Lecture 16 Flashcards
use proper terminology to describe primary cells and cell lines
primary culture/cells: cells derived directly from a particular organ/tissue
cell line with finite life span: after first sub culture
cell line with indefinite life span: some cells continue growing past hayflick limit
transformed cell lines: some cell lines become tumorigenic
list and describe differences between primary cells and cell lines, and explain the reasons for those differences
primary cultures are considered more representative of tissue specific characteristics than the cell lines. primary cultures are heterogeneous, and the longer the sub culture the more homogenous and less specialized the cells become. cells lose characteristics associated with tissue of origin during adaptation to culture conditions
describe technical consideration for generating and maintaining primary cell cultures
Technical: tissue source can come from biopsies or animal embryos.
isolation technique (fine dissection, mechanical disaggregation, enzymatic disaggregation).
identification of desired cell types.
determination of nutritional requirements.
propagation and selection.
remove fat
chop tissue finely
seed first culture at very high concentration
use rich medium and serum
list potential sources for primary cells and describe advantages and disadvantages of the sources
human tissue sources: fetus, tissue biopsies, surgically removed tissues, naturally derived living tissue (placenta)
animal tissue sources: mouse embryos, chick embryos
animal tissue often better b/c harvested more intentionally and sterile
describe different methods that can be used to generate primary cell cultures
fine dissection: chopping down to explant size
mechanical disaggregation: sieving, syringing, pipetting. only for very soft tissues (brain, young embryo)
warm trypsin: shorter, can be rough and lose cells
cold trypsin: longer, loses less cells
collagenase: long time, good for sensitive cells
describe general consideration for generating and maintaining primary cell cultures
General: need to think about species of donor, age of donor, and choice of tissue. tissue from mature animals yields less dividing cells. some cell types are terminally differentiated and unlikely to survive
details of primary explant, how its done, pros and cons
tissue is chopped, washed, and seeded onto culture surface with small volume of serum medium, incubate until outgrowth.
good for small tissue amounts, but some tissues don’t adhere well. high failure rate
details of enzymatic digestion. types?
cells dissociate from ECM. Viability and yield are critical for success, use dissociation aids (chelators or enzymes) most commonly trypsin or collagenase.
collegenase splits collagens and uncoils fiber fragments. there are four types. good for sensitive tissues, takes a long time
trypsin is a pancreatic serine protease, can be pure or crude. pure = less toxic but crude = more effective. warm or cold; warm is faster but can damage. cold minimizes damage and has higher yeild, but longer time
pancreatin is super crude mixture from pancreas, super effective but also kills cells
details of mechanical disaggregation. pros and cons
outgrowths from primary explants are slow and highly selective. mechanical disaggregation is quicker and less labor intensive than enzymatic digestion, avoids proteolytic damage, but is only effective on few soft tissues like brain
