Primary Cell Culture Flashcards
1
Q
Primary Culture vs Cell lines
A
- P: cells derived directly from tissue that have a finite lifespan, have interpatient variability, divide and/or carry out normal functions
- C: identical, divide constantly, and can theoretically divide forever (HeLa) - gene expression is also not normal
2
Q
Examples of cells: Non-haemopoietic and haemopoietic
A
- Non - liver, muscle, skin, nerves, fibroblasts, endothelial cells
Haem: stem/progenitor cells, T and B cells, monocytes, osteoblasts, dendritic cells, neutrophils, platelets etc
3
Q
How do we disaggregate the cells?
A
- allow to migrate out of an explant
- mechanical dissociation (break them apart by continually pipetting in and out)
- enzymatic dissociation (trypsin, collagenase, hyaluronidase, protease, DNAase)
- exception - haemopoietic cells are already disaggregated
4
Q
Where do we get stem cells from?
A
- bone marrow aspirate
- umbilical cord blood
- mobilised peripheral blood - need to mobilise stem cells in peripheral blood by giving them growth factor
5
Q
Haemopoeisis
A
- STEM CELLS divide and differentiate, changing characteristics to become EARLY PROGENITORS. (still looks the same)
- Then differentiate into LATE PROGENITORS (still look the same)
- Become IMMATURE PRECURSORS and start to become a bit more recognisable
- They then differentiate into their final stage such as RBCs, neutrophils, platelets.
- all controlled by cytokines and other growth factors
- as they get further into the process, the more committed they become
6
Q
Stem cells
A
- pluripotent - gives rise to all lineages
- self-renew
- rare cells
- responsible for engraftment (red cell growth)
7
Q
Progenitor cells
A
- undifferentiated
- not distinguished by morphology
- committed to one or more lineages
- detected in colony-forming assays
8
Q
Precursor cells
A
- immature but recognisable
- cells starting to differentiate
- few final divisions before mature cells
9
Q
Haematopoietic growth factors
A
- polypeptide growth factors (cytokines)
- bind to cell surface transmembrane receptors
- stimulate growth and survival of progenitors
10
Q
Recreating in vivo situation
A
- need to put growth factors in, or cells that will produce the growth factors
- stem cells will not just differentiate into all the different types of cells by themselves
11
Q
Cells are supported by a microenvironment
A
- stromal cells (fibroblasts, macrophages, endothelial cells, adipocytes)
- EC matrix
- Adhesion receptros
- cytokines (positively/negatively acting)
- inhibitors
12
Q
How can we tell the difference between stem cells from mature cells?
A
- CD34 is an antigen on the surface of stem cells, but lost after maturity
- lin (lineage specific) negative - anything on the mature cells that is not present on the stem cells - e.g. CD4
- can label Rh123 with a fluorochrome. majority of stem cells are not active and are sitting out of cycle. The stem cells wont light up, however later on when they start dividing, the cells will light up with Rh.
13
Q
How do we purify the stem cells?
A
- erythrocyte lysis - get rid of RBCs
- density gradient centrifugation - remove neutrophils
- adherence depletion - bone marrow onto plastic, sticky cells will leave solution - macrophages and fibroblasts
- antibody depletion - use antibodies to get rid of cells we dont want
- antibody selection (PUREST) - use antibodies to keep cells we do want - using magnetic bead or fluorescent tag
14
Q
What environment do we culture cells in?
A
- laminar flow cabinet - sterile air inside
- suspend in medium in culture flask such as agar
15
Q
Colony assays
A
- progenitors grow to form clonies of mature cells
- from 32 to 100s/1000s of cells in a colony
- progenitors are hence called “colony forming units” - CFU
- semi-solid medium (agar, methylcellulose)
- add growth factors
