Experimental Pharmacology

Sterilization Methods

Steam under pressure: 121°C or 132°C for various time intervals. Sterilization stays 20-30 minutes.

Dry heat: simple oven, 1 hour at 171°C, 2 hours at 160°C or 16 hours at 121°C.

Filtration: for small amount of solution, with HEPA filters with 0.22 (nothing pass) -0.45 (some viruses pass) μm pore size.

UV radiation: variable exposure to 254 nm wavelength.

Ionizing radiation: variable exposure to microwave or gamma radiation.

Gas vapor sterilants: Ethylene oxide.

Formaldehyde vapor: used in lab when the filter is replaced.

Hydrogen peroxide vapor.

Plasma gas: highly ionized hydrogen peroxide gas.

Chlorine dioxide gas: variable.

Chemical sterilants: Peracetic acid: 0.2%. Glutaraldehyde: 2%.

Pros and cons:

Steam and Dry Heat: the most common methods for most materials.

Cons: NOT good for heat-sensitive, toxic or volatile chemicals.

Filtration: remove bacteria and fungi from air or solutions, like HEPA filters.

Cons: unable to remove

viruses and some small bacteria (mycoplasma).

Ethylene oxide: the most common gas vapor sterilant.

Ø Cons: flammable and explosive, potential carcinogenic.

Formaldehyde gas: carcinogenic.

Ø Autoclave: simple bench-top autoclave that generates 100 kPa (1 atm, 15 lb/in.2) may be sufficient, but a larger model with a timer and a choice of pre-sterilization and post-sterilization evacuation and temperature recording give more capacity and greater flexibility in use. Offers the opportunity to comply with good laboratory practice (GLP).

Chemical monitoring: involves the use of heat-sensitive chemical that changes color when they are exposed to certain conditions. Process indicators are placed on the outside of the instrument packages before sterilization. They respond to heat only.

In vitro assay· Tumor progression Starts with the mutation of a particular cell and it grows uncontrollably. Some of the cells starts growing in a particular tissue, increasing the amount of the tumor and

starts a second step: angiogenesis, formation of new vessels. From them, there’s extravasation or cells can use lymphatic vessels and starts lymphatic metastases.

In vitro, tumor cells can be studied:

• Cell proliferation assay (Coulter counter, cell cycle analysis, iCelligence system).
• Cytotoxic assay (MTT, iCelligence).
• Cell migration assay (Boyden’s chamber assay).

Most thing saw in tumors can also be seen in atherosclerotic plaques: accumulation of SMC.

Cell proliferation assay:

Cells are grown in tissue culture dish in the presence of medium containing 10% FCS (fetal calf serum).

Seeding the cells in 6, 24 or 48 well trays for the experiment:

• All the procedure will be performed under sterile condition in the biohazard hood.
• Aspirate the medium containing FCS (it stops trypsin) with sterile Pasteur connected to vacuum system.
• Wash once with sterile PBS (phosphate buffer saline).
• Add 2 ml of trypsin for detaching the cells from the substrate.

Add EDTA, if cells stay attached.

1. Transfer the cells into a 15 ml tube containing 3 ml of medium with 10%FCS.
2. Count the cells with hemocytometer or coulter counter.

What is cell counting?

Quantification in cell culture is required for experimental analysis.

Although we can estimate the growth of a culture from its appearance under the microscope, proper quantitative experiments are difficult to analyze and reproduce unless are counted before and after each experiment.

Cell counting: various methods for the quantification of cells in cellular biology & medicine.

By counting the cells in a known volume of culture, the density can be determined.

Hemocytometer

Manual cell count four squares than make the average.

Cells/ml = average cell number x dilution factor x 10000.

Coulter counter

Particles, liquid and electric current are pumped through an orifice of an extract diameter.

The particles cause changes in the electric current and these changes are

The coulter counter is used to monitor and count particles. It provides the number of cells in 0.5 ml of isoton solution.

An aliquot of 0.1 ml of cell suspension is added to 9.9 ml of isoton solution in a plastic becker.

To determine the total number of cells present in the becker, multiply the number of cells obtained from the coulter counter by 20 (0.5 ml / 10 ml).

To determine the number of cells present in 1 ml of cell suspension, multiply the number of cells obtained from the coulter counter by 200 (0.1 ml / 1 ml).

Pharmacological test

Add tested compounds on day 1.

Perilic alcohol: it's a monoterpenes, limonene derivative. It can be found in essential oils and in several plants including mint.

The naturally occurring monoterpenes R(+) limonene and S(-) perillyl alcohol have been shown to both prevent the development of carcinogenesis in a variety of experimental rodent models and to cause regression of rat mammary carcinomas. In several mammalian species,

including humans, limonene and perillyl alcohol are rapidly metabolized to perillic acid and dihydroperillic acid (major products) as well as to the methyl esters of the two acids (minor products). Counting cells after 3 days: number of treated cells is half than control. Proliferation cell number at time 0 minus cell number at time 72h. Wrong, because cell number start from 20k, not 0, so inhibition of cell proliferation is 20k/60k = 0.33 → 67%. IC : inhibitory concentration of 50%. It represents the concentration of a compound or drug required for reducing 50 the cell proliferation, or any particular biological events, by 50%. Compound 4 IC is 12.2 μM, is reasonable efficacy, but at some point, it stops working, so it’s very potent, but not 50 so effective. It’s not cytotoxic, but cytostatic: stops growth, but not kills. · iCelligence system It’s an impedance-based real-time cell analysis system, which allows label-free, dynamic monitoring of cellular events. The

Il sistema misura l'impedenza elettrica attraverso micro-elettrodi integrati sul fondo delle piastre di coltura tessutale delle E-Plates. La misurazione dell'impedenza fornisce informazioni quantitative sullo stato biologico delle cellule, inclusi il numero di cellule, l'adesione, la vitalità e la morfologia.

10/12· Analisi del ciclo cellulare con citofluorimetro

Anche chiamato FACS, la macchina aspira le cellule e misura l'intensità di fluorescenza di ogni singola cellula. Ogni cellula passa attraverso un capillare molto stretto e viene investita da un laser, al fine di emettere fluorescenza che verrà rilevata. Ogni campione ha almeno 100.000 cellule, un'analisi adeguata richiede 20.000 cellule per campione.

Il risultato è una curva, in un grafico in cui Y è il numero di cellule e X è l'intensità di luminescenza, su una scala logaritmica: descrive quante cellule hanno una certa fluorescenza.

Prima, le cellule devono essere incubate con ioduro di propidio (PM: 668,4 Da). Di solito le cellule non sono permeabili ad esso, quindi devono essere fissate e permeabilizzate per consentire la penetrazione.

The agent pass inside and reach the DNA, since it's an intercalating agent. It's a stechiometric interaction. It's a condition only of necrotic cells, so NOT for apoptotic ones. It becomes fluorescent only when it interacts with the DNA, so it can be used to stain cells. Every cell has some fluorescence, but adding this agent it can be increased.

In the same sample, there are cells and an excess of propidium iodide, but the machine measures only the fluorescence from the cells, not the one in the sample.

The fluorescence intensity depends on how much DNA is present in cells, so it depends on the phase in which are the cells. In tumor cells, there's a large amount of cells in G2/M because they are quickly duplicating. Apoptotic bodies still contain DNA but not the whole diploid amount of normal cells.

To know how many cells are in the different phase of cell cycle, software can calculate the AUC (area under the curve) and extrapolate the cell number in the different phases.

phases.CV = coefficient of variation. %CV is also an important parameter. It calculates the variability of our analysis. SMC analysis. 0.4% FCS: not sufficient amount to let cell proliferate (but enough to keep them alive). 10% FCS: cells start to grow, cells are moving from G0/G1 phase to S phase. Something that can interfere with the cell cycle is added. Ever = immunosuppressing drug (mTOR inhibitor), Everolimus is the full name. It can also block cell proliferation. A very small amount of this drug can almost completely block proliferation. It blocks the passage from G0/G1 to S: increasing of the first signal, decreasing of S signal and second signal. Using a drug blocking the M phase (chemotherapeutic drug for example) the picture will be different: cells accumulating in the M phase so the first signal decreases and the second one increases (they become more or less the same). Another possibility is that the drug can also induce apoptosis: increase of % of cells in sub-G0/G1 phase. Another

1. The marker used to stain apoptotic cells is annexin V, which binds phosphatidylserine (PS) in the inside of the cell membrane.
2. During apoptosis, PS flips out and is exposed on the cell surface, so it can be bound by annexin V.
3. Once the binding is formed, there's a fluorescence signal that can be detected. So only apoptotic cells will have a fluorescence signal.
4. Same data can be visualized as single dots, every dot is a single cell, with a particular intensity of the fluorescence.
5. There isn't the number of cells, but two schemes of fluorescence: one of propidium iodide (necrotic cells) and one of annexin V (apoptotic cells).
6. In this experiment, cells aren't permeabilized, but just incubated with the two agents, so propidium iodide can pass the membrane only of the cells in necrotic conditions.
7. There are also cells positive for both agents.
8. Variable dots: not positive for annexin V neither for propidium iodide.
9. The cytofluorimeter detect these cells because everyone emits some fluorescence.

fluorescence.- Cytotoxic assay -To measure toxicity of a compound on cells: how many cells are alive and how many are dying?

· MTT assay

The chemical compound is usually added after 1 day of cells growth.

MTT is a cheap chemical compound that can be reduced by the mitochondrial reductase into formazan salt. The reduction is effective when there’s a lot of mitochondrial, so the cells are really vital. If the cells start dying, there’s also a lower amount of enzyme and a lower formation of the salt, that takes 3 hours. The salt precipitates and gives the cells a purple color, while MTT gives a yellow one. It cells form the formazan, they are still alive, while they remain yellow if they’re dying. But salt precipitates are difficult to see inside cells, so in order to obtain a homogeneous color we dissolve cells and the formazan salt will be homogenous in solution. Isopropanol is used to dissolve the salts. The solvent must be the s