Cellular pathology: Primary cell culture techniques

Question 1

What is primary cell culture?

Answer 1

• Technique that involves the extraction of cells directly from primary tissues or cell suspensions. These cells are then grown in a controlled environment, in vitro.

Question 2

What is a cell line and what is the main differenence between it and primary cell culture?

Answer 2

• Technique that involves growing cells under controlled conditions that are generally different to their natural environment
• The main difference between cell line and primary cell culture is that cell lines involve the growth of transformed cells while primary cell culture is growth of cells extracted directly from tissue

Question 3

What other differences are there between primary cell culture and cell lines?

Answer 3

• In primary cell culture you get interpatient variability, cells you extract from the same area in different patients will be different compared to one another, while in cell lines each cell is exactly the same
• Cells grown using primary cell culture have a finite lifespan while cells grown using cell lines are “immortal”
• Cells grown using primary cell culture have ability to grow and/or differentiate into different cell types while cells grown using cell lines can only grow and reproduce the same cell type
• Cells grown using primary cell culture have normal functions while cells grown using cell lines might not have normal functions because they’re transformed cells

Question 4

Name some examples of non-haemopoietic sources for primary cell culture

Answer 4

• Liver
• Muscle
• Skin

Question 5

Name some examples of haemopoietic sources for primary cell culture

Answer 5

• Progenitor cells
• T and B cells
• Macrophages

Question 6

What are the different ways in which cells can be disaggregated?

Answer 6

• Place cells in culture and allow them to migrate out of an explant
• Mechanical dissociation e.g. mincing, sieving, pipetting
• Enzymatic dissocation e.g. trypsin, collagenase, hyaluronidase, protease or DNAase

Question 7

Why don’t Haemopoietic cells need to be disaggregated?

Answer 7

• Because they they are already in a single cell suspenesion

Question 8

Where within the body can haemopoietic stem cells be extracted in order for use in primary cell culture?

Answer 8

• Bone marrow aspirate
• Umbilical cord blood
• Mobilised peripheral blood - Stem cells normally aren’t in peripheral blood so person has to be treated with growth factors so stem cells move from marrow into peripheral blood

Question 9

What type of bone marrow does hameopoiesis occur in?

Answer 9

• Red bone marrow

Question 10

Where can red bone marrow be extracted from in children?

Answer 10

• Red bone marrow is present in all bones
• Also present within liver and spleen

Question 11

Where can red bone marrow be extracted from in adults?

Answer 11

• Red bone marrow only present in bones of axial skeleton:
  
  • Skull
  • Vertebrae
  • Ribs
  • Sternum
  • Pelvis
• Also present in ends of long bones such as the femur

Question 12

Where specifically within the bone marrow does haemopoiesis occur?

Answer 12

• Occurs in the endosteum

Question 13

Briefly describe the process of haemopoiesis

Answer 13

• Stem cells first become early progenitors and amplify as they do so
• Those early progenitors then become late progenitors and amplify even further
• The late progenitors then become immature precusors. It’s at this stage that the cells begin to look different from the cells at earlier stages which all look the same as each other
• Finally, the immature precusors then become mature cells types, e.g. red cells, neutrophils and platelets

Question 14

What is it that causes cells in the process of haemopoiesis to progeress to the next stage?

Answer 14

• Growth factors

Question 15

What are some characteristics of stem cells?

Answer 15

• Pluripotent - Can give rise to all lineages
• Have ability to self-renew
• Rare cells
• Responsible for engraftment

Question 16

What are some characteristics of progenitor cells?

Answer 16

• Undifferentiated
• Unable to distinguish between different types of progenitor cell as well as to stem cells
• Committed to one or more lineages
• Detected in colony-forming assays

Question 17

What are some characteristics of precusor cells?

Answer 17

• Immature but recognisable cells
• Cells that have started to differentiate
• Only require a few final divisions before they become mature cells

Question 18

What cell type are haematopoietic growth factors?

Answer 18

• Polypeptides (cytokines)

Question 19

How do haematopoietic growth factors induce their effects?

Answer 19

• They bind to cell surface transmembrane receptors

Question 20

Are haematopoietic growth factors specific for each of the different lineages seen in haematopoiesis?

Answer 20

• Some growth factors are specific to a particular lineage e.g. Erythropoietin (EPO) only works to produce red cells from red cell progenitors
• Some growth factors are able to work on multiple lineages and at multiple stages of haematopoiesis e.g. Interluekin 3 (IL-3)

Question 21

Apart from stem cells, what other things are present within bone marrow? For each thing mentioned give some examples

Answer 21

• Stromal cells: E.g. fibroblasts, macrophages, endothelial cells and adipocytes
• Extracellular matrix proteins (on stem cell surface): E.g. Collagen I, II and III, Lamin
• Adhesion receptors (also on stem cell surface): E.g. Integrins, Selectins and Lectins
• Cytokines: E.g. IL-3, IL-3, IL-6
• Inhibitors: TGF-β and TNF-α

Question 22

Why can CD43 be used to distinguish between stem cells and progenitor/mature cells?

Answer 22

• Because stem cells are negative for CD34 while multi-potential and lineage-committed progenitor cells, as well as mature cells, are positive for CD34

Question 23

What is Lin and why can it be used to distinguish between stem cells and mature cells?

Answer 23

• Lin is any type of lineage specific marker used to identify whether a particular cell is part of a lineage
• Lin can be used to distinguish between stem cells and mature cells because stem cells are Lin negative and mature cells are Lin positive

Question 24

Give an example of a lineage specific marker (Lin) and what mature cell they are able to identify

Answer 24

• CD4 is used to identify T lymphocytes

Question 25

What is rhodamine 123 and what cell types is it used to distinguish between?

Answer 25

• Rhodamine 123 is a fluoresecent dye used to stain mitochondria
• It’s used to distinguish between stem cells in G0, which stain dull, and any cell types actively going through cell cycle, e.g. progeniotor cells, which stain bright.
  *

Question 26

Why do stem cells in G₀ stain dull for rhodamine 123?

Answer 26

• It’s because the stem cells in G₀ don’t contain any mitochondria. This is because they aren’t actively going through the cell cycle and so don’t require any energy

Question 27

What is 5-Fluorouracil (5-FU)?

Answer 27

• It’s a drug that affects cells actively going through cell cycle and so can be used to distinguish between cycling cells and non-cycling cells, cells in G₀

Question 28

What are some of the method that can be used to purify/enrich haematopoietic stem cells from a sample of red bone marrow?

Answer 28

• Erythrocyte lysis
• Density gradient centrifugation
• Adherence depletion - Use plastic which particular cells will adhere to, e.g. macrophages, and seperate those cells from cells that don’t adhere to plastic
• Antibody depletion - use of specific antibodies which bind to particular mature cell antigens allowing them to be seperated from stem cells
• Antibody selection - use of spefic antibodies which bind to particular stem cell antigens allowing for them to be extracted

Question 29

Why are progenitors called colony forming units (CFUs)?

Answer 29

• Because progenitors can grow to form colonies of mature cells

Question 30

How do you produce a colony assay?

Answer 30

• Place single cell suspension of bone marrow into a semi-solid medium (agar or methylcellulose) and then add growth factors
• You then let it incubate for 7-14 days and let the colonies grow

Question 31

What are the different types of colony forming unit that you can create a colony assay of?

Answer 31

• 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 32

Analysis of colony assays can be biological or non-biological, give some examples of non-biological analysis that can be carried out on colony asssays

Answer 32

• You can look at cell morphology of each of the cells within each of the colonies that grow
• You can use fluorescence-activated cell sorting (FACS) to sort the cells within a colony into two or more containers based upon the specific light scattering and fluorescent characteristics of each cell.

Question 33

Give some examples of biological analysis that can be carried out on colony asssays

Answer 33

• You can look at:
  
  • Growth rate
  • Plating efficiency
  • Function

Question 34

What are some applications of primary cell culture of haematopoietic stem cells?

Answer 34

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