Histotypic culture involves growing cell lines in a three-dimensional matrix at high density to form tissue-like structures. Common techniques include using gels, sponges, hollow fibers, spheroids, and rotating chambers to provide a 3D environment that allows cells to organize similarly to how they would in tissues. This allows for the study of processes like drug penetration and cell differentiation that are not possible with traditional 2D cultures. While histotypic cultures provide a model for certain tissue functions, they also face challenges like loss of cell differentiation over time.

1. Histotypic Culture
BHAVYASHREE P V
II M.SC BIOTECHNOLOGY
ALVA’S COLLEGE
MOODBIDRI

2. Three main types of techniques
• Organ culture- In this type of culture, the whole organs or small fragments of
the organs with their spatial and intrinsic properties are used in culture.
• Histotypic culture- The propagated cell lines grown in three dimensional
matrix to high density represent histotypic cultures.
• Organotypic cultures- Cells of different lineages are combined in
experimentally determined ratios and spatial relationships to recreate a
component of the organ under study.

3. Histotypic culture:
Growth and propagation of cell lines in three-dimensional matrix
to high cell density
It is possible to use dispersed monolayers to regenerate tissue
like structures

4. Commonly used techniques are:
Gel and sponge techniques
Hollow fibers
Spheroids
Rotating chamber systems
Immobilization of living cells in alginate
Filter well inserts
Cultures of neuronal aggregates

5. Gel and sponge techniques
• Leighton first demonstrated that both normal and
malignant cells penetrate cellulose sponge
• The gel or sponge are used which provide the matrix
for the morphogenesis and cell growth.
• The cell penetrates these gels and sponges while
growing.
• Collagen gel provides a matrix for the
morphogenesis of primitive epithelial structures.
• Many different types of cell can be shown to
penetrate such matrices and establish a tissuelike
histology
• The kidney epithelial cell line MDCK responds to
paracrine stimulation from fibroblasts by producing
tubular structures, but only in collagen gel

6. Matrigel.
• Matrigel is a commercial product derived from the
extracellular matrix of the Engelbreth–Holm–Swarm
(EHS) mouse sarcoma
• That has been used for coating plastic but can also be
used in gel form.
• It is composed of laminin, collagen, fibronectin, and
proteoglycans with a number of bound growth factors,
although it can be obtained in a growth factor depleted
form
• Used as a substrate for epithelial morphogenesis formation
of capillaries from endothelial cells and in the study of
malignant invasion.
• It’s a complex and not completely defined matrix and can
also inhibit some morphogenetic events, such as
hepatocyte growth factor (HGF)-induced tubulogenesis of
MDCK cells

7. Hollow fiber techniques
• Hollow fibers are used which helps in more
efficient nutrient and gas exchange
• In recent years, perfusion chambers with a
bed of plastic capillary fibers have been
developed to be used for histotypic type of
cultures
• The cells get attached to capillary fibers and
increase in cell density to form tissue like
structures

8. Contd…
• The fibers are gas- and nutrient permeable and support
cell growth on their outer surfaces.
• Medium, saturated with 5% CO2 in air, is pumped
through the centers of the capillaries, and cells are
added to the outer chamber surrounding the bundle of
fibers.
• The cells attach and grow on the outside of the
capillary fibers, fed by diffusion from the perfusate,
and can reach tissuelike cell densities.
• It’s an ideal system for studying the synthesis and
release of biopharmaceuticals and are now being
exploited on a semi-industrial scale
• Eg, in cultures, choriocarcinoma cells release more
human chorionic gonadotrophin than they would in
conventional monolayer culture and colonic carcinoma
cells produce elevated levels of CEA

9. Spheroids
• The re-associaton of dissociated cultured cells
leads to the formation of cluster of cells called
spheroids
• Similar to the reassembling of embryonic cells
into specialized structures
• Principle: spheroid cultures is that the cells in
heterotypic or homotypic aggregates have the
ability to sort themselves out and form groups
which form tissue like architecture
• The 3-D structure of spheroids allows the
experimental study aspects of drug penetration
and resistance to radiation or chemotherapy
that are dependent on intercellular contact.

10. Contd…
• The role of 3-D spatial configurations in gene expression in
cell populations and the assessment of cytotoxic treatment.
• Treatment end points include growth delay, determination
of the proportion of spheroids cured by treatment, and
colony formation in monolayer after disaggregation of
treated spheroids
• Use of spheroids to study the penetration of cytotoxic
drugs, antibodies, or other molecules used in targeted
therapy
• Application that is not possible in single-cell suspensions
or monolayer cultures. Spheroids have proved useful in the
study of cell killing by biologically targeted radionuclides

11. Rotating chamber systems
• Miniperm bioreactor:
• A conventional roller bottle in two-compartment
chambers.
• This kind of design is the MiniPERMTM, a two
compartment cylinder with cells in the smaller
compartment and medium in the larger, separated
by a semipermeable membrane.
• It is rotated to ensure mixing, and the medium can
be sparged or replaced without disturbing the cells
or product.
• E.g., monoclonal antibodies, the geometry of the
chamber and the slow rotation tend to favor
aggregate formation and enhance product
formation.

12. Rotatory cell culture system(RCCS)
• This bioreactor is available from Synthecon, Inc., as a disposable
RCCS unit, or a reusable STLV (slow turning lateral vessel).
• Cylindrical vessel of bioreactor is completely filled with cell
suspension, and then continuously rotated to maintain cells in a free
fall state.
• Very low shear stress forces
• Minimal contact with vessel wall
• Stimulated zero gravity
• Quick production of speroids
• Culture multiple cell types
• Produce more differentiated complex epithelial shape
• Intrigued by the concept of growing cells in microgravity, in the
1980s NASA
• Gas exchange occurs from the cell-containing cylinder through a
central silicone membrane
• When the rotation stops, the aggregates sediment and the medium
can be replaced.
• To determine the effects of microgravity on cells with application
to the space program this culture vessel has also provided a suitable
bioreactor for bulk culture of tissue engineering constructs

13. Immobilization of living cells in alginate
• Composed of alternating molecules of M and G, and
divalent cations bind strongly between separate G blocks
and initiate the formation of an extended alginate gel
network.
• At present, numerous cell types can be genetically
engineered to produce specific proteins of choice. By
encapsulating such cells in alginate, a valuable vehicle is
obtained for delivering specific recombinant proteins to
the organism.
• . Thus, such alginate ‘‘bioreactors’’ may have an important
therapeutic potential for the treatment of a number of
diseases, in which the alginate may prevent the
encapsulated cells from being destroyed by the immune
system.

14. Filter Well Inserts
• Filter well inserts are a commercialization of a filter-
based culture system the origins of which go back to
the 1950s and used in various forms since then.
• A filter substrate provides an environment for
studying cell interaction, stratification, polarization,
and tissue modeling
• Polarity and functional integrity can be established
as in thyroid ,intestinal and kidney, epithelium.
• Filter cultures allow generation of stratified
epidermis
• They have used them to study invasion by
granulocytes or malignant cells
• One of the major advantages of filter well inserts is
that they allow the recombination of cells at very
high, tissuelike densities, with ready access to
medium and gas exchange, but in a multireplicate
form.

15. • Filter well inserts available in a variety of
translucent or transparent materials, including
polycarbonate, PTFE, and polyethylene
teraphthalate, and ranging in size from 6.5 mm
to 9 cm, suitable for 24-well, 12-well, and 6-
well plates, or larger dish
• Filters can be obtained precoated with collagen,
laminin, fibronectin, or Matrigels.

16. Cultures of Neuronal Aggregates
• Aggregating cultures of fetal brain cells have been
extensively used to study neural cell differentiation
• The aggregating cells follow the same developmental
sequence as observed in vivo, leading to an organoid
structure consisting of mature neurons, astrocytes, and
oligodendrocytes. A prominent neuropil is also formed
• In tumor biology, the aggregates can be used to study
brain tumor cell invasion in vitro

17. Tissue Equivalents and Tissue Engineering

18. Advatages and disadvantages
• Advantages
Development of a cell line over several generations
Scale-up is possible
Absolute control of physical environment
Homogeneity of sample
Less compound needed then in animal models
• Disadvantages
 Cells may lose some differentiated characteristics
 Hard to maintain
 Only grow small amount of tissue at high cost
 Dedifferentiation
 Instability, aneuploidy

19. References
• R.Ian Freshney,(2005),Culture Of Animal Cells: A Manual Of Bacis
Techniques, 5th edition,Jhon Wiley & Sons,New Jersey,pp 436-450
• R.Ian Freshney,(2005),Culture Of Animal Cells: A Manual Of Bacis
Techniques, 6th edition,Jhon Wiley & Sons,New Jersey,pp 486-492

20. Gel
• Gel and sponge technique-