Primary Cell Culture Flashcards
What is a primary cell culture?
- Technique by which cells from primary tissues or cell suspensions are grown under controlled conditions in vitro
- Cells will divide and or differentiate like normal cells, carrying out their normal function
Provide some examples of non-haemopoietic primary cultures
Liver, Muscle, Skin, Nerves, Fibroblasts, Endothelial
Provide some examples of haemopoietic primary cultures
- Stem, Progenitor cells
- T and B cells
- Monocytes, Macrophages
- Osteoblasts
- Dendritic cells
- Neutrophils
- Eosinophils, Basophils, Mast cells
- Erythrocytes
- Megakaryocytes
What can primary cultures be established from?
- Haemopoetic cells
- Giving rise to mature blood cell types
- Non-haematopoietic cells
What are the differences between cell cultures and cell lines?
- Primary cell cultures are directly derived from tissues
- Cell lines are transformed cells which may be genetically identical
- Interpatient variabillity
- In a cell line every cell is identical
- Finite lifespan (cant consistently maintain O2 and growth medium)
- A cell line will divide and reproduce itself exactly the same
- Cells divide and/or differentiate
- A cell line will divide and reproduce itself exactly the same
- Cells carry out normal funciton
- Because cell lines are transformed it may not have a normal function
By what process do we manipulate tissues to get a single cell suspension for primary cell cultures?
Disaggregation
List some disaggregation techniques
- Cells migrate out of explant
- Mechanical dissociation (mincing, sieving, pipetting)
- Enzymatic dissociation (trypsin, collagenase, hylaluronidase)
What cell does not need to be disaggregated?
Haematopoietic cells do not need to be disaggregated as they are already in single cell suspension
What are sources of stem cells?
- Bone marrow aspirate
- Umbilical cord blood
- Mobilised peripheral blood
Where does haematopoiesis occur in children?
Red bone marrow
Liver
Spleen
Where does haematopoeisis occur in adults?
In adults (after 20 years), the bone marrow retreats and haematopoiesis only occur in:
- End of long bones (e.g femur, humerus)
- Skull
- Vertebrae
- Sternum
- Pelvis
What is lifecycle of haematopoetic cells?
Pluripotent stem cells → Early progenitors → Late Progenitors → Immature Precursors → Mature Blood Cells
Why are you not able to see the lineage of progenitor cells?
- They are undifferentiated cells and therefore do not show any mature characteristics
- HOWEVER they are already commited to a specific lineage and make a specific type of cell
Stem Cells
pluripotent, give rise to all lineages, self-renew, rare cells, responsible for engraftment
Progenitor cells
Undifferentiated, not distinguished by morphology, committed to one or more lineages, detected in colony forming assays
Precursor cells
immature but recognisable, cells starting to differentiate, dew final divisions before forming mature cells
What is haemopoiesis?
process whereby immature precursor cells develop into mature blood cells
How do haematopoietic growth factors?
- Bind to cell surface transmembrane receptors
- Stimulate growth + survival of progenitors
What are stromal cells?
Provide some examples of stromal cells.
Stromal cells are cells in the bone marrow which form the environment for stem cells
- Fibroblasts, Macrophages, Endothelial cells, adipocytes
What do stromal cells produce on their surface?
Produce ECM (extracellular matrix) proteins on their surface:
- Collagen I, II, III, IV
- Laminin
- Fibronectin
- Hemonectin
- Thrombospondin
- Proteoglycans
What do stromal cells contain on their surface?
Adhesion receptors
- Integrins
- Selectin
- CD44
- Lectins
What cells will produce cytokines and inhibitors?
Stromal cells
How are stem cells in interaction with stromal cells?
The stem cells will have equivalent receptors to adhesion receptors which will recognise cytokines and inhibitors produced by stromal cells.
What are different techniques to identify the haematopoesis stages that are not cell culture?
- Phenotype
- Fluorescent stains/ cytotoxic drugs
- Assays
Phenotypic techniques to identify haematopoetic cells
- Stem cells and progenitor cells (early+ late) are all posotive for CD34, mature blood cells are negative CD34
- A stem cells are Lin negative, mature cells are Lin positive
How do fluorescent stains/ cytotoxic drug techniques work to identify haematopoetic cells?
- Rh123 is a fluorescent dye = stains mitochondria. Stem cells out of the cycle will not stain for that. Actively cycling cells will stain bright
- 5-FU is a cytotoxic drug and will only affect cycling cells. Cells out of cycle wil be resistant to this drug. Adding this drug will kill cycling cells
Assay techniques to identify haematopoietic cells
- Some assays can recognise and grow early stem cells
- Colony forming unit assays for lineage commited progenitors
How to we undertake cell processing?
- Haematopoietic stem cells do not require processing as they are already in a single cell suspension
- There are various techniques which will enrich and purify stem cells ready for culture
-
Erythrocyte lysis
- Enrichment of stem cells
-
Density gradient centrifugation
- This will remove some cells and enriches for others
-
Adherance depletion
- Bone marrow on a sticking culture, remove non-adherant cells through pipetting
-
Antibody depletion
- Can use antibodies for Lin antigens, these will recognise mature cells and deplete
-
Antibody selection
- Can use antibodies for stem cells and select them via flow cytometry/ magnetic beads
- Label bone marrow with CD34 (e.g)
-
Erythrocyte lysis
What is a colony assay?
- This is where progenitors will grow to form colonies of mature cells
- Progenitors are also called CFUs (colony forming units)
- Progenitors will grow from 30 to several hundreds and thousands
How is a colony forming assay formed?
- Take single cell suspension and grow it with growth factors in a semi solid medium (agar-methylcellulose) in an incubator for 7-14 days
- The progenitor cells will replicate to produce a colony of more mature cells (most one lineage only )
- This can be seen down a microscope and can be identified as cell types
What are some applications of primary cell cultures?
Research - basic haematopoeisis and carcinogenesis
Testing for toxicity of chemotherapeutic agents and carcinogens
Generate cells for stem cells transplantation and manipulation
