Primary Culture Techniques Flashcards
Describe primary cell cultures.
They are cells derived directly from tissues.
There’s a lot of inter-patient variability, because we’re taking cells directly from patients.
These culture have a finite lifespan. The cells divide and/or differentiate further.
They then carry out normal functions.
Essentially, with primary culture techniques, we are recreating in-vivo conditions.
Compare primary cell cultures to cell lines.
PRIMARY CELL CULTURE:
- cells derived directly from tissues
- interpatient variability
- cells have a finite lifespan
- cells divide and/or differentiate
- cells carry out normal functions
CELL LINES:
- spontaneously derived from tumours or after genetic manipulation
- cells are identical
- cells are theoretically immortal
- cells divide but don’t differentiate
- might have abnormal gene expression, so may not have fully normal function
List some examples of primary cultures.
NON-HAEMOPOIETIC:
- Liver
- Muscle
- Skin
- Nerves
- Fibroblasts
- Endothelial cells
HAEMOPOIETIC:
- Stem, Progenitor cells
- T and B cells
- Monocyte, Macrophages
- Osteoblasts
- Dendritic cells
- Neutrophils, Eosinophils, Basophils, Mast cells
- Erythrocytes
- Megakaryoctyes, Platelets
How do we get haematopoietic cell samples?
Haematopoietic are already in a suspension (they are already disaggregated) so we can take them directly from the body with no manipulation.
How do we get cell samples from tissues?
There are 3 ways:
1) You put the tissue into culture, and the cells are allowed to migrate out of an explant, divide and grow.
2) You can mechanically dissociate them (either by mincing, sieving,pipetting, etc.)
3) You can enzymatically dissociate them (using trypsins, collagenases, proteases, etc.) to break the bonds between the cells and disaggregate them.
What are some sources of stem cells?
- Bone marrow aspirate
- Umbilical cord blood
- Mobilised peripheral blood
For last point, normally people don’t have stem cells in peripheral blood, but if we pump growth factors into people then we can push the stem cells out into the peripheral blood and collect them.
Describe the areas from which we can extract stem cells.
For children, we can extract them from bones as all of them have red bone marrow. There are also stem cells present in the liver and spleen.
In an adult, stem cells are mainly harvested from the ends of long bones (like the femur and humerus). They can also be found in the skull, vertebrae, ribs sternum and pelvis.
Describe stem cells.
- they’re pluripotent - give rise to all lineages
- they self-renew
- they are rare cells
- they are responsible for engraftment
Describe progenitor cells.
- they are undifferentiated
- they are not distinguished by morphology
- they’re committed to one or more lineages
- they’re detected in colony-forming assays
Describe precursor cells.
- they’re immature but recognisable
- the cells are starting to differentiate
- they have a few final divisions before they become mature cells
Describe haematopoietic growth factors.
They are polypeptide growth factors (or cytokines; the words are interchangeable).
They bind to cell surface transmembrane receptors, and stimulate the growth and survival of progenitors.
They are, again, responsible at every stage of haemopoeisis.
They’re also important in keeping cells out of the cell cycle until they need to be brought in.
Describe how the different components of the stem cell niche interact.
Stromal cells (such as fibroblasts and macrophages) produce ECM (extra-cellular matrix) and adhesion molecules.
They are also responsible for the cytokines that act in the environment.
Describe the two antigen markers we can use to distinguish between populations of cells.
CD34: stem cells and progenitor cells are CD34+, while mature cells are CD34-
Lin: stem cells and progenitor cells are Lin-, while mature cells are Lin+
There is also something called Rhodamine 123, which is a fluorescent dye, and is only picked up by cycling cells. So, early stem cells will appear dull, but when they enter the cycle they are bright. The same concept applies to the drug 5-fluorouracil. When out of cycle, the cells are resistant to the drug, but when they enter the cycle they are sensitive to the drug.
How else can we distinguish different populations of cells?
We can use colony-forming assays, to distinguish between the different progenitors themselves, and also to distinguish them from other cell type populations.
List and describe some different cell processing methods.
For an ENRICHED SAMPLE:
- Erythrocyte lysis - lyse the cells
For a SLIGHTLY MORE PURE SAMPLE:
- Density gradient centrifugation – spin cells on gradient, to remove a certain type of cell
- Adherence depletion – put marrow on plastic, and keep the cells that stick to it
For a VERY PURE SAMPLE:
- Antibody depletion - deplete all cells that are Lin+ (mature cells)
- Antibody selection – positively select the cells that we want, either using fluorescent cell sourcing or using magnetic beads to pull them out
Describe the basis of colony assays.
Progenitors can grow to form colonies of mature cells. These can range from 32 to hundreds or thousands of cells in a colony.
Thus, progenitors are called “Colony Forming Units” - CFUs.
again. this is used to identity progenitor cells.
How would we perform a colony assay?
We use a semi solid medium, usually agar or methylcellulose, and we add growth factors. Then, we incubate the sample for 7-14 days, waiting for the progenitors to divide and differentiate to form colonies that we can see down a microscope.
Hence, we can quantitate how many progenitor cells we originally had in our suspension that we put into our culture.
List some CFUs and their lineage.
CFU-G - granulocyte progenitor
CFU-E + BFU-E - erythroid progenitors
CFU-Mk - megakaryocyte progenitor
CFU-GM - granulocyte/monocyte progenitor
CFU-GEMM - granulocyte/erythroid/monocyte/megakaryocyte progenitor
CFU-bas - basophil progenitor
CFU-eo - eosinophil progenitor
We can see CFUs of single cell types.
We can also see CFUs of multiple cell types, meaning that they came from a progenitor at an earlier stage than the other cell types – a progenitor that still has the ability to differentiate down different lineages (2 or 4).
What are some applications of primary cell culture techniques?
- Research – they allow us to observe/test basic haemopoiesis and carcinogenesis
- We can test toxicity of chemotherapeutic agents and carcinogens
- We can generate cells for stem cell transplantation /manipulation
