Preparing Primary Tissue Samples or Frozen Samples
One of the most
critical steps in successful cell isolation is the preparation of the single
cell suspension. Cell loss may occur during isolation if the sample contains
cell clumps during the cell separation process. Additionally, the clumps may
interfere with the proper labeling of the cells that the process is meant to
target. By following a specialized protocol for harvesting and preparing the
single cell suspensions from primary or frozen tissue before the process of
cell separation, these issues can be avoided.
Preparing Frozen Samples
These steps are designed to discuss the proper procedures
for handling frozen samples and preparing them for single cell suspension.
vial of cells needs to be thawed by swirling it in a water bath of 37 degrees Celsius
and then transferring the thawed cells into an aseptic 50 mL testing or conical
vial is then rinsed using 1 mL of culture medium that contains 10 % FBS, which
will recover the remaining cells and then transfer the medium into a new tube.
the 50 mL tube with culture medium with 10 % FBS and gently invert to mix the
contents of the tube.
the tube at 300 x g for exactly 10 minutes at room temperature for the
collection of the cells.
the supernatant and discard carefully, so as not to disturb the pelleted cells.
A gentle tap on the tube will resuspend the pellet.
that appear clumpy will require DNase I to be added to yield a final
concentration of 100 µg/mL. Swirl the tube while adding the DNase I drops to
the cell suspension. The tube is ten incubated at room temperature for 15
25 mL of the culture medium with 2 % FBS is added to the mix while inverting
the solution and centrifuge again. Discard the supernatant and resuspend the
pellets, as before.
that still appear clumpy will be passed through a 30 µm to 70 µm cell strainer
to a fresh conical tube and rinsed three time with the culture medium that
contains 2 % FBS and strained.
the cell suspension is ready for cell counting or other application, like cell
Preparing Primary Tissue Samples
To prepare a single cell suspension, the cells need to be
dissociated from the sample.
a sterile dish with culture medium or a dissociation enzyme to mince and
harvest the tissue.
the sample through a cell strainer size of either 100 µm or 70 µm to a fresh
2 or 3 mL of culture medium over the tissue and cells that remain in the
strainer. Repeat this step and then discard strainer and remaining tissue.
culture medium is added to the conical tube up to 40 mL or 50 mL total volume
and collect the cells using centrifugation.
the supernatant and discard, while trying not to disturb the pellet. Resuspend
the pellet gently.
optional washing step can be used at this stage, which would include the adding
of 25 mL of culture medium, inverting the cell pellet and solution mix, and
centrifuge. The supernatant would be removed and discarded, and the pellet
resuspended at this stage.
the cell suspension would be ready for cell counting or other applications.
The cell strainers are
vital in this process, because they allow the cells to be separated from the
tissue and a single-cell solution to be formed. Cell strainers are designed to
fit on the 50 mL conical tubes, so they are easy to strain. They are designed
to specifically allow only certain sized material to pass through and help to
remove cell clumps. Various styles and sizes are available for different uses,
depending on the application.
Cell strainers are essential to the process of single
cell suspension and help to create a sterile, easy, and quick way of
successfully separating cells.
– 100 µm
for removing aggregates from the sample material and can be used to cultivate
and produce cell spheroid cultures.
– 200 µm
for physical disruption of the tissue to gain primary cells from the tissue.
The tissue is disrupted on the cell strainer. A connector ring can be added
in a closed stated when working with a solution. It allows for the
preservation of the cell culture medium and keeps the material from drying
– 500 µm
for the physical shredding of tough tissue, such as skin, fat, muscles, lung,
or other similar tissues.
The various cell
strainer mesh sizes can be stacked to sieve various aggregate sizes. The sieves
can be turned upside down to allow for flushing the material back into a new
tube for further analysis and testing. These disposable cell strainers are a
more effective way of creating the single cell solution than a gauze filtration
system and help to make the process quicker and easier.