primary cell culture

Question 1

primary cell culture?

Answer 1

• cells derived directly from tissues
• Interpatient variability
• Finite lifespan
• Cells divide and/or differentiate
• Cells carry out normal functions

Question 2

cell line?

Answer 2

• 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

Question 3

examples of primary cultures?

Answer 3

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

Question 4

what is meant by disaggregation of cells?

Answer 4

Cells allowed to migrate out of an explant

Mechanical dissociation (mincing,sieving,pipetting)

Enzymatic dissocation (trypsin,collagenase, hyaluronidase, protease, DNAase)

Question 5

which cells are an exception to disaggregation?

Answer 5

Haemopoietic cells

-they do not need to be disaggregated, they already are in a single cell suspension

Question 6

why is the assay read out more complicated for primary cell culture?

Answer 6

cells differentiate, so we have to be more sophisticated, the assay read out is more complicated

Question 7

summary of Primary Culture of Haemopoietic Stem Cells

Answer 7

Cell source

Cell characteristics

Cell processing

Culture conditions

Assay read-outs

Applications

Question 8

name some sources of stem cells

Answer 8

Bone marrow aspirate
Umbilical cord blood
Mobilised peripheral blood

Question 9

where is bone marrow located?

Answer 9

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

Question 10

Endosteum?

Answer 10

middle of bone, where haemopoeisis is occurring (mesh like)

Question 11

Cell characteristics - process of differentiation

Answer 11

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

Question 12

is the process of differentiation tightly controlled?

Answer 12

Tightly controlled by positive and negative growth factors

Question 13

as the process of differentiation goes along what happens?

Answer 13

Amplification as process goes along

Question 14

are stem cells normally in the cycle?

Answer 14

stem cells normally not in the cycle, but stimulated they either self renew or differentiate

Question 15

what do cells go through?

Answer 15

a period of commitment

Question 16

what does CFU and CFU-GM stand for

Answer 16

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

Question 17

BFU?

Answer 17

burst forming unit

Question 18

describe characteristics of stem cells

Answer 18

continually renewing, don’t run out of them

• pluripotent- give rise to all lineages
• self-renew
• rare cells
• responsible for engraftment

Question 19

describe characteristics of progenitor cells

Answer 19

Undifferentiated
Not distinguished by morphology
Committed to one or more lineages
Detected in colony-forming assays

Question 20

describe characteristics of precursor cells

Answer 20

Immature but recognisable
Cells starting to differentiate
Few final divisions before mature cells

Question 21

what are haematopoetic growth factors and their role?

Answer 21

they are polypeptide growth factors (cytokines) that bind to cell surface transmembrane receptors

stimulate growth and survival of progenitors

Question 22

what is meant by phenotype?

Answer 22

antigens on the cell surface

Question 23

which cells are CD34+ and Lin-?

Answer 23

stem cells and early/late progenitor cells

Question 24

which cells are CD34- and Lin+?

Answer 24

Mature cells

Question 25

what is 5-FU?

Answer 25

a cytotoxic drug that only affects cyclin cells | -we can add it into cultures and kill off cells

Question 26

how can we identify cells at different stages of haematopoeisis?

Answer 26

Phenotypes

Question 27

describe the microenvironment of stem cells?

Answer 27

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

Question 28

examples of stromal cells?

Answer 28

fibroblasts, macrophages, endothelial cells, adipocytes

Question 29

examples of adhesion receptors?

Answer 29

integrins and selectins

Question 30

examples of cytokines and inhibitors?

Answer 30

IL-1, IL-3, IL-6 | MIP-1alpha

Question 31

what is meant by cell processing?

Answer 31

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

Question 32

what is an assay?

Answer 32

a procedure used to measure/quantify the amount of a target entity

Question 33

different types of assays?

Answer 33

Biological assays Non-biological assays Morphology Growth rate FACS Plating efficiency Function

Question 34

how do CFU's form (colony assays)?

Answer 34

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

Question 35

how do you make a colony assay and what is their purpose?

Answer 35

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

Question 36

what can can see with the naked eye?

Answer 36

BFUs

Question 37

name some different colony assays?

Answer 37

``` 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 ```

Question 38

Applications of primary cell culture?

Answer 38

Research – basic haemopoiesis and carcinogenesis Testing toxicity of chemotherapeutic agents and carcinogens Generate cells for stem cell transplantation/manipulation

Question 39

define primary cell culture

Answer 39

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.