activity in cell culture
excessive protein expression may alter gene function
characteristics of stable transfection
continuous expression
integration of plasmid into host genome > stable cell lines
expression levels vary across clonal lines
selection of “clonal” lines in which all cells express same level of the transgene
persistent gain of function or loss of function
potential compensation of cellular process of interest
