Cell Culture Flashcards
Primary Cultures
cells taken directly from the animal
develop similar characteristics to cells in vivo
labour intensive / limited life span
Cell-specific markers in neural cultures
GFAP - astrocytic marker B tubulin III - neuronal marker MBF - oligodendritic marker nestin - porgenitor cell bisbenzamide - nuclear stain
Cell lines
Immortal divid rapidly and dont need to attach to subsrate for growth
e.g immortal/viral oncogene / tumour derived
easy to prepare
long life span
less work
lack invivo characteristics
Transfection methods
Ca+2 phosphate/ DNA co-precipitation Lipofection electrophoration nucleofection microinjection biolistics
Ca+2 phosphate / DNA co-precipitation
Ca+2 is pos DNA is neg
fused together applied to cells ca+2 pumps DNA into cells
common and cheap low transfer rate 1-5%
Lipofection
Cationic liposomes encapsulate DNA
fuses with membrane DNA enters cell 4-5% can be done in presence of serum
Electroporation
Cell membrane opened using electric current DNA enters
need a electoporator
Nucleofection
high voltage applied opens both the nuclear and cell membranes
more damaging to cells (60-80% transport efficiency)
Microinjection
injection of DNA using glass capillaries
used for large neurons of invertebrates
Biolistics
Naked DNA delivered to cell using gene gun
DNA adhered to gold/tungsten particles
Transduction methods
Lentivirus
herpes
AAV
adenovirus
Lentiviral vector system
consists of the viral vector the packaging vector and the envelope vector
Organotrophic / slice cultures
Culturing an explant (slice) of neural tissue
Slices typically 100-400 µm thick
Tissue is usually harvested postnatally (P0-10)
Advantages:
- can be maintained for long time in vitro
- tissue retains in vivo organisation and connections
- functional circuits can be studied and manipulated
Culturing neural stem cells two methods
neurospheres
monolayers
Neurospheres
Neurosphere = non-adherent spherical cluster of cells
Neurosphere culture assay by Reynolds & Weiss Science,
1992
Cell suspension prepared from tissue harvested at time of
proliferation in vivo (prior to differentiation)
Two phases in vitro:
(i) Expansion stage - requires growth factors (EGF, FGF)
for proliferation
(ii) Differentiation stage - on substrate
