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2 - Cellular Pathology > primary cell culture > Flashcards

primary cell culture Flashcards

1
Q

primary cell culture?

A
  • cells derived directly from tissues
  • Interpatient variability
  • Finite lifespan
  • Cells divide and/or differentiate
  • Cells carry out normal functions
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2
Q

cell line?

A
  • transformed cell that is spontaneously transformed or genetically manipulated
  • theoretically immortal
  • divides and reproduces itself exactly the same
  • may not have normal function because its been transformed
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3
Q

examples of primary cultures?

A

Non-haemopoietic

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

Haemopoietic

  • Stem, Progenitor cells
  • T and B cells
  • Monocyte
  • Macrophages
  • Osteoblasts
  • Dendritic cells
  • Neutrophils, Eosinophils, Basophils, Mast cells
  • Erythrocytes
  • Megakaryoctyes, Platelets
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4
Q

what is meant by disaggregation of cells?

A

Cells allowed to migrate out of an explant

Mechanical dissociation (mincing,sieving,pipetting)

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

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5
Q

which cells are an exception to disaggregation?

A

Haemopoietic cells

-they do not need to be disaggregated, they already are in a single cell suspension

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6
Q

why is the assay read out more complicated for primary cell culture?

A

cells differentiate, so we have to be more sophisticated, the assay read out is more complicated

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7
Q

summary of Primary Culture of Haemopoietic Stem Cells

A

Cell source

Cell characteristics

Cell processing

Culture conditions

Assay read-outs

Applications

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8
Q

name some sources of stem cells

A

Bone marrow aspirate
Umbilical cord blood
Mobilised peripheral blood

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9
Q

where is bone marrow located?

A

in children, all bones with red bone marrow and liver and spleen

in adults, after 20 yrs, the end of long bones like the humerus and femur, the ribs, sternum and pelvis

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10
Q

Endosteum?

A

middle of bone, where haemopoeisis is occurring (mesh like)

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11
Q

Cell characteristics - process of differentiation

A

early stem cells

early progenitors - look exactly the same as stem cells when looking down a microscope

late progenitors - also look the same, amplification happening

immature precursors - cells differentiating and committed to a certain lineage, look different to each other

mature cell types have very distinct morphology, easily distinguishable

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12
Q

is the process of differentiation tightly controlled?

A

Tightly controlled by positive and negative growth factors

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13
Q

as the process of differentiation goes along what happens?

A

Amplification as process goes along

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14
Q

are stem cells normally in the cycle?

A

stem cells normally not in the cycle, but stimulated they either self renew or differentiate

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15
Q

what do cells go through?

A

a period of commitment

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16
Q

what does CFU and CFU-GM stand for

A

CFU – colony forming unit
CFU-GM – colony forming unit (in culture) granulocytes and monocytes – this cell in culture has the ability to form granulocytes and monocytes

17
Q

BFU?

A

burst forming unit

18
Q

describe characteristics of stem cells

A

continually renewing, don’t run out of them

  • pluripotent- give rise to all lineages
  • self-renew
  • rare cells
  • responsible for engraftment
19
Q

describe characteristics of progenitor cells

A

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

20
Q

describe characteristics of precursor cells

A

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

21
Q

what are haematopoetic growth factors and their role?

A

they are polypeptide growth factors (cytokines) that bind to cell surface transmembrane receptors

stimulate growth and survival of progenitors

22
Q

what is meant by phenotype?

A

antigens on the cell surface

23
Q

which cells are CD34+ and Lin-?

A

stem cells and early/late progenitor cells

24
Q

which cells are CD34- and Lin+?

A

Mature cells

25
Q

what is 5-FU?

A

a cytotoxic drug that only affects cyclin cells

-we can add it into cultures and kill off cells

26
Q

how can we identify cells at different stages of haematopoeisis?

A

Phenotypes

27
Q

describe the microenvironment of stem cells?

A

Stromal cells form the environment that the stem cells are sitting in by producing growth factors and extracellular matrix

On the surface of stromal cells are various extracellular matrix proteins, e.g. collagen, laminin. They also have adhesion receptors on their surface, producing cytokines and inhibitors. Stem cells have equivalent receptors which will recognise those molecules
-known as a stem cell leash

28
Q

examples of stromal cells?

A

fibroblasts, macrophages, endothelial cells, adipocytes

29
Q

examples of adhesion receptors?

A

integrins and selectins

30
Q

examples of cytokines and inhibitors?

A

IL-1, IL-3, IL-6

MIP-1alpha

31
Q

what is meant by cell processing?

A

when examining bone marrow, you can use techniques that enrich or purify your stem cells

prcoessing methods include:
Erythrocyte lysis
Density gradient centrifugation
Adherence depletion
Antibody depletion
Antibody selection

use antibodies that will recognise mature cells, like Lin. Or use positive selection, come in with antibodies (CD34) for the stem cells and pick them out

use magnetic beads that attach to antibodies and then pull them out with a magnet

32
Q

what is an assay?

A

a procedure used to measure/quantify the amount of a target entity

33
Q

different types of assays?

A

Biological assays Non-biological assays
Morphology Growth rate
FACS Plating efficiency
Function

34
Q

how do CFU’s form (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”. Once in the colony assay, they divide and form colonies of mature cells, which are distinguishable

35
Q

how do you make a colony assay and what is their purpose?

A

Put suspension into culture to work out how many CFU there are

Semi-solid medium (agar, methylcellulose)
- so the cells remain there

Growth factors
-stromal cells in the ‘in vivo’ environment produce GF’s to support the process, so we need to replicate this

Look through a microscope and identify and count the CFU’s

36
Q

what can can see with the naked eye?

A

BFUs

37
Q

name some different 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
38
Q

Applications of primary cell culture?

A

Research – basic haemopoiesis and carcinogenesis

Testing toxicity of chemotherapeutic agents and carcinogens

Generate cells for stem cell transplantation/manipulation

39
Q

define primary cell culture

A

a technique where:

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.

2 - Cellular Pathology (24 decks)

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