Sterile Cell Culture Technique


This is a short demonstration of the techniques
used to propagate animal cells. The first step is to visually inspect you
culture, remove it from the incubator and look at it.
The cells should be stuck to the surface of the culture vessel. there should be no apparent
mould or cloudiness and the colour of the medium should be unchanged.
Put the flask onto the microscope platform, look carefully at the culture. Check that
there are enough cells, approximately 80% of the flasks surface should be covered. Also
check that the cells are well grown, and that nothing is contaminating them.
This is a Class 2 microbiological safety cabinet. Air from outside of the laboratory is drawn
through the front and underneath the plates at the bottom. It goes up a space in the back
and is pushed down from the top through a big filter which removes dust and micro-organisms
so that the air which flows down to the working space is relatively clean.
To clean the cabinet we use 70% Ethanol which is a very safe surface disinfectant. Spray
generously onto the cleaning paper and disinfect all the inside surfaces of the cabinet. Make
sure all of the surfaces are cleaned, and ensure you work into the corners.
Once the inside is clean you begin preparing all of the equipment you are going to use.
Put on a pair of gloves and sterilise them by spraying generously with Ethanol. Make
sure the entire surface of the gloves are covered.
The gloves protect your sample from micro-organisms on your skin. Once your gloves are sterile
don’t touch your hair or skin or you’ll get contamination.
Sterilising all of the equipment using Ethanol prior to placing it in the cabinet is one
of the key ways of preventing contamination. Arrange your equipment in the cabinet, to
the right if you’re right handed. Don’t bring too many items into the cabinet as this can
disrupt the air flow. The purpose of cell culture is to remove the
old culture media that the cell has been growing in and replace it with new media. This is
because the nutrients become exhausted and it also gives the cells more room to divide.
The
first thing we need to do is remove the old media using a pipette. The pipette is sterile
so handle it using the plastic wrapper. Load the pipette into a filler and remove the plastic
sleeve. Be careful that the pipette doesn’t touch anything or you’ll risk contamination.
Using one hand remove the lid from the tissue culture vessel and place the pipette inside
the liquid, and use the top button of the pipette filler to suck the liquid up, and
then dispose of it into the disinfectant. Replace the top as soon as you can to minimise
the risk of contamination from the air. Remove the pipette from the filler and dispose of
it into the bin provided. The next step is to add a buffer solution
which doesn’t have any nutrients. Load a pipette into the filler handling it with the plastic
wrapper. The buffer solution is used to wash off any residue of the old culture medium
since the process that we use to detach the cells can be affected if there’s any medium
left. Draw 10 millilitres of buffer solution into
the pipette and wash the buffer down the surface that the cells are stuck to, rinsing over
the cells. Draw the buffer solution back out and dispose
of it in the disinfectant. For health and safety reasons and to prevent any release
of biological material into the environment, all biological material must be disposed of
in disinfectant. The next step uses a molecule called Trypsin,
a digestive enzyme that’s found in all mammals. It’s used to disrupt the proteins which stick
the cells to the plastic flask. Load a pipette into the filler handling it with the plastic
wrapper. Again, use one hand to remove the cap from the Trypsin and hold the cap in the
same hand. Put the Trypsin on the surface of the cell
culture vessel that the cells are growing on and replace the lid.
The Trypsin is a digestive enzyme and works most effectively at 37 degrees Centigrade.
Put the flask into an incubator for three to four minutes. Don’t leave the cells in
the Trypsin for too long or you’ll remove the cells surface signalling proteins.
After incubation visually inspect the flask on the microscope. The cells should be beginning
to detach, ideally they should appear spherical but still be attached to the plastic. If they’re
all floating free they’ve been Trypsinised too much.
Shock the flask by tapping to dislodge the cells. Examine the cells again to ensure that
they’re floating free of the plastic. Remember to sterilise the flask with Ethanol
before placing it back in the cabinet. To arrest the Trypsinisation and prevent damage
to the cells we add fresh culture medium which provides lots of protein to mop up the Trypsin.
Take a pipette and open the container using one hand. Fill the pipette and pour the fresh
medium down the back wall of the flask which washes the cells to the bottom.
Using the same pipette remove all of the liquid from the flask
and using one hand remove the cap of a centrifuge
tube and put the liquid in before replacing the cap.
Open the centrifuge and load the sample. Make sure that there’s a counterweight before shutting
the lid. Spin the sample at 1800 RPM for 4 minutes, be careful not to over spin or the
cells will be damaged. Take the sample from the centrifuge and inspect
it to ensure that the cells are in a pellet at the bottom. Sterilise it using Ethanol
before returning it to the cabinet. Next prepare a new flask of culture medium
for the cells to grow in. Take a new flask and load a pipette into the filler.
Fill the pipette with fresh media and transfer it to the flask. Remove the lid with one hand
and replace it as soon as possible to reduce the risk of contamination.
The next step is to re-suspend the cells using some fresh culture medium. Invert the flask
over the disinfectant in one fluid motion to remove all the culture media and avoid
dislodging the pellet, then check that the pellet is still there.
Take a fresh pipette and attach it to the filler. Fill the pipette with fresh media.
Flush the liquid in and out of the pipette until the pellet is broken up and is a single
cell suspension again. To avoid and uneven cell culture repeat the process until there
are no lumps floating around in it. Take approximately 30% of the cell suspension
and place it in the culture medium, then tighten the cap.
Examine the culture using the microscope to make sure the cell density is high enough.
Put the new cell culture into the incubator at 37 degrees.
The last step is to clean up. All liquids go into the disinfectant for at least two
hours prior to disposal. The Trypsin and fresh culture medium are replaced
in the fridge. All inside surfaces of the cabinet are cleaned
and ready for the next user. Replace the cabinet cover.
Lastly, any general waste and plastic-ware that is contaminated with biological material
is disposed of in the bio-hazard waste bin.

Author: Kennedi Daugherty

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