Thyroid Tissue-Organotypic Culture
Using a New Approach for Overcoming
the Disadvantages of Conventional
The organ culture of thyroid tissue has been applied
to the studies of thyroid biology (Bussolati et al.
Cau et al.
, 1976; Young and Baker, 1982). However,
the conventional organ culture system can not
retain viable three-dimensional (3D) thyroid follicles
containing both thyrocytes and C cells for a term long
enough to investigate their biological behavior, as the
tissue becomes necrotic progressively. Although the
conventional method allows thyrocytes to grow out
only in a monolayer from the tissue periphery placed
on culture dishes, it cannot enable them to organize
and maintain 3D follicles due to the lack of a 3D microenvironment
of extracellular matrix (ECM) (Toda et al.
, 1996, 2001). Thyroid follicles in vivo
in an interfollicular ECM, supported by a
dense network of fenestrated capillaries (Fujita and
Murakami, 1974). This suggests that both ECM and
sufficient oxygen supply are important for the maintenance
of follicular structure and function. By simulating
this in vivo
microenvironment of follicles, we
have established a new organotypic culture using a 3D
collagen gel culture of thyroid tissue fragments with
improved oxygenation through air exposure (Toda et
al., 2002). This system maintains very well the 3D follicle
structures containing both thyrocytes and C cells
for more than 1 month. Furthermore, the new follicle
formation from preexisting follicles (mother folliclederived
folliculogenesis) takes place actively in the
peripheral zones of each tissue fragments in our
system (Toda et al.
, 2003). We herein describe a useful
tool for the long-term organ culture of thyroid tissue.
In relation to our method, we propose a new approach
to cell type-specific culture systems on the basis of in
vivo microenvironments of various cell types.
II. MATERIALS AND
Materials, reagents, and equipment are as follows:
(1) 6-month-old porcine or human thyroid, (2) Eagle's
MEM (EMEM, Cat. No. 05900, Nissui Pharmaceutical
Co., Ltd., Tokyo, Japan), (3) dispase I solution (bacterial
neutral protease, 1000 protease U/ml in EMEM,
Cat. No. GD 81020, Goudoh-Shusei, Tokyo, Japan), (4)
Ham's F12 (Cat. No. 05910, Nissui Pharmaceutical Co.,
Ltd.), (5) fetal bovine serum (FBS, Cat. No. F9423, Lot
No. 92K2301, Sigma Chemical Co., MO), (6) gentamicin
(Gentamicin, Shering-Plough Co., Ltd., Osaka,
Japan), (7) complete medium (Ham's F-12 medium
supplemented with 10% FBS and 50µg/ml gentamicin),
(8) acid-soluble type I collagen (Cellmatrix type
I-A, Nitta Gelatin Co. Ltd., Osaka, Japan), (9) reconstructive
buffer (2.2g NaHCO3
and 4.77g HEPES in
NaOH), (10) a special culture dish of
which the bottom is made with nitrocellulose membrane
(30 mm diameter, Millicell-CM, Cat. No. PICAM
3050, Millipore, Bedford, MA), (11) 90-mm-diameter bacterial dish (Cat. No. SH-2OS, Terumo Co., Ltd.,
Tokyo, Japan), and (12) stainless-steel mesh (840µm,
Cat. No. Testing Sieve 840, Ikemoto Rikakogyo Co.,
Ltd., Tokyo, Japan). All materials, reagents, and equipment
used in culture must be sterile.
A. Initial Preparation of Tissue for
the Organotypic Culture of the Thyroid
- For porcine thyroid, tissues must be kept in icecold
EMEM from the slaughterhouse to laboratory.
Wash the tissue three times with ice-cold EMEM and
remove connective and adipose tissues.
- For human thyroid, obtain tissues from surgical
materials with permission and follow the same procedures
as in step 1.
- For obtaining C cell-rich follicles, use tissue
derived from only the middle to upper third of the
lateral lobe of porcine and human thyroids, as C cells
are mainly restricted to this area.
B. Preparation of Culture Assembly for
- Mince thyroid tissue into small fragments (~2mm)
with sterile scissors.
- Transfer the tissue fragments (5g) into a 100-ml
beaker containing 50 ml dispase I solution and incubate
at 37 °C for 1 h.
- Remove the dispase I solution by filtrating the fluid
through mesh (840 µm).
- Further mince the tissue remaining on the mesh into
smaller pieces (~0.5 ram) with sterile scissors.
- Transfer the fragments into a 50ml-test tube, wash
the tissue fragments with ice-cold EMEM by pipetting,
and then centrifuge the tissue suspension at
186g for 5rain at room temperature. Repeat these
- The thyroid tissue fragments obtained as a pellet
after the final centrifugation are subjected to the following
In comparison with the conventional organ culture,
our culture system is characterized by the following
items. (1) Minced tissues are placed in a 3D collagen
gel. (2) They are supplied sufficient oxygen through air
exposure. The two conditions result in allowing the
tissue fragments to situate under a 3D air-liquid (A-L)
interface, but not under a submerged state. In this system, the tissues are kept moist and fed with the
culture medium that percolated by capillary action
from the medium-containing outer dish, through the
acellular layer, and into the cellular layer (Toda et al.
2000, 2002, 2003). Likewise, the culture cells can be
stimulated by various reagents added to the culture
medium of the outer dish. In addition, exposing the
cellular layer to various concentrations of oxygen
permits the embedded cells to be supplied those of
oxygen. Figure 1 illustrates our organotypic culture
|FIGURE 1 Scheme of thyroid tissue-organotypic culture system.
Minced tissues embedded in type I collagen gel (cellular layer) are
placed on the acellular gel (acellular layer) in the inner dish (1). The
inner dish (1) is put in the outer dish (2) with culture medium.
In this way, tissues in the cellular layer are localized under air
exposure-induced oxygenation. Tissues are kept moist and are fed
by culture medium that percolates by capillary action from the
medium-containing outer dish through the acellular layer and into
the cellular layer.
C. Analyses of Organotypic Culture Cells
- Prepare the collagen gel solution as follows
(Elsdale and Bard, 1972). First, mix 8 volumes of acidsoluble
type I collagen with 1 volume of 10× concentrated
Ham's F-12 medium by gently pipetting in a test
tube. Second, add I volume of reconstructive buffer to
the mixture and pipette it gently. Keep this mixture
- Place 2ml cold collagen gel solution in a special
dish containing the nitrocellulose membrane and
immediately warm the dish to 37 °C for at least 30min
in a 5% CO2 incubator for gel formation. The collagen
gel layer is called the acellular layer. Preparation of this
layer should be completed before the beginning the
steps in Section III,A.
- Pore 1 ml cold collagen solution into a test tube
containing the tissue fragments obtained as a pellet.
Then, gently and fully mix 1 ml cold collagen gel solution
with the tissue fragments (a total of 0.5g). The
initial amount of 5 g tissue results in the preparation of
10 culture dishes.
- Place 1ml collagen gel solution containing the
fragments on the acellular layer and immediately
warm the dish to 37°C for at least 30min in a 5% CO2 incubator to allow gel formation. The resultant overlayer
is the cellular layer. The culture dish prepared in
this way is referred to as the "inner" dish.
- After at least 30min, when the gel is fully firm,
place the inner dish in a larger "outer" dish (90mm in
diameter) containing 10ml complete medium.
- Place this culture assembly in a conventional
culture incubator, thereby exposing the cellular
layer to a humidified air atmosphere supplemented
with 5% CO2 at 37°C. In this way, the tissue fragments
are situated under a microenvironment that consists
of both type I collagen and air exposure-induced
oxygenation. We call this culture condition a 3D A-L
Culture cells can be observed by phase-contrast
microscopy. The collagen gel layer containing viable
cells is scraped easily from the culture assembly. The
layer can be treated similar to the various tissues
resected from the body and used for analyzing the cellular
behavior as follows. (1) The cellular layer gels are
fixed in 4% formalin, processed routinely, and embedded
in paraffin. The deparaffinized and frozen sections
are applied easily to histochemistry, immunohistochemistry,
and in situ
hybridization. (2) To examine
the fine structure of the cells, transmission electron
microscopy is carried out using cellular layer gels
fixed in 2.5% glutaraldehyde and prepared by a
standard method. (3) Biochemical and genetic analyses
of the cells can be carried out by the various
methods described in this volume.
D. Examples of Thyroid
|FIGURE 2 Histology of thyroid tissues (a) and
for calcitonin (b) in the organotypic
culture. (a) At 40 days
in culture, viable thyroid follicles
enclosed by thyrocytes contain
colloid substance in their
lumens (F). (b) At 30 days in culture,
consisting of both thyrocytes and C cells (arrowheads)
are clearly maintained. Scale bar: 100µm.
In this system, viable 3D follicles within thyroid
tissue fragments are maintained for more than 1
month. These follicle structures consist of thyrocytes
and C cells with their specific differentiation (Fig. 2).
In the tissue periphery, thyrocytes undergo actively
growth and mother follicle-derived thyroid folliculogenesis.
Likewise, isolated or clustered thyrocytes,
which were localized in the tissue periphery at the
starting time of the culture, reconstruct follicles. C cells
show no proliferative ability and cannot grow even
with the stimulation of various concentrations of free
calcium. Most endothelial cells of capillaries disappear
until 7 days in culture (for further details, see Toda et
al., 1990, 1992, 1993, 1997, 2002, 2003).
In relation to our culture system, it seems that the
time has come to reconsider conventional culture
methods in a cell type-specific way. The microenvironments
of many cell types of the body are subdivided
mainly into the following three types. The first
is that of parenchymal and stromal cell types of solid
organs, e.g., thyroid, adrenal, and liver. The second is
that of surface-lining cell types on which sufficient
liquid is not overlayed, e.g., those of the skin, cornea,
respiratory, and digestive tracts. The third is the
microenvironment of surface-lining cell types on
which enough fluid is overlayed, e.g., those of the
cardiovascular system and cerebral ventricle. With
respect to the first microenvironment, the following is
our notion regarding that of the in vivo
stroma by which various cell types are supported. The extravasucular space consists of ECM and tissue fluid
percolated from blood vessels. The tissue fluid blended
by nutrients and air molecules infiltrates into ECM and
results in formation of the moist stroma. The moist
microenvironment is different from the intravascular
one, which has the sufficient liquid of blood. Our
culture system seems to simulate simply the first
microenvironment of extravascular stroma (Toda et al.
2001, 2002, 2003). Thus, our system is suitable for culturing
various cell types other than surface-lining cell
On the basis of the in vivo
various cell types, we propose the following three
culture systems in a cell type-specific manner. (1) The
usual monolayer culture under a submerged condition
with enough medium is suitable for culturing the
surface-lining cell types of endothelial cells, ependymocytes,
and so on (Tokunaga et al.
, 1991). (2) The
A-L interface culture is useful for culturing the
surface-lining cell types of epidermis, cornea, respiratory,
and digestive tracts (Nishimura et al.
Yamada et al.
, 1999; Sugihara et al.
, 2001; Ootani et al.
2003). (3) A three-dimensional collagen gel culture
with an A-L interface is suitable for culturing
parenchymal and stromal cell types of solid organs
(Toda et al.
, 2000, 2002, 2003).
The organ culture method described in this article
is very simple and it is reproduced easily by beginners.
The only one pitfall regarding our method is as
follows: the collagenase solution should not be used to
loosen ECM of thyroid tissue because the collagen gel
layer is often digested by the remaining collagenase,
even after washing the tissue fragments three times.
Thus, dispase I solution is recommended.
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