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SEM 4:Cellular Pathology > Primary Cell Culture Technqiues > Flashcards

Primary Cell Culture Technqiues Flashcards

1
Q

What is primary cell culture techniques?

A

Being able to grow cells and tissues in culture as close to the in vivo environment as possible

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2
Q

What are the primary cell cultures?

A
  • Cells derived directly from tissues
  • Interpatient variability
  • Finite lifespan
  • Cells divide and/or differentiate
  • Cells carry out normal functions
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3
Q

Differences between primary cell cultures and cell lines

A

Cell lines are transformed cells that have been manipulated or induced compared to primary cell cultures that are directly derived from tissues.
Cell lines produce exactly the same cell compared to primary cell cultures that divide and/or differentiate.

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4
Q

Two types of primary cultures

A
  • Non-haemopoietic

- Haemopoietic

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5
Q

Examples of cells from non-haemopoietic cells

A 
Liver 
Muscle 
Skin
Nerves 
Fibroblasts 
Endothelial cells
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6
Q

Examples of cells from haemopoietic cells

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

How are cells disaggregated for primary cell culturing?

A
  1. Cells are allowed to migrate out of an explant.
  2. Then, they undergo mechanical dissociation (mincing, sieving, pipetting).
  3. Then, they undergo enzymatic dissociation (trypsin, collagenase, hyaluronidase, protease, DNAse)
    This is done to produce a single cell dimension
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8
Q

Which cells do not undergo disaggregation and why not?

A

Haemopoietic cells as they are already in a single cell dimension so do not need to be disaggregated.

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9
Q

What is one disadvantage of primary cultures?

A

Assay readout is more complicated than just counting the number of cells.

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10
Q

What are some sources of stem cells?

A
  • Bone marrow aspirate
  • Umbilical cord blood: as stem cell-enriched
  • Mobilized peripheral blood: treated with GF then the stem cells move out of marrow into peripheral and used to treat stem cells for proliferation.
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11
Q

Why is bone marrow harder to acquire in adults?

A

Bone marrow retreats so are only harvested at the ends of long bones like the femur, humerus. Also from, the skull, vertebrae, ribs, sternum, and pelvis.

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12
Q

Where is bone marrow aspirate found in children?

A
  • All bones with red bone marrow

- Liver and spleen

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13
Q

Simplified structure of the bone

A

Periosteum - Hard part on the outside

Endostium - Where haemopoiesis occurs

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14
Q

What does a smear show on the bone marrow?

A
  • Can only densely packed, immature cells
  • Blood vessels -> pink
  • Fats -> white blobs
  • Concentrated areas of active haemopoiesis
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15
Q

Describe the process of haemopoiesis

A
  1. Stem cells
  2. Early progenitors: down the microscope, they look exactly the same as stem cells.
  3. Late progenitors: down the microscope, they look exactly the same as stem cells.
  4. Immature precursors: start to look different as they are committing to a specific lineage and look different.
  5. Mature cell types: easily distinguishable; reason they need to be cultured to do something.
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16
Q

How does haemopoiesis occur?

A
  • When stimulated, stem cells either self-renewing or differentiating.
  • Also a source of stem cells.
  • Amplification and differentiation control the development
  • Controlled by +/- GF for this process
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17
Q

What is the period of commitment?

A

It is a period that cells go through where they commit to a specific lineage. Either the Myeloid stem cell can form 4 different types of mature cells or the Lymphoid stem cells which can form only B-lymphocytes or T-lymphocytes.

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18
Q

Features of stem cells

A
  • Pluripotent - give rise to all lineages
  • Self-renew
  • Rare cells
  • Responsible for engraftment
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19
Q

Features of progenitor cells

A
  • Undifferentiated
  • Not distinguished by morphology
  • Committed to one or more lineages
  • Detected in colony-forming assays
20
Q

Features of precursor cells

A
  • Immature but recognizable
  • Cells starting to differentiate
  • Few final divisions before mature cells
21
Q

What are hematopoietic growth factors and what are they used for?

A
  • Polypeptide growth factors such as cytokines
  • Bind to cell surface transmembrane receptors
  • Stimulate growth and survival of progenitors or inhibit
22
Q

Examples of cell types and their mature cells

A
  • Megakaryocyte Erythroid Progenitor forms a erythrocytes and platelets
  • Granulocyte-Macrophage Progenitor forms the WBC - basophils, eosinophils etc.
  • Common lymphoid progenitor forms the T-lymphocytes, B-lymphocytes, and NK cells
23
Q

What forms the micro-environment that stem cells live on?

A

The stromal cells form fibroblasts, macrophages, endothelial cells, and adipocytes that the stem cells live on/in.
Also, have the extracellular matrix (ECM) formed by collagen I, III, IV; Laminin, Fibronectin, Haemonectin, Thrombospondin, and proteoglycans (GAGs)

24
Q

What are adhesion receptors?

A

Cells that produce cytokines and inhibitors

  • Integrins
  • Selectins
  • CD44
  • Lectins
25
Q

Examples of cytokines

A
  • IL-1
  • IL-3
  • IL-6
  • IL-11
  • G-CSF
  • GM-CSF
  • SCF
  • LIF
  • b-FGF
26
Q

Examples of inhibitors

A
  • MIP-1alpha; TGF-beta; TNF-alpha; INF-gamma
27
Q

What is a stem cell niche?

A

The associations of SC in bone marrow. Stem cells have equivalent receptors to match the stromal cells and extracellular matrix.

28
Q

How can cells (at different differentiation stems) be identified?

A

Use cultures to identify the antigens, drugs etc.

29
Q

What are the antigens on each cell?

A

Progenitor cells (early and late) are positive to CD34+
Mature RBC are CD34-
Stem cells have Ln- but a mature red cell is +.

30
Q

What is Rodemin?

A

It is a fluorescent dye that labels mitochondria.

31
Q

What is 5FU?

A

It is a drug that only affects cycling cells and is killed off when used in cultures.

32
Q

What is cell processing?

A

Cell processing is used to when something has a lot of cells as well so it can enrich or purify the culture for example bone marrow.
Don’t need to do it for haemopoietic SC as they are already in a single cell suspension

33
Q

What are the processing methods that are used?

A
  • Erythrocyte lysis
  • Density gradient centrifugation
  • Adherence depletion: enrich by sticking down and remove
  • Antibody depletion
  • Antibody selection - CD34+ - labelled to pull out cells etc.
34
Q

What do purified CD34+ cells look like?

A
  • Very immature with big nuclei and small cytoplasm
35
Q

How are “Colony Forming Units” formed?

A
  1. Progenitors grow to form colonies of mature cells.
  2. From 32 to hundreds or thousands of cells in a colony.
  3. Thus progenitors are called “Colony Forming Units”.
    Burst Forming Units can be seen with a naked eye.
36
Q

What are colony assays?

A
  1. Put suspension into a semi-solid medium (agar, methylcellulose).
  2. Add growth factors to act as stromal cells.
  3. Incubate for 7-14 days.
  4. Use microscope to identify the CFU.
37
Q

What are laminar flow cabinets?

A
  • Produce filtered air is used so the air stays sterile
38
Q

What cells are looked for in a colony-forming assay?

A
  • Stem cells
  • Progenitors
  • Immature precursors
  • Mature cells
39
Q

CFU-G

A

Granulocyte progenitor

40
Q

CFU-E and BFU-E

A

Erythroid progenitors

41
Q

CFU-Mk

A

Megakaryocyte progenitor

42
Q

CFU-GM

A

granulocyte/monocyte progenitor

43
Q

CFU-GEMM

A

Granulocyte/erythroid/monocyte/megakaryocyte progenitor

44
Q

CFU-bas

A

basophil progenitor

45
Q

CFU-eo

A

eosinophil progenitor

46
Q

How to identify a BFU?

A
  • Can see the Hb so know it is an erythroid colony

- “Burst” apart looks like several colonies all close together

47
Q

What are the applications of primary cell cultures?

A
  • Research - basic haemopoiesis and carcinogenesis
  • Testing the toxicity of chemotherapeutic agents and carcinogens
  • Generate cells for stem cell transplantation/manipulation - make sure it isn’t toxic for stem cells.

SEM 4:Cellular Pathology (13 decks)

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