primary cell culture Flashcards
primary cell culture?
- cells derived directly from tissues
- Interpatient variability
- Finite lifespan
- Cells divide and/or differentiate
- Cells carry out normal functions
cell line?
- transformed cell that is spontaneously transformed or genetically manipulated
- theoretically immortal
- divides and reproduces itself exactly the same
- may not have normal function because its been transformed
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
what is meant by disaggregation of cells?
Cells allowed to migrate out of an explant
Mechanical dissociation (mincing,sieving,pipetting)
Enzymatic dissocation (trypsin,collagenase, hyaluronidase, protease, DNAase)
which cells are an exception to disaggregation?
Haemopoietic cells
-they do not need to be disaggregated, they already are in a single cell suspension
why is the assay read out more complicated for primary cell culture?
cells differentiate, so we have to be more sophisticated, the assay read out is more complicated
summary of Primary Culture of Haemopoietic Stem Cells
Cell source
Cell characteristics
Cell processing
Culture conditions
Assay read-outs
Applications
name some sources of stem cells
Bone marrow aspirate
Umbilical cord blood
Mobilised peripheral blood
where is bone marrow located?
in children, all bones with red bone marrow and liver and spleen
in adults, after 20 yrs, the end of long bones like the humerus and femur, the ribs, sternum and pelvis
Endosteum?
middle of bone, where haemopoeisis is occurring (mesh like)
Cell characteristics - process of differentiation
early stem cells
early progenitors - look exactly the same as stem cells when looking down a microscope
late progenitors - also look the same, amplification happening
immature precursors - cells differentiating and committed to a certain lineage, look different to each other
mature cell types have very distinct morphology, easily distinguishable
is the process of differentiation tightly controlled?
Tightly controlled by positive and negative growth factors
as the process of differentiation goes along what happens?
Amplification as process goes along
are stem cells normally in the cycle?
stem cells normally not in the cycle, but stimulated they either self renew or differentiate
what do cells go through?
a period of commitment
what does CFU and CFU-GM stand for
CFU – colony forming unit
CFU-GM – colony forming unit (in culture) granulocytes and monocytes – this cell in culture has the ability to form granulocytes and monocytes
BFU?
burst forming unit
describe characteristics of stem cells
continually renewing, don’t run out of them
- pluripotent- give rise to all lineages
- self-renew
- rare cells
- responsible for engraftment
describe characteristics of progenitor cells
Undifferentiated
Not distinguished by morphology
Committed to one or more lineages
Detected in colony-forming assays
describe characteristics of precursor cells
Immature but recognisable
Cells starting to differentiate
Few final divisions before mature cells
what are haematopoetic growth factors and their role?
they are polypeptide growth factors (cytokines) that bind to cell surface transmembrane receptors
stimulate growth and survival of progenitors
what is meant by phenotype?
antigens on the cell surface
which cells are CD34+ and Lin-?
stem cells and early/late progenitor cells
which cells are CD34- and Lin+?
Mature cells
what is 5-FU?
a cytotoxic drug that only affects cyclin cells
-we can add it into cultures and kill off cells
how can we identify cells at different stages of haematopoeisis?
Phenotypes
describe the microenvironment of stem cells?
Stromal cells form the environment that the stem cells are sitting in by producing growth factors and extracellular matrix
On the surface of stromal cells are various extracellular matrix proteins, e.g. collagen, laminin. They also have adhesion receptors on their surface, producing cytokines and inhibitors. Stem cells have equivalent receptors which will recognise those molecules
-known as a stem cell leash
examples of stromal cells?
fibroblasts, macrophages, endothelial cells, adipocytes
examples of adhesion receptors?
integrins and selectins
examples of cytokines and inhibitors?
IL-1, IL-3, IL-6
MIP-1alpha
what is meant by cell processing?
when examining bone marrow, you can use techniques that enrich or purify your stem cells
prcoessing methods include: Erythrocyte lysis Density gradient centrifugation Adherence depletion Antibody depletion Antibody selection
use antibodies that will recognise mature cells, like Lin. Or use positive selection, come in with antibodies (CD34) for the stem cells and pick them out
use magnetic beads that attach to antibodies and then pull them out with a magnet
what is an assay?
a procedure used to measure/quantify the amount of a target entity
different types of assays?
Biological assays Non-biological assays
Morphology Growth rate
FACS Plating efficiency
Function
how do CFU’s form (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”. Once in the colony assay, they divide and form colonies of mature cells, which are distinguishable
how do you make a colony assay and what is their purpose?
Put suspension into culture to work out how many CFU there are
Semi-solid medium (agar, methylcellulose)
- so the cells remain there
Growth factors
-stromal cells in the ‘in vivo’ environment produce GF’s to support the process, so we need to replicate this
Look through a microscope and identify and count the CFU’s
what can can see with the naked eye?
BFUs
name some different 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
Applications of primary cell culture?
Research – basic haemopoiesis and carcinogenesis
Testing toxicity of chemotherapeutic agents and carcinogens
Generate cells for stem cell transplantation/manipulation
define primary cell culture
a technique where:
cells from primary tissues or cell suspensions are grown under controlled conditions, in vitro
proliferation and/or differentiation can be supported, although cultures have a finite life span. Cultures can be used for experimental, diagnostic or therapeutic purposes.
