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CellularPathologyCOPY-aimen n-use this > Primary Cell Culture Techniques > Flashcards

Primary Cell Culture Techniques Flashcards

1
Q

Where are cells obtained from in primary cell cultures?

A

Cells derived directly from tissues; contrast to cell line which has either spontaneously been transformed or genetically manipulated

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2
Q

Describe the difference in primary cell cultures between patients

A 
Interpatient variability (cell lines are absolutely identical; reproducibility)
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3
Q

How long do cells from primary cell cultures survive?

A

Finite lifespan - cell lines are immortal

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4
Q

How do primary cell cultures differ from cell lines?

A

Cells divide and/or differentiate (cell lines are clones)
Cells carry out normal functions
(cell lines are non-functional)

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5
Q

Give examples of non-haematopoietic primary cultures

A

Non-hematopoietic

  • Liver
  • Muscle
  • Skin
  • Nerves
  • Fibroblasts
  • Endothelial cells
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6
Q

Name examples of haemopoietic primary cultures

A 
Stem, Progenitor cells
T and B cells
Monocyte, Macrophages
Osteoblasts
Dendritic cells
Neutrophils, Eosinophils,Basophils, Mast cells
Erythrocytes
Megakaryocytes, Platelets
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7
Q

What is the purpose of cell disaggregation?

A

Cells allowed to migrate out of an explant

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8
Q

What is cell disaggregation?

A

Breaking up of a total (aggregate), integrated whole, into smaller elements, parts, or units, usually for easier handling

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9
Q

What are the different ways of disaggregating cells?

A

Mechanical dissociation (mincing,sieving,pipetting)

Enzymatic dissociation (trypsin,collagenase, hyaluronidase, protease, DNAase)

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10
Q

What cells don’t require disaggregation?

A

Exception – Haemopoietic cells don’t need to be disaggregated as they already are

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11
Q

What are the sources of stem cells?

A
  • Bone marrow aspirate
  • Umbilical cord blood (enriched)
  • Mobilised peripheral blood
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12
Q

Why may stem cells be found in the peripheral blood?

A

Normally stem cells aren’t found in peripheral blood but treating patients with growth factors causes them to move out

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13
Q

What happens in red bone marrow?

A

Red bone Marrow is where hematopoiesis occurs

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14
Q

Where is red bone marrow found?

A

Children contain red bone marrow in all bones; Adult red bone marrow retreats and only occurs in specific areas

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15
Q

Outline the specific ares red bone marrow is found in children?

A

all bones, liver and spleen

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16
Q

Where is red bone marrow found in adults (20+)?

A

Ends of long bones (femur, humerus)

skull, vertebrae, ribs, sternum,, pelvis

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17
Q

Describe the different layers of bone

A

Hard periosteum around the outside, on the endomysium (inside) is where haematopoiesis is occurring

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18
Q

Where are blood vessels found in bone?

A

Blood vessels travel through the middle - haemopoiesis occurs here

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19
Q

Describe the structure of bone

A

Hard exterior and a fibrous mesh in the middle where haematopoiesis occurs
3D environment

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20
Q

How does a bone marrow film and blood film differ?

A

Peripheral blood stains show a variety of maturity in cells present
whereas bone marrow has densely packed areas of immature cells

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21
Q

Describe the features of a bone marrow slide under a mciroscope

A

In a concentrated area of active haematopoiesis

Pink blobs are blood vessels
White areas are fat

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22
Q

What are the stages of cell development from stem cells?

A
  1. Stem cells
  2. Early progenitors
  3. Late progenitors
  4. Immature precursors
  5. Mature cell types - RBC, WBC
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23
Q

Describe the structure of early and late progenitors

A

Early & late progenitors are identical to stem cells as they are undifferentiated

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24
Q

What happens to immature precursors?

A

Immature precursors begin to differentiate and commit to a certain lineage - can identify and tell them apart from one another and their homogenous precursors

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25
Q

What makes mature cell types identifiable?

A

Mature cell types have very distinct morphology under the microscope - easily distinguishable

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26
Q

How is stem cell development regulated?

A

This process is tightly regulated by negative and positive growth factors

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27
Q

Describe proliferation of stem cells during development

A

Amplification increases throughout the process

28
Q

What part of the cell cycle are stem cells in?

A

Stem cells are normally in G0, when activated they either self renew or differentiate

29
Q

Describe the morphology of stem cell sand progenitors

A

Stem cells and progenitors look identical under a microscope but start to differentiate further into immature precursors

30
Q

What are CFU?

A

CFU - colony forming unit

E.g. CFU-GM has the ability to differentiate into a granulocyte and monocyte

31
Q

What are BFU?

A

BFU - burst forming units

32
Q

Outline the features of stem cells

A

Pluripotent- give rise to all lineages
Self-renew
Rare cells
Responsible for engraftment

33
Q

Describe features of Progenitor cells

A

Undifferentiated
Not distinguished by morphology
Committed to one or more lineages
Detected in colony-forming assays

34
Q

What are the distinctive features of precursor cells?

A

Immature but recognisable
Cells starting to differentiate
Few final divisions before mature cells

35
Q

What are haematopoietic growth factors?

A

Polypeptide growth factors (cytokines)

36
Q

How are haematopoietic growth factors activated?

A

Bind to cell surface transmembrane receptors

Stimulate growth and survival of progenitors.

37
Q

Describe growth factor specificity in haematopoiesis

A

Some growth factors are very specific to a lineage
e.g. EPO for red cells ILF for eosinophils

But other growth factors work all over the place across lineages at different stages e.g. IL3

38
Q

What forms the bone marrow microenvironment?

A 
Stromal cells
Extracellular matrix (ECM)
Adhesion Receptors
Cytokines
Inhibitors
39
Q

Name examples of stromal cells

A
  • fibroblasts
  • macrophages
  • endothelial cells
  • adipocytes
40
Q

Describe the composition of the stem cell environment

A

Stromal cells form the environment the stem cells are sitting in and on their surface contain various extracellular matrix proteins, adhesion receptors

Stromal cells also produce cytokines and inhibitors

41
Q

What structures form the extracellular matrix (ECM)?

A
  • collagen I, III, IV
  • laminin
  • fibronectin
  • hemonectin
  • thrombospondin
  • proteoglycan (GAGs)
42
Q

Name some adhesion molecules

A
  • ontegrins
  • selectins
  • CD44
  • Lectins
43
Q

Describe the stem cell niche

A

Stem cell niche - each stem cell corresponds to a stromal cell

44
Q

How can we identify cells in different stages of haematopoiesis

A

Can use the following factors to identify cells during different stages of haematopoiesis:

  • Phenotype (cell surface antigens)
  • Drugs & dyes
  • Cell cultures
45
Q

How can we use CD34 antigen to identify different haematopoietic cells?

A

Both early and late progenitors are positive for CD34 but mature cells are CD34 negative

46
Q

What is LIN?

A

LIN - lineage specific marker

47
Q

How is LIN used to identify how mature stem cells are?

A

Early and late progenitors are LIN-

Mature cells are LIN+ as they are specific to certain lineages

48
Q

What is Rhodamine 1,2,3?

A

Rhodamine 1,2,3 is a fluorescent dye that stains mitochondria - used in flow cytometry

49
Q

How is rhodamine used to identify cells in cycle?

A

Out of cycle stem cells won’t stain for rhodamine as they have no mitochondria present

50
Q

What is 5-fluorauracil?

A

5-fluorouracil is a cytotoxic drug that only affects cycling cells, out of cycle cells are resistant

51
Q

When are colony forming unit assays used?

A

Colony forming unit assays for lineage committed progenitors

52
Q

Which assay type is used to identify mature cells?

A

Staining and functional assays used for mature cells

53
Q

Outline cell processing methods

A
  • Erythrocyte lysis
  • Density gradient centrifugation
  • Adherence depletion
  • Antibody depletion (mature cells) e.g. CD34
  • Antibody selection (stem cells) e.g. CD34
54
Q

Describe the structure of CD34 stem cells

A

Purified CD34 stem cells fluorescently dyed and put through a flow cytometer and pulled out

Can see they’re very immature - huge nuclei and very little cytoplasm

55
Q

Outline the culture conditions of colony assays

A
  • 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
56
Q

Outline how colony assays are undertaken

A
  1. Semi-solid medium
    (agar, methylcellulose)
  2. Plus growth factors
  3. Incubate for 7-14 days
  4. Identify under microscope
57
Q

Why are petri dish cultures carried out more than once?

A

2 petri dishes as all experiments carried out in duplicate; reproducibility

58
Q

Why is sterile water added to colony assays?

A

Sterile water to prevent drying out

59
Q

What apparatus are colony assays produced in?

A

Carried out in a laminar flow cabinet - air has been filtered to provide a sterile environment before cultures are incubated

60
Q

Give examples of common colony assays

A 
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
61
Q

What are some of the non biological assays?

A

Non biological Assays

  • Morphology
  • FACS
62
Q

Outline why biological assays are carried out

A
  • Growth rate
  • Plating efficacy
  • Function
63
Q

What are the applications of colony assays?

A

Research – basic haemopoiesis and carcinogenesis

Testing toxicity of chemotherapeutic agents and carcinogens

Generate cells for stem cell transplantation/manipulation

64
Q

What is a primary cell culture?

A

Primary culture is a technique by which cells from primary tissues or cell suspensions are grown under controlled conditions, in vitro

65
Q

What are cell cultures used for?

A

Cultures can be used for experimental, diagnostic or therapeutic purposes.

CellularPathologyCOPY-aimen n-use this (13 decks)

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