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)
