Primary Cell Culture Techniques

Question 1

1. What is primary cell culture?

Answer 1

Primary cell culture techniques allow you to grow cells directly from the body. They are put into situations in vitro and the in vivo situation is recreated as closely as possible.

Question 2

2. The following questions are about primary cell culture:
   - Where are the cells derived from?
   - Is there interpatient variability
   - What is the lifespan of these cells?
   - What do the cells do?

Answer 2

• Directly from tissue
• Interpatient Variability
• Finite Lifespan
• Cells carry out normal functions e.g. differentiate

Question 3

3. Give 6 examples of non-haemopoietic primary cell cultures?

Answer 3

Liver 
Muscle
Skin
Nerves
Fibroblasts
Endothelial cells

Question 4

4. Give examples of Haemopoietic primary cell cultures?

Answer 4

Stem Cells
Progenitor cells
T and B cells Monocyte
Macrophages
Osteoblasts
Dendritic cells
Neutrophils
Eosinophils
Basophils
Mast cells
Erythrocytes
Megakaryocytic
Platelets

Question 5

5. What is the disaggregation of cells and what are the two types of disaggregation?

Answer 5

You put the tissue into culture and cells will move out - cells allowed to migrate out of an explant.
There are two types:
1. Mechanical Dissociation: Mincing,Sieving, Pipetting
2.Enzymatic Dissociation (Trypsin, Collagenase, hyaluronidase, Protease,DNAase)

Question 6

5. What is the exception to disaggregation?

Answer 6

Haemopoietic Cells

They are already are

Question 7

7. What are three sources of stem cells?

Answer 7

1. Bone Marrow
   Aspirate
2. Umbilical Cord Blood
3. Mobilised Peripheral Blood

Question 8

8. What is the different of stem cel sources in children and adults?

Answer 8

In children:

• All bones with red bone marrow
• Liver and Spleen

In Adults :

• Ends of long bones like the femur and humerus
• Skull
• Vertebrae
• Ris
• Sternum
• Pelvis

Question 9

9. What is Haematopoiesis?

Answer 9

The Differentiation of stem cells

Question 10

10. The following questions refer to haematopoiesis:
    - As stem cells differentiate , do they also amplify in size or no?
    - Are the cells self-renewing?What does this mean?
    - What are 4 main cells that form after stem cells differentiate?
    - At what cell stage do they become more recognisable? At what stage can they be identified from their appearance?
    - What is needed at every stage?

Answer 10

• -They do amplify
• Yes, the cells are self-renewing. Means they make more identical cells
• Stem Cells -> Early Progenitors > Late Progenitors -> Immature Precursors -> Red Cells
• Immature Precursors is when they become more recognisable
• Last mature cell stage - they can be identified from their appearance
• Growth Factors

Question 11

11. In primary cell culture, which stage of the cell differentiation is assayed for?

Answer 11

Progenitor Cells

Question 12

12. What does it mean if cells are labelled CFU and BFU?

Answer 12

CFU = colony forming unit, unit that is used to estimate the number of bacteria/fungal cells
BFU- burst forming units,

Question 13

13. List some key features of stem cells?

Answer 13

• > Pluripotent
• > Self-renew
• > Rare
• > Responsible for engraftment ( blood-forming cells you received on transplant day start to grow and make healthy blood cells)

Question 14

14. List some key features of progenitor cells?

Answer 14

• > Undifferentiated
• > Undistinguishable by morphology alone
• > Starting to become committed
• > Need assays

Question 15

15. List some key features of immature precursors?

Answer 15

• > Starting to differentiate

- > immature but recognisable

Question 16

16. How do growth factors act in Haematopoiesis?

Answer 16

1. Polypeptide growth factors (Cytokines)
2. They bind to cell surface transmembrane receptors
   3.Stimulate growth and survival of progenitors
   (important in every stage)

Question 17

17. We can separate growth factors into either specific to their lineage and growth factors that are non-specific. Give examples of each

Answer 17

Growth factors are specific to their lineage:

• EPO GF is specific to erythrocyte lineage
• IL5 is responsible for eosinophil lineage

Growth factors that are non-specific to their lineage?

• IL3
• SCF

Question 18

18. What 5 main components does the microenvironment consist of?

Answer 18

• > Stromal Cells
• > Extracellular Matrix
• > Adhesion Receptors
• > Cytokines
• > Inhibitors

Question 19

19. Give examples for each section of the microenvironment ?

Answer 19

Stromal Cells:

• Fibroblasts
• Macrophages
• Endothelial Cells
• Adipocytes

ECM:

• > Collagen I,III,IV
• Laminin
• Fibronectin
• Hemonectin
• Thromboospondin
• Proteoglycans

Adhesion Receptors :

• Integrins
• Selectins
• CD44
• Lectins

Cytokines:

• IL-,3,6,11,F
• G-CSF
• GM-CSF
• SCF
• b-FGF

Inhibitors

• MIP-1alpha
• TDF-alpha
• TNF-alpha
• INF-y

Question 20

20. What are the three main ways you can differentiate between cells?

Answer 20

1. Antigen Markers
2. Fluorescent Dye
3. Assays

Question 21

21.How would you use Antigen Markers to differentiate between cells?

Answer 21

CD34:
Stem Cells and Progenitor Cells : CD34+
Mature Cells : CD34-

Lin:
Stem Cells/Progenitors : Lin-
Mature Cells : Lin+

Question 22

22.How would you use Fluorescent Dye to differentiate between cells?

Answer 22

Rhodmine 123:

• Only picked up by cycling cells
• Early stem cells will appear dull but if in the cycle-will appear bright

Cytotoxic Drugs: 5-fluoro-uracil:

• Only picked up by cycling cells so will be killed by them
• Cells out of the cycle are resistant to this drug

Question 23

23. Explain the 5 methods to differentiate between stem cells depending on purity of stem cells ?

Answer 23

1. Erythrocyte lysis (least pure)
   􏰁 Gives enriched population of stem cells
2. Density gradient centrifugation
   􏰁 Spin cells on gradient
   􏰁 remove certain cells
3. Adherence depletion
   􏰁 Bone marrow put onto plastic
   􏰁 Some stick to plastic so can be harvested
4. Antibody depletion
   􏰁 Get rid of certain cells
   􏰁 Deplete all cells that are Lin+
5. Antibody selection (most pure)
   􏰁 Positively select cells that are CD34+ 􏰁 Early stem cells: big nucleus

Question 24

24. Why are progenitors called CFU?

How do you get progenitors to form colonies?

Answer 24

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
-Put early cell into culture
-Stimulate it to divide into mature cell

Question 25

25. List the 7 types of colony assays?

Answer 25

1. CFU-G granulocyte progenitor
2. CFU-E + BFU-E erythroid progenitors
3. CFU-Mk megakaryocyte progenitor
4. CFU-GM granulocyte/monocyte progenitor
5. CFU-GEMM granulocyte/erythroid/monocyte/megakaryocyte progenitor
6. CFU-bas basophil progenitor
7. CFU-eo eosinophil progenitor

Question 26

26. List the different biological and non-biological assays?

Answer 26

Non-Biological:

• Morphology
• FACS- Fluorescence Activated Cell Sorting (more quantitive)

Biological:

• Growth Rate
• Plating Efficiency
• Function

Question 27

27.What are the stages of a colony assay?

Answer 27

1. Done on a semi-solid medium
   􏰀 Agar/methylcellulose
   􏰀 Add growth factors
2. Incubate for 7-14 days
3. Single cells will divide and differentiate and form colonies
4. Can be identified using a microscope
5. Can quantitate number of cells in original suspension
   o Done in laminar flow cabinet -Sterile environment

Question 28

28. What are the three main categories for applications of colony assays?

Answer 28

1. Experimental
2. Diagnostic
3. Therapeutic

Question 29

29.List three applications for colony assays?

Answer 29

Research – basic haemopoiesis and carcinogenesis

Testing toxicity of chemotherapeutic agents and carcinogens

Generate cells for stem cell transplantation/manipulation