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The organotypic model consists of three approaches for the original structural
and functional interactive relationships of the organ.
The three approaches are: (1) Organ Cultures (2) Histotypic Cultures and (3)
Organotypic Cultures.

The cell cultures are widely used in the laboratories world over for various
purposes. In vitro studies with isolated cells are useful for understanding of many
cell functions such as transcription, translation, cell proliferation, respiration and
glycolysis. Thus for the study of biology and many functions, the cells grown in
conventional and monolayer cultures may be adequate.

However, for the study of integrated cellular functions or organ

functions, isolated cells will be not be of much use, as explained
below:

Cellular Interactions in Organ Functions:

content is associated with the risk of O2induced toxicity e.g. nutrient metabolite
exchange is severely affected.
Growth and Differentiation:
In general, the organ cultures do not grow except some amount of proliferation
that may occur on the outer cell layers.

Advantages of Organ Cultures:

i. Provide a direct means of studying the behaviour of an integrated tissue in the
laboratory.

ii. Understanding of biochemical and molecular functions of an organ/tissue

becomes easy.

Limitations of Organ Cultures:

i. Organ cultures cannot be propagated, hence for each experiment there is a need
for a fresh organ from a donor.

ii. Variations are high and reproducibility is low.

iii. Difficult to prepare, besides being expensive.

Techniques of Organ Culture:

The most important requirement of organ or tissue culture is to place them at
such a location so that optimal nutrient and gas exchanges occur.

This is mostly achieved by keeping the tissue at gas- limited interface

of the following supports:
i. Semisolid gel of agar.

ii. Clotted plasma.

iii. Micro-porous filter.

iv. Lens paper.

v. Strip of Perspex or Plexiglas.

In recent years, filter-well inserts are in use to attain the natural geometry of
tissues more easily.

Procedure for Organ Culture:

The basic technique of organ culture consists of the following stages:
1. Dissection and collection of the organ tissue.

2. Reduce the size of the tissue as desired, preferably to less than I mm in

thickness.

3. Place tissue on a support at the gas medium interface.

4. Incubate in a humid CO2 incubator.

5. Change the medium (M199 or CMRL 1066) as frequently as desired.

6. The organ culture can be analysed by histology, autoradiography and

immunochemistry.

Organ Culture on Stainless Steel Support Grid:

Small fragments of tissue can be cultured on a filter laid on top of a stainless steel
grid (Fig. 40.1).

Organ Culture on Filter-well Inserts:

Filter-well inserts have become very popular for organ cultures. This is mainly
because the cellular interaction, stratification and polarization are better in these
culture systems. Further, the recombination of cells to form tissue like
densities, and access to medium and gas exchange are better.

The four different types of filter wells for growing tissues in the form of cell layers
are depicted in Fig. 40.2.
i. Growth of cell layer on top of filter (Fig. 40.2A).

ii. Growth of cell layers on matrix (collagen or matrigel) on top of filter (Fig.
40.2B).

iii. Cell layers grown on the interactive cell layers placed on the underside of filter
(Fig. 40.2C).

iv. Cell layer grown on the matrix with interactive cell layer on the underside of
the filter (Fig. 40.2D).

Filter well-inserts with different materials (ceramic, collagen, and nitrocellulose)

are now commercially available for use in culture laboratories.

Filter-well inserts have been successfully used to develop functionally integrated

thyroid epithelium, stratified epidermis, intestinal epithelium and renal (kidney)
epithelium.

2. Histotypic Cultures:
Growth and propagation of cell lines in three- dimensional matrix to high cell
density represent histotypic cultures. The advantage with this culture system is
that dispersed monolayer cultures can be used to regenerate tissue-like
structures. The commonly used techniques in histotypic cultures use gel and
sponge hollow fibers and spheroids.

Gel and Sponge Technique:

The cells (normal or tumor) in culture can penetrate gels (collagen) or sponges
(gelatin) which provides a matrix for morphogenesis of primitive cells. This
approach has been used for the development of mammary epithelium, and some
tubular and glandular structures.

Hollow Fibers Technique:

In recent years, perfusion chambers with a bed of plastic capillary fibers have
been developed. The advantage of using hollow fibers in histotypic cultures is that
nutrient and gas exchange is more efficient. As the cells attached to capillary
fibers grow, there occurs an increase in cell density to form tissue-like structures.

Many workers claim that the behaviour of high-density cells formed on hollow
fibers is comparable to their in vivo behaviour. For instance, choriocarcinoma
cells grown in hollow fiber cultures release more chorionic gonadotrophin than in
a conventional monolayer. Hollow fiber culture techniques are regarded as ideal
systems for the industrial production of several biologically important
compounds. Work is progressing in this direction.

Three Dimensional Cultures:

Spheroids in Histotypic Culture:
Spheroids represent the clusters of cells usually formed by the re-association of
dissociated cultured cells. It is known for some years that the dissociated
embryonic cells reassemble to form a specialized structure. The basic principle of
using spheroids in histotypic culture is that the cells in heterotypic or bomotypic
aggregates are capable of sorting out themselves into groups to form tissue-like
architecture. The major drawback of spheroids is the limitation in the diffusion
and exchange of nutrients and gases.

Multicellular Tumor Spheroids (MCTS):

Multicellular tumor spheroids provide an in vitro proliferating model for studies
on tumor cells. The three dimensional structure of MCTS allows the experimental
studies related to drug therapy, penetration of drugs, resistance to radiation etc.

Further, MCTS have also been used to study several biological

processes:
i. Regulation of cell proliferation and differentiation.

ii. Immune responses.

iii. Cell death.

iv. Cell invasion.

v. Gene therapy.

The main advantage of three dimensional cell cultures (in the form of MCTS) is
that they provide a well-defined geometry of cells planar or spherical which is
directly related to the structure and function. It is now well accepted that the
MCTS behave like the initial avascular stages of solid tumors in vivo. However,
beyond a critical size ( 500 mm), most of the MCTS develop necrosis (death of
cells) at the centre surrounded by viable cells. A diagrammatic representation of
MCTS in comparison with tumor is depicted in Fig. 40.3.
Technique of MCTS production:
Single-cell suspension obtained from trypsinized monolayer cells or
disaggregated tumor is inoculated into the medium in magnetic stirrer flasks or
roller tubes. As the incubation is carried out for about 3-5 days, aggregates of
cells representing spheroids are formed. It is observed that spheroid formation is
more efficient under static conditions on stationary and non-adhesive surfaces.
For this reason, agar/agarose-coated culture dishes to which cells do not adhere
are frequently used to initiate spheroid formation.

Once the spheroids are formed, they are transferred to 24 well plates for analysis.
Spheroid growth is quantified by measuring their diameters regularly. This can
be done by using a microscope eyepiece micrometer or an image analysis
scanner. Good growth of spheroids is observed when grown in wells.

Transfectant mosaic spheroids:

It is now possible to produce spheroids from cells that have been transfected with
different genes. Mosaic spheroids are formed by mixing transfected and non-
transfected spheroids in the desired proportion.

MCTS co-cultures:
MCTS can be produced from heterogenous cells also, forming MCTS co- cultures.
This is comparable to heterologous spheroids (in short heterospheroids)
consisting of tumor cells in combination with host cells.

Some of the MCTS co-cultures are listed:

i. MCTS and immune cells.

ii. MCTS and fibroblasts.

iii. MCTS and endothelial cells.

Heterospheroids with heterotypic cell interaction serve as good models for

studying several in vivo processes e.g. inflammation. MCTS co-cultures are very
useful in tissue modelling and tissue engineering, the details of which are given
later.

Applications of Spheroids or MCTS:

Spheroids have a wide range of applications. Some of the important
ones are listed:
i. Serve as models for a vascular tumor growth.

ii. For the study of gene expression in a three dimensional configuration of cells.

iii. To determine the effect cytotoxic drugs, antibodies, radio nucleotides used for
therapeutic purposes.

iv. To study certain disease processes e.g. rheumatoid arthritis.

v. For the development of gene therapies for several diseases e.g. cancer.

vi. To evaluate radiation effects on target tissues.

vii. For the development of tissues and tissue models.

3. Organotypic Cultures:
Organotypic culture basically involves the combination of cells from different
lineages in a determined ratio to create a component of an organ. With the
advances in the organotypic culture techniques, it is now possible to develop
certain tissues or tissue models.

i. Skin equivalents have been created by co-culturing dermis with epidermis with
interviewing layers of collagen.

ii. Models for prostate and breast.

iii. Models for control of growth and differentiation of lung.