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Primary cell culture Flashcards

1
Q

What is primary cell culture?

A

They are cells derived directly from tissues.

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2
Q

what is the difference between primary cell culture and cell line in terms of life span

A

Primary cell culture are normal cells; they will divide, differentiate and then die (finite life span)

IN CONTRAST TO
Cell line which is immortal

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3
Q

what conditions are being recreated with primary cell culture techniques

A

recreating in vivo conditions

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4
Q

what are the KEY differences between primary cell culture and cell line

  • where they are derived from?
  • identical or non-identical?
  • lifespan?
  • do they divide/differentiate?
  • function: normal or abnormal?
A

PRIMARY CELL CULTURE:

  • cells derived directly from tissues
  • interpatient variability
  • cells have a finite lifespan
  • cells divide and/or differentiate
  • cells carry out normal functions

CELL LINES:

  • spontaneously derived from tumours or after genetic manipulation
  • cells are identical
  • cells are theoretically immortal
  • cells divide but don’t differentiate
  • might have abnormal gene expression, so may not have fully normal function
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5
Q

primary cultures can be NON-HAEMOPOIETIC
OR
HAEMOPOIETIC

list some examples of non-haematopoetic primary cultures

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

primary cultures can be NON-HAEMOPOIETIC
OR
HAEMOPOIETIC

list some examples of haematopoetic 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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7
Q

how do we get cell samples from tissues

3 different ways

A

1) You put the tissue into culture, and the cells are allowed to migrate out, divide and grow.
2) You can mechanically dissociate them (either by mincing, sieving, pipetting, etc.)
3) You can enzymatically dissociate them (using trypsins, collagenases, proteases, etc.) to break the bonds between the cells and disaggregate them.

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8
Q

haematopoetic cells (eg- t and B cells) cannot be extracted from tissues in the same way as non-haematopoetic cells (eg- liver).

So, how do we get haematopoietic cell samples?

A

Haematopoietic are already in a suspension (they are already disaggregated) so we can take them directly from the body with no manipulation.

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9
Q

What are some sources of stem cells?

A
  • Bone marrow aspirate
  • Umbilical cord blood
  • Mobilised peripheral blood
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10
Q

what is a method used to generate stem cells for transplantation

A

normally people don’t have stem cells in peripheral blood, but if we pump growth factors into people then we can push the stem cells out into the peripheral blood and collect them.

This is a method used to generate stem cells for transplantation.

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11
Q

Describe the areas from which we can extract stem cells in:

  • children
  • adults
A

Children:

  • bones
  • liver
  • spleen

Adult:

  • ends of long bones (like the femur and humerus)
  • skull
  • vertebrae
  • ribs
  • sternum
  • pelvis
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12
Q

Describe stem cells properties (4)

A
  • they’re pluripotent - give rise to all lineages
  • they self-renew
  • they are rare cells
  • they are responsible for engraftment
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13
Q

describe progenitor cells properties (4)

A
  • undifferentiated
  • not distinguished by morphology
  • committed tone or more lineages
  • detected in colony forming assays
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14
Q

describe precursor cells properties (3)

A
  • immature but recognisable
  • cells starting to differentiate
  • few final division before mature cells
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15
Q

2 fates for stem cells

A

can differentiate

OR

renew themselves

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16
Q

what is the difference between a non-biological assay and a biological assay

A

non biological assay shows:
- morphology (appearance down a microscope)

-FACS

biological assay shows:

  • growth rate
  • cell function
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17
Q

what is FACS

A

flow cytometer used in non biological assays to see whats on the cells inside and outside using fluorescent antibodies

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18
Q

Describe haematopoietic growth factors and their function

A

They are polypeptide growth factors (or cytokines; the words are interchangeable).

They bind to cell surface transmembrane receptors, and stimulate the growth and survival of progenitors OR inhibit

They are, again, responsible at every stage of haemopoeisis.
They’re also important in keeping cells out of the cell cycle until they need to be brought in.

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19
Q

why are progenitors called CFU?

A

Progenitors can grow to form colonies of mature cells. These can range from 32 to hundreds or thousands of cells in a colony.

Thus, progenitors are called “Colony Forming Units” - CFUs.
This is used to identify progenitor cells.

note: some CFUs are specific for a lineage but others will work across lineages

20
Q

name this CFUs lineage (what this CFU aka progenitor will go on to become and produce colonies of):

CFU-G

A

granulocyte progenitor

21
Q

name this CFUs lineage (what this CFU aka progenitor will go on to become and produce colonies of):

CFU-E + BFU-E

A

erythroid progenitor

22
Q

name this CFUs lineage (what this CFU aka progenitor will go on to become and produce colonies of):

CFU-Mk

A

megakaryocytic progenitor

23
Q

name this CFUs lineage (what this CFU aka progenitor will go on to become and produce colonies of):

CFU- GM

A

granulocyte/monocyte progenitor

24
Q

name this CFUs lineage (what this CFU aka progenitor will go on to become and produce colonies of):

CFU-GEMM

A

granulocyte/erythroid/monocyte/megakaryocyte progenitor

25
Q

name this CFUs lineage (what this CFU aka progenitor will go on to become and produce colonies of):

CFU-Bas

A

basophil progenitor

26
Q

name this CFUs lineage (what this CFU aka progenitor will go on to become and produce colonies of):

CFU- eo

A

eosinophil progenitor

27
Q

what is found in bone marrow (6)

CAISES

A

stem cells

stromal cells

extracellular matrix

adhesion receptors

cytokines

inhibitors

28
Q

these are all found in bone marrow:

stem cells

stromal cells

extracellular matrix

adhesion receptors

cytokines

inhibitors

how do all of the above interact/relate to each other?

A

stromal cells forms the environment that the stem cells are sitting in

ECM and adhesion receptors and present on the surface of stromal cells

cytokines and inhibitors and produced by stromal cells

29
Q

examples of stromal cells found in bone marrow

FAME

A

Fibroblasts
Macrophages
Endothelial cells
Adipocytes

30
Q

examples of extracellular matrix components found in bone marrow

A 
Collagen I, III, IV
Laminin 
Fibronectin 
Hemnonectin 	Thrombospondin 
Proteoglycans (GAGs)
31
Q

examples of adhesion receptors found in bone marrow

A

Integrin
Selectin
CD44
Lectins

32
Q

examples of cytokines found in bone marrow

A 
Interleukins 
	G-CSF
	GM-CSF
	SCF
	LIF 
B-FGF
33
Q

examples of inhibitors found in bone marrow

A

MIP-1A
TGF-B
TGF-A
INF-Y

34
Q

Describe the two antigen markers we can use to distinguish between populations of cells

A

for stem cells and progenitor cells we use:
• CD34+
• Lin-

for mature cells we use:
• CD34-
• Lin +

35
Q

Rhodamine 123 is a method used in flow cytometry. Describe its use and meaning.

A

Rhodamine 123, is a fluorescent dye which stains mitochondria, and is only picked up by cycling cells.

Early stem cells will appear dull, but when they enter the cycle they are bright.
Out of cycle = appear dull
In cycle = stain bright

36
Q

What can we use to differentiate between cells at different stages?

A
  • Phenotype
  • Fluorescent staining (rhodamine 123)
  • Cytotoxic drugs (5-fluorouracil)
  • Culture
37
Q

describe how cytotoxic drugs such as 5-fluorouracil works to differentiate between cells at different stages

A

When out of cycle, the cells are resistant to the drug, but when they enter the cycle they are sensitive to the drug.

Out of cycle = resistant to drug
In cycle = kills off cells that are cycling

38
Q

what are the 5 different methods used to differentiate between stem cells?

A

DEPENDS ON HOW PURE YOU WANT THE CELL

  • Erythrocyte lysis (LEAST PURE)
  • Density gradient centrifugation
  • Adherence depletion
  • Antibody depletion (to get a VERY PURE sample)
  • Antibody selection (to get a VERY PURE sample)
39
Q

what is the most and least pure method to differentiate between stem cells

A

LEAST PURE = erythrocyte lysis

MOST PURE = antibody depletion OR antibody selection

40
Q

there are 5 different methods used to differentiate between stem cells:

  • Erythrocyte lysis
  • Density gradient centrifugation
  • Adherence depletion
  • Antibody depletion
  • Antibody selection

briefly describe each of these

A

erythrocyte lysis
- produces enriched population of stem cells

density gradient centrifugation
- spin cells on gradient which removes certain cells

adherence depletion
- bone marrow put into plastic and the ones that stick are harvested

antibody depletion
- removes certain cells; deplete all cells that are Lin+ (mature cells)

antibody selection
- positively selects cells that are CD34+ (stem and progenitor cells)

41
Q

which method would you use to differentiate between mature cells and progenitor/stem cells?

  • which method for mature cells only
  • which method for stem cells and progenitor cells
A

mature cells
- antibody selection

stem cells/progenitor cells
- antibody depletion

42
Q

describe how colony assays are made

A
  • inject single cell suspension of bone marrow on a semi-solid medium
  • incubate for 7-14 days
  • single cells will divide and differentiate into colonies
  • can be identified using a microscope
  • can quantify number of cells in original suspension
43
Q

what conditions are needed to carry out a colony assay

A

done in laminar flow cabinet = sterile environment

44
Q

what medium can be used in a colony assay

A

semi solid medium:

  • agar
  • methylcellulose
45
Q

list the applications of stem cells

A

basic research on stem cells

testing toxicity of chemotherapeutic agents and carcinogens

generate stem cells for stem cell transplantation or manipulation

CELLULAR PATHOLOGY (10 decks)

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