This document discusses the culture of specialized epithelial cells. It describes various types of epithelial cells and their functions. It discusses challenges in culturing epithelial cells, such as removing contaminating fibroblasts. Various methods are described for obtaining pure epithelial cell cultures, including selective attachment, feeder layers, selective media, cloning, and physical separation techniques. Characterization of epithelial cell cultures involves assessing markers like cytokeratins and junctional complexes. The document also discusses culturing of mammary epithelial cells and tumor cells from breast tissue, as well as mesenchymal stem cells from bone marrow.

1. Culture of specialised cells
Dr. Radhakrishna G Pillai
Department of Life Sciences
University of Calicut

2. Epithelial cells
• Various layers of cells that either
– coat surfaces on the exterior of the organism or
– line internal organs, ducts, or secretary acini
• They may act as a total barrier, such as
– the epidermis, with minimum permeation of polar
substances, or
– a regulated barrier
• for example, in the intestine and the lung, where
• selected substances are able to cross the plasma membrane
or the whole epithelium via specific transporters
• Basal to apical polarization is fundamental to the
normal function of all epithelia.

3. Epithelial cells
• Epithelia are associated with the major functional role
of many tissues, such as
– hepatocytes and liver metabolism
– epidermal keratinocytes and the barrier properties of skin
– pancreatic acinar cells and digestive enzyme secretion
• have been a focus of interest in the development of in
vitro models for many years
• Because most epithelia are renewable;
– they have proliferating precursor compartments and stem
cells capable of self-renewal and
– hence form attractive models for studying the regulation of
cell proliferation and differentiation

4. Epithelial cells- cancer
• Regenerative nature makes epithelial cells sites for
malignant transformation in vivo, and
• the most common solid tumors are the
– carcinomas of lung, breast, colon, prostate, and bladder,
– derived from the epithelial cells of these tissues
• Epithelial cell systems have therefore been adopted as
appropriate models for studies of
– Carcinogenesis and
– Differentiation
– on the assumption that malignancy results, at least in part, from
a failure to differentiate
• This has produced many excellent models for differentiation
and,
• some of the clearest examples of stem cell maturation,

5. Epithelial cells -Histology
• Epithelial cell layers are separated from other cellular compartments (e.g.,
connective tissue, capillaries) by a basement membrane made up of
collagen, laminin, fibronectin, and proteoglycans, and the reconstitution
of this basement membrane in vitro has been featured in many attempts
to grow functional epithelium
The basement membrane is
usually a joint product of the
epithelium and the underlying
stroma and, together with soluble
factors from the stroma, serves to
regulate the differentiated
function of the epithelium as
well as providing physical support
and a barrier separating epithelial
and stromal compartments

6. Functions of epithelial cells
• Epithelial cells are closely associated in vivo
• regulated permeability and transport functions; to maintain this structural
integrity,
• they are usually joined by desmosomes -the mechanical junctions connected to
the intermediate filament cytoskeleton that hold epithelium together and are
characteristic markers of epithelial identity
• Where barrier properties are particularly crucial (e.g., kidney ductal epithelium,
or secretory acini), the desmosomes are accompanied by tight junctions forming
a junctional complex that is quite specific to epithelium
• The presence of these junctional complexes, together with cytokeratin
intermediate filaments provide very useful and specific markers for recognizing
epithelial cells in vitro

7. Culturing epithelial cells
• As epithelial layers are closely associated in vivo and are strongly
self-adherent
• they tend to survive better in vitro as clusters or sheets of cells
• Dissociation techniques that have been found to be most
successful tend to exploit this observation and do not try to reduce
the population to a single cell suspension
• For this reason, cultures have been derived either by gentle
mechanical disaggregation or collagenase digestion in preference
to trypsinization
• Collagenase appears to give good survival as it does not
completely dissociate the epithelium but frees it from the
surrounding stroma
• The clusters of epithelium formed by this technique can be
collected by
– allowing them to sediment through the more finely dispersed
fibroblastic stromal cells or
– by filtration through nylon gauze

8. Removing fibroblasts
• it is very difficult to eliminate all the fibroblasts by
physical methods
– more rapid growth rate
– they will tend to overgrow the culture
• This is caused by the stimulation of the fibroblasts by
• platelet-derived growth factor (PDGF) and
• by the cytostatic effect on the epithelium of
transforming growth factor type (TGF-), released from
platelets during the preparation of serum.
• It is also due partly to the design of the media, many of
which were developed for fibroblastic cells

9. Removing fibroblasts
Selective Attachment
• seed the cell mixture into a flask for a short time (e.g., 30 min
or 1 h) and then transfer the unattached cells into a fresh
flask
• This is repeated at intervals of 1–3 h up to 24 h or 48 h
• it is often found that the fibroblasts tend to stick down first,
whereas the epithelial cells remain in suspension and attach
in the later-seeded flasks
• This is probably due to a combination of circumstances
– some mechanical (the size of the epithelial aggregates) and
– some physiologic (the greater need for extracellular matrix
regeneration for epithelial attachment)
• selective adhesion methods such as these have limited
success but may be of value in combination with other
techniques or for short periods of culture

10. Removal of fibroblasts
• Selective Detachment
• enzymes such as dispase can release epithelial
sheets before the fibroblasts [
• like selective attachment
– it is effective in certain conditions
– for example, releasing epithelial patches of colonic
epithelial cells
– it is not universally successful and may select or alter the
epithelium released
• EDTA can also be used to remove fibroblasts
selectively from mixed cultures of keratinocytes

11. Removing contaminating fibroblasts
• Substrate Modification
• To exploit the principle of selective attachment, some groups
have attempted to modify the substrate to favor epithelial
attachment
• Collagen coating, particularly native or undenatured collagen,
has been used to select epidermal cells and breast epithelium
• Collagen and laminin separately or combined, particularly in
Matrigel, have also been found to encourage the expression
of the differentiated phenotype in many epithelial cells
although this may limit their proliferative capacity
• Becton Dickinson produces a modified plastic, Primaria, that
has a net positive charge claimed to favor epithelial growth in
preference to fibroblasts

12. Removal of contaminating cells
Feeder Layers
• The most popular substrate modification is to preplate with a
monolayer of fibroblasts, or other cells, that can be irradiated to
prevent their further growth
• A preformed layer of irradiated or mitomycin C-treated 3T3 cells
enhances the survival of many epithelial cells, including
keratinocytes, cervical epithelium, and breast epithelium
• This appears to repress the further growth of fibroblasts
• This may be caused, partly, by the ability of epithelium to
– attach to the fibroblasts
– normal fibroblasts are unable to do so
– also probably caused by release by the feeder cells of paracrine
factors that enhance epithelial survival and may block the action of
TGF
• It is probably the most generally successful method of enhancing
epithelial growth and inhibiting fibroblastic overgrowth

13. Physical methods of separation
• It is possible to separate many cell types by physical methods,
such as density gradient centrifugation, centrifugal
elutriation, and flow cytometry
• Of these, flow cytometry has the greatest resolution, but it
has a relatively low yield;
• centrifugal elutriation gives the highest yield
– only effective when there are clear distinctions in cell size
– The purification achieved is seldom complete but may suffice for
the generation of a purified population for immediate use
– it seldom has a lasting effect, as the contaminating stromal cells
usually proliferate more rapidly in standard media with serum
supplementation
• Both of these methods are technically complex to use and
involve expensive equipment, but they have been used very
successfully with many different cell systems

14. Obtaining pure culture
• Magnetic separation has become increasingly effective
• Specific antibodies conjugated to iron-containing coated beads have been used to isolate
different cell types magnetically, either by a positive sort for epithelium [
• a negative sort for contaminating fibroblasts [
• A cell suspension, previously incubated with antibody-conjugated beads, is passed down a
glass cylinder,
• the cells bound to the beads are trapped at the side of the tube by placing an electromagnet
outside the cylinder
Turning off the current allows the beads to
be eluted with the attached cells, which
can be separated from the beads by
trypsinization
Some systems (e.g., Miltenyi) used
magnetizable microbeads that permit
subsequent culture without requiring
removal of the beads and have antibody
conjugations suitable for both positively
sorting epithelial cells and negatively
sorting fibroblasts

15. Selective Culture
• The ideal method of purifying a population of cells is by cloning
• Used in conjunction with a feeder layer, this method permits many
epithelial cells to be cloned quite successfully
• unfortunately the culture may senesce before sufficient cells are
generated
• If relatively few cells are required, if the clones can be pooled, or if the line
has been immortalized, cloning may prove to be the ideal method
• Selective media have become one of the principal methods for growing
epithelial cells preferentially
• Several media have been developed capable of supporting different types
of epithelial cells
• They are serum-free, eliminating TGF- and PDGF, which favor fibroblastic
growth, and they often incorporate hormones and growth factors, such as
hydrocortisone, isoproterenol (isoprenaline), and epidermal growth factor
(EGF), which stimulate epithelial proliferation
• They have the advantage that they do not depend on one selective event
but continue to exert a selective pressure in a nutritionally optimized
environment. Many of these media are available commercially

16. Characterisation
• Validating selected epithelial cell lines requires the adoption of specific
criteria for identifying the cells as epithelial
• The time honored method is by morphology, as most epithelial cultures
have a characteristic tight pavement like appearance with cells growing in
well-circumscribed patches
• However, not all epithelia grow in this manner; some show greater
plasticity in shape, particularly when derived from tumors
• some mesenchyme derived cells such as endothelium and mouse embryo
fibroblasts like 3T3 cells (which are probably primitive mesenchyme rather
than committed fibroblasts) can look quite epithelial at confluence
• reliable epithelial identification depend on the recognition of certain
specific markers
• The intermediate filament proteins have long been recognized for their
tissue specificity
• Among these the cytokeratin group are found predominately in epithelia
• cytokeratins are rarely seen in other cells

17. Characterisation
• the cytokeratins are a large group with considerable diversity
• different anti-cytokeratin antibodies may have differing specificities, enabling distinctions to
be made among different types of epithelia or between different stages of differentiation
• When the culture reaches confluence, desmosome junctions, which are also specific to
epithelia, can be detected by electron microscopy and desmosomal proteins, like
desmoplakin can be demonstrated by immunostaining
• Where cultures of ductal cells are able to differentiate, it may also be possible to see
junctional complexes with desmosomes and tight junctions in a characteristic association
• Because of this ability to form tight junctions, and their ability to transport water and ions,
epithelial monolayers sometime generate so-called domes, formed when the cell layer
blisters off the substrate because of fluid and ion transport from the medium to the
subcellular space . This activity is characteristic of ductal and secretory epithelia
• A number of cell surface antigens have been shown to be specific to epithelium. These are
often from one group of transmembrane mucin like glycoproteins and include epithelial
membrane antigen (EMA)
• human milk fat globule (HMFG)-1 and HMFG-2
• These antigens are most strongly expressed in differentiated ductal epithelium, where they
may become polarized to the apical surface, but present to varying degrees in many
different epithelia. There are also a number of more specific markers such as involucrin in
keratinocytes

18. Culturing mammalian mammary epithelial
cells
• Different types of epithelial cells at different stages of
differentiation
• Immunological markers are used against specific phenotypes
• Epithelial keratins are useful in immunological detection as they
are expressed in culture also
• Different mammalian epithelial cells express different keratins
• Luminal epithelial cells express 8 and 18
• Basal cells express keratins 5 and 14 – not express K 8 and 18
• Polymorphic epithelial mucin (PEM) expressed by luminal
epithelial cells
• Common leukocytic leukemia antigen expressed by basal epithelial
cells
• In vivo breast epithelial cells interact with other cell (fibroblasts,
adipose cells, matrix components) types; normal culture
conditions do not model this complexity

19. Mammary epithelial cells- different types

20. Culturing tumour cells from mammalian
breast
• Culturing mammary tumour epithelial cells were found
difficult than normal HMEC
• They do not lead to outgrowth of cells displaying
tumour -associate phenotypes
• Specialised culture conditions developed for the growth
of tumour derived HMEC that display phenotypes of
tumour cells
– Specialised media formulations
– Low Ca, nutrient and oxygen concentration
– Specialised methods of tissue digestion
• There is no one standard procedure allowing
demonstrable tumour cells from primary tumours

21. Tissue processing
• Tissue digestion with enzymes – break down stroma and free
epithelial organoids
• Filter to remove the digested stroma
• Fibroblasts cold be obtained from the filtrate
• MCDB 170 medium is used for primary culture
• Fibroblasts and blood vessel associated cells will not grow
well in MCDB 170 medium
• MCDB 170 medium developed for the clonal growth of
epithelial cells
• Sub cultured when large epithelial patches are present, but
before confluence
• Density of seeding and attachment will influence the time
required (aprox 7-14 days)
• To generate multiple secondary culture and to retain primary
culture – only 50% cells are removed by partial trypsinization

22. Mesenchymal stem cells
• Mesenchymal stem cells (MSCs) are multipotent stem cells
• also termed Mesenchymal Stromal Cells
• have the potential to self-renew and differentiate into a
variety of specialised cell types such as osteoblasts,
chondrocytes, adipocytes, and neurons
• MSCs are easily accessible, expandable, immunosuppressive
and they do not elicit immediate immune responses
• Therefore, MSCs are an attractive cell source for tissue
engineering and vehicles of cell therapy
• MSCs can be isolated from various sources such as adipose
tissue, tendon, peripheral blood, and cord blood
• Bone marrow (BM) is the most common source of MSCs

23. MSCs from Bone marrow
• MSCs have been successfully isolated and characterised
from many species including mouse, rat, rabbit, dog, sheep,
pig, and human MSCs isolated from bone marrow display
multilineage differentiation potentials
• Two main stem cell populations and their progenies,
haematopoietic stem cells and BM-MSCs, are the main
residents of bone marrow
• BM-MSCs are usually isolated and purified through their
physical adherence to the plastic cell culture plate
• Several techniques have been used to purify or enrich MSCs
including antibody-based cell sorting
• low and high-density culture techniques and
• positive and negative selection method
• frequent medium changes and, enzymatic digestion approach

24. Isolation and culture of MSCs
• All the available methods had some short falls:
• the standard MSCs culture method based on
plastic adherence has been confirmed to have
lower successful rate whereas
• the cell sorting approach reduced the osteogenic
potentials of MSCs
• Negative selection method leads to granulocyte–
monocyte lineage cells reappearing after 1 week
of culture

25. Human MSCs
• Low frequency of MSCs in primary tissue
• Expansion is critical in biological studies
• They could only be propagated a limited number of ties
• After that proliferation rate decreases
• Serum free or low serum media are used for expansion and culture
• Traditionally, two-dimensional (2D) adherent culture conditions
have been used as a standard technique for in vitro expansion of
MSCs
• In vitro culture of multicellular aggregates was originally described
for embryonic cells 70 years ago
• Because of their spherical shape, these multicellular aggregates
are now called multicellular spheroids, or spheroids
• Spheroids have been utilized in the field of oncology, stem cell
biology, and tissue engineering

26. Spheroid cell culture
• Different methods are used to develope spheroid culture and they include;
• The spinner flask method -constant agitation of high density cell suspension
to minimize cellular attachment to the solid surface and to maximize cell to
cell contact
• Liquid overlay technique uses agar to prevent attachment
• Early spinner flask and liquid overlay techniques result in a heterogeneous
population of spheroids
• Improved methods are developed to generate a more homogeneous
population of spheroids
• 96-well plates are now commercially available with low attachment surfaces
for single spheroid production per well (e.g., 96 Well Ultra-Low Attachment
Spheroid Plate from Corning in Corning,
• Another widely used technique for spheroid formation is the hanging drop
method, which eliminates surface attachment by placing the cell suspension
in a drop, allowing gravity to facilitate cellular aggregation at the bottom of
the drop
• These cells spontaneously attach to each other to form cell aggregates if the
possibility of surface attachment is abolished

27. Spheroid cell culture
• Another recent spheroid formation technique involves the use of chitosan
membranes to initiate the 2D to 3D transition
• Chitosan is a deacetylated derivative of a natural polysaccharide, chitin, and
is often paired with another glycosaminoglycan, hyaluronan, known to have
an impact on cell migration, proliferation, and matrix secretion
• Spheroidal cell culture has been used extensively in the field of oncology as
spheroidal cell culture exhibits both histological and physiological features
similar to those of solid tumors in the body
• Tumor spheroids synthesize ECM similar to original tumors in vivo, where
the capacity for ECM production is reduced in the same cells in 2D culture
conditions
• The response of cancer cells to therapeutic interventions in vivo is better
reproduced in in vitro spheroidal culture than in 2D adherent culture
• In evaluating the efficacy of radiation therapy, spheroid culture of cancer
cells produces a more comparable response to cells in vivo than cancer cells
in 2D culture
• Additionally, tumor spheroids might possibly mimic circulating tumor cell
aggregates

28. Spheroids in stem cell culture
• Spheroidal cell culture with pluripotent stem cells (PSCs), including embryonic stem
cells (ESCs), is specifically called embryoid body
• Utilization of embryoid bodies is a standard protocol to produce specific cell
lineages of interest in vitro, as the intercellular interactions of embryonic cells
occurring during embryogenesis are recapitulated in the 3D culture setting
• Similarly, spheroidal cell culture of neural stem cells (NSCs), or neurospheres, has
been used routinely for NSC isolation from embryonic and adult tissues and in
vitro expansion and differentiation of NSCs into neurons, oligodendrocytes, and
astrocytes
• Differentiation capability and potential of stem and progenitor cells are generally
enhanced in the 3D culture setting
• For example, salivary gland-derived progenitor cells can differentiate into
hepatocytic and pancreatic islet cell lineages, but these differentiations only take
place when the cells are cultured in 3D cell aggregates, not in 2D monolayer
• Neuronal differentiation of ESCs is enhanced in embryoid body culture compared
to 2D monolayer cell culture
• Moreover, in vitro reproduction of complex organ architecture, such as the optic
cup, is made possible only in 3D culture, in which the inherent tissue self-
organization capability of ESCs is maximized

29. Gonadal tissue
• Major players in growth and differentiation in the testis and
ovaries are different
• In severe cases of disorders of sex development (DSD), which
are due to mosaic sex chromosome aneuploidy, individuals
often present at birth with an uncertain phenotypic gender,
the differences are blurred
• Meiotic cell division is a unique feature of germ cell
development and an early morphological sign of sex
differentiation in the developing gonads
• initiation of meiosis involves the action of retinoic acid (RA),
which in fetal ovaries mediates the up-regulation of
stimulated by retinoic acid gene 8 (Stra8) that is required for
pre-meiotic DNA replication
• Such variation from somatic cells or tissues makes the
culturing of Gonadal tissue different

30. • elective termination of pregnancy during the
first trimester
• one of the terminations were for reasons of
fetal abnormality, and all fetuses appeared
morphologically normal
• used for hanging-drop culture

31. Culture techniques
• primary human germ cells are difficult to culture outside the somatic niche,
and
• the available testicular cancer cell lines are all isolated from fully developed
Testicular germ cell tumours (TGCTs) including a range of embryonal
carcinoma lines
• The lack of a suitable model system to investigate the early progression
from Carcinoma in situ (CIS) cells to invasive tumours has also made it
difficult to determine the role of specific pathways in the pathogenesis of
testicular cance
• Several different approaches to culture human testis tissue have previously
been employed, including ex vivo cultures of adult tissue and fetal testis
tissue on membranes
• xenografting of fetal, pre-pubertal and adult testis tissue into nude mice
• Xenografting of human testis cancer cell lines into nude mice, including JKT-
1
• Hanging drop cultures are the widely used culture approach for both
embryonic (including embryonic stem cells) and adult tissues, and have
been previously successfully applied to culture intact fetal mouse testes and
adult murine seminiferous tubules

32. Hanging drop culture
• Hanging drop cultures are the widely used culture approach for both
embryonic (including embryonic stem cells) and adult tissues,
• previously successfully applied to culture intact fetal mouse testes and
adult murine seminiferous tubules (
• This culture approach has multiple benefits, including
• three-dimensional tissue architecture maintenance,
• facilitation of efficient gas exchange and
• requirement for only small media and supplement volumes
• Importantly, these cultures are particularly effective at preserving ex
vivo functional integrity and signalling activity
• a suitable avenue through which to study the effects of specific
treatments on a range of human orchidectomy specimens, from relatively
normal testis tissue, to tissue containing CIS and seminoma tumours

33. Hanging drop culture
• cultures of normal testis tissue and CIS can be maintained for
up to 14 days without signs of increased apoptosis, while the
organisation of the seminiferous epithelium is preserved and
germ cells continued proliferation
• Cultures of seminoma samples can be maintained for up to 7
days, with histology indicating that samples remain consistent
with expected tumour morphology and that proliferating
seminoma cells are present for at least 3 days
• activin A treatment of hanging drop cultures induces specific
gene and protein alterations of relevance to TGCTs
• These outcomes illustrate the value of this approach for
investigating responses to growth factors or chemical
treatments that may ultimately be applied to alter the in
vivo development and growth of testicular germ cell tumours

34. Lymphocyte culture
• Isolation of Human T Lymphocytes
• Blood from a healthy donor is used
• Allow the blood to cool to room temperature (~30 min) before proceeding to the
next step.
• Density gradient centrifugation by layering blood over a density gradient media in a
round-bottom polystyrene tube
• Centrifuge the tubes at 500 x g for 45 minutes at room temperature
• Peripheral blood mononuclear cells (PBMC) are separated from the other cells in
blood
• The PBMC layer appears, from the top down, as the first cloudy band.
Carefully remove the clear yellow-colored upper
phase of the blood, above the PBMC layer, and
Recover the PBMC layer to a 15 mL or 50 mL conical
tube
Wash the PBMC twice with PBS, centrifuging cells at
500 x g for 5 minutes each time. The supernatant
will be somewhat cloudy after each wash

35. Purification of the cells
• PBMC transferred to a T-75 culture flask in 20 mL RPMI 1640 media
• The media will containing 10% foetal bovine serum (FBS), 1%
penicillin/streptomycin, and 1 μg/mL phytohemagglutinin (PHA)
• Incubate at 37°C and 5% CO2 for at least 1 hour, and up to 24 hours
• Allows monocytes, which will be adherent to the flask surface, to be separated
from the lymphocytes that remain in suspension.
• If a short incubation (1 hour) is used at this step, it is acceptable to use RPMI 1640
media containing 10% FBS and 1% penicillin/streptomycin without supplementing
with PHA
• Carefully remove all of the media from the flask, add it to a 50 mL conical tube, and
centrifuge at 500 x g for 5 minutes
Resuspend the cell pellet, which now primarily
contains lymphocytes, and transfer the cells to a
new T-75 flask containing 25 mL RPMI 1640 media
containing 10% FBS, 1% penicillin/streptomycin,
and 1 μg/mL PHA
The cells will grow as suspension culture