What is primary cell culture techniques?
Being able to grow cells and tissues in culture as close to the in vivo environment as possible
What are the primary cell cultures?
- Cells derived directly from tissues
- Interpatient variability
- Finite lifespan
- Cells divide and/or differentiate
- Cells carry out normal functions
Differences between primary cell cultures and cell lines
Cell lines are transformed cells that have been manipulated or induced compared to primary cell cultures that are directly derived from tissues.
Cell lines produce exactly the same cell compared to primary cell cultures that divide and/or differentiate.
Two types of primary cultures
- Non-haemopoietic
- Haemopoietic
Examples of cells from non-haemopoietic cells
Liver Muscle Skin Nerves Fibroblasts Endothelial cells
Examples of cells from haemopoietic cells
Stem, progenitor cells T and B cells Monocyte, Macrophages Osteoblasts Dendritic cells Neutrophils, Eosinophils, Basophils, Mast Cells Erythrocytes Megakaryocytes, Platelets
How are cells disaggregated for primary cell culturing?
- Cells are allowed to migrate out of an explant.
- Then, they undergo mechanical dissociation (mincing, sieving, pipetting).
- Then, they undergo enzymatic dissociation (trypsin, collagenase, hyaluronidase, protease, DNAse)
This is done to produce a single cell dimension
Which cells do not undergo disaggregation and why not?
Haemopoietic cells as they are already in a single cell dimension so do not need to be disaggregated.
What is one disadvantage of primary cultures?
Assay readout is more complicated than just counting the number of cells.
What are some sources of stem cells?
- Bone marrow aspirate
- Umbilical cord blood: as stem cell-enriched
- Mobilized peripheral blood: treated with GF then the stem cells move out of marrow into peripheral and used to treat stem cells for proliferation.
Why is bone marrow harder to acquire in adults?
Bone marrow retreats so are only harvested at the ends of long bones like the femur, humerus. Also from, the skull, vertebrae, ribs, sternum, and pelvis.
Where is bone marrow aspirate found in children?
- All bones with red bone marrow
- Liver and spleen
Simplified structure of the bone
Periosteum - Hard part on the outside
Endostium - Where haemopoiesis occurs
What does a smear show on the bone marrow?
- Can only densely packed, immature cells
- Blood vessels -> pink
- Fats -> white blobs
- Concentrated areas of active haemopoiesis
Describe the process of haemopoiesis
- Stem cells
- Early progenitors: down the microscope, they look exactly the same as stem cells.
- Late progenitors: down the microscope, they look exactly the same as stem cells.
- Immature precursors: start to look different as they are committing to a specific lineage and look different.
- Mature cell types: easily distinguishable; reason they need to be cultured to do something.
How does haemopoiesis occur?
- When stimulated, stem cells either self-renewing or differentiating.
- Also a source of stem cells.
- Amplification and differentiation control the development
- Controlled by +/- GF for this process
What is the period of commitment?
It is a period that cells go through where they commit to a specific lineage. Either the Myeloid stem cell can form 4 different types of mature cells or the Lymphoid stem cells which can form only B-lymphocytes or T-lymphocytes.
Features of stem cells
- Pluripotent - give rise to all lineages
- Self-renew
- Rare cells
- Responsible for engraftment
Features of progenitor cells
- Undifferentiated
- Not distinguished by morphology
- Committed to one or more lineages
- Detected in colony-forming assays
Features of precursor cells
- Immature but recognizable
- Cells starting to differentiate
- Few final divisions before mature cells
What are hematopoietic growth factors and what are they used for?
- Polypeptide growth factors such as cytokines
- Bind to cell surface transmembrane receptors
- Stimulate growth and survival of progenitors or inhibit
Examples of cell types and their mature cells
- Megakaryocyte Erythroid Progenitor forms a erythrocytes and platelets
- Granulocyte-Macrophage Progenitor forms the WBC - basophils, eosinophils etc.
- Common lymphoid progenitor forms the T-lymphocytes, B-lymphocytes, and NK cells
What forms the micro-environment that stem cells live on?
The stromal cells form fibroblasts, macrophages, endothelial cells, and adipocytes that the stem cells live on/in.
Also, have the extracellular matrix (ECM) formed by collagen I, III, IV; Laminin, Fibronectin, Haemonectin, Thrombospondin, and proteoglycans (GAGs)
What are adhesion receptors?
Cells that produce cytokines and inhibitors
- Integrins
- Selectins
- CD44
- Lectins
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Cellular Growth Regulation53
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Cell Death and Cell Damage48
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Mechanisms of Oncogenesis60
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Oncogenes and Tumour Suppressor Genes52
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Tumour Angiogenesis, Invasion, and Metastasis37
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Primary Cell Culture Technqiues47
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Cell Culture Techniques46
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Microscopy32
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MRI and MRS71
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Ultrasonagraphy62
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X-ray, CT, PET41
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Flow Cytometry - Introduction35
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Flow Cytometry - Applications15
