BIOCHEMISTRY &

MOLECULAR
BIOLOGY

KULTUR SEL HEWAN

KULTUR SEL DAN
JARINGAN
• Cell culture is the process by which prokaryotic,
eukaryotic or plant cells are grown under controlled
conditions. But in practice it refers to the culturing
of cells derived from animal cells.
• In vitro cultivation of organs, tissues & cells at defined
temperature using an incubator & supplemented
with a medium containing cell nutrients & growth
factors is collectively known as tissue culture
• Cell culture was first successfully undertaken by
Ross Harrison in 1907
• Roux in 1885 for the first time maintained
embryonic chick cells in a cell culture
Totipotency is the ability of a single
cell to divide and produce all the
differentiated cells in an organism,
including extraembryonic tissues.
Pluripotency = “having more than
one potential outcome.”
Pluripotent stem cells can give rise to any
fetal or adult cell type. However, a single cell
or a conglomerate of pluripotent cells cannot
develop into a fetal or adult animal because
they lack the potential to organize into an
embryo.

Plasticity of biological systems refers to the

ability of living organisms to change their
'state' in response to any stimuli and applying
the most appropriate, adaptive response.
This occurs at any level of complexity:
molecular, cellular, systemic and behavioural.
(1) A Drop in pH. The rate of fall and absolute level should be considered. Most cells
stop growing as the pH falls from pH 7.0 to pH 6.5 and start to lose viability
between pH 6.5 and pH 6.0, so if the medium goes from red through orange to
yellow, the medium should be changed.

(2) Cell Concentration. Cultures at a high cell concentration exhaust the

medium faster than those at a low concentration. This factor is usually
evident in the rate of change of pH, but not always.

(3) Cell Type. Normal cells (e.g., diploid fibroblasts) usually stop dividing at a high
cell density because of cell crowding, growth factor depletion, and other reasons.
The cells block in the G1 phase of the cell cycle and deteriorate very little, even if left
for two to three weeks or longer. Transformed cells, continuous cell lines, and some
embryonic cells, however, deteriorate rapidly at high cell densities unless the
medium is changed daily or they are subcultured.

(4) Morphological Deterioration. This factor must be anticipated by regular

examination and familiarity with the cell line. If deterioration is allowed to progress
too far, it will be irreversible, as the cells will tend to enter apoptosis
 Unhealthy Cells
• Vacuolation and granulation in
bronchial epithelial cells (BEAS-
2B) due, in this case, to medium
inadequacy.
• The cytoplasm of the cells
becomes granular, particularly
around the nucleus, and
• vacuolation occurs.
The cells may become more
refractile at the edge if cell spreading
is impaired.
Subculture gives the
opportunity to expand the
cell population, apply
further selective pressure
with a selective medium,
and achieve a higher
growth fraction and allows
the generation of replicate
cultures for characterization,
preservation by freezing, and
experimentation.
 Why sub culturing?
• Once the available substrate surface is covered by cells
(a confluent culture) growth slows & ceases.
• Cells to be kept in healthy & in growing state have to be
sub- cultured or passaged
• It’s the passage of cells when they reach to 80-90%
confluency in flask/dishes/plates
• Enzyme such as trypsin, dipase, collagenase in combination
with EDTA breaks the cellular glue that attached the cells to
the surface
• Media changed from nutrient rich to nutrient poor –
induces withdrawal from the cell cycle giving the cells 3
choices:
- Die (apoptosis)
- Senescent (become quiescent)
- Differentiate
The need to
subculture a
monolayer is
determined by the
following criteria:
(1)Density of Culture
(2)Exhaustion of
Medium
(3)Time Since Last
Subculture
(4)Requirements
for
Other Procedures.
BASIC ASEPTIC CONDITIONS

• If working on the bench use a Bunsen flame to heat the air

surrounding the Bunsen
• Swab all bottle tops & necks with 70% ethanol
• Flame all bottle necks & pipette by passing very quickly through
the hottest part of the flame
• Avoiding placing caps & pipettes down on the bench; practice
holding bottle tops with the little finger
• Work either left to right or vice versa, so that all material goes to
one side, once finished
• Clean up spills immediately & always leave the work place neat & tidy
 Effects of Biological
Contamination’s

• They competes for nutrients

with host cells
• Secreted acidic or alkaline by-
products ceses the growth of
the host cells
• Degraded arginine & purine
inhibits the synthesis of histone
and nucleic acid
• They also produces H2O2 which is
directly toxic to cells

Source of Contamination: Bacteria, fungi, mould, yeast, mycoplasma, etc.

1. Perhitungan jumlah sel (Cell
counting) dengan
Hemositometer, Coulter counter,
Flow cytometer
2. Pengamatan siklus pertumbuhan
3. Determinasi biokimia
dan sitometri
4. Analisis DNA – 4,6-diamidino-2-
phenylindole (DAPI)
5. Analisis Protein: spektrofotometri
– metode Lowry, Bradford
 The level of cellular confluency is
important as the cells change
their growth with changing
densities.
• Low-density cells (10-20%)
usually grow slower than 50%
confluent cells.
• If the plate is completely grown
by cells, they will tend to grow
much slower again. And the
changes of growth rate will
influence their genetic program,
As a general guide, from a confluent flask of cells: 1:2 split behavior in experiments and
should be 70-80% confluent and ready for an experiment in 1
to 2 days. 1:5 split should be 70-80% confluent and ready for transfection efficiency.
an experiment in 2 to 4 days. 1:10 split should be 70-80% • The best cellular confluency rate is
confluent and ready for sub-culturing or plating in 4 to 6 days.
approximately 90%.
 cryogenic freezing

KRIOPROTEKSI
(DMSO/gliserol 5-20%)
an perlahan 
sel terdehidrasi, hidup
an cepat  sel mati karena adanya Kristal es internal