1. Primary Culture Techniques Flashcards
What is a primary cell culture? – compare to cell lines
- Taken directly from tissues (vs cell line: taken from tumours or genetically manipulated)
- Interpatient variability (cell lines: identical – homogenous population of cells)
- Finite lifespan (cell line: theoretically immortal – get it out of liquid nitrogen, grow it and put it back in again)
- Cells divide and/or differentiate (cell line: divide but doesn’t differentiate – produce daughter cells)
- Cells carry out normal functions (cell line: have aberrant gene expression not normal cells)
- Cells are studied in conditions that are close to in vivo conditions to see how they would normally function in the body
What are non-haemopoietic and haemopoietic 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 you to obtain cells from tissue?
- Cells allowed to migrate out of an explant (putting the cells into culture ~ progenitor cells eventually migrate away from tissue and divide, - these can then be put into another culture).
- Mechanical dissociation from tissue (mincing, sieving, pipetting to make single cells suspension)
- Enzymatic dissocation – enzymes break bonds between cells to make a single cell suspension (e.g. trypsin, collagenase, hyaluronidase, protease, DNAase)
- Exception = Haemopoietic cells - do not need to be disaggregated as they already are
What are the sources of stem cells? – also in children and in adults (after 20yrs)
- Bone marrow aspirate
- Umbilical cord blood
- Mobilised peripheral blood – mobilising stem cells out of bone marrow and into peripheral blood and harvest the blood
- In children - in all bones with red bone marrow, liver and spleen
- In adults - Ends of lond bones like femur, humerus, skull, vertebrae, ribs, sternum and pelvis (main source)
Describe the differentiation of haemopoietic stem cells.
- Cells divide and differentiate (haemopoiesis) amplification and differentiation
- Stem cells -> early progenitors (still look the same as stem cells) -> late progenitors (still look the same) -> immature precursors (start to take on characteristics of mature cells – you can start to identify them) -> terminally differentiated (e.g. red cells, neutrophils, platelets)
- Cells (pluripotent) become more committed to a lineage as they move on to right/progress along differentiation
- Process tightly controlled by growth factors
What can stem cells divide to do?
• Normally stem cells (low in number) mostly out of cycle in bone marrow, can divide to:
- self-renew (make more of themselves) - differentiate
What are the characteristics of progenitors?
- Undifferentiated
- Not distinguished by morphology
- Committed to one or more lineages
- Detected in colony-forming assays
Describe the differentiation of pluripotent stem cell into lymphoid and myeloid stem cells.
- Lymphoid stem cell – can become T or B lymphocyte
- Myeloid stem cell – can become RBC, neutrophil, monocyte, megakaryote (platelets)
- Progenitors and immature precursors – also committed to lineage, some more specifically than others
What are haemotopoietic growth factors and what do they do?
- They are responsible for every stage of haemopoiesis
- Polypeptide growth factors (cytokines)
- Bind to cell surface transmembrane receptors
- Stimulate growth and survival of progenitors
- In primary culture, growth factors or cells that make GFs must be added (in cell line growth factors not needed, as cells immortal)
- Some GFs are specific for lineage or work across, some specific to certain stage, etc
What are different microenvironments made up of for stem cells?
- Stem cells are sitting in close association with other cells in the bone – stromal cells (e.g. fibroblasts, macrophages, endothelial cell, adipocytes)
- Stromal cells make:
- Extracellular matrix
- Adhesion receptors
Also responsible for… - Cytokines
- Inhibitors
- Some stromal cells can make growth factors
How can you differentiate between different cell types?
- By the use of antigens
- Drugs
- Assays
- Morphological stains
What are haemotopoietic growth factors and what do they do?
- They are responsible for every stage of haemopoiesis
- Polypeptide growth factors (cytokines)
- Bind to cell surface transmembrane receptors
- Stimulate growth and survival of progenitors
- In primary culture, growth factors or cells that make GFs must be added (in cell line growth factors not needed, as cells immortal)
- Some GFs are specific for lineage or work across, some specific to certain stage, etc
Expand on the use of drugs to differentiate cell types.
- Rh123 (Rhodamine 123) – can be labelled with fluorochrome ~ This is a mitochondrial stain
- So only cell in cycle (e.g. progenitors) will be positive with this dye
(• 5-FU (5-fluorouracil) – cytotoxic drug
- Stem cells out of cycle resistant to drug ~ allows purification of stem cells)
Expand on the use of drugs to differentiate cell types.
- Rh123 (Rhodamine 123) – can be labelled with fluorochrome ~ This is a mitochondrial stain
- So only cell in cycle (e.g. progenitors) will be positive with this dye
(• 5-FU (5-fluorouracil) – cytotoxic drug
- Stem cells out of cycle resistant to drug ~ allows purification of stem cells)
What are different cell processing methods? – How to get stem cells out of marrow (enrichment/purification)?
- Erythrocyte lysis – enrichment of SC population
- Density gradient centrifugation – spinning the cells on a gradient makes it purer
~ Enrichment of cells - Adherence depletion – some cell types will just sit down on the plastic (like fibroblasts and macrophages) they are placed on and just harvest the rest
~ Enrichment - Antibody depletion – purification flow cytometry
• magnetic beads to take out unwanted cells (negative selection) - Antibody selection – purification
• positively select for stem cells (fluorescence or magnetic beads on antibody)
What are colony assays?
- Progenitors grow to form colonies of mature cells (with right GFs)
- From 32 to hundreds/thousands of cells in colony
- Thus progenitors are called ‘colony forming units’ (CFU)
What is the method of colony assays?
• This is all done in a laminar flow cabinet (in a sterile environment)
- Put single cell suspension of bone marrow/purified stem cells into semi-solid medium (e.g. agar, methylcellulose) – allows culture to stay in colonies and not ‘swim around’
- Apply growth factors
- Incubate 7-14 days
- Progenitors form colonies – you can quantitate how many progenitor cells we originally had in our colony
Give examples of the different colony assays. What does looking at teh colonies under a mircoscope tell?
• Different colony assays for different types, e.g.:
- CFU-G (neutrophilic) granulocyte progenitor – specific to granulocytes
- CFU-GM granulocyte/monocyte progenitor – able to form mature granulocytes or monocytes
- CFU-E + BFU-E erythroid progenitors ~ BFU = burst forming unit
- CFU-GEMM - granulocyte/erythroid/monocyte/megakaryocyte progenitor - (so a bit further back in hierarchy than above, less committed, as it is able to form 4 cell types)
- CFU-bas – specific to basophil progenitor
- CFU-eo – specific to eosinophil progenitor
•Looking at image on microscope allows us to identify cell type and quantify (number of colonies formed of that cell type)
What are the applications of primary culture techniques?
• Fall into 3 categories:
1. Experimental
2. Diagnostic
3. Therapeutic
• Research – basic haemopoiesis and carcinogenesis
• Testing toxicity of chemotherapeutic agents and carcinogens – e.g. to check that we are not affecting normal stem cells
• Generate cells for stem cell transplantation/manipulation – expand stem cells in culture by adding GFs, or adding genes into stem cells, etc
