Primary Cell Culture Techniques Flashcards
- What is primary cell culture?
Primary cell culture techniques allow you to grow cells directly from the body. They are put into situations in vitro and the in vivo situation is recreated as closely as possible.
- The following questions are about primary cell culture:
- Where are the cells derived from?
- Is there interpatient variability
- What is the lifespan of these cells?
- What do the cells do?
- Directly from tissue
- Interpatient Variability
- Finite Lifespan
- Cells carry out normal functions e.g. differentiate
- Give 6 examples of non-haemopoietic primary cell cultures?
Liver Muscle Skin Nerves Fibroblasts Endothelial cells
- Give examples of Haemopoietic primary cell cultures?
Stem Cells Progenitor cells T and B cells Monocyte Macrophages Osteoblasts Dendritic cells Neutrophils Eosinophils Basophils Mast cells Erythrocytes Megakaryocytic Platelets
- What is the disaggregation of cells and what are the two types of disaggregation?
You put the tissue into culture and cells will move out - cells allowed to migrate out of an explant.
There are two types:
1. Mechanical Dissociation: Mincing,Sieving, Pipetting
2.Enzymatic Dissociation (Trypsin, Collagenase, hyaluronidase, Protease,DNAase)
- What is the exception to disaggregation?
Haemopoietic Cells
They are already are
- What are three sources of stem cells?
- Bone Marrow
Aspirate - Umbilical Cord Blood
- Mobilised Peripheral Blood
- What is the different of stem cel sources in children and adults?
In children:
- All bones with red bone marrow
- Liver and Spleen
In Adults :
- Ends of long bones like the femur and humerus
- Skull
- Vertebrae
- Ris
- Sternum
- Pelvis
- What is Haematopoiesis?
The Differentiation of stem cells
- The following questions refer to haematopoiesis:
- As stem cells differentiate , do they also amplify in size or no?
- Are the cells self-renewing?What does this mean?
- What are 4 main cells that form after stem cells differentiate?
- At what cell stage do they become more recognisable? At what stage can they be identified from their appearance?
- What is needed at every stage?
- -They do amplify
- Yes, the cells are self-renewing. Means they make more identical cells
- Stem Cells -> Early Progenitors > Late Progenitors -> Immature Precursors -> Red Cells
- Immature Precursors is when they become more recognisable
- Last mature cell stage - they can be identified from their appearance
- Growth Factors
- In primary cell culture, which stage of the cell differentiation is assayed for?
Progenitor Cells
- What does it mean if cells are labelled CFU and BFU?
CFU = colony forming unit, unit that is used to estimate the number of bacteria/fungal cells
BFU- burst forming units,
- List some key features of stem cells?
- > Pluripotent
- > Self-renew
- > Rare
- > Responsible for engraftment ( blood-forming cells you received on transplant day start to grow and make healthy blood cells)
- List some key features of progenitor cells?
- > Undifferentiated
- > Undistinguishable by morphology alone
- > Starting to become committed
- > Need assays
- List some key features of immature precursors?
- > Starting to differentiate
- > immature but recognisable
- How do growth factors act in Haematopoiesis?
- Polypeptide growth factors (Cytokines)
- They bind to cell surface transmembrane receptors
3.Stimulate growth and survival of progenitors
(important in every stage)
- We can separate growth factors into either specific to their lineage and growth factors that are non-specific. Give examples of each
Growth factors are specific to their lineage:
- EPO GF is specific to erythrocyte lineage
- IL5 is responsible for eosinophil lineage
Growth factors that are non-specific to their lineage?
- IL3
- SCF
- What 5 main components does the microenvironment consist of?
- > Stromal Cells
- > Extracellular Matrix
- > Adhesion Receptors
- > Cytokines
- > Inhibitors
- Give examples for each section of the microenvironment ?
Stromal Cells:
- Fibroblasts
- Macrophages
- Endothelial Cells
- Adipocytes
ECM:
- > Collagen I,III,IV
- Laminin
- Fibronectin
- Hemonectin
- Thromboospondin
- Proteoglycans
Adhesion Receptors :
- Integrins
- Selectins
- CD44
- Lectins
Cytokines:
- IL-,3,6,11,F
- G-CSF
- GM-CSF
- SCF
- b-FGF
Inhibitors
- MIP-1alpha
- TDF-alpha
- TNF-alpha
- INF-y
- What are the three main ways you can differentiate between cells?
- Antigen Markers
- Fluorescent Dye
- Assays
21.How would you use Antigen Markers to differentiate between cells?
CD34:
Stem Cells and Progenitor Cells : CD34+
Mature Cells : CD34-
Lin:
Stem Cells/Progenitors : Lin-
Mature Cells : Lin+
22.How would you use Fluorescent Dye to differentiate between cells?
Rhodmine 123:
- Only picked up by cycling cells
- Early stem cells will appear dull but if in the cycle-will appear bright
Cytotoxic Drugs: 5-fluoro-uracil:
- Only picked up by cycling cells so will be killed by them
- Cells out of the cycle are resistant to this drug
- Explain the 5 methods to differentiate between stem cells depending on purity of stem cells ?
- Erythrocyte lysis (least pure)
Gives enriched population of stem cells - Density gradient centrifugation
Spin cells on gradient
remove certain cells - Adherence depletion
Bone marrow put onto plastic
Some stick to plastic so can be harvested - Antibody depletion
Get rid of certain cells
Deplete all cells that are Lin+ - Antibody selection (most pure)
Positively select cells that are CD34+ Early stem cells: big nucleus
- Why are progenitors called CFU?
How do you get progenitors to form colonies?
Progenitors grow to form colonies of mature cells
From 32 to hundreds or thousands of cells in a colony
Thus progenitors are called “Colony Forming Units” - CFU
-Put early cell into culture
-Stimulate it to divide into mature cell
- List the 7 types of colony assays?
- 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
- List the different biological and non-biological assays?
Non-Biological:
- Morphology
- FACS- Fluorescence Activated Cell Sorting (more quantitive)
Biological:
- Growth Rate
- Plating Efficiency
- Function
27.What are the stages of a colony assay?
- Done on a semi-solid medium
Agar/methylcellulose
Add growth factors - Incubate for 7-14 days
- Single cells will divide and differentiate and form colonies
- Can be identified using a microscope
- Can quantitate number of cells in original suspension
o Done in laminar flow cabinet -Sterile environment
- What are the three main categories for applications of colony assays?
- Experimental
- Diagnostic
- Therapeutic
29.List three applications for colony assays?
Research – basic haemopoiesis and carcinogenesis
Testing toxicity of chemotherapeutic agents and carcinogens
Generate cells for stem cell transplantation/manipulation
