Techniques

WORK IN PROGRESS

Cell Culture is a fundamental technique in biotechnology.

Generally the cells are grown and then an appropriate gene can be inserted by transfecting with a virus or reagent.

The key thing in cytology is that cells are alive, and fickle! Try to keep them happy! 5% CO2 and 37C for human cells. Other cells may require different circumstances.

Common cell cultures include HeLa (immortal) and HEK293 (easily transfectable) for humans. With non immortal lines, the more times you split the cell cultures the more likely they are to die off. Ideally you only split the cells the minimum number of times.

When you receive a sample of cells note the [i]passage[/i] number, this is the number of times that they have been [i]passaged[/i], or split, and re-homed.

The primary concern for culturing cells is contamination from the environment such as spores or bacteria.

Ideally all operations will take place inside a sterile environment. Preferably a laboratory with positive pressure and a fumehood.

The environment needs to be cleaned with a biocide such as Virkon and then further cleaned with Absolute Ethanol (100% EtOH - not 100% proof with is only 50% EtOH)

The operator themselves needs not to contaminate the working space - ideally a button-up elasticated lab coat although a face mask and disposable gloves at a minimum. The gloves also need to be cleaned with EtOH

Most of the equipment for cell culture is sealed and sterile so only the package needs a quick spritz with EtOH before being introduced to the fume hood.

The actual culturing happens within disposable flasks which have a lining enabling the cells to adhere to the bottom. Their screw top also contains a breathable membrane.

[img]http://www.thermofisher.com/content/dam/tfs/LPG/LCD/LCD%20Product%20Images/Cell%20Culture%20Vessels%20and%20Microplates/Cell%20Culture%20Flasks/F65417~p.eps/jcr:content/renditions/cq5dam.thumbnail.450.450.png[/img]

The flask needs to contain a growth medium for the cells. There are several kinds which vary depending on the kind of cell cultured.

There are various additives such as serums which can augment the growth phase of the cells.

Fume Hood / Laboratory Flasks Serology pipette Serum Growth Medium Transfection Reagent (lipofectamine) 6 Well Plate Transfection Plate –

Before transfecting cells, you need to determine the concentration of your cells. This is measured in cells per milliltre.

Generally the media is removed from the cells and disposed of. 1ml of the enzyme trypsin is added and left for a minute or two. This enzyme cuts the cell cultures bonds so they start to detatch and split up.

After the cells are released, more growth media is added, this neutralises the trypsin too. The cells can now be passaged - drawn up the pipette and released 2-3 times to break them up.

You are now ready to count the cells. Take a 200ul pipette and place a sample onto your cytometer. The cytometer is a special microscope slide with a 100ul well and etched lines. Put a slide over it.

Look at the cells underneath a microscope. 20x or 40x is enough, don't go for 100x-200x!

You can count the cells using the grid to help. A clicker-counter is very helpful. By counting one quarter and multiplying by 4 you can get an average.

If there are too many cells, you need to dilute your sample, so by adding another ml of medium you halve the concentration.

Then again you must count the cells. Once there the correct number of cells (this depends on your experiment) you are free to proceed.

Generally you get to know your cells growth rates. So if you have 10 cells per ul and know that they will be 20 cells per ul in 24 hours tomorrow AND your experiment means that you need to transfect at 15cells per ul then you know that you can setup your plates and then transfect them in 18 hours.

Cytometer/M'scope Automatic cytometer

–

Samples can be obtained form companies. Should be enough for a pass.

-how to -screening cells