Lecture 3: Ex Vivo Models

Question 1

What are tissue explants?

Answer 1

Small blocks of tissue from animals or human biopsy samples

Question 2

What are some uses of tissue explants?

Answer 2

• Maintain tissue architecture and ECM
o Closer to in vivo physiology than isolated cultured cells
• Can be used to obtain cultures of primary cells
• Can be used to perform experiments

Question 3

How to maintain tissue explants?

Answer 3

• No need for perfusion
• Oxygenated physiological medium

• Temperature controlled organ bath
o E.g. blood vessel rings, small segments of intestine

• Maintained on collagen sub-stratum
o Tissue attachment
o Microchannels to allow cells more contact with medium

• Another approach:
o Isolation of tissue microstructures with functional groups of cells
o Example: Islets of Langerhans: α, β, and δ cells

Question 4

Explain the reverted gut sac model

Answer 4

You can take any segment of the intestine. They are excised from rats and they are everted using a glass rod, you
flush the intestinal tissue you have with it and then you put the gloss rod into part of tissue and you tie it with a knot and whatever is beyond the knot, you trim off. Then, C, use the rod to turn it over and make it inside out. Once it is completely everted around the rod, you cut the part and gently push it off the rod to have a fully everted gut sac. Then you tie one end and you inject your test substance into the other end and tie the other end. So, now you have
a sausage looking intestine with media on the inside. You take this and put it in more media which should be a different media. You
take the sac and incubated in an oxygenated physiological media at 37 degrees for any desired period of time. The picture on the right is a water bath to keep it at 37 degrees with 95% oxygen. On the
outside, you put your test nutrient or your drug, so the solution is on the outside bc this is an everted gut sac. Inside, is the side where it
would normally be in contact with the body. Now the outside represents the lumen. Inside the sac is called the serosal fluid bc it is like the serum and the outside the sac is called the mucosal fluid.
You put your test substance in the flask and then you can analyze how much of your test substance actually was transported across the
epithelium into the serosal fluid. At the end of the experiment, you can collect the serosal and the mucosal fluids to see how much of the test substance is left in the mucosal fluid and how much of it got transported to the serosal fluid, has any of it been metabolized, transformed, or transported in any way.

Question 5

Explain the ussing chamber

Answer 5

This also applies to the intestinal tissue. This can also be used to study the transport of ions across the intestinal epithelium or lots of other molecules. Here the chamber consists of two halves. The piece of intestine is opened, and you have two sides of it basolateral and the apical sides of the intestine and they are separated. When you fill the both sides of the chamber with physiological solution, the transport of ions across the sides will generate a voltage change and you can measure that voltage change to measure the transport of these ions based on the voltage chain. You can also use this technique to study transport of drugs, nutrients, etc. across the intestine.

Question 6

What are isolated perfused organs?

Answer 6

• Directly from animals
• Cannulation of suitable blood vessels
• Whole organ; various cell types, maintain tissue architecture
• Complex system/apparatus
• Short-term

Question 7

How to maintain isolated perfused organs?

Answer 7

• Perfused via blood vessels with physiological medium
o Salts, nutrients
o 37oC
o 95% O2/5% CO2
o Can also use whole blood

Question 8

What do we analyze in isolated perfused organs?

Answer 8

Can analyse perfusate and tissue

Question 9

What are the advantages of isolated perfused organs?

Answer 9

• various cell types

- studying the whole organ without the influence of other organs.

Question 10

What are the disadvantages of isolated perfused organs?

Answer 10

• complex process

- kept alive for short time

Question 11

Explain the Langerdorff heart

Answer 11

• Used to study basic cardiac functions, pharmacological investigations

• Reverse perfusion:
o Cannulation of aorta
o Aortic valve closes
o –> Perfusate flows through coronary blood system

Question 12

What can we measure/analyze/explore?

Answer 12

• Media
• Cells

• Cell products
o Metabolites
o Enzyme activities
o Secretion of hormones, cytokines, etc o Cell signalling molecules

• Cell behavior
o Proliferation
o Differentiation 
o Apoptosis
o Migration
o Motility

Question 13

What are the 3 important aspects of microscope?

Answer 13

• magnification
• resolution
• contrast

Question 14

What is magnification?

Answer 14

How bigger you are making it look

Question 15

What is resolution?

Answer 15

minimum distance between 2 clearly identifiable object points

Question 16

What is contrast?

Answer 16

Most objects are transparent so we need contrast to identify them.

Question 17

How does light microscope work?

Answer 17

• Light passes through specimen
• Light emitted from specimen travels through objective
to detector
• Upright microscope vs. Inverted microscope

Question 18

Upright vs Inverted microscope

Answer 18

upright microscope (conventional microscope): objectives are above the stage or above the samples. You have to put your cells on a slide cover them with a coverslip and you have to lower the objective on the slide. This one is limited to very thin samples (80mm). when you move your slide to the right, the image goes to the left.

inverted microscope: the objectives are below the stage. On this one, bc we don’t have to use a slide or coverslip, we can take our entire culture flask and put it on the stage of the microscope and look at the cells that are on the bottom without having them take off the flask. This way is way faster. Not limited to thin samples. The sample moves the way it should, if you are sliding the slide to the left, the image will also go to the left.

Question 19

What is electron microscopy?

Answer 19

• Very high magnification – up to 10,000,000
• Can see individual macromolecules
• Disadvantage: sample must be in vacuum

Question 20

What is a disadvantage for electron microscopy?

Answer 20

sample must be in vacuum so we cannot look at live cells

Question 21

How does the electron microscope work?

Answer 21

we are not shining a light; we are sending a beam of electrons which will give us really high magnifications (compared to 2000x in light microscope). This is bc the magnification of an image is dependent of the wavelength of what you are shining through the image. So, electrons have a wavelength up to a 10,000,000x shorter than the wavelength of the visible light. With this high resolution, we are able to see individual macromolecules. The main disadvantage is that your sample has to be in a vacuum so, I can’t look at living cells.

Question 22

How does the fluorescence microscopy work?

Answer 22

• Fluorescence: when a material is excited with a light of a particular wavelength and emits light of a different wavelength
• Specimen irradiated by light at an appropriate wavelength
• Light emitted by specimen is passed through series of filters
• Use a fluorescent probe (unless natural fluorescence)

Question 23

What are the disadvantages of fluorescent microscopy?

Answer 23

when whole specimen fluoresces –> unfocused background

Question 24

How do you overcome the disadvantage of fluorescent microscopy?

Answer 24

confocal microscopy

Question 25

Explain the the mechanism of fluorescent microscope

Answer 25

We choose the wavelength that we want and let it pass through a filter and the filter will chose the actual wavelength that I want to look at. You take the molecule and you hit it with a photon. Photon number 1 hits the molecule and it is being absorbed and the molecule jumps to a higher level and the lose some energy and come to a lower level and jump back down to the ground level and this is the energy that is emitted as photon number 2 which has lower energy and corresponds to a different color with a smaller frequency and a longer wavelength (less energy). The molecule loses its energy by emitting heat etc. and goes to a lower level but then it goes to its ground state by emitting photon with lower frequency and higher wavelength.

Question 26

What eliminates the out-of-focus light in confocal microscope?

Answer 26

pinholes

Question 27

Widefield vs Confocal microscopes

Answer 27

In widefield microscope the light source will shine all of the visible wavelengths into the excitation filter and this filter will select the one wavelength that I want, which is going to hit the dichroic mirror and that beam of the wavelength that I want will hit the specimen, the specimen will fluoresce and send back a lower wavelength all the way up to the detector. Dichroic mirror lets certain things pass and certain things reflect. in a confocal microscope, the light source is a laser, it goes through the pinhole that will block out any light that is out of focus. They both have excitation and emission filter. Excitation filter will choose the wavelength which I want to excite the specimen and emission filter will detect the wavelength that I want. This way we are able to separate the specimen into slices and we are looking at one focal plane at a time. And this is how we can look at sharp images. One drawback of confocal is that since most of the light is filtered out by the pinhole system, the amount of fluorescent light that researches the detector is a lot dimmer, so we need much more sensitive detectors.

Question 28

What is the light source of widefield?

Answer 28

lamp, LED, laser

Question 29

What is the light source of confocal?

Answer 29

laser

Question 30

Which one is cheaper? Widefield or Confocal

Answer 30

widefield

Question 31

Which one has greater image quality? Widefield or confocal

Answer 31

confocal

Question 32

Which one has shorter exposure time? Widefield or confocal

Answer 32

widefield

Question 33

Which one is better for thick samples? widefield or confocal

Answer 33

confocal

Question 34

What is histochemistry?

Answer 34

o Identification of chemical compounds in biological samples o Study of their distribution o Combines microscopy + other techniques (histological staining) o E.g.: H&E staining to distinguish nuclei vs cytosol

Question 35

What does histochemistry require?

Answer 35

H&E staining to distinguish nuclei vs cytosol

Question 36

In H&E staining, which one stains the nuclei and which one stains the cytosol?

Answer 36

H will stain nuclei | E stain the cytosol

Question 37

What is immunohistochemistry?

Answer 37

``` • Refines histochemistry by using antibodies specific to particular proteins • Information on o The presence of these proteins o Their subcellular location o Their movement within the cells ```

Question 38

What kind of information can we get from immunocytochemistry?

Answer 38

o The presence of these proteins o Their subcellular location o Their movement within the cells

Question 39

What are the disadvantages of immunocytochemistry?

Answer 39

o Often cells must be removed from ECM --> immunocytochemistry

Question 40

What is the principle behind immunocytochemistry?

Answer 40

* Tag the protein of interest with a specific antibody (Ab) * Ab linked to fluorescent probe (e.g. FITC, rhodamine) * --> direct labeling – low sensitivity * Tag the protein of interest with a specific Ab (IgG) * Use a secondary Ab that binds to first Ab (anti-IgG from another animal) * Secondary Ab is attached to probe * --> Indirect labeling

Question 41

Explain direct labelling and give an example

Answer 41

Tag the protein of interest with a specific antibody (Ab) | • Ab linked to fluorescent probe (e.g. FITC, rhodamine)

Question 42

Explain indirect labelling and give an example.

Answer 42

* Tag the protein of interest with a specific Ab (IgG) * Use a secondary Ab that binds to first Ab (anti-IgG from another animal) * Secondary Ab is attached to probe

Question 43

Which one has low sensitivity? Direct labelling or indirect labelling?

Answer 43

direct labeling

Question 44

What is an advantage to indirect labelling?

Answer 44

you don’t have to make a fluorescent-linked antibody for every single one of your specific proteins.

Question 45

How to use immunocytochemistry with fluorescence microscope?

Answer 45

• After treatment with Ab, can generate cell image using fluorescence or confocal microscopy • Can tag ≥ 2 proteins with different probes o Colocalization, interactions

Question 46

Explain the "Colocalization of Ferroportin-1 with Hephaestin on the Basolateral Membrane of Human Intestinal Absorptive Cells" study

Answer 46

FPN-1 transports iron from inside the cell to the outside the cell and it is on the basolateral membrane of enterocytes. Heph could be also involved in iron transport. Are they found together on the membrane? They used Caco2 cells. These images are the same, but we are looking at them through different filters. All the green is FPN that is fluorescing green, red is plasma membrane and we take these two images and combine them, and we get yellow. The yellow is where these two are found together so we can see that most FPN is on the plasma membrane. Once they showed that the two proteins were found on the plasma membrane, they merged the images to see if they are found together.

Question 47

How did they tag proteins in the "Colocalization of Ferroportin-1 with Hephaestin on the Basolateral Membrane of Human Intestinal Absorptive Cells" study

Answer 47

• Ferroportin-1 (FPN-1): iron exporter • Hephaestin (Heph): multi-copper oxidase • Objective: characterize the cellular localization of FPN-1 and Heph in a human intestinal absorptive cell model • Cells were then fixed with 2% formaldehyde and permeabilized • Incubated with primary antibodies anti-FPN-1 and anti-Heph • Incubated with Alexa Fluor 546 (red) conjugated goat anti- rabbit IgG • Plasma membrane is stained with Alexa Fluor 594 conjugated WGA • Inserts placed on glass slides

Question 48

Explain immunocytochemistry in terms of live cell imaging

Answer 48

o Cells attached to solid support o Cells permeabilized to allow Ab and probe to enter o Most fluorophores can cause phototoxicity

Question 49

Explain live cell imaging

Answer 49

o Probe must be able to enter the cell without damaging structural integrity o Must be detectable while cell is alive

Question 50

Explain nanotechnology: quantum dots

Answer 50

o Semiconductors 2 – 10 nm diameter (~50 atoms) o Fluoresce when excited by laser o Signal lasts longer quantum dots are semi-conductor particles, wide spectrum, the light that they emit will depend on their size (blue to deep red). Emission spectra is narrow so there is less overlap between structure so we can use many more different probes and wavelengths to look at more molecules.

Question 51

Explain fluorophores over an example

Answer 51

o E.g. GFP § Cloned in 1990s from jellyfish Aequora Victoria § Expose to blue – UV lightàfluoresces bright green § The gene can be introduced to cells by transfection, fused to protein of interest § --> Protein always expressed with GFP § --> Can follow protein expression, location & movement GFP (Green fluorescent protein) is used to study the dynamic processes within the living cells. You take the gene that codes for GFP you introduce it to cells bc transfection. The cells is going to start making that protein so you are able to follow the expression of that protein and the processes related to it.

Question 52

Explain flow cytometry

Answer 52

• Cells usually treated with a fluorescent probe • Cells pass through laser beam, scatter the light and fluoresce • Scattered and fluorescent light is diverted to detectors using beam splitters and filters • Computer analysis: subpopulations of cells

Question 53

What can we measure with flow cytometry

Answer 53

o Size o Granularity o Internal complexity o Relative fluorescence intensity

Question 54

Give some examples where we can use flow cytometry

Answer 54

o Proportion of cells that express a particular antigen o Viability, proliferation, necrosis, apoptosis, senescence, differentiation o DNA & RNA content, membrane potential, ROS generation o Cell sorting by FACS