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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
What makes mature cell types identifiable?
Mature cell types have very distinct morphology under the microscope - easily distinguishable
26
How is stem cell development regulated?
This process is tightly regulated by negative and positive growth factors
27
Describe proliferation of stem cells during development
Amplification increases throughout the process
28
What part of the cell cycle are stem cells in?
Stem cells are normally in G0, when activated they either self renew or differentiate
29
Describe the morphology of stem cell sand progenitors
Stem cells and progenitors look identical under a microscope but start to differentiate further into immature precursors
30
What are CFU?
CFU - colony forming unit | E.g. CFU-GM has the ability to differentiate into a granulocyte and monocyte
31
What are BFU?
BFU - burst forming units
32
Outline the features of stem cells
Pluripotent- give rise to all lineages Self-renew Rare cells Responsible for engraftment
33
Describe features of Progenitor cells
Undifferentiated Not distinguished by morphology Committed to one or more lineages Detected in colony-forming assays
34
What are the distinctive features of precursor cells?
Immature but recognisable Cells starting to differentiate Few final divisions before mature cells
35
What are haematopoietic growth factors?
Polypeptide growth factors (cytokines)
36
How are haematopoietic growth factors activated?
Bind to cell surface transmembrane receptors Stimulate growth and survival of progenitors.
37
Describe growth factor specificity in haematopoiesis
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
What forms the bone marrow microenvironment?
``` Stromal cells Extracellular matrix (ECM) Adhesion Receptors Cytokines Inhibitors ```
39
Name examples of stromal cells
- fibroblasts - macrophages - endothelial cells - adipocytes
40
Describe the composition of the stem cell environment
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
What structures form the extracellular matrix (ECM)?
- collagen I, III, IV - laminin - fibronectin - hemonectin - thrombospondin - proteoglycan (GAGs)
42
Name some adhesion molecules
- ontegrins - selectins - CD44 - Lectins
43
Describe the stem cell niche
Stem cell niche - each stem cell corresponds to a stromal cell
44
How can we identify cells in different stages of haematopoiesis
Can use the following factors to identify cells during different stages of haematopoiesis: - Phenotype (cell surface antigens) - Drugs & dyes - Cell cultures
45
How can we use CD34 antigen to identify different haematopoietic cells?
Both early and late progenitors are positive for CD34 but mature cells are CD34 negative
46
What is LIN?
LIN - lineage specific marker
47
How is LIN used to identify how mature stem cells are?
Early and late progenitors are LIN- Mature cells are LIN+ as they are specific to certain lineages
48
What is Rhodamine 1,2,3?
Rhodamine 1,2,3 is a fluorescent dye that stains mitochondria - used in flow cytometry
49
How is rhodamine used to identify cells in cycle?
Out of cycle stem cells won’t stain for rhodamine as they have no mitochondria present
50
What is 5-fluorauracil?
5-fluorouracil is a cytotoxic drug that only affects cycling cells, out of cycle cells are resistant
51
When are colony forming unit assays used?
Colony forming unit assays for lineage committed progenitors
52
Which assay type is used to identify mature cells?
Staining and functional assays used for mature cells
53
Outline cell processing methods
- Erythrocyte lysis - Density gradient centrifugation - Adherence depletion - Antibody depletion (mature cells) e.g. CD34 - Antibody selection (stem cells) e.g. CD34
54
Describe the structure of CD34 stem cells
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
Outline the culture conditions of colony assays
- 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
Outline how colony assays are undertaken
1. Semi-solid medium (agar, methylcellulose) 2. Plus growth factors 3. Incubate for 7-14 days 4. Identify under microscope
57
Why are petri dish cultures carried out more than once?
2 petri dishes as all experiments carried out in duplicate; reproducibility
58
Why is sterile water added to colony assays?
Sterile water to prevent drying out
59
What apparatus are colony assays produced in?
Carried out in a laminar flow cabinet - air has been filtered to provide a sterile environment before cultures are incubated
60
Give examples of common colony assays
``` 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
What are some of the non biological assays?
Non biological Assays - Morphology - FACS
62
Outline why biological assays are carried out
- Growth rate - Plating efficacy - Function
63
What are the applications of colony assays?
Research – basic haemopoiesis and carcinogenesis Testing toxicity of chemotherapeutic agents and carcinogens Generate cells for stem cell transplantation/manipulation
64
What is a primary cell culture?
Primary culture is a technique by which cells from primary tissues or cell suspensions are grown under controlled conditions, in vitro
65
What are cell cultures used for?
Cultures can be used for experimental, diagnostic or therapeutic purposes.

CellularPathologyCOPY-aimen n-use this (13 decks)

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