Primary cell culture technique

Question 1

What does primary cell culture technique allow?

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

Examples of primary culture

Answer 2

• Non-haemopoetic
• Liver
• Muscle
• Skin
• Nerves
• Fibroblasts
• Endothelial cells
• Haemopoietic – already in a cell suspension, do not have to be manipulated
	○ Stem, Progenitor cells
	○ T and B cells
	○ Monocyte, Macrophages
	○ Osteoblasts
	○ Dendritic cells
	○ Neutrophils, Eosinophils,Basophils, Mast cells
	○ Erythrocytes
Megakaryoctyes, Platelets

Question 3

Different type of way for the disaggregation of cells

Answer 3

1. Put tissue into culture and cells will move out
   ○ Cells allowed to migrate out of an explant
2. Mechanical dissociation (mincing, sieving, pipetting)
3. Enzymatic dissocation (trypsin,collagenase, hyaluronidase, protease, DNAase)
   -Exception – Haemopoietic cells – Do not need to be disaggregated – They already are (in a cell suspension so do not need to use manipulation techniques)

Question 4

What are examples of sources of stem cells?

Answer 4

• Bone marrow aspirate

* Umbilical cord blood

Question 5

What is a source of stem cells in children?

Answer 5

○ all bones with red bone marrow

Question 6

What is a source of stem cells in adults?

Answer 6

○ Ends of long bones e.g. femur, humerus
○ Skull
○ Vertebrae
○ Ribs
○ Sternum	
○ Pelvis – main area for harvesting stem cells

Question 7

What happens to cells in haematopoiesis?

Answer 7

• Cells become more differentiated as well as more amplified as they progress

Question 8

What is the stage in haematopoiesis that is assayed for and what are the cells labelled as?

Answer 8

• Progenitor stage is the one that is assayed for

-Cells are labelled as CFU or BFU

Question 9

Stem cells

Answer 9

○ Pluripotent
○ Self-renew
○ Rare
○ Responsible for engraftment

Question 10

Progenitor

Answer 10

○ Undifferentiated
○ Undistinguishable by morphology alone
○ Starting to become committed
○ Need assays

Question 11

What do haematopoietic(Cytokines) growth factors bind to and stimulate?

Answer 11

• Bind to cell surface transmembrane receptors
• Stimulate growth and survival of progenitors
-Important in every stage

Question 12

What are some growth factors specific to and not specific to?

Answer 12

Some growth factors are specific to their lineage and Other growth factors are non-specific

Question 13

What are stromal cells?

Answer 13

Stem cells that are sitting in close association to other cells. Examples:
	§ Fibroblasts
	§ Macrophages
	§ Endothelial cells
        § Adipocytes

Question 14

What is the extracellular matrix produced by?

Answer 14

Produced by stromal cells
Examples:
	§ Collagen
	§ Laminin
	§ Fibronectin
	§ Hemonectin
	§ Thrombospondin
        § Proteoglycans

Question 15

What are adhesion receptors produced by ?

Answer 15

Produced by stromal cells
Examples:
	§ Integrins
	§ Selectins
	§ CD44
        § Lectins

Question 16

What are the 3 ways to differentiate between cells?

Answer 16

1. Antigen markers
2. Fluorescent dye
3. Assays

Question 17

Example of antigen markers and differences in stem cells and progenitor cells

Answer 17

○ CD34
	§ Stem cells and progenitor cells: CD34+
	§ Mature cells: CD34-
		○ Lin
	§ Stem cells/progenitors: 
          Lin-
        § Mature cells: Lin+

Question 18

Examples of fluorescent dye and differences in stem cells and progenitor cells

Answer 18

○ Rhodomine 123
§ Only picked up by cycling cells
i. Cells in cycle will be positive for this dye
1) Early stem cells would appear dull but if in cycle will appear bright
a. Cytotoxic drug: 5-fluoro-uracil
i. Only taken up by cycling cells so will be killed
ii. Cells out of cycle are resistant to this drug

Question 19

Different methods to differentiate between stem cells

Answer 19

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

Question 20

What do progenitors grow to form and thus what are progenitors called?

Answer 20

Progenitors grow to form colonies of mature cells

-Thus progenitors are called “colony forming units”

Question 21

Steps in colony assay

Answer 21

• Put early cell into culture

* Stimulate it to divide into mature cell

Question 22

What are the types of colony assay?

Answer 22

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
   CFU-eo eosinophil progenitor

Question 23

What are non-biological assays used for?

Answer 23

• Morphology

* FACS – Fluorescence Activated Cell Sorting (more quantitative)

Question 24

What are biological assays used for?

Answer 24

• Growth rate
• Plating efficiency
• Function

Question 25

What are the stages of colony assay?

Answer 25

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
     • Done in laminar flow cabinet
		○ Sterile environment

Question 26

What are applications of cell culture?

Answer 26

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