Cell Biology Assignment Help-Using mammalian cell cultures to measure toxicity
Cell Biology
Using mammalian cell cultures to measure toxicity
Students will be given the opportunity to design and carry out a series of experiments into the toxicity of laboratory compounds using mammalian cultured cells.
Students will work in their mini-groups of three with each group looking at the toxicity of different compounds, including alcohols (ethanol and methanol), a range of “over the counter” medicines (e.g. salicylic acid (Aspirin), paracetamol and Anti-histamine) and antibiotics. The available options will be discussed during the first workshop.
Over the three weeks of the projects students will have to consider the following.
Week 1 – Planning, designing and initial optimisation of experiment
Groups should decide which cell type they wish to look at and why, we have CHO cells (hamster) and LTK cells (mouse), they may wish to look at responses in more than one cell type and see if there are any differences. They should consider that they need a confluence of cells of around 50-60% to be able to assess the effects of their compounds.
In this first session they should also decide what compound they wish to look at and at what range of concentrations they wish to look at, students should look at a concentration that gives variable responses. They should also consider what will happen when they add there compound to their culture the following week, will there be a dilution to take into account. They should also consider the number of replicates, size of multi-well dish to use and controls required. You should also generate a list of lab equipment that you will require during week 2, you should discuss these requirements with staff.
During this session staff will show you how to make a cell suspension and you will be able to practice how to count cells using the haemocytometer, this will give you an insight into the number of cells available from a stock flask of cells and should help you with designing your experiment.
Week 2 – Analysis of cell seeding densities, preparation of experimental samples and optimisation of toxicity assays
Students should then plate out their cells for adding their compound, they should have considered in their planning the number of replicates required and the controls they will need. They should use this time in the lab to make up their stock solutions, this could be Molar concentrations or % they need to think about what the compound they have is and how they wish to use it. Once they have done this and correctly added their compounds they should arrange with staff who will fix their cells on the Friday of this week to initially assess the effects of the compounds on their cells.
Any toxicity can be quantified by using cell viability assays, cells are fixed using paraformaldehyde ready for staining for crystal violet the following week.
Week 3 – Measurement of cellular toxicity, review of results and redesign
Using the plates from the previous week, each group will stain their plates with crystal violet, after washing the cells with PBS to remove excess stain. The stain can be extracted from the living cells with ethanol and then the level of crystal violet in the toxin containing wells compared to the control wells using a spectrophotometer. Each group should then analyse their data, review the effects of their selected compound in relation to their experimental design and how they may redesign their experiment to take into account what they have found.
Additional information to help in preparation for the project
mammalian cell culture. The ability to grow isolated mammalian (including human) cells in vitro is a key technique in a number of areas of biology, including investigation of biochemical pathways, the production of protein drugs by recombinant DNA technology, cancer research, stem cell isolation and biomedical diagnostics.
Introduction to mammalian cell culture
Cells can be isolated from most mammalian tissues and grown in culture. Most mammalian cells require a solid surface on which they must adhere to survive, grow and multiply. Sometimes cells can be persuaded to retain, at least for a time, many of the differentiated properties that they possessed in vivo, for example, the synthesis of extracellular matrix components such as collagen and fibronectin by connective tissue cells, or the ability of isolated muscle cells to form or maintain contractile muscle fibres. More often, cells simply lose their differentiated characteristics in culture because the factors which maintained them in vivo are missing in the artificial in vitro environment. However, with the recent interest in stem cells there is now a huge research effort directed towards identifying combinations of growth and cell-cell attachment factors that will enable cells to maintain their differentiation status in vitro and eventually to generate specific tissues and organs for medical treatment, for example, replacement skin, nerves, muscle and livers, perhaps even hearts.
Initial cultures of isolated mammalian cells are termed primary cell cultures. Cells from the tissue adhere to the culture vessel surface and will divide until they cover the surface. These cultures then require to be subcultured, in which a proportion of the culture is transferred to a new culture vessel. The majority of such primary cell lines can only be sub-cultured a finite number of times (i.e. can undergo a certain number of cell divisions) before they die. However some cell cultures, especially those that are derived from cancer tissue, are able to divide and be maintained in culture indefinitely. In many cases, primary cell lines from normal tissue will acquire the ability to grow indefinitely. However, the properties of these immortal transformed cell lines are often very different from their primary cell line counterparts or from normal tissue in vivo.
Mammalian cell cultures are usually maintained in containers made from plastic that has been specially treated to encourage cell attachment. A large variety of containers is available, ranging from flasks, dishes and tubes to multi-well plates and coverslips.
The culture medium contains a mixture of salts, amino acids, glucose and vitamins, plus bovine serum (or a synthetic substitute) which contains essential hormones, attachment and growth factors. The antibiotics penicillin and streptomycin are routinely added to suppress the growth of contaminating bacteria. Phenol red is also added as a pH indicator dye. At neutral pH the medium will be a red-pink colour which changes to orange and finally bright yellow in acidic conditions, or to purple in very alkaline conditions. Cultures are normally grown in a special incubator at 37 °C in an atmosphere of 5% CO2 in air.
It is very important that cultures are manipulated under good aseptic conditions to avoid microbiological contamination. Even when penicillin and streptomycin are added to the culture medium as described above, they will not necessarily be able to suppress growth of all bacteria (it would depend on the types and level of contamination) and they will have no effect on fungi. It is not safe to routinely supplement cell culture media with anti-fungal agents as these are generally also somewhat toxic to the cultured cells since both are eukaryotes and have very similar metabolic systems. Thus to eliminate or at least reduce contamination risks to a minimum, a laminar flow cabinet, which blows a sterile air stream over the work surface, is normally used for cell culture operations together with sterile instruments and reagents.
Most non-transformed adherent cell cultures grow as a monolayer (i.e. a single cell layer). They will continue to grow and divide until the flask floor is covered, after which growth ceases as the cells become inhibited from further division by close contact with their neighbours. At this point, or preferably a bit before, the culture needs to be subcultured. The extent of growth of cultures can be roughly estimated by assessing the confluency. Confluency refers to the proportion of the flask floor that is covered by cells expressed as a percentage.
Subculturing involves removing cells from one flask (the parental or stock flask) and putting some of them into a new flask (the daughter flask) with fresh nutrient medium. For adherent cells this procedure requires their release (often termed “stripping”) from the flask surface, often using trypsin (a protease), to make a stock cell suspension. The new flasks are usually inoculated with cells from this stock suspension at a specific plating density i.e. a particular number of cells per unit area of the flask floor.
The number of cells in the stock cell suspension can be determined by counting a sample of cell suspension in a haemocytometer. The cells are mixed with a dye called methylene blue which, due to its molecular size, is excluded from living (viable) cells but is able to enter membrane damaged dead or dying cells to stain the cytoplasm and nuclei blue.
Using tissue culture to study cytotoxicity
In vitro cultured mammalian cells are excellent systems to carry out initial experiments to test the potential toxic effects of chemicals and drugs. Such experiments, although they may not completely replace whole animal testing, provide important preliminary data about the concentration at which the drug or chemical will show toxic effects and also allow investigation of the mechanisms of toxicity. In vitro cultured cells are exposed to varying doses of the compound under investigation for varying periods of time. A number of different parameters can be assessed – these include the effect of the substance on cell metabolism (e.g. by measuring the activity of certain enzymes), on cell viability (e.g. by looking at the permeability of the plasma membrane using dyes such as trypan blue), or on cell survival (e.g. by looking at cell numbers after a culture period often with a stain such as crystal violet).
