ANIMAL CELL CULTURE TECHNIQUE

2. Secondary cell cultures
 When a primary culture is sub-cultured, it becomes
secondary culture or cell line. Subculture (or passage)
refers to the transfer of cells from one culture vessel to
another culture vessel.
 Subculturing- Subculturing or splitting cells is
required to periodically provide fresh nutrients and
growing space for continuously growing cell lines. The
process involves removing the growth media, washing
the plate, disassociating the adhered cells, usually
enzymatically. Such cultures may be called secondary
cultures.

3.  Cell Line
 A Cell Line or Cell Strain may be finite or continuous
depending upon whether it has limited culture life span or
it is immortal in culture. On the basis of the life span of
culture, the cell lines are categorized into two types:
 a) Finite cell Lines - The cell lines which have a limited
life span and go through a limited number of cell
generations (usually 20-80 population doublings) are
known as Finite cell lines. These cell lines exhibit the
property of contact inhibition(monolayer-cell growing),
density limitation and anchorage dependence. The growth
rate is slow and doubling time is around 24-96 hours.

4.  b) Continuous Cell Lines - Cell lines transformed under
laboratory conditions or in vitro culture conditions give rise to
continuous cell lines. The cell lines show the property of ploidy
(aneupliody(abnormal chr no) )absence of contact inhibition
and anchorage dependence. They grow in monolayer or
suspension form. The growth rate is rapid and doubling time is
12-24 hours.
 c) Monolayer cultures - When the bottom of the culture vessel
is covered with a continuous layer of cells, usually one cell in
thickness, they are referred to as monolayer cultures.
 d) Suspension cultures - Majority of continuous cell lines grow
as monolayers. Some of the cells which are non-adhesive e.g.
cells of leukemia or certain cells which can be mechanically kept
in suspension, can be propagated in suspension.

5.  Normal or transformed: Transformed cell lines
usually have an increased growth rate and higher
plating efficiency, are continuous, and require less
serum in media, but they have undergone a permanent
change in their phenotype through a genetic
transformation.

6.  There are certain advantages in propagation of cells by
suspension culture method.
 These advantages are:
(a) The process of propagation is much faster.,
(b) The frequent replacement of the medium is not
required.,
(c) Suspension cultures have a short lag period,
(d) treatment with trypsin is not required,
(e) a homogenous suspension of cells is obtained,
(f) the maintenance of suspension cultures is easy and bulk
production of the cells is easily achieved.,
(g) scale-up is also very convenient.

7.  The cell lines are known by:
a) A code e.g. NHB for Normal Human Brain.
b) A cell line number- This is applicable when several
cell lines are derived from the same cell culture source
e.g. NHB1, NHB2.
c) Number of population doublings, the cell line has
already undergone e.g. NHB2/2 means two doublings.

10. COMMANLY USED CELL LINES
 Cell lines are an invaluable scientific tool. They allow
us to dissect the internal workings of tissues in a
controlled environment without the ethical
implications of working with whole organisms.
 Starting with the first successful immortal cell line
HeLa, the number of available cell lines has since
diversified into a plethora(large amt) of options. Just
like model organisms, the cell lines we work with
define our scientific tribes.

12. Number 1: HeLa
 Unlike the other cell lines above, HeLa is named after
an individual, an American women called Henrietta
Lacks. Shortly after establishment of this cell line,
HeLa cells were used to proliferate the famous polio
vaccine, and they continue to be the most widely used
cell line in research labs worldwide.
 According to the British newspaper The Guardian,
HeLa “has led to hundreds, if not thousands, of new
pieces of knowledge, and helped to shape the way
medicine moved in the second half of the 20th century
and the first decade of this one”.

13. Number 2: HEK 293
 HEK293, or human embryonic kidney-derived
epithelial cells, are arguably one of the most widely
used cell lines in cell biology research. HEK293 is a
rapidly dividing, robust line cell with a good reputation
for post-translational modification of its
heterologously expressed proteins.
 Therefore, it’s hardly surprising that it is often the cell
line of choice in transient and stable transformation
experiments, for protein expression and production,
and even in electrophysiological experiments.

14. Number 3: Insert your favorite
immortal human cell line here!
 Some may consider it cheating to use a cell line
collection instead of a single cell line. But depending
on your favorite object of study, you may end up using
a collection such as those from the NCI-60 NCI-60
Human Tumor Cell Lines Screen (5).
 Alternatively, you might be interested in working with
Jurkat or HL-60 (white blood cells), MCF-7 (breast
cancer), Saos-2 cells (bone cancer), PC3 (prostate
cancer) or many others.

15. Number 4: Chinese hamster ovary
cells
 Chinese hamster ovary cells (CHOs) are clearly ovary-
derived cells, but this time, we are taking mammalian
cells. Similarly to Sf9 cells, they can exist both as
adherent or suspension cells in culture.
 CHO cells are used in various applications such as
recombinant protein production and studies of
the epidermal growth factor receptor .

16. Number 5: Sf9 insect epithelial
cells
 Derived from the ovaries of the fall armyworm moth (Spodoptera
frugiperda), these cells are probably related to all insect cell lines
in labs worldwide . Sf9cells can be cultured as adherent or
suspension cells. Most cell lines are adherent cells, which grow
only on the surfaces of culture vessels.
 This limits the amount of cells you can expect to obtain from
each culture. Similarly to E.coli, suspension cells can grow in the
entire volume of the medium, thus increasing the amount of
cells that can be harvested from a vessel.
 Furthermore, and because of the high volume: cell number ratio,
suspension cultures allow a much more effective use of medium
than adherent cultures. Sf9/baculovirus systems are typically
preferred for large-scale protein production including industrial
manufacture of mammalian proteins, including the vaccine for
cervical cancer CERVARIX® .

17. KARYOTYPE
o The study of chromosomes, their structure and
their inheritance is known as Cytogenetics.
o Each species has a characteristic number of
chromosomes and this is known as karyotype.

18. What each of the human chromosomes
look like

20. In other words…
 Chromosomes are digitally arranged so that they
are matched with their homologue or “partner”
chromosome.
 Homologue chromosomes are the same size, shape,
and carry the same genes, and one is inherited from
each parent.
 They are numbered according to size.

21. Sex determination with karyotype
 This karyotype has 23
exact pairs, which
means the person is
female.
 Note that #23
chromosomes are both
X.

22. Normal human male
 Note that #23
chromosomes are
X and Y.

23. Is this person female or male?

24. Trisomy 21
 Abnormality
shown in
karyotype
 Note that there
are three copies
of #21
chromosome.
 This person has
Down Syndrome.

25. Monosomy X
 Abnormality
shown in
karyotype
 Note this person
only has 1 copy
of the X
chromosome.
 This female has
Turner’s
syndrome.

26. XXY Male (Extra X)

27. How are DNA samples obtained for
karyotypes?

28. Amniocentesis: obtaining amniotic fluid
which has cells from the fetus

29. Chorionic villi sampling: removing cells
from the chorion with fetal tissue

30. Characterization of a cell line
 Characterization of a cell line is vital for determining
its functionality and in proving its authenticity as pure
cell line.
 Special attention must be paid to the possibility that
the cell line has become cross-contaminated with an
existing continuous cell line or misidentified because
of mislabeling or confusion in handling DNA profiling.

31.  The various important factors for cell line characterization are:
 It leads to authentication or confirmation that the cell line is not
cross-contaminated or misidentified
 It is confirmation of the species of origin
 It is used for correlation with the tissue of origin, which comprises
the following characteristics:
 Identification of the lineage to which the cell belongs
 Position of the cells within that lineage (i.e., the stem, precursor, or
differentiated status)
 For determination whether the cell line is transformed or not:
 Whether the cell line is finite or continuous?
 Whether the cell line expresses properties associated with
malignancy?
 It indicates whether the cell line is prone to genetic instability and
phenotypic variation
 Identification of specific cell lines within a group from the same
origin, selected cell strains, or hybrid cell lines, all of which require
demonstration of features unique to that cell line or cell strain

32. Parameters of Characterization
Biochemical
 Enzymes: Three parameters are available in enzymatic
characterization:
 The constitutive level (in the absence of inducers or
repressors)
 The induced or adaptive level (the response to inducers and
repressors)
 Isoenzyme(aa seq diff) polymorphisms(any forms)
 Intermediate filament proteins : These are among the
most widely used lineage or tissue markers. Glial fibrillary
acidic protein (GFAP) for astrocytes(star shape-Glial Cells-
CNS) and desmin(muscle specific-protein) for muscle are
the most specific, whereas cytokeratin(found in
cytoskeleton of muscle) marks epithelial cells and
mesothelium.

33. GENETIC
 Unique Markers: Unique markers include specific
chromosomal aberrations (e.g., deletions,
translocations, polysomy), major histocompatibility
(MHC) group antigens (e.g., HLA in humans), which
are highly polymorphic, and DNA fingerprinting or
STR DNA profiling.
 Transformation: The transformation status forms a
major element in cell line characterization.

34.  Chromosome Content: Chromosome content or karyotype is one of
the most characteristic and best-defined criteria for identifying cell
lines and relating them to the species and sex from which they were
derived. Chromosome analysis can also distinguish between normal
and transformed cells because the chromosome number is more stable
in normal cells (except in mice, where the chromosome complement of
normal cells can change quite rapidly after explantation into culture).
 Chromosome Banding: This group of techniques was devised to
enable individual chromosome pairs to be identified when there is
little morphological difference between them. For Giemsa banding, the
chromosomal proteins are partially digested by crude trypsin,
producing a banded appearance on subsequent staining.
Trypsinization is not required for quinacrine banding(visualizing
condensed chr). The banding pattern is characteristic for each
chromosome pair.

36.  Chromosome Analysis
 The following are methods by which the chromosome
complement may be analyzed:
 Chromosome count : Count the chromosome number
per spread for between 50 and 100 spreads. (The
chromosomes need not be banded.)
 Karyotype : Digitally photograph about 10 or 20 good
spreads of banded chromosomes. Image analysis can
be used to sort chromosome images automatically to
generate karyotypes.

37.  Chromosome counting and karyotyping allow species
identification of the cells and, when banding is used,
distinguish individual cell line variations and marker
chromosomes. However, karyotyping is time-
consuming, and chromosome counting with a quick
check on gross chromosome morphology may be
sufficient to confirm or exclude a suspected cross-
contamination.