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Fundamentals of P > Primary cell culture techniques > Flashcards

Primary cell culture techniques Flashcards

1
Q

Define purpose of primary cell culture techniques

A

Being able to grow cells in culture to recreate an environment like in vivo​

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2
Q

Primary cell culture vs cell line

A 
Cells derived directly from tissues​
Interpatient variability​
Finite lifespan​
Cells divide and/or differentiate​
Cells carry out normal functions

WHERAS cell lines

Cell line is transformed – manipulated or spontaneous
Will always live​
Have no function​
Identical to parent cell

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3
Q

Non-haemopoietic examples of primary cultures

A 
Liver ​
Muscle​
Skin​
Nerves​
Fibroblasts​
Endothelial cells
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4
Q

Haemopoietic examples of primary cultures

A 
Stem, Progenitor cells​
T and B cells​
Monocyte, Macrophages​
Osteoblasts​
Dendritic cells​
Neutrophils, Eosinophils,Basophils, Mast cells​
Erythrocytes​
Megakaryoctyes, Platelets
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5
Q

Describe disaggregation of cells and any exceptions

A

Cells allowed to migrate out of an explant​
​Mechanical dissociation
​Enzymatic dissocation
​Exception – Haemopoietic cells – Do not need to be disaggregated – already are in single cell suspension

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6
Q

Sources of stem cells​ - 3

A

Bone marrow aspirate​
Umbilical cord blood​
Mobilised peripheral blood – if treated with growth factors, stem cells move from BM to peripheral blood

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7
Q

Stem cells in children - locations

A

All bones w/red bone marrow

Liver and spleen

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8
Q

Stem cells in adults (20 yrs >) - locations

A 
End of long bones like femur, humerus
Skull
Vertebrae
Ribs
Sternum
Pelvis
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9
Q

Site of haematopoiesis

A

Endosteum

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10
Q

Description of stem cells to red cells, neutrophils, platelets

A

Stem cells - early progenitors - late progenitors - immature progenitors - RBC, neutrophils + platelets

At immature precursors, cells start to look different = commiting to a lineage = look different to homogenous group of earlier cells

Controlled by negative and positive GF

Stem cells not in cycle, make a decision when stimulated = self-renew or differentiate

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11
Q

CFU vs BFU

A

CFU = colony forming unit = ability in culture
CFU may transition into a precursor cell that still retains proliferative potential

BFU = burst forming unit, more immature = enormous bursts

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12
Q

Stem cells features

A

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

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13
Q

Progenitor cells features

A

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

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14
Q

Precursor cells features

A

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

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15
Q

Describe stimulation of growth/survival of progenitors

A

Polypeptide growth factors (cytokines)

Bind to cell surface transmembrane receptors

Stimulate growth and survival of progenitors

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16
Q

Formation of erythrocytes/platelets - description of pathway w/names of growth factors

A

Haematopoietic stem cells (using IL-3, GM-CSF, M-CSF)

→ common myeloid progenitor (using IL-3, SCF, TPO)

→ megakaryocyte erythroid progenitor

using EPO to get to erythrocytes

OR

use IL-11 + TPO to make platelets

17
Q

Formation of basophil/mast cells - pathway description w/names of GFs

A

Haematopoietic stem cells (using IL-3, GM-CSF, M-CSF)

→ common myeloid progenitor (using GM-SCF)

→ granulocyte macrophage progenitor (using IL-11, TPO)

→ myeloblasts (using G-CSF, GM-CSF, IL-3)

→ Basophil/mast cells

18
Q

Formation of eosinophils - pathway description w/names of GFs

A

Haematopoietic stem cells (using IL-3, GM-CSF, M-CSF)

→ common myeloid progenitor (using GM-SCF)

→ granulocyte macrophage progenitor (using IL-11, TPO)

→ myeloblasts (using GM-CSF, IL-3, IL-5)

→ eosinophils

19
Q

Formation of neutrophils - pathway description w/names of GFs

A

Haematopoietic stem cells (using IL-3, GM-CSF, M-CSF)

→ common myeloid progenitor (using GM-SCF)

→ granulocyte macrophage progenitor (using IL-11, TPO)

→ myeloblasts (using G-CSF, GM-CSF, IL-6, SCF)

→ neutrophils

20
Q

Formation of macrophages - pathway description w/names of GFs

A

Haematopoietic stem cells (using IL-3, GM-CSF, M-CSF)

→ common myeloid progenitor (using GM-SCF)

→ granulocyte macrophage progenitor (using GM-CSF, M-CSF)

→ monocytes (using IL-6, SCF, G-CSF)

→ macrophages

21
Q

Formation of T-lymphocytes - pathway description w/names of GFs

A

Haematopoietic stem cells (using IL-17)

→ common lymphoid progenitor (using IL-2, IL-7)

→ T-lymphocytes

22
Q

Formation of B-lymphocytes - pathway description w/names of GFs

A

Haematopoietic stem cells (using IL-17)

→ common lymphoid progenitor (using IL-3, IL-4, IL-7)

→ B-lymphocytes

23
Q

Formation of natural killer cells - pathway description w/names of GFs

A

Haematopoietic stem cells (using IL-17)

→ common lymphoid progenitor (using IL-15)

→ natural killer cells

24
Q

4 examples of stromal cells

A

Fibroblasts
Macrophages
Endothelial cells
Adipocytes

25
Q

Description of microenvironment of ECM

A 
Collagen I, III, IV
Laminin
Fibronectin
Hemonectin
Thrombospondin
Proteoglycans
26
Q

4 examples of adhesion receptors

A

Integrins
Selectins
CD44
Lectins

27
Q

9 examples of cytokines

A 
IL-1
IL-3
IL-6
IL-11
G-CSF
GM-CSF
SCF
LIF
b-FGF
28
Q

4 examples of inhibitors

A

M1P-1 alpha
TGF-beta
TNF-alpha
INF-gamma

29
Q

List 5 bone marrow processing methods

A 
Erythrocyte lysis
Density gradient centrifugation
Adherence depletion
Antibody depletion
Antibody selection
30
Q

Describe formation of CFUs

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

31
Q

Describe method for colony assays

A

Semi-solid medium (agar, methylcellulose)
Plus growth factors as stromal cells would normally support HaemP
W/the single cell suspension of bone marrow
Incubate for 7-14 days
Culture to work out how many CFU = identify as different cell types

32
Q

List all possible colony assays and the relevant progenitor (8)

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
33
Q

Describe 3 applications of primary cultures

A

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

34
Q

Define what is a primary cell culture technique

A

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

Proliferation and/or differentiation can be supported, although cultures have a finite life span. Cultures can be used for experimental, diagnostic or therapeutic purposes

35
Q

What can primary cultures be established from and effect

A

Primary cultures can be established from hematopoietic cells giving rise to all the mature blood cell types, and non-hematopoietic tissues.

Fundamentals of P (62 decks)

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