Primary Cell Culture

Question 1

What is a primary cell culture?

Answer 1

• Technique by which cells from primary tissues or cell suspensions are grown under controlled conditions in vitro
• Cells will divide and or differentiate like normal cells, carrying out their normal function

Question 2

Provide some examples of non-haemopoietic primary cultures

Answer 2

Liver, Muscle, Skin, Nerves, Fibroblasts, Endothelial

Question 3

Provide some examples of haemopoietic primary cultures

Answer 3

• Stem, Progenitor cells
• T and B cells
• Monocytes, Macrophages
• Osteoblasts
• Dendritic cells
• Neutrophils
  
  • Eosinophils, Basophils, Mast cells
• Erythrocytes
• Megakaryocytes

Question 4

What can primary cultures be established from?

Answer 4

• Haemopoetic cells
  
  • Giving rise to mature blood cell types
• Non-haematopoietic cells

Question 5

What are the differences between cell cultures and cell lines?

Answer 5

• Primary cell cultures are directly derived from tissues
  
  • Cell lines are transformed cells which may be genetically identical
• Interpatient variabillity
  
  • In a cell line every cell is identical
• Finite lifespan (cant consistently maintain O2 and growth medium)
  
  • A cell line will divide and reproduce itself exactly the same
• Cells divide and/or differentiate
  
  • A cell line will divide and reproduce itself exactly the same
• Cells carry out normal funciton
  
  • Because cell lines are transformed it may not have a normal function

Question 6

By what process do we manipulate tissues to get a single cell suspension for primary cell cultures?

Answer 6

Disaggregation

Question 7

List some disaggregation techniques

Answer 7

• Cells migrate out of explant
• Mechanical dissociation (mincing, sieving, pipetting)
• Enzymatic dissociation (trypsin, collagenase, hylaluronidase)

Question 8

What cell does not need to be disaggregated?

Answer 8

Haematopoietic cells do not need to be disaggregated as they are already in single cell suspension

Question 9

What are sources of stem cells?

Answer 9

• Bone marrow aspirate
• Umbilical cord blood
• Mobilised peripheral blood

Question 10

Where does haematopoiesis occur in children?

Answer 10

Red bone marrow

Liver

Spleen

Question 11

Where does haematopoeisis occur in adults?

Answer 11

In adults (after 20 years), the bone marrow retreats and haematopoiesis only occur in:

• End of long bones (e.g femur, humerus)
• Skull
• Vertebrae
• Sternum
• Pelvis

Question 12

What is lifecycle of haematopoetic cells?

Answer 12

Pluripotent stem cells → Early progenitors → Late Progenitors → Immature Precursors → Mature Blood Cells

Question 13

Why are you not able to see the lineage of progenitor cells?

Answer 13

• They are undifferentiated cells and therefore do not show any mature characteristics
  
  • HOWEVER they are already commited to a specific lineage and make a specific type of cell

Question 14

Stem Cells

Answer 14

pluripotent, give rise to all lineages, self-renew, rare cells, responsible for engraftment

Question 15

Progenitor cells

Answer 15

Undifferentiated, not distinguished by morphology, committed to one or more lineages, detected in colony forming assays

Question 16

Precursor cells

Answer 16

immature but recognisable, cells starting to differentiate, dew final divisions before forming mature cells

Question 17

What is haemopoiesis?

Answer 17

process whereby immature precursor cells develop into mature blood cells

Question 18

How do haematopoietic growth factors?

Answer 18

1. Bind to cell surface transmembrane receptors
2. Stimulate growth + survival of progenitors

Question 19

What are stromal cells?

Provide some examples of stromal cells.

Answer 19

Stromal cells are cells in the bone marrow which form the environment for stem cells

• Fibroblasts, Macrophages, Endothelial cells, adipocytes

Question 20

What do stromal cells produce on their surface?

Answer 20

Produce ECM (extracellular matrix) proteins on their surface:

• Collagen I, II, III, IV
• Laminin
• Fibronectin
• Hemonectin
• Thrombospondin
• Proteoglycans

Question 21

What do stromal cells contain on their surface?

Answer 21

Adhesion receptors

• Integrins
• Selectin
• CD44
• Lectins

Question 22

What cells will produce cytokines and inhibitors?

Answer 22

Stromal cells

Question 23

How are stem cells in interaction with stromal cells?

Answer 23

The stem cells will have equivalent receptors to adhesion receptors which will recognise cytokines and inhibitors produced by stromal cells.

Question 24

What are different techniques to identify the haematopoesis stages that are not cell culture?

Answer 24

• Phenotype
• Fluorescent stains/ cytotoxic drugs
• Assays

Question 25

Phenotypic techniques to identify haematopoetic cells

Answer 25

* **Stem cells and progenitor cells** (early+ late) are all **posotive for CD34,** mature **blood cells are negative CD34** * A stem cells are **Lin negative**, mature cells are **Lin positive**

Question 26

How do fluorescent stains/ cytotoxic drug techniques work to identify haematopoetic cells?

Answer 26

* **Rh123** is a **fluorescent dye** = stains mitochondria. Stem cells out of the cycle will not stain for that. Actively cycling cells will stain bright * **5-FU** is a **cytotoxic drug** and will only affect cycling cells. Cells out of cycle wil be resistant to this drug. Adding this drug will kill cycling cells

Question 27

Assay techniques to identify haematopoietic cells

Answer 27

* Some assays can recognise and grow early stem cells * Colony forming unit assays for lineage commited progenitors

Question 28

How to we undertake cell processing?

Answer 28

* Haematopoietic stem cells do not require processing as they are already in a single cell suspension * There are various techniques which will enrich and purify stem cells ready for culture * **Erythrocyte lysis** * Enrichment of stem cells * **Density gradient centrifugation** * This will remove some cells and enriches for others * **Adherance depletion** * Bone marrow on a sticking culture, remove non-adherant cells through pipetting * **Antibody depletion** * Can use antibodies for Lin antigens, these will recognise mature cells and deplete * **Antibody selection** * Can use antibodies for stem cells and select them via flow cytometry/ magnetic beads * Label bone marrow with CD34 (e.g)

Question 29

What is a colony assay?

Answer 29

* This is where progenitors will grow to form colonies of mature cells * Progenitors are also called CFUs (colony forming units) * Progenitors will grow from 30 to several hundreds and thousands

Question 30

How is a colony forming assay formed?

Answer 30

1. Take single cell suspension and grow it with growth factors in a semi solid medium (agar-methylcellulose) in an incubator for 7-14 days 2. The progenitor cells will replicate to produce a colony of more mature cells (most one lineage only ) 3. This can be seen down a microscope and can be identified as cell types

Question 31

What are some applications of primary cell cultures?

Answer 31

Research - basic haematopoeisis and carcinogenesis Testing for toxicity of chemotherapeutic agents and carcinogens Generate cells for stem cells transplantation and manipulation