Types of Cell Culture

DR. NIDA DASTAGIR

 Animal tissue
Culture
Category-A Category-B
Cultures that allow Cultures in which cell–
cell–cell interactions cell communication or
and encourage interactions are lost or
communication or the
signaling between signaling between
cells. them is missing.
Animal Cell Culture
CATEGORY A CATEGORY B
Organ Culture Monolayer Cultures
Histotypic Culture Suspension Cultures
Organotypic Culture
• A culture of native tissue that
retains most of the in vivo
histological characteristics
• Whole embryonic organs or
small tissue fragments are
cultured in vitro in such a
manner that they retain their
tissue architecture
Organ
Culture
• Culturing cells for their re-
aggregation to yield tissue-like
structure
• Individual cell lineages are
initially derived from an organ
and then cultured separately to
high density in a 3D matrix to
study interactions and signaling
between homologous cells
Histotypic
Culture
• Cells from different origins are
mixed together in specific
proportions and spatial
relationships so as to re-form a
component of an organ
• The recombination of different
cell types to yield a more defined
tissue or organ
Organotypic
Culture
Monolayer Culture
• An anchorage-dependent culture of usually one cell in thickness with
a continuous layer of cells at the bottom of the culture vessel.

Suspension Culture
• Cells are nonadhesive and can be mechanically kept in suspension
 Primary Cell Culture
The first culture (a freshly isolated cell culture) directly obtained from animal or human tissue
These cells are typically slow growing, heterogeneous and carry all the features of the tissue of their origin.
They have the same karyotype as the original tissue.
Once subcultured, primary cell cultures can gives rise to cell lines
Have a limited life span, i.e. the cells cannot be maintained indefinitely.
An increase in cell numbers - exhaustion of the substrate and nutrients - influence cellular activity -
accumulation of high levels of toxic metabolites - inhibition of cell growth.
This stage is called the confluence stage (contact inhibition), when a secondary culture or a subculture
needs to be established to ensure continuous cell growth.
Primary Cell Culture
ANCHORAGE-DEPENDENT OR ADHERENT
CELLS
Adherent cells are those cells which require
attachment for growth and are also called
anchorage-dependent cells
These cells are capable of attaching on the
surface of the culture vessel.
These types of cells are often derived from the
tissues of organs, for example from the kidney,
where the cells are immobile and embedded
in connective tissue.
ANCHORAGE-INDEPENDENT OR
SUSPENSION CELLS
Suspension cells do not require attachment or
any support for their growth and are also
called anchorage-independent cells.
All suspension cells are isolated from the
blood system, for example white blood cell
lymphocytes, and are suspended in plasma.
Secondary Cell Culture
First passaging of cells - a switch to a different kind of culture system, or the first culture
obtained from a primary culture.
This is usually carried out when cells in adherent cultures occupy all the available substrate or
when cells in suspension cultures surpass the capacity of the medium to support further growth,
and cell proliferation begins to decrease or ceases completely
To maintain optimal cell density for continued growth and to encourage further proliferation, the
primary culture has to be subcultured.
Difference Between Primary and Secondary Culture
Techniques for Development of Primary Cell
Culture

• Mechanical disaggregation
• Enzymatic disaggregation
• Primary explant techniques
Mechanical disaggregation
It is necessary to disaggregate soft tissues such as soft tumors.
Involves slicing or harvesting tissue and subsequent harvesting of spill out cells.
Methods are sieving, syringing and pipetting.
Inexpensive, rapid and simple
Risk of cell damage.
Mechanical disaggregation is only used when the viability of the cells in the final yield is not very
important.
Enzymatic disaggregation
Efficient disaggregation of cells with high yield by using enzymes such as trypsin, collagenase
and others.
Enzyme based disaggregation allows hydrolysis of fibrous connective tissue and the
extracellular matrix.
Currently, the enzymatic method is extensively used as it offers high recovery of cells without
affecting the viability of cells.
 Primary Explant Technique
Disaggregation of small quantities of tissue for which mechanical and enzymatic disaggregation are not
suitable
Tissue is initially suspended in basal salt solution and then chopped properly and washed by settling.
Tissue fragments are uniformly distributed over the growth surface.
This is followed by the addition of a suitable medium and then incubation for 3–5 days. Old medium is
replaced by fresh medium unless desired growth or considerable outgrowth of the cells is not achieved.
Once optimum growth is achieved the explants are separated and transferred to new culture vessels which
contain fresh medium.
A major drawback - the poor adhesiveness of certain tissues on the growth surface (substrate material),
which can create problems in the selection of cells for desirable outgrowth.
Utilized frequently for culturing embryonic cells, in particular glial cells, fibroblasts, myoblasts and epithelial
cells.
Cell Lines
Cell line is defined as,
A permanently established cell culture which will propagate forever, provided the continuous
supply of suitable fresh medium and the availability of space for the cells to propagate.
The propagation of a culture after the first subculture.
When primary culture is sub cultured it results in the development of a cell line.
Cell lines differ from cell strains in that they become immortalized.
A cell line consist of cells with a uniform genetic make-up
The cell line obtained after selection or cloning is called a cell strain, which does not have an
infinite life, since they die after a number of divisions.
Cell Line
Development
Types of Cell Lines
Finite Cell Lines:
Cell lines that lose their ability to divide after a limited period of time are finite cell lines.
Finite cell lines contain cells which can divide 20–100 times (i.e. population doubling by 20–100
times) before losing their capability to divide.
The extent of population doubling is dependent on several factors, such as cell lineage, cell type,
origin, species, culture environment, etc.
Population doubling for human cell lines is between 50–100 times, whereas murine cell lines
divide 20–30 times before extinction.
Types of Cell Lines
Continuous/Infinite Cell Lines:
Treatment of cells with carcinogens (chemicals), oncogenic viruses, etc, results
in changes in phenotypical characteristics, in particular morphology, which can
alter cells and lead to the development of cells that grow faster than normal
cells.
Cell lines obtained from these altered cells have infinite life spans and referred
as continuous cell lines.
These cell lines are immortal, transformed and tumorigenic
 Terminologies-Cell Lines
Immortalization. Achieving a state of cell culture when cells proliferate continuously.
Attachment efficiency. The proportion of cells that actually adhere to the surface of the culture vessel
within a given time after inoculation.
Passaging. The transfer of cells from one culture vessel to another. A more specific term is sub-culturing
where the cells are first subdivided before being transferred into multiple cell culture vessels.
Split ratio. Divisor of the dilution ratio of a cell culture.
Population doubling Level: The population doubling (PD or PDL) number is the estimated number of
doublings that the cell population has undergone since isolation.
Passage number. The number of times the culture has been sub-cultured.
Doubling Time
of Different
Cell Lines
Cell Lines
Cell lines should display and maintain functional features as close to the primary cells as
possible.
Since cell lines are genetically manipulated, this may alter their phenotype, native functions and
responsiveness to stimuli.
Serial passage of cell lines can further cause genotypic and phenotypic variation over an
extended period of time, and genetic drift can also cause heterogeneity in cultures.
Therefore, cell lines may not adequately represent primary cells and may provide different
results
Standard Nomenclature of Cell Lines
New cell lines should be given a code or designation [e.g., normal human brain (NHB)]; a cell
strain or cell line number (if several cell lines were derived from the same source; e.g., NHB1,
NHB2, etc.); and, if cloned, a clone number (e.g., NHB2-1, NHB2-2, etc.)
The source and clone number represents the basic criteria for nomenclature.
It is usually followed by assigning codes or designations to cell lines for their further
identification, e.g HeLa-S3 represents a human cervical tumor cell line.
MG-63 cell line - It is the 63rd sample of a tumor that produces a high amount of interferon
beta. Therefore, its nomenclature is ‘human tumor-63’, or in Dutch, ‘menselijk gezwell-63’ or
MG-63.
Applications of Cell Lines
• Vaccine production.
• Examining drug metabolism.
• Cytotoxicity.
• Antibody production.
• Investigating gene function.
• Development of artificial tissues (e.g artificial skin).
• Production of biological compounds (e.g therapeutic proteins).
Contamination of Cell Lines
Contamination with other cell lines and mycoplasma is a major problem in cell culture.
Cross-contamination was explored by Nelson-Rees in early 1970s.
Contamination of one cell line with a new one results in mixed cultures or occasionally complete
overgrowth of the original cells by the contaminating line.
Nelson-Rees used chromosome banding (a procedure in which condensed chromosomes are
stained to produce a visible karyotype) to prove that numerous immortal cell lines, earlier
supposed to be unique, were in fact HeLa cell lines.
He also demonstrated the fact that contamination with HeLa cells is responsible for the
outgrowth of other cell lines
He demonstrated clearly that most of the of cell lines being investigated globally and distributed
by cell banks were contaminated with HeLa cells.
 Contamination of Cell Lines
Contamination considerable challenge for the animal tissue culture industry.
During cell line contamination, contaminants, in particular rapidly proliferating cells, take over a
whole cell line before its own growth takes place.
HeLa cells are a frequent contaminant and, moreover, other contaminants such as mycoplasma
can continue undetected in cell cultures for a long period of time.
Prolonged exposure to contaminants can cause widespread changes in gene expression and cell
behavior.
According to certain reports, 15%–35% of cell lines submitted to cell banks were likely to be
contaminated with mycoplasma.
 Selection of Cell Lines
Several factors are considered during the selection of cell lines, such as
Origin of the cell line (human or non-human cell line; human cell lines are more vulnerable to different
types of contamination).
Type of cell line (finite or continuous).
Types of cells (normal or transformed).
Growth patterns.
Cloning efficiency.
Cell number under specified culture conditions (saturation density).
Population doubling time.
Availability of cell line.
Availability of growth factors or media for its maintenance.
Physical expression of traits, or characteristics.
Characterization of Cell Lines
Must ensure that the cell line obtained is appropriate or acceptable for their designed
experiment or purpose.
To reveal changes in a cell line, karyotyping is the most recommended approach.
It can demonstrate that a cell line has a normal karyotype, and the cell line can then be used for
various research purposes.
Karyotyping is a simple test that can reveal changes in a cell line.
Molecular assays for copy number variation or RNA profiling will also be indicative of changes,
but are more costly.
It is also advisable to capture an image of the cell line in culture at different cell population
densities and perform basic characterization (e.g. calculating the population doubling time for
that cell line) soon after arrival
 Maintenance of Cell Lines
Cellular Morphological Examination:
 Cells should be examined routinely to check for the presence of any other contaminant.
 Morphological examination is essential to investigate and differentiate the natural cellular organization and the
physiological state of the cells from the contaminated.

Media Replacement:
 Regular changing of the medium is required to maintain cell lines in culture; however, the frequency of changing the
medium always varies.
 For example, proliferating cells require more nutrients in comparison to non-proliferating cells.
 The rate of cellular growth and metabolism decides the interval between the changing, or addition of fresh, medium.
Maintenance of Cell Lines
Cell Density: Cultures with a high density of cells use medium faster than those with low density,
thus medium need to be changed more frequently for high densities of cells.
Fluctuations in pH:
 Changes in pH should be monitored carefully, since a decrease in pH may be associated with a decline in
the growth rate of the cells.
 The optimal pH for the growth the cells is 7, and a decline in pH (6.5) can retard the growth of cells.
 Further decline in pH (usually to between 6.0– 6.5), may stop the growth of the cells, and if the low pH
persists cells will start losing their viability.

Type of cell.
 Feeder cells such as embryonic stem cells and tumorigenic cells such as transformed cell lines
(continuous cell lines) grow fast and thus require a greater supply of nutrients.
 Thus, rapidly growing cells require more frequent medium changes than normal cells
Stem Cell
In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can
differentiate into various types of cells and proliferate indefinitely to produce more of the same
stem cell.
Stem cells are unspecialized cells of the human body.
They are able to differentiate into any cell of an organism and have the ability of self-renewal.
Stem cells exist both in embryos and adult cells.
There are several steps of specialization.
Developmental potency is reduced with each step, which means that a unipotent stem cell is not
able to differentiate into as many types of cells as a pluripotent one.
Stem Cells
Totipotent stem cells
Totipotent stem cells are able to divide and differentiate into cells of the whole organism.
Totipotency has the highest differentiation potential and allows cells to form both embryo and
extra-embryonic structures.
One example of a totipotent cell is a zygote, which is formed after a sperm fertilizes an egg.
These cells can later develop either into any of the three germ layers or form a placenta.
After approximately 4 days, the blastocyst’s inner cell mass becomes pluripotent. This structure
is the source of pluripotent cells.
 Stem Cells
Pluripotent stem cells (PSCs) form cells of all germ layers but not extraembryonic structures, such as the
placenta. Embryonic stem cells (ESCs) are an example.
ESCs are derived from the inner cell mass of preimplantation embryos.
Another example is induced pluripotent stem cells (iPSCs) derived from the epiblast layer of implanted
embryos.
One of the methods to assess their activity and spectrum is the teratoma formation assay.
Teratomas are benign tumors characterized by their rapid growth in vivo and their haphazard mixture of
tissues, belonging to all three germ layers responsible for the formation of organs, teeth, hair, muscle,
cartilage, and even bone.
Teratoma formation assay is widely viewed in stem cell research as the “gold standard” for assessing
pluripotency.
iPSCs are artificially generated from somatic cells, and they function similarly to PSCs
Stem Cells
Multipotent stem cells:
Multipotent stem cells have a narrower spectrum of differentiation than PSCs, but they can
specialize in discrete cells of specific cell lineages.
One example is a haematopoietic stem cell, which can develop into several types of blood cells.
After differentiation, a haematopoietic stem cell becomes an oligopotent cell.
Its differentiation abilities are then restricted to cells of its lineage
Stem Cells
Oligopotent stem cells:
Oligopotent stem cells can differentiate into several cell types.
A myeloid stem cell is an example that can divide into white blood cells but not red blood cells.
Unipotent stem cells:
Unipotent stem cells are characterized by the narrowest differentiation capabilities and a special
property of dividing repeatedly.
These cells are only able to form one cell type, e.g. dermatocytes.
Properties of Stem Cells
Self-renewal:
The ability to go through numerous cycles of cell growth and cell division, known as cell
proliferation, while maintaining the undifferentiated state

Potency:
The capacity to differentiate into specialized cell types.
In the strictest sense, this requires stem cells to be either totipotent or pluripotent—to be able
to give rise to any mature cell type, although multipotent or unipotent progenitor cells are
sometimes referred to as stem cells.
Stem Cells in Culture
Three types of stem cells are commonly expanded in culture.
Embryonic stem cells (ESCs),
Adult-stem cells
Induced pluripotent stem cells (iPSCs)
 Embryonic Stem Cells (ESC)
ESCs - first isolated from mouse embryos in 1981.
In 1998, ESC were isolated from human - hESC.
hESCs have an ability to differentiate into all body cells, including beta cells of the islets of
Langerhans, neural cells, cardiomyocytes, and hepatocyte-like cells.
The pluripotent capabilities of hESCs have given hope to millions of patients who are suffering from
diabetes, Parkinson’s disease, cardiovascular disease, and liver diseases.
One of the challenges of the hESCs was the method of isolation of stem cells from the human
embryo, as hESCs can only be obtained from the inner cell mass (ICM) of human embryos.
After ICM isolation, the stems cells are grown to generate the ESCs using feeder layers, extracellular
matrices, proteins, peptides, and synthetic polymers.
The isolation of ICM requires destruction of human embryos which has raised serious ethical
concerns.
Adult Stem Cells
Adult stem cells are undifferentiated found throughout the body after embryonic development,
that multiply by cell division to replenish dying cells and regenerate damaged tissues.
The primary roles of adult stem cells in a living organism are to maintain and repair the tissue in
which they are found.
Unlike embryonic stem cells, somatic stem cells differentiate into the cell type from which they
originate.
These cells are used by the body to maintain and repair the originating tissue.
Scientists are still uncovering other areas of the body that contain these quiescent stem cells
and are attempting to understand their role.
Induced Pluripotent Stem Cells (iPSC)
In 2006, Shinya Yamanaka, together with Kazutoshi Takahashi, discovered that it is possible to
reprogram multipotent adult stem cells to the pluripotent state.
This process avoided endangering the foetus’ life in the process.
These cells are somatic cells which have been reprogrammed back to a pluripotent stem cell
state by introducing few genes such as the SOKM (Sox2, Oct4, Klf4 and c-Myc) or SOLN (Sox2,
Oct4, Lin28, Nanog) that are mainly expressed in embryonic stem cells, by retrovirus-mediated
transduction
This new form of stem cells was named iPSCs.
After this success, the method opened a new field in stem cell research with a generation of
iPSC lines that can be customized and biocompatible with the patient.
Retroviral-mediated transduction induces pluripotency
in isolated patient somatic cells
Stem Cell Culture
Stem cell culture - allow the establishment and the maintenance of phenotypically well defined
and karyotypically stable cells.
Induced pluripotent stem cells are considered a major candidate for future cell treatment
research due to their self-renewal and differentiation potential and the lack of ethical concerns.
 Stem Cell Culturing
Three types of stem cells are commonly expanded in culture.
◦ Embryonic stem cells (ESC),
◦ adult-stem cells,
◦ induced pluripotent stem cells (iPSC).

Standard culture of hPSCs involves exposure to media enriched with growth factors found in fetal bovine
serum (FBS) or defined serum replacements.
In addition, standard hPSC culture systems utilize support cells such as an inactivated mouse embryonic
fibroblast (MEF) feeder layer to support growth and prevent differentiation.
These cells provide necessary intercellular interactions, extracellular scaffolding and factors creating a
robust and stable hPSC culture environment.
Stem Cell Culturing
Embryonic cells were always grown on a layer of mouse embryonic
fibroblast (MEF) cells.
Before they can be used as feeder cells, the MEF cells must be
mitotically inactivated using either UV light or treatment with the
chemical mitomycin-C.
This prevents the MEF cells from overwhelming the slow-growing stem
cells in the culture.
Methods to culture ES cells without feeder layers have been developed
recently.
This method requires coating tissue culture plates with BD Matrigel
(60% laminin, 30% collagen IV, and 8% entactin), a gelatinous protein
mixture isolated from mouse tumor cells.
Preparation of mitotically inactivated MEF cells for use
as feeder cells.