Biosynthetic Cell and Organ Culture Methods 219
219
18
Biosynthesis of Mucin Cell and Organ Culture Methods
for Biosynthetic Study
Anthony P. Corfield, Neil Myerscough,
Alexandra W. C. Einerhand, B. Jan-Willem Van Klinken,
Jan Dekker, and Christos Paraskeva
1. Introduction
The study of the biosynthesis has been greatly assisted by the use of cultured cells
and tissue explants in short-term culture. Cells are available from a wide range of
tissue sources, and this chapter focuses on the use of intestinal cells and tissue. Human
colonic cell lines have been widely used in biosynthetic studies and the relationship of
some lines to stages in the adenoma-carcinoma sequence is of particular interest,
allowing study of the expression of mucin during the development and progression of
disease (1–3). Recently the importance of proliferation, differentiation, and apoptosis
has attracted attention to the use of culture systems for the study of cell behavior in
normal and disease processes (4,5). In the same way, tissue obtained from patients at
surgery or as biopsies can be placed in short-term primary or organ culture to study
similar changes in disease (6,7).
Improvements in the study of glycoproteins, especially mucins, have been achieved
through the use of defined human mucosal cells that can be grown in long-term culture
(1,2). Radioactive tracer methods allow relatively small numbers of cells and tissue
fragments (biopsies) to be analyzed, and cell culture also gives access to larger amounts
of the mucins produced by the individual cell lines (8,9).
Each of the model systems described for the study of the synthesis and secretion of
mucin has distinct advantages and drawbacks. Ideally we would wish for a clonal cell
line, which has all the typical characteristics of the mucin-producing cells in vivo.
Because no such cell line exists for any of the mucin producing cells, we must settle
for either tissue explants, in which the mucin-producing cells have retained their natu-
ral tissue context, or isolated clonal cells from carcinomas. The first system is natu-

rally short-lived, which affects reproducibility, whereas the latter system is far more
reproducible; however, these cells will have irreversible genetic changes that distin-
From:
Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A. Corfield © Humana Press Inc., Totowa, NJ
220 Corfield et al.
guish them from the cells in vivo. On the other hand, the cell lines may consist of only
one cell type, which can be an advantage.
Study of the colon has the advantage that the sequence of changes during carcino-
genesis in the colonic epithelial cells has been documented particularly well. This
implies that cells can be isolated from each stage of malignant growth: starting from
healthy tissue, from which normal epithelial cells can be brought into primary culture
and display only minimal growth in vitro, via the adenoma stage that maintains inter-
mediary characteristics to the full blown carcinoma cells, which usually grow quite
well (10). This sequence of changes in the epithelium allows precise studies of how
the changes in cells influence the mucins that are produced. Several of the end prod-
ucts of malignant transformation of colonic epithelium have yielded valuable cell lines,
such as the goblet cell-like LS174T and the enterocyte-like Caco-2 cell lines, which
are widely used to study the synthesis and regulation of mucin in detail (11,12). The
PC/AA cell lines, originally derived from a single, large, colonic tubular adenoma,
have been used at early premalignant, intermediate premalignant, adenocarcinoma,
and mucinous carcinoma stages (1,2,8,9,13). Furthermore, the HRA-19 colorectal cell
line (3) is valuable because it can be cloned to give all three differentiation pathways
to columnar, goblet, and endocrine cells.
The methods described here cover the use of cellular systems for the study of the
biosynthesis of mucin which includes cultured cells (primary cultures and cell lines) and
organ culture. The data refer to human colorectal cells and tissue, but similar systems
and principles apply to a wide range of other tissues where mucins are produced.
2. Materials
1. Human gastrointestinal (GI) biopsies or surgical tissue from stomach (corpus and an-

trum), duodenum, jejunum, ileum, colon (ascending, transverse, descending, sigmoid,
and rectum), or gallbladder. Tissue obtained at surgery is dissected to isolate the mucosal
layer and cut into small sections of approx 2–4 mm
3
. Endoscopically obtained biopsies
are used without further manipulations.
2. GI tissue explants of rat or mouse from stomach (corpus and antrum), duodenum, jejunum,
ileum, colon (proximal, mid, and distal), or gallbladder (only in mouse). Full thickness
explants, including the muscle layers are used cut to give segments of 2–4 mm
2
of mu-
cosal surface.
3. Cell lines:
a. Swiss 3T3 cells obtained from the American Tissue Type Culture Collection (ATCC,
Manassas, VA, cat. no. CCL92).
b. LS174T, colonic adenocarcinoma cell line (the parental line obtained from ATCC,
cat. no. CL 188).
c. Caco-2, colonic adenocarcinoma cell line (from Prof. G. J. Strous, Laboratory for
Cell Biology, Utrecht, The Netherlands).
d. A431, epidermoid carcinoma cell line (ATCC, cat. no. CRL 1555).
4. Fetal bovine serum (FBS). Batch testing is essential.
5. Cell culture media
a. Standard growth medium: Dulbecco’s modified Eagles medium (DMEM) containing
2 mM glutamine, 1 µg/mL of hydrocortisone sodium succinate, 0.2 U/mL of insulin,
100 IU/mL of penicillin, 100 mg/mL of streptomycin, and 20% of FBS.
Biosynthetic Cell and Organ Culture Methods 221
b. Washing medium: The same as the standard growth medium, but with 5% FBS, double
the concentration of penicillin and streptomycin, and 50 µg/mL of gentamicin.
c. Digesting solution: The same as the washing medium but containing 5% FBS together
with 1.5 mg/mL of collagenase (Worthington type 4, Lorm Diagnostics, Reading,

UK) and 0.25 mg/mL of hyaluronidase (type 1; Sigma, Poole, UK).
d. 3T3 Conditioned medium: DMEM is supplemented with 10% FBS, 2 mM glutamine,
100 IU/mL of penicillin, 100 µg/mL of streptomycin, and put onto 24-h postconfluent
3T3 cell layers for 24 h. After conditioning, the medium is filtered through a 0.2-mm
filter (Nalgene, Milton Keynes, UK) and further supplemented to give 20% FBS,
1 µg/mL of hydrocortisone sodium succinate, and 0.2 U/mL of insulin.
e. Cell growth medium: Dulbecco’s modified Eagles medium (DMEM, Gibco/BRL,
Parsley, Scotland) containing 100 IU/mL of penicillin, 100 µg/mL of streptomycin,
3.7 g/L of NaHCO
3
, and nonessential amino acids (sterile 100X stock solution, Gibco/
BRL), supplemented with 20% of FBS (LS174T cells) or 10% of FBS for Caco-2 and
A431 cells.
f. Mouse thymocyte culture medium: RPMI medium (Gibco/BRL) supplemented with
10% FBS, 2 mM of glutamine, 100 IU/mL of penicillin, 100 µg/mL of streptomycin
and 2 mM of glutamine.
6. Collagen (human placental type 4; Sigma) at 1 mg/mL is prepared in 1 part glacial acetic
acid to 1000 parts sterile tissue culture grade distilled water and, and stored at 4°C.
7. Dispase solution: Dispase (grade 1; Boehringer, Lewes, UK) is prepared in DMEM con-
taining 10% FBS, sterile filtered, and stored at –20°C.
8. MF-1 mice (Olac, Bicester, UK).
9. Trypsin 0.1% by weight in 0.1% EDTA in PBS.
10. Acridine orange (Sigma).
11. Organ culture medium:
a. Grid cultures: DMEM, containing 10% FBS, 10 mM of sodium bicarbonate, 2 mM of
glutamine, 100 µg/mL of streptomycin, 50 IU/mL of penicillin, 50 µg/mL of gentami-
cin, and 20 mM of HEPES, pH7.2
b. Submerged cultures: Eagle’s minimal Essential medium (EMEM, Gibco/BRL),
supplemented with: non-essential amino acids (sterile 100X stock solution, Gibco/
BRL), 100 IU/mL of penicillin, 100 µg/mL of streptomycin, 2 mM of

L
-glutamine.
Incubate under 95% O
2
/5% CO
2
.
12. Carbogen gas: 5% O
2
/5% CO
2
.
13. Culture dishes (Costar, Cambridge MA).
14. Airtight capped, transparent tubes (3–5 mL) with round bottom (Sarstedt, Nümbrecht,
Germany).
15. Water bath at 37°C.
3. Methods
3.1. Preparation of Collagen Coated Culture Flasks and Swiss 3T3
Feeder Cells (
see
Notes 1–3)
1. To prepare collagen-coated flasks, coat tissue culture flasks (T2525 cm
2
) with a thin layer
of collagen solution (Subheading 2., item 6; 0.2 mg/flask), and allow to dry at room
temperature in a laminar flow hood for 2–4 h (see Note 4).
2. Grow Swiss 3T3 cells (Subheading 2., item 1) on collagen on plastic tissue culture flasks
in DMEM containing 10% fetal calf serum until they are 24 h postconfluent.
222 Corfield et al.
3. Lethally irradiate the cells with 60 Kgray (6 mrad) of radiation, or treat with 10 µg/mL of

mitomycin C (Sigma) for 2 h.
4. Wash the cells and produce a single-cell suspension using a Pasteur pipet (wide aperture
to avoid shearing of cells). The cells can either be used immediately as feeders or be
stored at 4°C as a single-cell suspension for up to 4 d (see Note 5).
3.2. Primary Culture-Enzyme Digestion (
see
Notes 3, 6, and 7)
1. Wash the tumor specimens (adenoma and carcinoma) four times in washing medium
(Subheading 2., item 5b) and cut with surgical blades to approx 1 mm
3
in a small volume
of the same medium.
2. Wash the tissue four times in washing medium, and place in digestion solution
(Subheding 2., item 5c). Pellet cells by centrifugation at 300g for 5 min. Roughly put 1 cm
3
in 20–40 mL of solution.
3. Rotate at 37°C overnight (12–16 h).
4. Mix the suspension by using a Pasteur pipet to improve the separation of the epithelial
elements from the stroma resulting from enzymatic digestion.
5. Filter the suspension through 50-mm mesh nylon gauze, or repeatedly allow to settle out
under gravity and collect the pellets. The large clumps of cells and epithelial tubules
(organoids that contain the majority of the epithelial cells) are separated from the single
cells (mostly from the blood and stroma) and cell debris.
6. Wash the cell pellets three times, and place in culture on collagen-coated T25 flasks in
the presence of Swiss mouse 3T3 feeders (approx 1 × 10
4
cells/cm
2
) at 37°C in a 5% CO
2

in air incubator (13). In some situations, 3T3-conditioned medium (Subheading 2., item
5d) can be used instead of adding mouse 3T3 cells directly to cultures (see Note 5).
3.3. Long-Term Culture of Adenoma Cell Lines (
see
Note 8)
1. Prepare culture conditions for adenoma cell lines as previously described for primary
cultures.
2. Carry out passage of adenoma cells as clumps of cells using sufficient dispase just to
cover the cells, and incubate for approx 30 min at 37°C. Remove the cells as a sheet, and
pipet with a Pasteur pipet to remove them from the flask and to break up the sheets into
smaller clumps of cells (13).
3. Wash the clumps of cells and replate under standard culture conditions. Reattachment of
cells may take several days, and during medium changing, any floating clumps of cells
must be centrifuged and replated with fresh medium.
3.4. Long-Term Culture of Clonal Carcinoma Cell Lines, Including
LS174T, Caco-2, and A431 Cell Lines (
see
Notes 8–10)
1. Grow the carcinoma cell lines in tissue culture plastics without collagen coating and 3T3 feeders
in DMEM supplemented with 10% FBS and 1 mM of glutamine (Subheading 2., item 5e).
2. Carry out passage as single cells using 0.1% trypsin in 0.1% EDTA (see Notes 9 and 10).
3.5. Apoptotic and Differentiating Cells
3.5.1. Isolation and Identification of Apoptotic and Differentiating Cells
(
see
Note 11)
1. During routine culture of cell lines, remove floating cells with the medium and pellet by
centrifugation. Most cell lines give rise to floating cells, the majority of which undergo
apoptosis.
Biosynthetic Cell and Organ Culture Methods 223

2. Stain the cells with acridine orange at 5 µg/mL in PBS, which stains the DNA and allows
visualization of the condensed chromatin of apoptotic cells. Stain cells for 10 min and
then observe with a fluorescent microscope using narrow-band FITC excitation (excita-
tion wavelength, 450–490 nm; and barrier filter, 520–560 nm). Count at least 300 cells.
3. Extract DNA from 10
6
cells and electrophorese on 2% (w/v) agarose gel containing 0.1
mg/mL of ethidium bromide at 40 V until the dye front has migrated 3 to 4 cm. Run the
DNA from an equivalent number of attached cells as a control, and use dexamethasone
treated mouse thymocytes as a positive control for DNA laddering (see Subheading 3.5.2.
and Note 11).
3.5.2. Preparation of Thymocyte Cell Cultures (
see
Note 11)
1. Sacrifice MF-1 mice (Subheading 2., item 8) at 2–3 mo, dissect out the thymus and
release thymocytes by pressing through sterile gauze.
2. Suspend the thymocytes in thymocyte culture medium (Subheading 2., item 5f) at a
density of 10
6
/mL and treate with 10
–7
M of dexamethasone.
3. Collect samples after 18 h incubation with dexamethasone.
3.6. Organ Culture of GI Tissue
3.6.1. Grid Cultures
(6)
(
see
Note 2)
1. Place the biopsies or explants, singly or up to six per dish, on lens tissue placed over a

stainless steel grid in culture dishes with a central well containing 2 mL of medium (Sub-
heading 2., item 11a). The orientation of the tissue is with the luminal surface uppermost.
2. Place dishes in an incubation oven connected with a continuous supply of carbogen gas
(Subheading 2., item 12) at 37°C.
3.6.2. Submerged Cultures
(14,15)
(
see
Notes 2, 12–15)
1. Place the biopsy or explants, up to a maximum of six, in a small, airtight capped tube in
100 µL of EMEM (Subheading 2., item 11b) (see Notes 16 and 17).
2. Blow carbogen gas into the tube and seal the carbogen gas atmosphere by replacing the cap.
3. Place the tube in a 37°C water bath.
4. Notes
1. The production and secretion of mucins by cultured colonic cells should be examined using
cells at different stages of confluency, because the stages of growth may alter the differen-
tiation properties of the cells and therefore the amount and type of mucin produced.
2. When using organ and primary cultures, it is important to consider the heterogenous nature
of the cell types in the tissue (i.e., stromal elements and lymphoid cells in addition to the
colonic epithelial cells). It may not be clear which cell type is producing the glycopro-
teins. Identification of specific cell-located glycoprotein expression can be further exam-
ined using histological methods with chemical, lectin, or antibody stains or in situ
hybridization to identify the cellular origin of the mucin of interest (Chapters 3 and 27).
3. The primary culture techniques described can be used for normal adult colon. However,
these are not as reproducible as those with the adenomas and carcinomas, and there are
more problems from contaminating stromal elements. There are at present no normal
adult colonic epithelial cell lines, only adenoma (1) and carcinoma cell lines such as PC/
JW/F1 (13), HT29 (10), LS174T (goblet cell like), and Caco-2 (enterocyte like) (11,12).
4. Collagen-coated flasks are necessary to obtain efficient attachment of primary cultures
and some adenomas and carcinomas to the flasks, and to retain the optimum differenti-