Primary cell culture techniques

Question 1

Define purpose of primary cell culture techniques

Answer 1

Being able to grow cells in culture to recreate an environment like in vivo​

Question 2

Primary cell culture vs cell line

Answer 2

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

WHERAS cell lines

Cell line is transformed – manipulated or spontaneous
Will always live​
Have no function​
Identical to parent cell

Question 3

Non-haemopoietic examples of primary cultures

Answer 3

Liver ​
Muscle​
Skin​
Nerves​
Fibroblasts​
Endothelial cells

Question 4

Haemopoietic examples of primary cultures

Answer 4

Stem, Progenitor cells​
T and B cells​
Monocyte, Macrophages​
Osteoblasts​
Dendritic cells​
Neutrophils, Eosinophils,Basophils, Mast cells​
Erythrocytes​
Megakaryoctyes, Platelets

Question 5

Describe disaggregation of cells and any exceptions

Answer 5

Cells allowed to migrate out of an explant​
​Mechanical dissociation
​Enzymatic dissocation
​Exception – Haemopoietic cells – Do not need to be disaggregated – already are in single cell suspension

Question 6

Sources of stem cells​ - 3

Answer 6

Bone marrow aspirate​
Umbilical cord blood​
Mobilised peripheral blood – if treated with growth factors, stem cells move from BM to peripheral blood

Question 7

Stem cells in children - locations

Answer 7

All bones w/red bone marrow

Liver and spleen

Question 8

Stem cells in adults (20 yrs >) - locations

Answer 8

End of long bones like femur, humerus
Skull
Vertebrae
Ribs
Sternum
Pelvis

Question 9

Site of haematopoiesis

Answer 9

Endosteum

Question 10

Description of stem cells to red cells, neutrophils, platelets

Answer 10

Stem cells - early progenitors - late progenitors - immature progenitors - RBC, neutrophils + platelets

At immature precursors, cells start to look different = commiting to a lineage = look different to homogenous group of earlier cells

Controlled by negative and positive GF

Stem cells not in cycle, make a decision when stimulated = self-renew or differentiate

Question 11

CFU vs BFU

Answer 11

CFU = colony forming unit = ability in culture
CFU may transition into a precursor cell that still retains proliferative potential

BFU = burst forming unit, more immature = enormous bursts

Question 12

Stem cells features

Answer 12

Pluripotent- give rise to all lineages
Self-renew
Rare cells
Responsible for engraftment

Question 13

Progenitor cells features

Answer 13

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

Question 14

Precursor cells features

Answer 14

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

Question 15

Describe stimulation of growth/survival of progenitors

Answer 15

Polypeptide growth factors (cytokines)

Bind to cell surface transmembrane receptors

Stimulate growth and survival of progenitors

Question 16

Formation of erythrocytes/platelets - description of pathway w/names of growth factors

Answer 16

Haematopoietic stem cells (using IL-3, GM-CSF, M-CSF)

→ common myeloid progenitor (using IL-3, SCF, TPO)

→ megakaryocyte erythroid progenitor

using EPO to get to erythrocytes

OR

use IL-11 + TPO to make platelets

Question 17

Formation of basophil/mast cells - pathway description w/names of GFs

Answer 17

Haematopoietic stem cells (using IL-3, GM-CSF, M-CSF)

→ common myeloid progenitor (using GM-SCF)

→ granulocyte macrophage progenitor (using IL-11, TPO)

→ myeloblasts (using G-CSF, GM-CSF, IL-3)

→ Basophil/mast cells

Question 18

Formation of eosinophils - pathway description w/names of GFs

Answer 18

Haematopoietic stem cells (using IL-3, GM-CSF, M-CSF)

→ common myeloid progenitor (using GM-SCF)

→ granulocyte macrophage progenitor (using IL-11, TPO)

→ myeloblasts (using GM-CSF, IL-3, IL-5)

→ eosinophils

Question 19

Formation of neutrophils - pathway description w/names of GFs

Answer 19

Haematopoietic stem cells (using IL-3, GM-CSF, M-CSF)

→ common myeloid progenitor (using GM-SCF)

→ granulocyte macrophage progenitor (using IL-11, TPO)

→ myeloblasts (using G-CSF, GM-CSF, IL-6, SCF)

→ neutrophils

Question 20

Formation of macrophages - pathway description w/names of GFs

Answer 20

Haematopoietic stem cells (using IL-3, GM-CSF, M-CSF)

→ common myeloid progenitor (using GM-SCF)

→ granulocyte macrophage progenitor (using GM-CSF, M-CSF)

→ monocytes (using IL-6, SCF, G-CSF)

→ macrophages

Question 21

Formation of T-lymphocytes - pathway description w/names of GFs

Answer 21

Haematopoietic stem cells (using IL-17)

→ common lymphoid progenitor (using IL-2, IL-7)

→ T-lymphocytes

Question 22

Formation of B-lymphocytes - pathway description w/names of GFs

Answer 22

Haematopoietic stem cells (using IL-17)

→ common lymphoid progenitor (using IL-3, IL-4, IL-7)

→ B-lymphocytes

Question 23

Formation of natural killer cells - pathway description w/names of GFs

Answer 23

Haematopoietic stem cells (using IL-17)

→ common lymphoid progenitor (using IL-15)

→ natural killer cells

Question 24

4 examples of stromal cells

Answer 24

Fibroblasts
Macrophages
Endothelial cells
Adipocytes

Question 25

Description of microenvironment of ECM

Answer 25

``` Collagen I, III, IV Laminin Fibronectin Hemonectin Thrombospondin Proteoglycans ```

Question 26

4 examples of adhesion receptors

Answer 26

Integrins Selectins CD44 Lectins

Question 27

9 examples of cytokines

Answer 27

``` IL-1 IL-3 IL-6 IL-11 G-CSF GM-CSF SCF LIF b-FGF ```

Question 28

4 examples of inhibitors

Answer 28

M1P-1 alpha TGF-beta TNF-alpha INF-gamma

Question 29

List 5 bone marrow processing methods

Answer 29

``` Erythrocyte lysis Density gradient centrifugation Adherence depletion Antibody depletion Antibody selection ```

Question 30

Describe formation of CFUs

Answer 30

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

Question 31

Describe method for colony assays

Answer 31

Semi-solid medium (agar, methylcellulose) Plus growth factors as stromal cells would normally support HaemP W/the single cell suspension of bone marrow Incubate for 7-14 days Culture to work out how many CFU = identify as different cell types

Question 32

List all possible colony assays and the relevant progenitor (8)

Answer 32

``` 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 33

Describe 3 applications of primary cultures

Answer 33

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

Question 34

Define what is a primary cell culture technique

Answer 34

Primary culture is a technique by which 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

Question 35

What can primary cultures be established from and effect

Answer 35

Primary cultures can be established from hematopoietic cells giving rise to all the mature blood cell types, and non-hematopoietic tissues.