Primary Cell Culture Techniques Flashcards
Where are cells obtained from in primary cell cultures?
Cells derived directly from tissues; contrast to cell line which has either spontaneously been transformed or genetically manipulated
Describe the difference in primary cell cultures between patients
Interpatient variability (cell lines are absolutely identical; reproducibility) `
How long do cells from primary cell cultures survive?
Finite lifespan - cell lines are immortal
How do primary cell cultures differ from cell lines?
Cells divide and/or differentiate (cell lines are clones)
Cells carry out normal functions
(cell lines are non-functional)
Give examples of non-haematopoietic primary cultures
Non-hematopoietic
- Liver
- Muscle
- Skin
- Nerves
- Fibroblasts
- Endothelial cells
Name examples of haemopoietic primary cultures
Stem, Progenitor cells T and B cells Monocyte, Macrophages Osteoblasts Dendritic cells Neutrophils, Eosinophils,Basophils, Mast cells Erythrocytes Megakaryocytes, Platelets
What is the purpose of cell disaggregation?
Cells allowed to migrate out of an explant
What is cell disaggregation?
Breaking up of a total (aggregate), integrated whole, into smaller elements, parts, or units, usually for easier handling
What are the different ways of disaggregating cells?
Mechanical dissociation (mincing,sieving,pipetting)
Enzymatic dissociation (trypsin,collagenase, hyaluronidase, protease, DNAase)
What cells don’t require disaggregation?
Exception – Haemopoietic cells don’t need to be disaggregated as they already are
What are the sources of stem cells?
- Bone marrow aspirate
- Umbilical cord blood (enriched)
- Mobilised peripheral blood
Why may stem cells be found in the peripheral blood?
Normally stem cells aren’t found in peripheral blood but treating patients with growth factors causes them to move out
What happens in red bone marrow?
Red bone Marrow is where hematopoiesis occurs
Where is red bone marrow found?
Children contain red bone marrow in all bones; Adult red bone marrow retreats and only occurs in specific areas
Outline the specific ares red bone marrow is found in children?
all bones, liver and spleen
Where is red bone marrow found in adults (20+)?
Ends of long bones (femur, humerus)
skull, vertebrae, ribs, sternum,, pelvis
Describe the different layers of bone
Hard periosteum around the outside, on the endomysium (inside) is where haematopoiesis is occurring
Where are blood vessels found in bone?
Blood vessels travel through the middle - haemopoiesis occurs here
Describe the structure of bone
Hard exterior and a fibrous mesh in the middle where haematopoiesis occurs
3D environment
How does a bone marrow film and blood film differ?
Peripheral blood stains show a variety of maturity in cells present
whereas bone marrow has densely packed areas of immature cells
Describe the features of a bone marrow slide under a mciroscope
In a concentrated area of active haematopoiesis
Pink blobs are blood vessels
White areas are fat
What are the stages of cell development from stem cells?
- Stem cells
- Early progenitors
- Late progenitors
- Immature precursors
- Mature cell types - RBC, WBC
Describe the structure of early and late progenitors
Early & late progenitors are identical to stem cells as they are undifferentiated
What happens to immature precursors?
Immature precursors begin to differentiate and commit to a certain lineage - can identify and tell them apart from one another and their homogenous precursors
What makes mature cell types identifiable?
Mature cell types have very distinct morphology under the microscope - easily distinguishable
How is stem cell development regulated?
This process is tightly regulated by negative and positive growth factors
Describe proliferation of stem cells during development
Amplification increases throughout the process
What part of the cell cycle are stem cells in?
Stem cells are normally in G0, when activated they either self renew or differentiate
Describe the morphology of stem cell sand progenitors
Stem cells and progenitors look identical under a microscope but start to differentiate further into immature precursors
What are CFU?
CFU - colony forming unit
E.g. CFU-GM has the ability to differentiate into a granulocyte and monocyte
What are BFU?
BFU - burst forming units
Outline the features of stem cells
Pluripotent- give rise to all lineages
Self-renew
Rare cells
Responsible for engraftment
Describe features of Progenitor cells
Undifferentiated
Not distinguished by morphology
Committed to one or more lineages
Detected in colony-forming assays
What are the distinctive features of precursor cells?
Immature but recognisable
Cells starting to differentiate
Few final divisions before mature cells
What are haematopoietic growth factors?
Polypeptide growth factors (cytokines)
How are haematopoietic growth factors activated?
Bind to cell surface transmembrane receptors
Stimulate growth and survival of progenitors.
Describe growth factor specificity in haematopoiesis
Some growth factors are very specific to a lineage
e.g. EPO for red cells ILF for eosinophils
But other growth factors work all over the place across lineages at different stages e.g. IL3
What forms the bone marrow microenvironment?
Stromal cells Extracellular matrix (ECM) Adhesion Receptors Cytokines Inhibitors
Name examples of stromal cells
- fibroblasts
- macrophages
- endothelial cells
- adipocytes
Describe the composition of the stem cell environment
Stromal cells form the environment the stem cells are sitting in and on their surface contain various extracellular matrix proteins, adhesion receptors
Stromal cells also produce cytokines and inhibitors
What structures form the extracellular matrix (ECM)?
- collagen I, III, IV
- laminin
- fibronectin
- hemonectin
- thrombospondin
- proteoglycan (GAGs)
Name some adhesion molecules
- ontegrins
- selectins
- CD44
- Lectins
Describe the stem cell niche
Stem cell niche - each stem cell corresponds to a stromal cell
How can we identify cells in different stages of haematopoiesis
Can use the following factors to identify cells during different stages of haematopoiesis:
- Phenotype (cell surface antigens)
- Drugs & dyes
- Cell cultures
How can we use CD34 antigen to identify different haematopoietic cells?
Both early and late progenitors are positive for CD34 but mature cells are CD34 negative
What is LIN?
LIN - lineage specific marker
How is LIN used to identify how mature stem cells are?
Early and late progenitors are LIN-
Mature cells are LIN+ as they are specific to certain lineages
What is Rhodamine 1,2,3?
Rhodamine 1,2,3 is a fluorescent dye that stains mitochondria - used in flow cytometry
How is rhodamine used to identify cells in cycle?
Out of cycle stem cells won’t stain for rhodamine as they have no mitochondria present
What is 5-fluorauracil?
5-fluorouracil is a cytotoxic drug that only affects cycling cells, out of cycle cells are resistant
When are colony forming unit assays used?
Colony forming unit assays for lineage committed progenitors
Which assay type is used to identify mature cells?
Staining and functional assays used for mature cells
Outline cell processing methods
- Erythrocyte lysis
- Density gradient centrifugation
- Adherence depletion
- Antibody depletion (mature cells) e.g. CD34
- Antibody selection (stem cells) e.g. CD34
Describe the structure of CD34 stem cells
Purified CD34 stem cells fluorescently dyed and put through a flow cytometer and pulled out
Can see they’re very immature - huge nuclei and very little cytoplasm
Outline the culture conditions of colony assays
- 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
Outline how colony assays are undertaken
- Semi-solid medium
(agar, methylcellulose) - Plus growth factors
- Incubate for 7-14 days
- Identify under microscope
Why are petri dish cultures carried out more than once?
2 petri dishes as all experiments carried out in duplicate; reproducibility
Why is sterile water added to colony assays?
Sterile water to prevent drying out
What apparatus are colony assays produced in?
Carried out in a laminar flow cabinet - air has been filtered to provide a sterile environment before cultures are incubated
Give examples of common 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
What are some of the non biological assays?
Non biological Assays
- Morphology
- FACS
Outline why biological assays are carried out
- Growth rate
- Plating efficacy
- Function
What are the applications of colony assays?
Research – basic haemopoiesis and carcinogenesis
Testing toxicity of chemotherapeutic agents and carcinogens
Generate cells for stem cell transplantation/manipulation
What is a primary cell culture?
Primary culture is a technique by which cells from primary tissues or cell suspensions are grown under controlled conditions, in vitro
What are cell cultures used for?
Cultures can be used for experimental, diagnostic or therapeutic purposes.
