Making formalin-fixed cell line plugs for incorporation into paraffin blocks for research

Alan K. Meeker, August 2019

This protocol is for the generation of faux tissue specimens from cells grown in cell culture. Cells are harvested,
pelleted and the pellets are then formalin fixed, followed by standard paraffin embedding. The end result is a
paraffin block featuring one or more cell pellets that act as tissue mimics of standard FFPE tissue specimens.
These cell blocks are useful as validation controls for various staining procedures, such as immunostaining as
well as DNA or RNA in situ hybridization. They are especially handy when working up staining protocols for
antibodies eventually to be used in FFPE tissue samples, as the optimized conditions developed with the cell
line mimics often transfer to fixed tissues with minimal need for further optimization. The protocol was co-
developed in the laboratories of Angelo De Marzo, William Nelson and Alan Meeker and refined over a number
of years largely through the work of Michael Haffner and David Esopi.

General Considerations:
 The more cells you have, the better! For adherent cells, for the larger size cell pellets, this
protocol is not worth doing unless you have at least one confluent T175 (175 cm2) worth of cells.
There is variation in cell size and density, so for some cell lines even one T-175 may not give an
adequate cell yield; thus, if it is little added trouble or expense, it is best to shoot for two T175
flasks worth of cells. Alternatively, smaller size microfuge tubes can be used if cells are limiting,
or bulk “carrier” cells can be used in an admixture to support a sufficient pellet. See below for
more details on these variables.
 A useful option if you have too few cells is to admix your cells of interest with a bolus of carrier
cells (e.g. an easily grown cancer cell line). We have had very good success with this
modification. Best strategy is to select a carrier that is significantly different than your cells of
interest, especially for the particular marker(s) you will be trying to stain for, so that the two cell
types will be easily distinguished from one another following staining. Differences in cell size
can also make differentiating the two cell types easier. Also, selecting a carrier cell line that is
already known to form stable pellets is a good idea.
 Some cell lines naturally form thick and stable pellets, others don’t. There’s almost no way to tell
how well a cell line will pellet, how big the pellet will be, or how stable during downstream
processing until you try it. In general, it has been our experience that cells growing in suspension
culture (e.g. leukemia cells, cells grown as spheroids, etc.) are more likely to produce unstable
pellets. One solution to this is to mix the suspension cells with a well-behaved carrier cell line as
described above.
 The final step in the protocol involves slicing a microfuge tube with a razor blade or scalpel.
Exercise caution here to avoid personal injury! Also, use a very sharp blade – you don’t want to
compress the pellet when pressing down with the blade.
 We typically fix in 10% neutral buffered formalin at room temperature for 48 hours. Make sure
the formalin solution you use is fresh! This may not be optimal for all purposes. So far we have
also used ethanol to fix a cell pellet and it worked well, forming a stable pellet. In that case, the
same procedure was used, but 70% ethanol was substituted for formalin in the protocol, fixation
was done overnight at RT, then the 70% ethanol was carefully changed to 95% ethanol and
fixation continued for another 24 hours.
 The size of the microfuge tube can be changed, depending on the expected size of the cell pellet.
We generally use 0.5 ml PCR-type tubes. <0.5 ml size tubes can be used if the cell pellet is
small, 1.0 or 1.5 ml tubes can be used for very large pellets.
 Remember that when your fixed pellet goes in for processing with ethanol and xylene, it will
shrink significantly (by up to 50%). This means that if your initial pellet is 5 millimeters thick,
after processing it will only be about 2.5-3 mm. Again, more cells/bigger pellets to start with are
preferred.
Pros:
 Better preservation of cell architecture
 Greater density of cells
 Does not require the cells to be resuspended in hot agarose, minimizing damage and inhomogeneity in cell
density throughout the pellet

Cons
 Requires a lot of cells (but can use the carrier cell option described above)
 Requires cutting your tube with a razor- safety hazard

Procedure:
1. Make up a solution of 2% agarose in PBS and pipet ~200 uL of molten agarose into the bottom of a 0.5
mL microfuge tube (Other size tubes can be used. Smaller tubes are easier to cut open later, but give
smaller cell pellets). Once the agarose has hardened, overlay with PBS, close the tubes and store in the
refrigerator until needed. These are quite stable and we routinely keep a range of tube sizes in the frig.
2. Harvest the cells by scraping or trypsinization and pellet them as you normally would during cell culture
techniques.
3. Wash the pellet 1X with PBS.
4. Resuspend the pellet in an appropriate (very small!) volume of neutral-buffered 10% formalin (make
sure it is not past its expiration date!) and pipet it into the microfuge tube. The best volume depends on
the size of the pellet and the size of the tube you are putting the cells into. If the volume of resuspended
cells is more than will fit into the microfuge tube, perform the spin in step 5, aspirate off the cleared
supernatant and add the remaining resuspended cells and spin again.
5. Centrifuge the microfuge tube so that the cells form a fairly thick layer on top of the agarose. Use of a
swinging bucket rotor is recommended and will keep the pellet flat. The microfuge can be carefully
slid down to the bottom of a 15 ml conical tube which serves as an adapter and is then spun. We
routinely use a Damon/IEC HN-SII benchtop clinical centrifuge at ~ ½ x speed setting which
corresponds to an rcf of ~ 750-1,000 x g. To perform this, cut off the cap of the microfuge tube first.
After the spin, remove the microfuge tube and carefully aspirate off the clear supernatant.
6. Carefully add buffered 10% formalin down the side of the microfuge tube onto the cell pellet, trying
not to disturb the pellet.
7. I always spin the tube again, as in step 5, immediately after adding the formalin- just to make sure that
the pellet is as thick and compact as possible.
8. Put the entire microfuge tube into a 15 mL conical containing ~10 mL buffered formalin. Use a plastic
disposable transfer pipette to gently submerge the microfuge tube and gently wedge it in the 15 ml
conical so it will remain submerged in the formalin without floating to the top during fixation.
9. Leave at room temperature to fix for 48 hours.
10. Remove the microfuge tube from the 15 ml tube. (This may require the use of forceps. Plastic
inoculating loops work well here too) At this point, if needed, the microfuge tube can be transferred to a
new 15 ml conical tube of PBS and stored in the refrigerator for many days prior to continuing.
11. Remove the microfuge tube and Carefully cut off the bottom of the tube with a razor blade or scalpel..
Avoid cutting the cell pellet and take care not to deform the tube too much while doing this as the pellet
may be crushed. Use a fresh, very sharp blade.
12. Immerse a labeled plastic tissue cassette in PBS in a shallow container, such as a petri dish. Using the
blunt end of a wooden cotton applicator stick, pipette tip, or other similar implement, gently extrude the
fixed cell plug into the immersed cassette.
13. Check to see that the pellet does not protrude over the top of the cassette; if so, then carefully trim the
pellet with a razor blade. If you don’t do this then the pellet will be crushed when the lid of the cassette
is closed. Carefully close the cassette. The submerged cassette can be kept in this container at 4°C for up
to one week, but the sooner it is submitted for processing the better.
 14. Submit the cassette to a histology services lab for processing into a paraffin block.

Step 1

Snip off cap

2% agarose (in PBS)

cells

Embed
PBS

10%
formalin-
buffered
phosphate

Figure 1. Schematic diagram of the steps involved in making a fixed cell pellet for FFPE