Primary Culture Techniques

Question 1

Where do cells for Primary Cell Culture come from?

Answer 1

Cells are derived directly from Tissues.

Question 2

Describe the main features of a Primary Cell Culture:

Answer 2

“1. There is often Interpatient Variability.

2. The Cells have a Finite Lifespan.
3. Cells Divide and/or Differentiate.
4. Cells carry out Normal Functions.”

Question 3

Give examples of Haematopoietic Primary Cultures:

Answer 3

“1. Stem/Progenitor Cells.

2. T and B cells.
3. Monocytes,
   Macrophages.
4. Osteoblasts.
5. Dendritic Cells.
6. Neutrophils,
   Eosinophils,
   Basophils,
   Mast Cells.
7. Erythrocytes,
   Megakaryocytes,
   Platelets.”

Question 4

Why are Cells Disaggregated?

Answer 4

“To allow them to migrate out of an explant.

Hematopoietic Cells do not need to be disaggregated because they already are.”

Question 5

What are the two methods of Dissaggregation:

Answer 5

“Mechanical Dissociation:

• Mincing
• Sieving
• Pipetting

Enzymatic Dissociation:

• Trypsin
• Collagenase
• Hyaluronidase
• Protease
• DNAse”

Question 6

Where can Stem Cells be obtained from?

Answer 6

“1. Bone Marrow Aspirate.

2. Umbilical Cord Blood.
3. Mobilised Peripheral Blood.”

Question 7

Where is Red Bone Marrow found?

Answer 7

“Children:
- All Bones

Adults:
- Skull
- End of Long Bones
  Humerus
  Femur
- Vertebrae
- Ribs
- Sternum
- Pelvis"

Question 8

Where does Haemopoiesis occur?

Answer 8

“Haemopoiesis occurs in the middle of the Endosteum.

There are Densely packed areas of Immature Cells.”

Question 9

What does Bone Marrow look like?

Answer 9

“The White areas are Fat..

The Pink areas are Blood Vessels.”

Question 10

Describe the Stages of Cell Morphology?

Answer 10

“Early Progenitors:
- Look the same as Stem Cells.

Late Progenitors:
- Look the same as Early Progenitors and Stem Cells.

Immature Precursors:

• Cells start to look different to each other and their previous precursors.
• Mature cell types are fully committed and have distinct morphology. “

Question 11

What does CFU mean?

Answer 11

“Colony Forming Unit in culture.

- It is given on the basis of the cells ability to form colonies on culture.”

Question 12

Stem Cells:

Answer 12

“Pluripotent

• Give rise to all lineages.
• Self-renew.
• Rare Cells.
• They are in G0 and don’t perform any function.
• Responsible for engraftment.”

Question 13

Progenitor Cells:

Answer 13

“Undifferentiated:

• Not distinguished by morphology.
• Committed to one or more lineages.
• Detected in Colony-Forming Assays. “

Question 14

Precursor Cells:

Answer 14

“Immature:

• Recognisable.
• Cells starting to differentiate.
• Few final divisions before Mature Cells.”

Question 15

What are Haematopoietic Growth Factors?

Answer 15

“Polypeptide Growth Factors: Cytokines.

• Bind to cell surface transmembrane receptors.
• Stimulate Growth and Survival of progenitors.”

Question 16

What 4 Main Components make up the Microenvironment?

Answer 16

“1. Stromal Cells

2. Extracellular Matrix
3. Adhesion Receptors
4. Cytokines
5. Inhibitors”

Question 17

How can the Microenvironment of the Cells affect them?

Answer 17

“1. Stem Cells are always in association with Stromal Cells in the Bone Marrow.

2. Lin is a marker that is specific for a lineage:
   Stem Cells: Lin Negative (Lin -).
   Mature Cells: Lin Positive (Lin +).
3. CD34 is a membrane protein that aids cells in cell-cell adhesion:
   Progenitor Cells: Positive for CD34 (CD34 +).
   Mature Blood Cells: Negative for CD34 (CD34 -) .
4. CD4 (Cluster of Differentiation 4) is a glycoprotein found on the surface of immune cells:
   T Cells: Positive for CD4 (CD4 +).”

Question 18

What is used to Differentiate Stem Cells and Cycling Cells?

Answer 18

“1. Rhodamine 123 is a Fluorescent Dye Stain for Mitochondria:
- The Stem Cells will NOT stain for Rh123.

2. A Cytotoxic Drug called 5-FU (5 fluorouracil) only affects Cycling Cells:
   
   • If this is added into cultures it kills Cycling Cells.”

Question 19

List the main Processing Methods for Cells?

Answer 19

“1. Erythrocyte Lysis.

2. Density Gradient Centrifugation.
3. Adherence Depletion.
4. Antibody Depletion.
5. Antibody Selection.”

Question 20

What are Colony Forming Units?

Answer 20

“Colony Forming Units CFUs:

• Progenitors grow to form Colonies of Mature Cells.
• From 32 to hundreds of thousands of Cells in a Colony.
• Burst Cells can be seen without a Microscope.”

Question 21

How are CFUs formed In Vitro?

Answer 21

“1. Take a Single Cell Suspension of Bone Marrow.

2. Put the Suspension on semi-solid medium (agar or methylcellulose).
3. Add the appropriate Growth Factors.
4. Incubate for 7 - 14 Days.”

Question 22

List the Different CFUs and what they form:

Answer 22

“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 23

What are the potential Applications of working on CFUs?

Answer 23

“Research: Basic Haemopoiesis and Carcinogenesis.
Testing Toxicity: Of Chemotherapeutic agents and Carcinogens.
Generate Cells: For Stem Cell Transplantation/Manipulation.”

Question 24

Give examples of Non-Hematopoietic Primary Cultures:

Answer 24

“1. Liver.

2. Muscle.
3. Skin.
4. Nerves.
5. Fibroblasts.
6. Endothelial Cell.”