Clinical Science and Molecular Medicine (1976) 50, 425-429.
Biochemical and morphological studies on human
jejunal mucosa maintained in culture
C. L’HIRONDEL, W. F. D O E A N D T. J. PETERS
Royal Postgraduate Medical School, London
(Received 3 November 1975)
Laster, 1971;Falchuk, Gebhard, Sessoms & Strober,
1974). In our hands, this technique proved unreliable
and an alternative procedure was developed by
Mitchell, Mitchell &Peters (1974). In this technique,
fragments of tissue are maintained in culture with a
roller-tube technique. The procedure was found to be
more reliable, as judged by morphological criteria,
but only small fragments of tissue could be maintained in a viable form. Thus only limited biochemical and morphological studies could be performed.
The present paper describes a simplifiedtechnique
which combines these two procedures, enabling
culture of approximately 10 mg fragments of both
normal and pathological tissue to be performed. The
details of the technique, together with biochemical
and morphologicalstudies, are described. In particular, it has been shown that assay of tissue and
medium DNA concentrations provides a satisfactory reference for biochemical and enzymic
studies.
SOmmarY
1. The combination of a wire-mesh support with
the roller-tube technique is described as a procedure
for the culture of human jejunal mucosa in vitro.
2. The technique has been applied to fragments
(approximately 10 mg) of jejunal biopsies from both
normal subjects and patients with coeliac disease.
3. The cultured tissue has been shown by radioautography to incorporate [3H]leucineinto proteins
of the villus epithelial cells and [3H]thymidine into
nucleic acid, predominantly by the enteroblasts.
4. Although the tissue protein and DNA contents
fall during culture, it was found that the combined
tissue and medium DNA content remained constant
during culture and may be used as a reference for
enzyme and biochemical studies on cultured intestinal biopsies.
Key words: DNA synthesis, jejunum, protein synthesis, radioautography, tissue culture.
Introduction
Materials and methods
In 1969, Browning & Trier described a technique in
Culture technique
Equipment. Sterile polystyrene tubes (10 cm x 1.5
cm;Sterilin Ltd, Teddington, Middx., U.K.) were
placed horizontally in a rack rotating at 12 rev./h
inside an air-tight plastic box in a 37°C incubator.
pieoes of w i x mesh (22 gauge) covered with sterile
agar (2%, wlv, in 0-15 mol/l sodium chloride) were
wedged in the tubes (Fig. 1). The tubes were loosely
capped with 2.0 cmx2.3 cm caps (Oxoid Ltd,
London) and a constant flow of OzlCOz(95:5) was
passed through the box. The caps and wire mesh
which portions of human jejunal mucosa could be
maintained in culture for up to 48 h by use of a wiremesh support system. Good morphologicalpreservation of the tissue was obtained and this technique
has been used by Z. M. Falchuk and his colleaguesto
study biochemical and immunological phenomena
in tissues from normal subjects and from patients
with coeliac disease (Loeb, Strober, Falchuk &
Correspondence:Dr T.J. Peters, Department of Medicine,
Royal Postgraduate Medical School, Du Cane Road, London
W12 OHS.
425
426
:::gm;
C. L'Hirondel, W.F. Doe and T. J. Peters
(covered with
Test tube
wall tube
\
\\
rev./h
FIG.1. Diagrammatic section of the rotating culture tube.
The mesh covered with 2% agar is wedged into the tube. The
biopsy is placed on the medium-moistened mesh, villi
uppermost. The tube is rotated at 12 rev./h, ensuring adequate
oxygenation of the tissue and medium.
were sterilized by heating at 160°C for 1 h before use.
Culture media were sterilized by Millipore (0.22 pm)
filtration.
Media. Trowell's medium, as modified by Jos,
Lenoir, de Ritis & Rey (1975), containing (by vol.)
65% Trowel1 T8 medium, 20% NCTC 135 medium
and 15% foetal calf serum, was used. All these
reagents were purchased from Gibco Laboratories,
Paisley, Scotland. Penicillin, 100 i.u./ml, streptomycin sulphate, 100 pglml, and amphotericin B, 0.5
pglml, were added to the medium. A peptic-tryptic
digest of human serum albumin (0.5 mg/ml) as
recommended by Jos et al. (1975)was also included
in the culture medium. This latter was prepared by
dissolving 1 g of serum albumin (Sigma Chemical
Co., London) with 20 mg of pepsin (1 :60 OOO: Sigma)
in 40 ml of water. The pH of the mixture was then
adjusted to 1.8 by addition of hydrochloric acid
(0.1 mol/l). After incubation at 37°C for 16 h, the
pH was adjusted to 7.8 with sodium hydroxide (0.1
mol/l). Trypsin (type I, Sigma), 10 mg, and 10 mg of
chymotrypsin (type I, Sigma) were added and the
mixture was then incubated for a further 16 h. The
pH was adjusted to 7.0and, after heating at 100°C
for 1 h, the mixture was sterilized by Millipore (0-22
pm) filtration.
Procedure. Biopsy specimens were taken from the
proximal jejunum under fluoroscopic control with
either a Crosby or a multiple biopsy capsule. Speci.
mens were obtained from eleven control subjects and
five patients with untreated coeliac disease. Informed
consent was obtained from all subjects. After collection the biopsy was placed in a small volume of
medium in a sterile culture dish and, under a dissecting microscope, cut into small fragments approximately 2 mm3. Representative fragments were fixedin buffered formalin and processed for routine
histology. The fragments for culture were placed,
villi uppermost, on the agar-covered wire mesh and
placed in the roller tubes with 1 ml of medium. The
tubes were placed in a glass bottle, gassed with
O,/CO, (95:5,v/v) for 10 min and rapidly transferred to the 37°C incubator. The time between
firing the capsule and placing the tissue in the culture
tubes was less than 5 min. The initial gas flow in the
incubator was 20 l/min and after 15 min was reduced
to 60 ml/min for the remaining period of culture.
After culture the fragments were processed for
morphological and for biochemical studies. The
fragments were washed vigorously in 1 ml of sucrose
(0.3 mol/l) containing disodium EDTA (1 mmol/l,
pH 7.4) and ethanol (22 mmol/l) and then homogenized in 2 ml of sucrose medium with a tightfitting Dounce homogenizer (Peters, Heath, Wansbrough-Jones & Doe, 1975). The sucrose medium
wash was added to the culture medium for biochemical analyses.
Radioautographic studies
Deoxyribonucleic acid (DNA). Addition of 10
pCi of [3H]methylthymidine (30 Cilmol, The
Radiochemical Centre, Amersharn, Bucks., U.K.)
was made to the culture medium. After 17 h culture
the tissue was transferred to isotope-free medium
for a further 7 h. The tissue was fixed with 10%
formalin in sodium phosphate buffer, pH 7-0 (0-1
mol/l). After embedding, 5 pm sections were cut and
covered with Kodak A.R. 10 stripping film. After 4
weeks' exposure, the slides were processed with
Kodak D19 developer and with Fixol and then were
stained with Haematoxylin-Eosin.
Protein. Addition of 2 pCi of [4,5-3H]leucine (38
mCi/mmol, The Radiochemical Centre) was added
to the medium and after 23 h culture the biopsy was
transferred to isotope-free medium for a further 1 h.
After culture the tissue was processed as above.
Biochemical determinations
Protein. This was determined by a micro-modification of the method of Lowry, Rosebrough, Farr &
Randall (1951)with bovine serum albumin (Arrnour
Pharmaceuticals) as standard.
Cultured human jejunal mucosa
FIG. 2. Section of cultured control jejunal biopsy. Good morphological preservation of the tissue is apparent
although there is some blunting of the villi ( x 1050).
Fro. 3. Section of cultured coeliac mucosa obtained by jejunal biopsy. The essential features of the lesion with
villus atrophy are preserved. The brush border region of the enterocytes is more distinct than in uncultured biopsies
( x 1200).
FIG.4. Radioautograph of cultured control tissue prepared after incubation with tritiated leucine. Most intense
labelling is found in the enterocytes with similar staining of both crypt and villus-tip cells ( X 1050).
(Facing p. 426)
C. L’Hirondel, W. F. Doe and T.J . Peters
FIG.5 . Radioautograph of cultured control tissue prepared after incubation with tritiated thymidine for 17 11.
Only cells of the crypt region show significant incorporation into nuclei ( x 1050).
FIG.6. Radioautograph of cultured coeliac biopsy prepared after incubation with tritiated thymidine. There a re
increased numbers of labelled mitoses compared with the control tissue and the labelled cells have migrated
further u p the crypts. Note that no significant labelling of the interstitial cells has occurred ( x 1050).
(facingp. 427)
427
Cultured human jejural mucosa
DNA. This was determined by the fluorimetric
method of Le Pecq & Paoletti (1966), ribonuclease
(type IIIa, Sigma) being used to hydrolyse the
ribonucleic acid. Calf thymus DNA (Koch-Li&t
Laboratories Ltd, Colnbrook, Bucks., U.K.) was
used as standard. All estimations were carried out in
duplicate. Suitable blanks and standards were used
with each batch of assays.
ReSdtS
I
T
I
Medium DNA
0
I
I
I
I
3
6
12
24
Time(h)
Morphological studies
Eighty-seven biopsy fragments of mucosa (sixty
from normal persons and twenty-seven from untreated coeliac patients) were examined under the
dissecting microscope after culture. In every case,
there was good preservation of the biopsy specimen
after 24 h culture. In normal mucosa, the villi
tended to be broader and reduced in height but, with
coeliac mucosa, the appearance was unchanged
during culture.
Light-microscopy performed on five cultured
normal jejunal biopsies (Fig. 2) showed normal
architecture with some blunting of villi; the lamina
propria contained numerous irnmunocytes, the
crypts were well preserved without necrosis, the
enterocytes were normal and some goblet cells were
seen.In biopsies from patients with untreated coeliac
disease, showing villus atrophy (Fig. 3), the appearance after culture was similar to that before culture,
with absence of villi, hypercellular crypt regions and
nuclear irregularities in the enterocytes. There did,
however, appear to be a more prominent brush
border region in the enterocytes after culture.
FIG. 7. DNA content of tissue and of medium in cultured
jejunal biopsy fragments from both control and coeliac
mucosa. Results show mean values+ SE of thirteen determinations at each time-point.
DNA content
The DNA content of the biopsies and of the
cultured medium was assayed in fifty-two specimens
from nine subjects (five control subjects and four
patients with coeliac disease). Fig. 7 shows the
changes in the absoluteamount of DNA in the tissue,
the culture medium and the tissue plus medium
during the 24 h period.
In the tissue there was a 25% decrease of tissue
DNA during the first 3 h. This continued more
slowly throughout the culture period. In the medium,
there was a significant increase in DNA during the
culture period. This was greatest during the first 3 h,
corresponding to the period of most marked loss
from the tissue. The total DNA in tissue and medium
remained constant during culture.
Protein content
Radioautography
Radioautographs were prepared after incubation
of three control biopsies with [aH]leucine(Fig. 4).
The enterocytes showed evidence of intense protein
synthesis but the immunocytes and interstitial cells
showed less intensity of labelling than the epithelial
cells. DNA synthesis was studied by incubation of
two normal biopsies with [3H]thymidine. Fig. 5
shows that several crypt cells were labelled without
any incorporation into the immunocytes or interstitial cells. Similar studies in cultured coeliac tissue
(Fig. 6) showed increased nuclear labelling in the
crypt$ with labelled cells emerging from the crypt
Orifiws.
Table 1 shows the protein content of biopsy
fragments from four control subjects and four
TABLE1. Protein content of cultured
control and coeliac biopsies
Mean values+ SEM are shown. Thirteen
biopsies were assayed at each time.
Time (h)
0
3
6
24
Protein (jig)
290+ 60
274k 70
255+ 50
200+ 30
428
C. L’Hirondel, W. F. Doe and T. J. Peters
untreated coeliac patients during various periods of
culture. There is a progressive and approximately
linear loss of protein throughout the period of
culture so that by 24 h approximately one-third of the
protein has been lost.
Discussion
The technique described in detail in this paper for the
culture in oitro of jejunal mucosa from normal subjects or patients with untreated coeliac disease
combines the static culture technique of Browning &
Trier (1 969) with the roller-tube technique of
Mitchell et al. (1974), in that the tissue, supported by
wire mesh, is placed with culture medium in rotating
tubes. The procedure for tissue preparation has been
simplified and larger fragments of tissue can be
satisfactorily maintained in culture as judged by
morphological criteria.
It is essential to maintain full oxygenation of the
tissue and medium both before and during the culture
procedure (Trowell, 1959). Careful preparation of
the medium is also essential; this was done daily and
batches of reagents were used within 1 month of
purchase. The complex washing procedure for the
tissue on albumin gradients (Mitchell et al., 1974)
was abandoned in favour of careful sterilization of
the medium, use of antibiotics and sterile manipulations of the tissue. The volume of culture medium
(1 ml) was not critical as satisfactory results as judged
by morphological criteria were obtained over the
range 0.7-1.4 ml, and thus sampling from and additions to the medium can be made during culture.
The protein content of the tissue decreased by
approximately one third during 23 h culture. This is
less than the one-half of the decrease noted by
Mitchell et al. (1974) but the use of protein as a
reference for changes in tissue activities is not
satisfactory, as discussed previously (Mitchell et al.,
1974). The reason for the loss of DNA and protein
into the medium, particularly during the first 3 h of
culture, is uncertain. It may in part be explained by
trauma to the tissue duringcollectionand preparation
of the biopsy. The observation that the total DNA
in the culture tube (tissue plus medium) remains
constant throughout the 24 h culture period,
although the tissuelmedium distribution ratio alters,
indicates that total DNA is a suitable reference for
biochemical change during culture.
Morphological studies at the dissecting and light-
microscope level have all shown excellent preservation of tissue structure over a 24 h culture period.
The radioautographic studies with labelled protein
and DNA precursors indicate that complex biosynthetic abilities of the tissue are preserved. The
experiments with labelled leucine indicate that both
enterocytes and crypt cells (enteroblasts) are sites of
intense protein synthesis compared with the interstitial cells. This finding suggests that incorporation
of labelled leucine into tissue protein in the cultured
biopsies largely reflects protein synthesis by the
enterocytes and enteroblasts. The brush border
region of the enterocytes appears to be strongly
labelled, but whether it indicates a particularly high
turnover of brush border membrane is uncertain.
The radioautographic studies with labelled thymidine indicate, as found by Browning & Trier (1969),
that the cells of the crypt region are the most active
in DNA synthesis. Very few interstitial cells are
labelled, indicating that in quantitative terms
measurements of tritiated thymidine incorporation
into DNA in whole biopsies reflects essentially
enteroblast proliferation. In radioautographs prepared from cultured biopsies from patients with
untreated coeliac disease compared with control
tissue there are increased numbers of labelled nuclei
in the crypt region. Similar results were obtained by
Trier & Browning (1970). Biochemical studies (P. E.
Jones & T. J. Peters, unpublished work) have also
shown strikingly enhanced incorporation of tritiated
thymidine into DNA in cultured coeliac mucosa
compared with control tissue, Thus jejunal mucosa
cultured by the improved roller-tube technique
maintains its integrity and displays the usual
parameters used to characterize previous culture
techniques. In future publications detailed biochemical studies of the synthesis rates for protein,
DNA and glycoprotein by cultured normal and
coeliac mucosa will be reported.
Acknowledgments
We thank Professor C. C. Booth for his enthusiastic
interest and encouragement. We are grateful to Dr
P. E. Jones for his helpful suggestions and to Ms
Jean de Luca for typing the manuscript. The help of
Mr W. Hinks and Ms Y.E. Maddison in the morphological studies is gratefully acknowledged. This
work is supported by The Wellcome Trust and the
Medical Research Council.
Cultured human jejunal mucosa
References
BROWNING,
T.H. & TRIER,J.S. (1969) Organ culture of
mucosal biopsies of human small intestine. Journal of
Clinical Investigation, 48, 1423-1432.
FALCHUK,
Z.M., GEBHARD,
R.L., SESSOMS,C. & STROBER,
W.
(1974) An in vitro model of gluten-sensitive enteropathy.
Effect of gliadin on intestinal epithelial cells of patients
with gluten-sensitiveenteropathy in organ culture. Journal
of Clinical Investigation, 53,487-500.
Jos, J., LENOIR,G., DE RITIS,G. & REY,J. (1975) In vifro
pathogenetic studies of coeliac disease. Scandinavian
Journal of Gastroenterology, 10, 121-128.
LE PECQ,J-B. & PA OLE TI^. C. (1966) A new fluonmetric
method for RNA and DNA determination. Analytical
Biochemistry, 17, 100-107.
LOEB,P.M., STROBER,
W., F A L C H ~ K
Z.M.
,
& LASTER,L.
(1971) Incorporation of L-leucine I4C into immunoglobulins by jejunal biopsies of patients with coeliac sprue
429
and other gastrointestinal diseases. Journal of Clinical
Investigation, SO, 559-569.
LOWRY,O.H., ROSEBROUGH,
N.J., FARR,A.L. & RANDALL,
R.J. (1951) Protein measurement with the Folin-phenol
reagent. Journal of Biological Chemistry, 193, 265-275.
MITCHELL,
J.D., MITCHELL,
J. & PETERS,
T.J. (1974) Enzyme
changes in human small bowel mucosa during culture in
vitro. Gut, 15, 805-8 1 1.
PETERS,T.J., HEATH,J.R., WANSBROUGH-JONES,
M.H. &
DOE, W.F. (1975) Enzyme activities and properties of
lysosomes and brush borders in jejunal biopsies from
control subjects and patients with coeliac disease. Clinical
Science and Molecular Medicine, 48, 259-267.
TRIER,
J.S. & BROWNING,T.H. (1970) Epithelial cell renewal
in cultured duodenal biopsies in coeliac sprue. New England
Journal of Medicine, 283, 1245-1250.
TROWELL,
D.A. (1959) The culture of mature organs in a
synthetic medium. Experimental Cell Research, 16, 118147.