Techniques for Modeling Human Intestinal Cancer in Mice

October 22-26, 2000

Presented by

The Jackson Laboratory

Faculty: Catherine Booth, William F. Dove, Jeffrey I. Gordon, Richard B.
Halberg, Julie Ann O\'Shea, Thaddeus S. Stappenbeck, and Robert H.
Whitehead

Edited by: W. F. Dove, R. B. Halberg, L. Clipson,

and I. L. Riegel

The McArdle Laboratory for Cancer Research

University of Wisconsin-Madison

Send any corrections or additions to dove@oncology.wisc.edu

3.  **Cell Culture**

**Primary Culture Isolations**

######## Catherine Booth

**Paterson Institute**

**Requirements**

Mouse colons (4 per isolation)

Forceps

Scalpel blade holder

Size 22-scalpel blade

10 ml syringe

21-gauge needle

Plastic petri dishes 100x20mm

T25 cm^2^ tissue culture flasks (2 per isolation)

T75 cm^2^ tissue culture flask (1 per isolation)

Scalpel blade remover

5 ml Movette pipettes

24-well tissue culture plates (usually 4 per isolation)

## Sterile clear universals

5, 10, and 25 ml pipettes

Centrifuge

500 ml beaker

**2.5% FCS in DMEM** with 4500 mg/L glucose and pyridoxine HCl (Life
technologies 41965-039) plus

5 ml sodium pyruvate Sigma S8636

**0.25 U/ml insulin**

100U/ml Penicillin

25 μg/ml Gentomycin Sigma G1397

30 μg/mlStreptomycin Sigma S0890

**2% D-sorbitol:** DMEM with 2.5% FCS (and the above antibiotics etc)
plus 2% D-sorbitol (Sigma S1876), i.e., 10 g in 500 ml

**1XHBSS:** 900 ml DD water, 100 ml 10X HBSS (Sigma H4641), 1.8 ml
sodium bicarbonate (Sigma S8761), antibiotics as above.

**All reagents should be sterilized by filtration prior to use, and all
work must be carried out in a tissue culture cabinet after enzymatic
digestion.**

**Digestion Media:** This consists of 30 ml DMEM (as previously
described) with 2.5% FCS together with 75U/ml Collagenase type XI (Sigma
C9407) and Dispase 1 neutral protease at 20 μg/ml (Roche 1284 908). A
stock supply of Collagenase and Dispase is usually reconstituted in
1XHBSS and stored as frozen aliquots, which are then adjusted to the
correct concentration/volume with DMEM.

**Coating Of Plates:** At least 3 hr prior to plating out your crypts,
you should coat your plates with a 1:10 dilution of Vitrogen (Nutacon BV
0701) made up in DDH~2~0. The Vitrogen is then removed, and the plates
are allowed to air-dry in the tissue culture hood.

**METHOD**

**Digestion:**

**1.** Remove colons from mice and place them in 1XHBSS solution.

**2.** The colons are then flushed out with 1XHBSS by using your needle
and syringe in a plastic petri dish, to remove their contents.

**3.** Place the colons on a clean lid of a petri dish and remove any
attached mesenchymal tissue. Using your scalpel, slice the colons down
the center and cut them into 2 mm^2^ pieces (small enough so that they
can easily be agitated with your pipette).

**4.** Transfer the contents to a T25 flask and top up with about 30 ml
HBSS; agitate your solution with your 5 ml movette pipette; allow the
pieces of tissue to sink to the bottom of the flasks and remove HBSS.
(Your HBSS will now appear cloudy; as you repeat the process, the
solution will become clearer as it gets cleaner).

**5.** Repeat the above process 4-5 times, until your HBSS remains
relatively clear.

**6.** Remove the HBSS and empty the contents of your flask onto a petri
dish lid. Remove any excess liquid and then begin to finely chop the
tissue, using your scalpel. The chopping process may initially take you
a few minutes; continue to chop until the tissue has a smooth,
paste-like appearance. (The best way to chop is probably via a rapid
chopping action; you can be fairly aggressive with the tissue so do not
worry that you are damaging it; ultimately you want to chop the tissue
as small as you can, as quickly as you can).

**7.** Transfer your material into a T25 flask and add your solution of
digestion mix. Incubate in a water bath at 37°C for a period of about 1
hr 30 min. (We usually put the small flask into a beaker of water in the
water bath.)

**8.** Transfer the contents of your flask into a T75 flask and shake
fairly vigorously; by this stage you should start to see that the tissue
has started to be digested and that small clumps of crypts have started
to appear (Fig2a). You now need to keep a close eye on your tissue;
return it to the waterbath, gently shake the flask, and check your
isolation at 20-min intervals, until you can see that most of your
crypts have become liberated and your solution contains large numbers of
single crypts (Fig2b).

**(Note: You cannot wait until you have a complete suspension of single
crypts as not all crypts will become free at the same time; those that
become free first are at risk of damage from the enzyme from
over-digestion. You will have to decide at which point to stop your
digestion; this is usually when about 70% of the crypts are present as
singlets).**

# Sedimentation

**9.** After digestion, you need to sediment your isolation to remove
any undigested material; wash it in several changes of sorbitol, thereby
cleaning up your prep to remove contaminating factors. This is done as
follows:

**10.** Inside the tissue culture hood, empty the contents of your flask
into two 25-ml sterile universal tubes (set 1) and top up with 2%
Sorbitol solution (so that you have a working volume of about 20 ml per
tube). Allow any undigested material to sink to the bottom of the tubes
(usually this takes about 60 sec) and then transfer the supernatant to
clean tubes (set 2).

You can then allow the tubes containing the supernatant (set 2) to
settle for a minute to remove any undigested material that you may have
carried over to your new tubes. Once again, transfer the supernatant to
a clean set of tubes (set 3) and top up with 2% sorbitol; these tubes
are then ready for centrifuging.

**11.** Centrifuge your tubes (set 3) at 2-300 revs/min (or lowest speed
possible on your centrifuge) for 4 min; while these are spinning, check
the material left in your remaining tubes (sets 1and 2) for the presence
of single crypts. If there is largely only undigested material, discard
this material. If, however, there are quite a few single crypts present,
pool the contents of the tubes into one, top up with sorbitol, and
repeat the above-mentioned sedimentation step.

**12.** When you remove your tubes from the centrifuge, carefully
discard the supernatant (which will be cloudy in appearance at this
stage, but will become clear as you clean up your isolation), and
resuspend your pellet by gently flicking the base of the universal and
topping up with 2% sorbitol. Again at this stage you may see some large
undigested pieces of material that you have failed to remove previously;
allow these to sink to the bottom of the tube and transfer your
supernatant into clean tubes, top up with sorbitol, and centrifuge once
more.

**13.** You will need to repeat the centrifugation/sedimentation stage
approximately 5 times in total, until you are left with your pellet
surrounded by a clear supernatant.

The crypts are then ready to be resuspended and plated out. They are
usually plated out at between 800 and 1000 per ml in a 24-well plate. To
do this, you will need to serial dilute your crypt suspension and count
the crypts present; e.g., resuspend your pellet in 5-10 ml medium,
transfer 100 μl of this to a universal containing 900 μl medium, mix,
and remove 100 μl to count the number of crypts present. The number of
crypts you count can then be multiplied by 100 to give you the number of
crypts per ml you have.

Incubate your crypts at 7.5% carbon dioxide and 37°C temperature. Allow
the culture to attach for a period of 48 hr and then change your samples
by adding 1 ml of fresh medium to each well. Isolations can routinely be
cultured for a period of 15 days if the medium is supplemented with 0.5
ml fresh medium at 5-day intervals, or for 30 days when the isolations
are grown on irradiated 3T3 feeder layers. The addition of EGF and
transferrin can also encourage the growth of the primary cultures. Fig.
3 illustrates typical primary cultures.

**Figure 2a: Clumps Of Crypts**

![](media/image1.jpeg){width="6.748611111111111in"
height="5.583333333333333in"}

**Figure 2b: Single Crypts**

![](media/image2.jpeg){width="4.719444444444444in"
height="4.719444444444444in"}

**Figure 3: Cultures**

![](media/image3.jpeg){width="6.740277777777778in"
height="4.391666666666667in"}

**A CLONOGENIC ASSAY FOR IDENTIFYING GROWTH AND DIFFERENTIATION FACTORS
FOR COLONIC CRYPT EPITHELIUM**

R. H. Whitehead

Department of Gastroenterology

Vanderbilt University

Nashville, TN 37232

Introduction

Although the intestinal epithelium proliferates rapidly throughout life,
very little is known about the factors that control this process.
Because of the difficulty we have encountered in culturing colonic
epithelial cells, most of our knowledge about growth control of the
colon come from *in vivo* studies. The problem with these studies is
that it is impossible to be certain that you are seeing a primary effect
and not some secondary effect caused by the factor you have
administered.

It is well demonstrated that each crypt of the colonic mucosa contains a
small proportion of stem cells from which all of the cells of the mucosa
arise. In the hemopoietic system it has been possible to develop assays
for the stem cells and to identify the factors that induce both
proliferation and differentiation into the known mature cell phenotypes
of the blood and bone marrow.

A similar assay for colonic stem cells could provide a method for
identifying similar factors for this tissue. I will demonstrate a method
that I have devised for isolating crypts free from stroma and for
disaggregating these crypts into single cells. I will then use these
single cells in a clonogenic assay based on the hemopoietic stem cell
assay. We can use this assay to identify growth factors for the
proliferative cells of the colonic crypt.

**Requirements**

Scissors and forceps

Mouse colons (2 per student)

Phosphate-buffered saline

500-ml beakers

4% sodium hypoclorite

3 mM EDTA/ 0.05 mM DTT solution in PBS. (111.6 mg EDTA disodium salt
plus 7.7mg DTT in 100 ml PBS -- sterilized by filtration).

Screw-capped, 50-ml centrifuge tubes

15-ml centrifuge tubes

Centrifuge

0.3% Pancreatin (Sigma P1500) in PBS -- this suspension is incubated in
a 37^o^C waterbath for 3 hr before being filtered. It will still contain
a lot of insoluble material, so prefiltration or centrifugation will
make filter sterilization easier.

Difco Bacto-agar - 1% in H~2~O \[sterilized by autoclave; liquid setting
(15 lb pressure for 15 min)\]

Sigma Type VII agar -- 0.8% in H~2~O \[sterilized by autoclave; liquid
setting (15 lb pressure for 15 min)\]

X2 RPMI 1640

Fetal calf serum

6-well tissue culture plates

Sterile pipettes -- 10 ml and 5 ml.

Sterile plugged Pasteur pipettes

Hemocytometer

20-ml syringes

18-gauge needles

21-gauge needles

Microscope

0.2-μ sterile filters

Trypan blue

**METHOD**

-   Prepare 0.04% sodium hypoclorite (1 ml of 4% sodium hypoclorite in
    100 ml PBS) and 3 mM EDTA + 0.05 mM DTT in PBS (114 mg tetrasodium
    EDTA plus 7.7 mg DTT in 100 ml CA^++^-, Mg^++^-free PBS). Sterilize
    EDTA/DTT solution by filtration.

-   Mice are killed and the colons are removed.

-   The colons are opened and washed in 3 changes of PBS (Ca^++^, Mg^++^
    free).

-   The washed colons are transferred to 25 ml of 0.04% sodium
    hypoclorite solution in sterile PBS in a sterile tube and incubated
    for 20 min at room temperature.

-   The colons are rinsed once in sterile PBS and transferred to 25 ml
    of EDTA/DTT solution in a sterile, screw-capped, 50-ml centrifuge
    tube and incubated at room temperature for 90 min.

-   The EDTA solution is removed and replaced with 15 ml of sterile PBS.
    The lid is closed tightly and the tube is shaken VIGOROUSLY for
    15--30 sec.

-   The supernatant is transferred to a 15-ml centrifuge tube; fresh
    sterile PBS is added to the tube containing the colon and shaken.
    The process is repeated 3 times.

-   The tubes are centrifuged at 400 rpm for 5 min.

-   The PBS is removed and the crypts are resuspended in PBS and
    examined microscopically.

-   The crypt pellets are pooled and transferred to a tube containing 25
    ml of 0.3% Pancreatin (300 mg Pancreatin in 100 ml PBS, dissolved
    and sterilized by filtration) and incubated for 90 min at room
    temperature.

• \*\*During this period, the 1% agar and 0.8% agarose are melted and
held in a 40^o^C waterbath. The x2 medium plus 10% FCS is also placed in
the 40^o^C waterbath.

• When the 1% agar has cooled to 40^o^C, the underlay can be prepared by
mixing equal volumes of 1% agar and x2 medium plus FCS and pipetting 2
ml of the mixture into each well of a 6-well plate. The plate is then
kept at room temperature to allow the agar to set, and then used below
to plate the cell suspensions made in agarose/FCS.

-   After 90 min the cell suspension is diluted with an equal volume of
    PBS and transferred to centrifuge tubes.

-   The tubes are centrifuged at 1000 rpm for 5 min.

-   The supernatant is removed carefully because a lot of mucus is often
    present and the whole pellet can be taken up into the pipette if
    care is not taken.

-   The pellets are resuspended in the EDTA/DTT solution and centrifuged
    at 1000 rpm for 5 min.

-   The cell pellets are resuspended in growth medium and pooled.

-   The cell suspension is examined microscopically.

-   If the cells are in a single-cell suspension, a cell count is done
    with a hemocytometer.

-   Normally small clumps of cells are still present, so the cell
    suspension is passed gently 3 times through an 18-gauge needle,
    followed by 3 times through a 21-gauge needle.

-   The cell suspension is examined again.

-   If small clumps are still present, the cell suspension is filtered
    through a cotton wool plug in a Pasteur pipette. (n.b.: this can
    result in considerable cell loss). Recently we have used 40-μ nylon
    cell strainers from Falcon. These give good results as long as you
    pipette the cell suspension gently.

-   The cells in the cell suspension are counted.

-   Factors to be tested are added to labeled wells at 2x the final
    concentration to be tested, to allow for diffusion through the
    underlay.

-   The cell suspension is diluted in a mixture of equal volumes of 0.8%
    agarose and x2 medium plus FCS to give a final cell concentration of
    50,000 cells per ml.

-   The cell mixture is aliquoted into the wells of the plate containing
    the underlay and factors. This pipetting should be done carefully to
    avoid bubbles.

-   The plates are cooled at 4^o^C for 5 min as the type VII agarose
    sets only at 26^o^C and can remain liquid for long periods at room
    temperature.

-   After 5 min at 4^o^C, the plates are placed in a sealed container
    with a small container of H~2~O and incubated at 37^o^C for 3--4
    weeks.

-   The plates are examined, and colonies of 30 cells or more are
    scored. Clusters of \>15 cells can also be counted.

**Potential Problems**

> The major problem with this technique comes from the release of mucus
> from the goblet cells. This becomes a major problem only if the crypts
> and later the single cells are pipetted too vigorously and cells are
> ruptured. Gentle pipetting, gentle passage through the needles, and a
> wash with EDTA/DTT all help in obtaining a good single-cell suspension
> of high viability.

**Summary**

> This is the first method to describe the clonal growth of
> proliferative cells of the colonic crypt. At the present time we
> cannot assume that this is a stem cell assay, but we know that we are
> assaying cells capable of autonomous growth, and that is a property
> associated with stem cells in other tissues. This method allows us to
> study growth responses and hence can be used to identify novel growth
> factors for the proliferative cells of the colon. It will also allow
> us to identify factors that induce the differentiation of these
> proliferative cells into the known differentiated cell phenotypes
> found in the colonic mucosa. For these studies, the colonies can be
> picked from the agarose using a fine-bore Pasteur pipette. The
> colonies can be fixed for ultrastructural studies or can be
> disaggregated and transferred to a slide using a cytocentrifuge for
> immunohisto­chemical studies of cell phenotype. Ultimately the
> stem-cell nature of the cells in the colony can be tested by
> disaggregating the colony and cloning the cells in the colony assay
> once more.
>
> **Note:** This is an evolving technique. The method described here is
> not necessarily the optimal method. Further experimentation may make
> improvements in crypt disaggregation, culture methods and medium, and
> methods of assessing colony formation. Similarly, better methods for
> assessing colony phenotype are needed.

**References**

1.  Whitehead, R.H., Brown, A. and Bhathal, P. (1987) A method for the
    isolation and culture of human colonic crypts in collagen gels. In
    Vitro 23:436-442.

2.  Whitehead, R.H. and Van Eeden, P.E. (1991) A method for the
    prolonged culture of colonic epithelial cells. J. Tissue Culture
    Methods, 13: 103-106.

3.  Whitehead, R.H., VanEeden, P.E., Noble, M.D., Ataliotis, P. and Jat,
    P.S. (1993) The establishment of conditionally immortalized
    epithelial cell lines from both colon and small intestine of adult
    H-2Kb-tsA58 transgenic mice. PNAS, 90, 587-591.

4.  Whitehead, R.H. and Joseph, J.L. (1994) Derivation of conditionally
    immortalised cell lines containing the Min mutation from the normal
    colonic mucosa and other tissues of an F1 mouse (Immortomouse/Min)
    Epithelial Cell Biol,3, 119-125.

5.  Whitehead, R.H., Demmler, K., Rockman, S.P. and Watson, N.K. (1999)
    Clonogenic growth of epithelial cells from normal colonic mucosa
    from both mouse and human. Gastroenterology, 117, 858-865.

**Single Crypts**

![](media/image4.jpeg){width="6.75in" height="5.0625in"}

**Single Cells**

![](media/image5.jpeg){width="6.75in" height="5.0625in"}

**Colonies**![](media/image6.jpeg){width="6.733333333333333in"
height="5.05in"}