Tissue Culture and Immunohistochemistry

Question 1

What is found in a defined medium?

Answer 1

> Amino acids
Vitamins
Defined proteins factors e.g. insulin, growth factors, transferrin and hormones.
pH indicator (phenol red).

Question 2

Why is phenol red placed within the culture medium?

Answer 2

To gage at the pH of the culture ==> it is important to make sure that the pH remains within the physiological range (e.g. venous blood ==> pH should be 7.36)

Question 3

In what conditions are modern mammalian cell cultures grown in today?

Answer 3

Modern mammalian cell cultures are grown in these defined/undefined media under the following set of conditions:
> Humid conditions
> Temperature: 37 degrees celsius
> In the presence of elevated levels of CO2 (5% or 8%) to maintain pH level.

Question 4

What peice of equipment helps to maintain these mammalian cell cultures within these conditions?

Answer 4

Specialised incubators helps to keep these cell cultures within these conditions.

Therefore these specialised incubators help to control temperature, co2 levels and humidity as well.

Question 5

What else grows well within the same conditions as found within these incubators?

Answer 5

Bacteria, fungi etc.

Question 6

What are examples of defined media?

Answer 6

> Ham’s F12 medium

> Dulbecco’s Minimal Eagle’s medium

Question 7

In which of the 2 defined media are B27 supplements found?

Answer 7

Ham’s F12 medium

Question 8

What is Ham’s F12 medium used for?

Answer 8

Ham’s F12 medium is used for the growth of neuronal cultures.

Question 9

What are the basics of tissue culture?

Answer 9

> Aseptic technique is crucial - must not touch anything that comes into contact with the culture medium of the cells/tissues.
All cell/tissue culure must be carried out in a tissue culture hood. This is because the laminar flow, negative pressure and filters helps to keep the environment aseptic.

To avoid unnecessarily infecting the tissue culture, one must use alcohol liberally to clean your hands, surfaces etc.

If the tissue culture does become infected, then use antibiotics (e.g. pencillin) or antifungals (streptomycin) to combat infection. Pen/Step contains both.

Everything that becomes into contact with cells becomes a biological hazard, therefore they must be disposed in biohazard bags which are heated up in an autoclave (autoclaved) before disposal.

Question 10

What do you do with unwanted plates of cells?

Answer 10

Unwanted plates of cells mut be treated with 20% bleach or vikron and then the medium can poured down the skin.

Question 11

What is subculturing of cells? Why is it necessary?

Answer 11

Subculturing cells is also called cell splitting or passaging of cells.

Subculturing cells is important because dividing cells can be confluent (fuse together to form large meshes) when they cover the bottom of the plate. Subculturing is needed because contact inhibition prevents cells from dividing. So subculturing is necessary to essentially stop the cells from becoming senescent.

Question 12

How do you remove cells that have adhered to the bottom of the cell plate?

Answer 12

To remove adherent cells, you have to use phosptate buffered saline (PBS) (which contains trypsin) and EDTA. Removing adherent cells with these components is called trypsinisation.

Question 13

What is trypsinisation?

Answer 13

Trypsinisation is the process where trypsin cleaves the proteins that holds adjacent cells together (helped with gentle agitation).

Question 14

How does EDTA help with the removal of adherent cells?

Answer 14

EDTA chelates calcium and magnesium to form a chelate. This chelate inhibits
cadherin and integrin-dependent adhesion of cells.

Question 15

What do you do after you have tryspinised the cell culture?

Answer 15

After exposure to phosphate buffered saline and EDTA, the cells should become detached.

After they have become detached, the trypsin needs to become neutralised to stop it from digesting the cells away. To neutralise them, you need to either add a trypsin inhibitor or more routinely, you need to flood the cell culture with a protein solution e.g. an equal volume of 10% fetal calf serum.

After you have removed the effect of trypsin, you need to separate the cells from the trypsin solution. This is achieved by centrifugation.

The unwanted solution is removed from the cell pellet and becomes discarded.

Then the pellet is then resuspended in a suitable growth medium. You then count the number of cells present using a haematocytometer to give you an indication of cell density.

Cells can be plated into fresh plates/well at a desired density.

Question 16

What peice of equipment do you use to count the number of cells?

Answer 16

Haematocytometer.

Question 17

What are the different things that we can culture?

Answer 17

We can culture many different types of things:

1) Explants
2) Primary cultures (separating cell types)
3) Secondary cultures
4) Cell lines

Question 18

What is an explant?

Answer 18

An explant is a tissue that is derived from an organism and is transferred onto a growth medium for purposes of cell culture.

Question 19

What is a typical experiment involving an explant?

What would we measure?

Answer 19

A typical experiment involving an explant includes suspending peice of tissues onto a semi-solid matrix (e.g. collagen gel) small distances away from each other.

The influence of one tissue on the other can be determined.

For example, if an explant of the dorsal spinal cord is placed in a collagen gel with a peice of a floor plate tissue, the neurons found within the dorsal tissue will grow towards the floor plate tissue as the floor plate secretes netrin which is attractive to commisural neurons found within the dorsal tissue.

The observation of such activities led to the purification of guidance molecules e.g. netrin, semaphorins.

Question 20

What are primary cultures?

Answer 20

Primary cell cultures are dissociated cells grown in culture that have been directly isolated from a peice of tissue.

Question 21

How do you set up primary cultures?

Answer 21

To set up primary cultures, you have to treat the peiice of tissue with:

1) Proteolytic enzymes e.g. trypsin (derived from phosphate buffer saline) and collagenase to break down the ECM that holds these cells together and gently agitate them to help with this process.
2) EDTA to chelate calcium to inhibit cell adhesion through cadherin mediated contact.

As previously mentioned, PBS and EDTA are the most common dissociating solution that is used to help set up primary cultures,

Question 22

What is needed to culture primary neurons?

Answer 22

To culture primary neurons, you need an adherent substratum (foundation) e.g. collagen, laminin or poly-L-lysine.

Question 23

Primary neurons can either cultured in mixed cultures or they can be purified. What can happen if glial cells are outnumbered by neurons?

Answer 23

In mixed cultures, if glial cells are outnumbered by neurons, then glial cells will start to proliferate profusely.

Question 24

What can you do in longterm cultures to stop glial cells from dividing ?Why doesnt this affect neuronal cells?

Answer 24

Add mitotic inhibitors ==>this doesnt affect neuronal cells because neuronal cells are post-mitotic structures.

Question 25

In many cases, neurons thrive in the presence of glial cells. In which assays are glial cells necessary?

Answer 25

Glial cells are necessary in many cases e.g.:

1) when studying myelination and remyelination of neurons. Oligodendrocytes-neuron coculture is needed to allow the oligodendrocyte to myelinate the neuronal cells.
2) when studying synapse formation - astrocytes enhance synapse formation.

Question 26

How can we separate mixed primary cell cultures?

Answer 26

2 methods can be used to separate cells from a primary cell culture:

1) Pre-plating cells on a tissue-culture plastic
- certain cells (e.g. neurons) do not grow on tissue culture plastic but require surfaces coated with ECM proteins (e.g. collagen or laminin) or even sticky surfaces coated with poly-L-lysine to attach. Other cells e.g. astrocytes release their own ECM molecules that allows themto adhere to the untreated tissue culture plastic. By pre-incubating mixed cultures in an uncoated dish, non-adherent neuronal cells can be crudely removed from adherent cells.
2) Immunopanning ==>
A cell type specific antibody e.g. O4 is coated onto a tissue culture dish. Cells expressing the O4 antigen (e.g. oligodendrocyte precursor cells) adhere most strongly to those antibodies and become separated from the mixed population of cells. Separation occurs according to size by centrifugation through a percoll gradient.

FACS (Flourescence -activated cell sorting).

Question 27

What are secondary cultures?

Answer 27

Secondary cultures are cell cultures that are composed of the progeny of primary cells that have been allowed to divide in culture.

Expanded secondary cultures allow large numbers of mixed cell types for cellular and biochemical analysis.

However, the process of cell sensecence means that primary cells only divide for a finite number of times e.g. 25-40 times for human fibroblasts.

Therefore, immortalised cell lines are very commonly used in molecular and cellular neurobiology research as they can divide an infinite number of times but can still obey cell culture conditions e.g. contact inhibition as a result of confluency.

Question 28

What are some common non-neuronal cell lines?

Answer 28

Common examples of non-neuronal cell lines include:
1) 3T3 (Swiss and NIH) mouse fibroblast

COS  - monkey kidney cells 
CHO  chinese hamster ovary
MDCK dog epithelial cells 
HeLa - human epithelial cells 
L6 rat  myoblast cells 
293 human kidney (transformed)

Question 29

Which of the immortalised cell lines grows in suspension?

Answer 29

Only the HeLa cell lines grows in suspension - the rest of these cell lines grows in substrate.

Question 30

Unlike other dividing cells why is it difficult to introduce transgenes inside neurons in cultures. How do we know this?

Answer 30

We know this because:

1) Common methods (e.g. electroporation, lipofectamine-based methods) have low efficiences.
2) Retroviral vectors dont work because they act upon dividing cells (neurons arent a target because they are postmitotic in nature)

Question 31

What methods look promising in introducing transgenes into neurons in cell cultures?

Answer 31

Certain viruses e.g. herpesviridae, lentivirus or adenvirus based vectors appear to be good vectors for introducing transgenes into neurons in cultures but they seem to take a large amount of time to set up.

Question 32

If you cant introduce transgenes into neurons what can you use instead?

Answer 32

Instead of using neurons, you could use neuronal cell lines e.g. PC12 or Neuro21.

PC12 ==> Chromaffin cell from a rat adrenal gland tumour. It is an immortal cell lines that divides indefinitely.

Neuro2A - neuroblastoma cell lines.

Question 33

What can PC12 be convered into when exposed to NGF?

Answer 33

When exposed nerve growth factor, PC12 cells can develop into sympathetic neurons.

Question 34

How do PC12 cells present phenotypically?

Answer 34

PC12 cells have a cholinergic phenotype and present with functional autapses (synapses formed on to themselves with the normal target being absent).

Question 35

What are the advantages of neuronal cell lines instead of neurons?

Answer 35

Neuronal cell lines have the advantage that they can divide in culture so transgenes can be introduced and inherited to create a large population of transgene expressing cells - but can also be changed chemically into a neuron to be used in functional/cell biological studies.

Question 36

What assay is used to test for proliferation of cells?

Answer 36

DNA synthesis assay

Question 37

What is meant by a DNA synthesis assay?

Answer 37

In a DNA synthesis assay, the proportion of cells that are undergoing synthesis phase of the cell cycle are counted. This is achieved by using labelled nucleotides:
Either
1) tritiated thymidine which is radioactive in nature so can be detected
2) bromodeoxyuridine (Brdu) which can be detected via immunohistochemistry via the use of BrdU antibody.

Question 38

What assay is used to measure the number of cells present within a solution?

Answer 38

A colorimetric assay is used to count the number of cells.

Question 39

What compound is used in colorimetric assay to help count the number of cells?

Answer 39

Tetrazolium salt (MTT or MTS) which changes colour from bright yellow to brown when reduced by electrons from the electron transport chain in respiring cells. Therefore, the more brown the solution is, the more respiring cells there are.

Question 40

Apart from carrying out a colorimetric assay, what are some other ways of counting the number of cells found within a culture?

Answer 40

> Manually counting using a microscope either in situ or after trypsinisation.
FACs (flourescence activated cell sorting )

Question 41

What assay is used for apoptosis?

Answer 41

The TUNEL assay (nick-end labelling) is used for apoptosis.

Double ended nicks are symptomatic of DNA of cells undergoing apoptosis.

Question 42

How do you carry out TUNEL assay?

Answer 42

One can carry out TUNEL assay by adding DNA polymerase 1 together with flourescently tagged nucloetides to cultured neuronal cells - one can determine the cells that are undergoing apoptosis.

Also, by staining total nuclei e.g. by using propidium iodide, the proportion of cells undergoing apoptosis can be determined.

Question 43

What is the 2nd method of identifying apoptotic cells?

Answer 43

Apart from using TUNEL assays, DNA laddering can also be carried out to assay apoptotic cells.

Cells dyring by apoptosis cleave their DNA in regular lengths, producing a ladder like fragments. These ladder like fragments can become isolated and used to identify whether orn ot cells are undergoing apoptosis or not.

Question 44

What assays are used to determine cell survival?

Answer 44

Colorimetry assay - again using the MTT/MTS reagent.

Although the same assay as the proliferation assay is being used, what distinguishes between the 2 assays is that in the cell survival assay is that,we are looking at whether or not cell number has risen (proliferation) or fallen (apoptosis) from the starting number. This can be determined by the relative degree of browness proprtional to the number of respiring cells.

Question 45

What other specific markers for apoptosis can be assessed?

Answer 45

e.g. cleavage of PARP

Question 46

What are the assay requirements needed to informs us that cells are dying via apoptosis?

Answer 46

At least 2 different assays are needed to convice that cells are dying via apoptosis e.g. MTS + TUNEL etc.

Question 47

What is the most common cell culture medium?

Answer 47

DMEM containing 10% fetal calf serum (DMEM/10% FCS).

This is the standard medium for culturing most non-neuronal cell types e.g. fibroblasts, epithelial cells and astrocytes.

DMEM, IMDM and RPMI are often used to culture leukocytes as well.

Question 48

How often do cells proliferate when DMEM FCS 10% is used?

Answer 48

Every 24 hours

Question 49

What happens when a lower FCS% is used?

Answer 49

The cels will survive but will proliferate at a much lower rate.

Question 50

When would you want to use lower FCS concentrations e.g. 0.5%?

Answer 50

When you are looking at the effect of a growth factor on cell division.

There is no point in using 10% FCS because the FCS will stimulate cell proliferation. By using a lower percentage, any cell division can be attribued to the growth factor.