cell culture techniques and the mtt



This graph shows that the viable cells number and the absorbance are directly proportional. Therefore if there is a high number cells, then there will be a high absorbance and vice versa.

Part B: Provide short answers to the following questions (10 marks for each answer).

What are the major forms of contamination that can occur in a cell culture?

In tissue culture, microbial contamination can occur, with the major forms including bacterial, yeast, molds, fungi, mycoplasmas and sometimes protozoa. Normally, unless it keeps recurring, the species or infection types it not significant. Contaminant type, detection, location of last handling and the name of the operator are the factors that need to be recorded. If a certain infection type keeps recurring, it could be of benefit to identify it so that its origin can be found. Occasionally viral and cross contamination may also occur.

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How can contamination be prevented?

Living cultures should be checked regularly for contamination by the use of normal and/or phase-contrast microscopy by using fixed preparations fluorescent staining or PCR for mycoplasmas.

Cultures should not be routinely maintained in antibiotics. One culture set of each cell line should be grown without antibiotics for at least two weeks at a time continuously so that cryptic contaminations become obvious.

If a culture becomes contaminated, trying to decontaminate can make things worse, so decontamination should only be attempted if the culture is irreplaceable. However, this should be carried out under strict quarantine.

New lines should be quarantined until it is clear that they are totally uncontaminated. Also, after they have been isolated, via DNA profiling/fingerprinting they should be characterized.

Media and solutions should not be shared amongst cell lines or operators. One should also regularly check the characteristics of the cell line to prevent cross contamination.

What are the essential pieces of equipment required to set up a cell culture facility and describe their function?

The first essential piece of equipment required would be a laminar-flow hood, especially in a lab which is busy. This provides an easy and simple way for aseptic conditions.

A dry incubator may be necessary if a hot room is unavailable, which has forced air circulation, temperature control and a safety thermostat. This is required for cell growth as it can provide the optimum conditions for it. Culture Flasks can also be used to provide optimum cell growth conditions, as cells bind to a surface and grow due to the optimum conditions.

An inverted microscope may also be required as it's still essential to look at the cultures regularly. Often, the first sign of morphological change is culture deterioration, as well as a microbiological infections characteristic pattern.

An advantageous piece of equipment would be a cell counter when you have more than two cell lines. It can also determine cell size as well as distinguish between viable and non-viable cells, and between single cells and aggregates.

A flow cytometer can be used for cell populations' analysis. It does this via light scattering, absorbance readings and fluorescence, however, the cost of these is very high.

A haemocytometer may also be useful as it's a cheapest method for cell counting. It also allows determination of cell viability via dye exclusion.

Other consumable items which are essential include pipettes, sterile containers and disinfectants.

Describe the endpoints used to assess the response of cells to cytotoxic drugs and comment on their advantages and disadvantages.

Cytotoxicity testing is used before new drugs, cosmetics, food additives amongst many other things are released for public use. Most assays determine cellular level effects (cytotoxicity) as it's difficult to keep an eye on the systemic and the physiological effects in vitro. Cytotoxicity is defined depending on the study and whether the cells are killed or result in their metabolism being altered.

There are several tests used to assess cell response to cytotoxic drugs. Viability assays are also used where the viable cells proportion after a potential traumatic procedure is measured. These rely upon the measurement of membrane breakdown integrity via dye uptake or release. This effect is, however, instant and can't always predict survival.

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Assays which are based on cell proliferation can be used. This is where the cells counts compounds on cell proliferations have their effects determined after a few days in culture, where there is a requirement of a growth curve due to the ambiguousness of cell counts at a single point in time. This is only possible when the samples numbers are small. When sample numbers are large, a single point in time is used, where it is within the log phase of control cells. Effects observed should be evidenced over the whole growth cycle via a complete growth curve or an alternative assay such as the MTT assay.

Survival assays are also used where long term tests demonstrate survival rather than short term toxicity. Survival, which is regenerative capacity retention, is normally measured by plating efficiency. Over 10% of plating efficiency is accepted.

Another endpoint is Metabolic Cytotoxicity Assays, where increase is cell numbers, DNA/protein total amount as well as metabolic activity continuation is measured. This survival is known as metabolic retention or the cell populations' proliferative ability after the influence of toxicity's withdrawal.

Microtitration assays may also be used where large sample numbers are simultaneously used, but with a small number of cells within each sample. The agent has the entire population exposed to it, with viability found out by measurement of metabolic parameters, for example ATP concentration. However, MTT reduction is now used as the optimal end point for measurement of cell viability. The MTT assay is where a water soluble dye is converted by viable cells in to formazan. MTT is a yellow tetrazolium dye reduced by live cells into the purple formazan product.

Describe the procedures used for routine maintenance of cell cultures and their long term storage.

After initiation of a culture, it will at some point require a change in the medium, maybe even subculture if there is cell proliferation, which are important for maintenance. Periodical changes of the medium may also be required if during non-proliferating cultures due to spontaneous degradation of cells. Medium change varies depending on the cell line, and is dependent on the growth rate and metabolism. Cell lines that are transformed and grow rapidly are subcultured weekly with the medium change coming around four day after. Cell lines that grow slowly can be subcultured after a couple of weeks, although can be changed after three of four weeks also, whilst the medium can be changed weekly.

Culture needs to be examined with care to make sure there is no contamination. Signed of deterioration should also be checked for, which can suggest that the medium needs to be changed. Subcultures or changes in medium should prevent this deterioration.

There are four factors indicating that the medium needs to be changed; pH drop, cell concentration, cell type (stop dividing at high cell density), morphological deterioration.

Cells can be stored in refrigerators and freezers (cryopreservation), at the growth phase. A cold room may also be better in terms of space. Intracellular ice crystals formation being reduced is dependent upon by the cells optimal freezing for maximum viable recovery on thawing. This can be accomplished by a few methods. The first is to let water exit the cell via slow freezing, but it can't be so slow so there is ice crystal growth. Water can also be isolated via the use of a hydrophilic cryoprotectant. In micelles in ice, high salt concentration effects on the denaturation of proteins can be minimized by the storage of cells at the lowest temperature possible. Finally, ice crystal growth and formation of solute gradients generation during melting of intracellular residual ice can be minimized by rapid thawing.

Cryofreezers can be used where cells are stored in a liquid nitrogen freezer. When cells are below 70°C, they're quick transferred into a cryofreezer.